Making the best of the worst of times: traits underlying combined … · 2016-05-13 · Ruscus...
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Making the best of the worst of times: traits underlying combined shade and drought tolerance of Ruscus aculeatus and Ruscus microglossum (Asparagaceae) Alexandria Pivovaroff A,C , Rasoul Sharifi B , Christine Scoffoni B , Lawren Sack B and Phil Rundel B A Department of Botany and Plant Sciences, University of California, 2150 Batchelor Hall, Riverside, CA 92521, USA. B Department of Ecology and Evolutionary Biology, University of California, 621 Charles E Young Drive South, Los Angeles, CA 90095-1606, USA. C Corresponding author. Email: [email protected] Abstract. The genus Ruscus (Asparagaceae) consists of evergreen, woody monocot shrubs with modified photosynthetic stems (phylloclades) that occur in dry, shaded woodland areas of the Mediterranean Basin and southern Europe. The combined drought and shade tolerance of Ruscus species challenges the ‘trade-off model’, which suggests that plants can be either drought or shade adapted, but not both. To clarify the potential mechanisms that enable Ruscus species to survive in shaded environments prone to pronounced soil drought, we studied form–function relations based on a detailed trait survey for Ruscus aculeatus L. and Ruscus microglossum Bertol., focusing on gas exchange, hydraulics, morphology, anatomy, and nutrient and isotope composition. We then compared these trait values with published data for other species. R. aculeatus and R. microglossum exhibited numerous traits conferring drought and shade tolerance via reduced demand for resources in general and an ability to survive on stored water. Specific traits include thick phylloclades with low rates of maximum photosynthetic CO 2 assimilation, low stomatal conductance to water vapour (g s ), low respiration rate, low light compensation point, low shoot hydraulic conductance, low cuticular conductance, and substantial water storage tissue. Ruscus carbon isotope composition values of –33 ‰ were typical of an understory plant, but given the low g s could be associated with internal CO 2 recycling. Ruscus appears to be a model for extreme dual adaptation, both physiologically and morphologically, enabling its occupation of shaded sites within drought prone regions across a wide geographical range, including extremely low resource understory sites. Additional keywords: carbon isotopes, functional morphology, gas exchange, hydraulic conductance, Mediterranean climate, phylloclades, understory. Received 4 March 2013, accepted 14 July 2013, published online 28 August 2013 Introduction Plant ecological distributions are constrained by several factors including tolerance of environmental conditions such as light and water availability (Sack 2004; Niinemets and Valladares 2006; Hallik et al. 2009; Sterck et al. 2011). According to the ‘trade-off model’ hypothesised by Smith and Huston (1989), a plant’s adaptations can either allow it to tolerate low light or low water availability. However, many plant species have been reported to tolerate sites prone to strong combinations of drought and shade, including Ruscus aculeatus L. (Sack et al. 2003b), which occurs in dry, shaded understory habitats subjected to annual seasonal drought. Previous studies have shown several species can tolerate combined shade and drought in experiments (Sack 2004; Martínez-Tillería et al. 2012) and in the field (Caspersen 2001; Engelbrecht and Kursar 2003; Niinemets and Valladares 2006), but the physiological mechanisms contributing to this ability have not received detailed study. The ability of Ruscus species to survive very strong combinations of shade and drought in the field and in experiments (Sack et al. 2003b; Sack 2004) makes it a model for such dual adaptation. However, the species have received little detailed study, and previous work has emphasised its adaptation via phenology (de Lillis and Fontanella 1992; Martínez-Pallé and Aronne 1999), high biomass allocation to roots, and its apparent conservative resource use (Sack et al. 2003b). The objective of this research was to clarify the wide range of potential adaptations of Ruscus that contribute to its remarkable ability to survive and regenerate in shaded sites prone to occasional or seasonal soil drought. To achieve this objective, we studied 57 traits relating to gas exchange, hydraulics, morphology, anatomy, and nutrient and carbon isotope composition in two Ruscus species, R. aculeatus and Ruscus microglossum Bertol. (Fig. 1). We then compared trait values CSIRO PUBLISHING Functional Plant Biology http://dx.doi.org/10.1071/FP13047 Journal compilation Ó CSIRO 2013 www.publish.csiro.au/journals/fpb
Transcript of Making the best of the worst of times: traits underlying combined … · 2016-05-13 · Ruscus...
Making the best of the worst of times traits underlyingcombined shade and drought tolerance ofRuscus aculeatusand Ruscus microglossum (Asparagaceae)
AlexandriaPivovaroffAC RasoulSharifiBChristineScoffoniB LawrenSackBandPhilRundelB
ADepartment of Botany and Plant Sciences University of California 2150 Batchelor Hall RiversideCA 92521 USA
BDepartment of Ecology and Evolutionary Biology University of California 621 Charles E Young Drive SouthLos Angeles CA 90095-1606 USA
CCorresponding author Email alexandriapivovaroffemailucredu
Abstract The genusRuscus (Asparagaceae) consists of evergreen woodymonocot shrubs withmodified photosyntheticstems (phylloclades) that occur in dry shaded woodland areas of the Mediterranean Basin and southern Europe Thecombined drought and shade tolerance of Ruscus species challenges the lsquotrade-off modelrsquo which suggests that plants canbe either drought or shade adapted but not both To clarify the potential mechanisms that enable Ruscus species to survivein shaded environments prone to pronounced soil drought we studied formndashfunction relations based on a detailed traitsurvey for Ruscus aculeatus L and Ruscus microglossum Bertol focusing on gas exchange hydraulics morphologyanatomy and nutrient and isotope composition We then compared these trait values with published data for other speciesR aculeatus and R microglossum exhibited numerous traits conferring drought and shade tolerance via reduced demandfor resources in general and an ability to survive on stored water Specific traits include thick phylloclades with low rates ofmaximum photosynthetic CO2 assimilation low stomatal conductance to water vapour (gs) low respiration rate low lightcompensation point low shoot hydraulic conductance low cuticular conductance and substantial water storage tissueRuscus carbon isotope composition values of ndash33 permil were typical of an understory plant but given the low gs could beassociated with internal CO2 recycling Ruscus appears to be a model for extreme dual adaptation both physiologicallyand morphologically enabling its occupation of shaded sites within drought prone regions across a wide geographicalrange including extremely low resource understory sites
Additional keywords carbon isotopes functional morphology gas exchange hydraulic conductance Mediterraneanclimate phylloclades understory
Received 4 March 2013 accepted 14 July 2013 published online 28 August 2013
Introduction
Plant ecological distributions are constrained by several factorsincluding tolerance of environmental conditions such as lightand water availability (Sack 2004 Niinemets and Valladares2006 Hallik et al 2009 Sterck et al 2011) According to thelsquotrade-off modelrsquo hypothesised by Smith and Huston (1989) aplantrsquos adaptations can either allow it to tolerate low light or lowwater availability However many plant species have beenreported to tolerate sites prone to strong combinations ofdrought and shade including Ruscus aculeatus L (Sack et al2003b) which occurs in dry shaded understory habitatssubjected to annual seasonal drought
Previous studies have shown several species can toleratecombined shade and drought in experiments (Sack 2004Martiacutenez-Tilleriacutea et al 2012) and in the field (Caspersen 2001Engelbrecht and Kursar 2003 Niinemets and Valladares 2006)but the physiological mechanisms contributing to this ability
have not received detailed study The ability of Ruscus speciesto survive very strong combinations of shade and drought in thefield and in experiments (Sack et al 2003b Sack 2004) makes ita model for such dual adaptation However the species havereceived little detailed study and previous work has emphasisedits adaptation via phenology (de Lillis and Fontanella 1992Martiacutenez-Palleacute and Aronne 1999) high biomass allocation toroots and its apparent conservative resource use (Sack et al2003b)
The objective of this research was to clarify the wide range ofpotential adaptations of Ruscus that contribute to its remarkableability to survive and regenerate in shaded sites prone tooccasional or seasonal soil drought To achieve this objectivewe studied 57 traits relating to gas exchange hydraulicsmorphology anatomy and nutrient and carbon isotopecomposition in two Ruscus species R aculeatus and Ruscusmicroglossum Bertol (Fig 1) We then compared trait values
CSIRO PUBLISHING
Functional Plant Biologyhttpdxdoiorg101071FP13047
Journal compilation CSIRO 2013 wwwpublishcsiroaujournalsfpb
with those hypothesised to confer shade tolerance droughtavoidance or both Overall for 21 traits we had a priorihypotheses of a benefit for shade tolerance and for 24 traits wehad a priori hypotheses of a benefit for drought avoidance Wethen compared the traits for Ruscus with values compiled fromthe literature for (1) temperate and tropical broadleaf evergreenspecies (2) Mediterranean species and (3) woody angiospermsin general in order to put Ruscus trait values in a global contextThis approach involved measuring a large number of key aspectsof structure and function and when possible compiling specifichypotheses for traits potentially involved in the shade anddrought tolerance Ruscus species relative to comparatorspecies (Tables 1ndash5) According to the previous literature onshade and drought tolerance (for example Givnish 1988 Jones1992) these suites of traits in Ruscus are expected to directly orindirectly contribute to mechanisms operating across cell typesand levels of leaf organisation conferring combined shade anddrought adaptation
Thus on the general understanding of shade and droughttolerance traits we hypothesised that Ruscus species wouldhave mechanisms of drought adaptation including traitsenabling the delay of tissue dehydration and traits enablingmaintained function even as tissue dehydrates Such traitsinclude a high water-use efficiency (Wright and Westoby2003) as well as water storage tissue with high water storagecapacitance associated with low bulk leaf modulus of elasticityhigh relative water content at turgor loss point and low cuticularconductance (Sack et al 2003a Pasquet-Kok et al 2010Ogburnand Edwards 2012) Traits potentially contributing to shadetolerance include low rates of maximum photosynthetic CO2
assimilation per leaf area and per leaf mass low lightcompensation point low maximum rate of carboxylation lowmaximum rate of electron transport and more negative carbon
isotope ratios (Walters and Reich 1999) Traits that potentiallyconfer a combined drought and shade tolerance through a generalconservative and cost-efficient resource use include thick laminawith thick epidermis low respiration rates per area andmass lowstomatal conductance and low shoot hydraulic conductance(Sack et al 2003b) Given the exceptional biology of thesespecies ndash their extreme tolerance and their possession ofphylloclades ndash we also qualified additional traits in particularthe detailed anatomical traits such as cell sizes for which wecould not compile hypotheses due to the paucity of comparativedata in the published literature However such anatomical traitshave been argued to be strongly associated with environmentaladaptation in principle (Haberlandt 1914) and thus these datafor Ruscus are likely to be important as future studies providecomparative data for many species
Materials and methodsStudy species and site
Ruscus (Asparagaceae) is a genus of six species of evergreensclerophyllous woody shrubs native to western and southernEurope (including north to southern England) Macaronesianorth-west Africa and south-western Asia ranging east to theCaucasus (de Lillis and Fontanella 1992 Martiacutenez-Palleacute andAronne 1999) Ruscus is thus found in a wide range oftemperate forests as well as in Mediterranean-type climatescharacterised by wet cool winters and dry warm summersthat result in an annual seasonal period of low wateravailability or drought (Matalas 1963 Dracup 1991 Cowlinget al 1996) Ruscus exhibits phylloclades which are flattenedphotosynthetic stems that resemble leaves and are consideredintermediate organs that combine stem and leaf features (Fig 1Cooney-Sovetts and Sattler 1987) Ruscus aculeatus L is a
Table 1 Mean values for morphological traits se for Ruscus aculeatus and R microglossum with units and replicationFor given traits expectations are given forwhetherRuscus should have a higher or lower value relative to comparator species according to the hypotheses of shadeor drought adaptation Comparator species data were taken according to availability in the previously published literature and number of species and minimummean andmaximum trait values are provided ormean se if only thesewere available Sources of comparative data leaf area (Sack et al 2012) LMA (Wright
et al 2004) density (Niinemets 1999) LDMC (Vile 2005) and SWC (Vendramini et al 2002 Ogburn and Edwards 2012)
Morphological traits Units R aculeatus R microglossum Hypotheses Comparator speciesMean plusmn se N Meanplusmn se N Shade
adaptedDroughtadapted
(N) (min mean max)
Leaf area cm2 180 plusmn 006 92 193 plusmn 034 84 LowerA Dicots (485) 010 178 280Leaf mass per area (LMA) gmndash2 1228 plusmn 89 92 910 plusmn 09 84 Higher LowerABC Temperate broadleaf
evergreen (132)580 153 429
Tropical broadleafevergreen (72)
406 145 370
Density g cmndash3 039 plusmn 003 10 027 plusmn 001 10 Higher LowerABC Woody trees andshrubs (38)
009 041 133
Leaf dry mattercontent (LDMC)
g∙gndash1 0389plusmn 0016 6 0310plusmn 0005 6 HigherA LowerBC Shrubs (gt100) 0384plusmn 00084
Saturated watercontent (SWC)
g∙gndash1 159 plusmn 012 6 223 plusmn 005 6 HigherBC Evergreen trees andshrubs (6)
067 182 60
Succulents (45) 170 117 520
AAn expectation according to a hypothesis was confirmed for R aculeatusBAn expectation according to a hypothesis was confirmed for R microglossumCExpectations for these traits are based on drought tolerance conferred by water storage tissue (lsquosucculencersquo) the opposite expectations would arise for droughttolerance conferred by the ability to maintain turgor with dehydration
B Functional Plant Biology A Pivovaroff et al
Tab
le2
Meanva
lues
foran
atom
ical
traitsseforRuscusaculeatusan
dRm
icroglossumw
ithun
itsan
dreplication
Forgiventraitsexpectatio
nsaregivenforw
hetherRuscusshouldhave
ahigherorlowervaluerelativ
etocomparatorspeciesaccordingtothehypothesesofshadeordroughtadaptationCom
paratorspeciesdata
weretakenaccordingtoavailabilityinthepreviouslypublishedliteratureandnumbero
fspeciesand
minim
umm
eanandmaxim
umtraitvaluesareprovidedSources
ofcomparativ
edatatissuethicknessand
airspace
(SackandFrole20
06)Dm(Brodribbetal2
007)N
Sn
otstatistically
significant
atPlt005
Anatomicaltraits
Units
Ra
culeatus
Rm
icroglossum
Hypotheses
Com
paratorspecies
Meanplusmnse
NMeanplusmnse
NShade
adapted
Drought
adapted
(N)
(minm
ean
max)
Tissuethicknessesan
dairspace
Lam
ina
mm27
8plusmn334
529
6plusmn857
5HigherA
BHigherA
BTropicalevergreen
trees(10)
156
267
512
Cuticle
mm352
plusmn022
5329
plusmn023
5Higher
Higher
Tropicalevergreen
trees(10)
1254
60
105
Epiderm
ismm
202plusmn0513
523
plusmn0657
5HigherA
BHigherA
BTropicalevergreen
trees(10)
9751
40
173
Epiderm
iscellwall
mm341
plusmn011
5369
plusmn025
5Mesop
hyll
mm72
6plusmn179
583
0plusmn272
5Water
storage
mm88
7plusmn705
586
2plusmn587
5Present
AB
totalleaf
airspace
10
5plusmn138
520
8plusmn173
5
Celldimension
sEpiderm
iscellarea
mm2
447plusmn27
05
599plusmn35
35
Epiderm
iscellperimeter
mm78
9plusmn261
594
1plusmn278
5Mesop
hyllcellarea
mm2
657plusmn21
55
846plusmn42
65
Mesophyllcellperimeter
mm94
5plusmn172
511
0plusmn241
5
chloroplastarea
inmesop
hyll
cell
21
2plusmn149
521
1plusmn307
5
Mesop
hyllareatotalleaf
area
(AmesA)
247plusmn13
520
5plusmn045
5
Water
storagecellarea
mm2
3414
plusmn19
65
5972
plusmn84
75
Water
storagecellperimeter
mm22
1plusmn572
529
1plusmn19
15
Vasculartraits
Minim
umdistance
from
vein
toepidermis(D
m)
mm20
3plusmn18
05
370plusmn49
35
HigherB
Low
erA
Dicotyledons
(11)
129
233
428
Inter-veinaldistance
(IVD)
mm27
2plusmn27
55
509plusmn71
55
Fibrous
bundlesheath
cellheight
mm18
4plusmn127
517
5plusmn149
5Fibrous
bundlesheath
cellwidth
mm17
4plusmn066
516
3plusmn092
5Fibrous
bundlesheath
cellwall
thickness
mm457
plusmn041
5248
plusmn023
5
Average
theoreticalxylemconduit
cond
uctiv
ityin
themidrib
10
5mmolm
ndash2
sndash1MPandash
1124
plusmn025
5198
plusmn055
5
Average
theoreticalxylemconduit
conductiv
ityin
aninterm
ediary
vein
10
5mmolm
ndash2
sndash1MPa
ndash1
063
plusmn014
5119
plusmn038
5
Average
theoreticalxylemconduit
cond
uctiv
ityin
aminor
vein
10
5mmolm
ndash2
sndash1MPandash
1037
plusmn012
5028
plusmn006
5
(contin
uednextpa
ge)
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology C
typical understory species in forests in the Mediterranean basin(Fig 1 de Lillis and Fontanella 1992Martiacutenez-Palleacute andAronne1999 Sack et al 2003b)RuscusmicroglossumBertol is a hybridproduced by crossing species Ruscus hypoglossum L from theBlack Sea region andRuscus hypophyllumL fromNorthAfrica(Fig 1 Thomas 1992 USDA ARS National Genetic ResourcesProgram 2009)
Experiments were conducted on R aculeatus andR microglossum plants in the Mildred E Mathias BotanicalGarden at the University California Los Angeles from Juneto August 2009 Measurements were made in shaded understorysites on at least three individuals of each species Diurnal lightmeasurements were made on sunny days above the Ruscus plantcanopies with a quantum sensor (Li-190S Li-Cor BiosciencesLincoln NE USA) Individuals received full sunlight averaging~1500mmolmndash2 sndash1 photosynthetically active radiation (PAR)for ~1 h each day and otherwise experienced PAR of~50mmolmndash2 sndash1 interspersed with sunflecks averaging~190mmolmndash2 sndash1 Plants were irrigated as needed and allRuscus individuals were watered before the start of this studyto reduce any differences in water availability betweenindividuals
Phylloclade morphology
We determined leaf morphological traits on recently formedmature phylloclades Although methods were applied tophylloclades for convenience we retained the names ofmethods and traits as applied to leaves (eg leaf mass perarea) Phylloclade area was measured on excised samples withan areameter (Li-3100 Li-Cor Biosciences) Samples were driedin an oven at gt70C for more than 48 h to determine drymass andcalculate leaf mass per area (LMA) as dry mass divided by areaPhylloclade thickness was measured with electronic digitalcalipers (Fisher Scientific Pittsburgh PA USA) and densitywas calculated as mass per area divided by thickness (Witkowskiand Lamont 1991)
Phylloclade anatomy
We sampled phylloclades of each species and prepared cross-sections for anatomical measurements Phylloclades werepreserved in formalin acetic acid (37 formaldehyde glacialacidic acid 95 ethanol and deionised water in a 10 5 50 35mixture) We measured the transverse cross-sectional anatomyusing sections cut halfway along the phylloclade lengthembedded in LR White (London Resin Co London UK) cut05mm thick using a microtome (Ultracut E Reichert-JungUltracut E Leica Microsystems Arcadia CA USA) stainedwith 001toluidine blue in 1sodiumborate andviewedunderthe light microscope using a 20ndash40 objective (DMRB LeicaMicrosystems Wetzlar Germany)
We measured tissue thicknesses and cell dimensions in thelamina and dimensions of vascular bundles and of xylemconduits using Image J software (ver 142 q NationalInstitutes of Health Bethesda MD USA) on microscopeimages We measured thickness for the lamina cuticleepidermis epidermis cell wall mesophyll and water storagecompartment We averaged three measurements of each type foreach cross-section The phylloclade tissues were arranged
Tab
le2
(contin
ued)
Anatomicaltraits
Units
Ra
culeatus
Rm
icroglossum
Hyp
otheses
Com
paratorspecies
Meanplusmnse
NMeanplusmnse
NShade
adapted
Droug
htadapted
(N)
(minm
ean
max)
Meanmaxim
umxy
lem
cond
uit
diam
eter
inthemidrib
mm966
plusmn044
511
0plusmn078
5
Meanmaxim
umxy
lem
cond
uit
diam
eterinan
interm
ediaryvein
mm831
plusmn055
5949
plusmn072
5
Meanmaxim
umxy
lem
cond
uit
diam
eter
inaminor
vein
mm715
plusmn072
5719
plusmn048
5
AAnexpectationaccordingto
ahypothesiswas
confi
rmed
forRa
culeatus
BAnexpectationaccordingto
ahypothesiswas
confi
rmed
forRm
icroglossum
D Functional Plant Biology A Pivovaroff et al
Tab
le3
Meanva
lues
forga
sexchan
getraitsseforRuscusaculeatusan
dRm
icroglossumw
ithun
itsan
dreplication
Forgiventraitsexpectatio
nsaregivenforw
hetherRuscusshouldhave
ahigherorlowervaluerelativ
etocomparatorspeciesaccordingtothehypothesesofshadeordroughtadaptationCom
paratorspeciesdata
weretakenaccordingtoavailabilityinthepreviouslypu
blishedliteratureandnu
mberofspeciesand
minim
umm
eanandmaxim
umtraitvaluesareprov
idedSourcesofcomparativ
edataA
areaA
massRareaR
mass
g s(W
rightetal2
004)LCP(W
altersandReich
1999)Ag
s(G
uliasetal2
003)VcmaxJ
max(W
ullschleger19
93)g m
in(K
erstiens
1996)NSn
otstatistically
significant
atPlt005
Gas-exchang
etraits
Abb
reviation
Units
Ra
culeatus
Rm
icroglossum
Hypotheses
Com
paratorspecies
Meanplusmnse
NMeanplusmnse
NShade
adapted
Droug
htadapted
(N)
(minm
ean
max)
Light-saturated
rateof
photosyn
thesisperarea
Aarea
mmolm
ndash2sndash
1522
plusmn133
451
plusmn047
6Low
erAB
Tem
peratebroadleaf
evergreens
(78)
2629
42
2265
Light-saturated
rateof
photosyn
thesisperm
ass
Amass
nmolCO2gndash
1sndash
1004
3plusmn001
14
0050plusmn0005
6Low
erAB
Tem
peratebroadleaf
evergreens
(78)
2287
46
209
Respiratio
nrateperarea
Rarea
mmolm
ndash2sndash
1004
4plusmn000
98
0156plusmn0015
10Low
erAB
Low
erAB
Tem
peratebroadleaf
evergreens
(50)
0320
93
170
Respiratio
npermass
Rmass
nmolCO2gndash
1sndash
1356
E-04plusmn702
E-05
8171E-03plusmn166E-04
10Low
erAB
Low
erAB
Tem
peratebroadleaf
evergreens
(50)
3337
06
157
Maxim
umstom
atal
conductanceperarea
g smmolm
ndash2sndash
133
plusmn000
74
35plusmn0006
6Low
erAB
Low
erAB
Tem
peratebroadleaf
evergreens
(48)
491
423
09
Light
compensationpo
int
LCP
mmol
photonsm
ndash2sndash
1485
plusmn0
4361
plusmn0
6Low
erAB
Tropicalevergreenshade-
tolerators(15)
16582
4
Intrinsicwater
use
efficiency
Ag
smm
olmol
ndash1
154plusmn8
414
2plusmn19
06
HigherA
BMediterraneanspecies
(78)
2335
94
142
Ratio
ofintercellularto
ambient[CO2]
CiC
a032
7plusmn010
64
0431plusmn0064
6
Maxim
umrateof
carbox
ylation
Vcmax
mmolm
ndash2sndash
126
6plusmn318
424
7plusmn542
5Low
erAB
Tem
peratehardwood
species(19)
114
711
9
Maxim
umrateof
electron
transport
J max
601plusmn635
550
6plusmn786
5Low
erAB
Tem
peratehardwood
species(19)
291
042
37
Leafcuticularcond
uctance
g min
mmolm
ndash2sndash
1037
9plusmn008
210
0295plusmn0082
12Low
erAB
Vascularplants(201)
01
1321
07Stem
cuticular
conductance
g min
mmolm
ndash2sndash
1009
5plusmn002
56
0030plusmn0003
4
AAnexpectationaccordingto
ahypothesiswas
confi
rmed
forRa
culeatus
BAnexpectationaccordingto
ahy
pothesiswas
confi
rmed
forRm
icroglossum
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology E
Tab
le4
Meanva
lues
forhy
drau
licstraitsseforRuscusaculeatusan
dRm
icroglossumw
ithun
itsan
dreplication
Forgiventraitsexp
ectatio
nsaregivenforw
hetherRuscusshouldhave
ahigh
erorlowervaluerelativ
etocomparatorspeciesaccordingtothehy
pothesesofshadeordrough
tadaptationCom
paratorspeciesdata
weretakenaccordingtoavailabilityinthepreviouslypu
blishedliteratureandnu
mberofspeciesand
minim
umm
eanandmaxim
umtraitvaluesareprov
idedorm
eanstandarderrorifon
lythesewereavailable
Sources
ofcomparativ
edataK
shoot(SackandHolbroo
k20
06)Y
tlpR
WCtlpP
oe
(Bartlettetal2
012)Cft(Scoffon
ietal2
008)NSnotstatistically
significant
atPlt005
Hydraulicstraits
Abbreviation
Units
Ra
culeatus
Rm
icroglossum
Hyp
otheses
Com
paratorspecies
Meanplusmnse
NMeanplusmnse
NShade
adapted
Drought
adapted
(N)
(minm
ean
max)
Sho
othy
draulic
cond
uctance
Kshoot
mmolm
ndash2sndash
1
MPandash
1216
plusmn010
10269
plusmn013
11Low
erAB
Low
erAB
Tem
peratewoo
dyangiosperm
s(38)
8plusmn1
Tropicalwoody
angiosperm
s(49)
13plusmn15
Osm
oticpotentialatfull
turgor
p oMPa
ndash128
plusmn010
6ndash065
plusmn003
6HigherA
BHigherA
BC
Evergreen
woo
dyspecies
(182)
-34ndash
183ndash
049
Turgo
rloss
point
Ytlp
MPa
ndash184
plusmn010
6ndash113
plusmn007
6HigherA
BHigherA
BC
Evergreen
woo
dyspecies
(158)
-425
ndash220ndash
054
Modulus
ofelasticity
eMPa
110plusmn141
6588
plusmn053
6Low
erABC
Evergreen
woo
dyspecies(139)
3561
71
734
Relativewater
contentat
turgor
loss
point
RWCtlp
88
8plusmn109
689
3plusmn085
6HigherA
BC
Evergreen
woo
dyspecies(19)
718
12
911
Relativecapacitanceatfull
turgor
Cft
MPandash
10067plusmn0008
60102plusmn0010
6HigherA
BC
Evergreen
species(6)
0040
0066
0113
Relativecapacitanceat
turgor
loss
Ctlp
MPandash
10104plusmn0015
60089plusmn0003
6
Predawnwater
potential
Ypre
MPa
ndash045
plusmn008
4ndash088
plusmn008
4Middaywater
potential
Ymid
MPa
ndash141
plusmn008
3ndash129
plusmn028
4
AAnexpectationaccordingto
ahy
pothesiswas
confi
rmed
forRa
culeatus
BAnexpectationaccordingto
ahypothesiswas
confi
rmed
forRm
icroglossum
CExpectatio
nsforthesetraitsarebasedon
drou
ghttolerance
conferredby
waterstoragetissue(lsquosucculencersquo)the
oppositeexpectations
wou
ldarisefordroughttoleranceconferredby
theabilitytomaintainturgor
with
dehydration
F Functional Plant Biology A Pivovaroff et al
symmetrically with adaxial and abaxial layers of mesophyllepidermis and cuticle above and below a central achlorophyllouswater storage tissue (Fig 2) The per cent air space in each of theadaxial and abaxial mesophyll and in the water storagecompartment was estimated to the nearest 5 and the totalphylloclade airspace was calculated
PAir space in each tissue
fraction of leaf cross section occupied by that tissue100
eth1THORN
As indices of cell size the cross-sectional areas andperimeters were measured for three cells in the epidermis(adaxial and abaxial) the mesophyll (adaxial and abaxialie above and below the water storage tissue) and thewater storage tissue The area occupied by chloroplastswithin a mesophyll cell was measured for three cells(adaxial and abaxial) and the percent cross-sectionalchloroplast area was calculated as the ratio of chlorophyllarea divided by mesophyll cell area
We calculated the surface area of mesophyll cells per leafarea (AmesA) as described by Sack et al (2013a) a measure ofthe area available for CO2 uptake formesophyll cell layers Giventhe lack of palisade-form cells we modelled all mesophyll cellsas spheres for these calculations
We also measured vascular anatomy to quantify traits relatedto the efficiency of water transport within and outside the xylemWe measured the inter-veinal distance (IVD) and also theminimum distance from edge of bundle sheath to epidermis(Dm) as the hypotenuse between the distance between veinsand the distance to the epidermis (Brodribb Feild and Jordan2007)
Dm frac14ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
IVD2
2thorn ethdistance from bundle sheath edge to epidermisTHORN2
s
eth2THORN
We averaged IVD and Dm from three values for each cross-section
For all anatomical traits except those relating to the centralwater storage tissue measurements were made both adaxial andabaxial halves and values for the two halveswere averagedwhennot significantly different (at P lt 005 in paired t-tests) exceptthey were summed for total AmesA
To characterise the midrib for three typical fibrous bundlesheath cells we measured the cross-sectional heights widthsand cell wall thicknesses calculating mean values for each traitfrom three measurements per cross-section To characterise thexylem anatomy and theoretical conductivity of xylem conduitsfor a typical conduit within the midrib an intermediary veinand a minor vein of each sampled phylloclade we treated theconduit as an ellipse and determined the major and minor axisdiameters We calculated the theoretical hydraulic conductivityof the xylem conduit using Poiseuillersquos equation for ellipsesbased on conduit dimensions (Lewis and Boose 1995 CochardNardini and Coll 2004)
Tab
le5
Meanvalues
fortissue
compo
sition
traitsseforRuscusaculeatusan
dRm
icroglossumw
ithun
itsan
dreplication
andsign
ificanceof
differencesbetw
eenspecies(t-tests)
Forgiventraitsexpectatio
nsaregivenforw
hetherRaculeatus
shouldhave
ahigherorlowervaluerelativ
etocomparatorspeciesaccordingtothehypothesesofshadeordroughtadaptationCom
paratorspecies
dataweretakenaccordingtoavailabilityinthepreviouslypublishedliteratureandnumberofspeciesand
minim
umm
eanandmaxim
umtraitvaluesareprovidedSourcesofcomparativ
edatad
13C(K
oumlrneretal
1991)NmassNarea(W
righ
tetal2
004)N
Sn
otstatistically
significant
atPlt005
Tissuecompositio
ntraits
Abbreviation
Units
Ra
culeatus
Rm
icroglossum
Hypotheses
Com
paratorspecies
Meanplusmnse
NMeanplusmnse
NShade
adapted
Drought
adapted
(N)
(minm
ean
max)
Npermass
Nmass
194
plusmn006
9192
plusmn007
9Low
erHigherA
BTem
peratebroadleaf
evergreen(129)
0581
19
231
Nperarea
Narea
238
plusmn007
9175
plusmn006
9Low
erHigherA
BTem
peratebroadleaf
evergreen(129)
0727
1733
63
Cpermass
Cmass
44
4plusmn048
943
6plusmn052
9Carbonto
nitrogen
ratio
CN
231plusmn068
922
9plusmn058
9Carbonisotoperatio
d13C
permilndash33
32plusmn029
9ndash33
05plusmn020
9Low
erAB
Higher
Tem
peratespecies(35)
ndash27
64
ndash26
03
ndash24
09
AAnexpectationaccordingto
ahypothesiswas
confi
rmed
forRa
culeatus
BAnexpectationaccordingto
ahy
pothesiswas
confi
rmed
forRm
icroglossum
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology G
K t frac14 pa3b3
64hetha2 thorn b2THORN eth3THORN
where a and b are the major and minor axes of the ellipse and his water viscosity at 25C
Gas-exchange measurements responses to light and CO2and cuticular conductance
In July and August 2009 photosynthetic light response curvesandCO2 response curvesweremeasuredusing aLi-6400portablephotosynthesis system (Li-Cor Biosciences ) with light provided
Ruscusaculeatus
Ruscusmicroglossum
01 mm
Fig 2 Lamina and midrib cross-sections of Ruscus aculeatus and R microglossum phylloclades (05mm thick) Note that bothspecies exhibit shade tolerance features such as absence of palisade tissue as well as drought tolerance features such as large waterstorage compartment and thick-walled epidermis andfibrousbundle sheath cells surrounding thexylemandphloemfor bothmajor andminor veins especially prominent in R aculeatus which is native to drier habitats
(a) (b)
Fig 1 (a)Ruscus aculeatus and (b)Rmicroglossumgrowing at theMildredEMathiasBotanicalGardenNote thatwhat atfirst glance appear to be leaves are infact phylloclades
H Functional Plant Biology A Pivovaroff et al
by a red-blue light source (6400ndash02B no SI-710 Li-CorBiosciences ) Gas-exchange measurements were made on atleast 1ndash2 phylloclades per individual
For light-response curves phylloclades were acclimatedfor at least 5min at 1500mmolmndash2 sndash1 PAR at temperatures of2527C with RH maintained at ~50 and CO2 concentrationof 400mmolmolndash1 Then phylloclades were measured for netCO2 assimilation per leaf area at PAR steps of 1600 1400 12001000 800 600 500 400 300 200 100 50 and 0mmolmndash2 sndash1with 180ndash240 s stabilisation at each irradiance step Wedetermined light-saturated photosynthetic rate per area andmass (Aarea and Amass) dark respiration rate per area and mass(Rarea and Rmass) ie the negative A at zero PAR maximumstomatal conductance per area (gs) light compensation point(LCP) as the x-intercept intrinsic water use efficiency (WUEAareags) and the ratio of intercellular to ambient CO2
concentration (CiCa) at 100mmolmndash2 sndash1 PAR We thenharvested the phylloclades measured for gas exchange todetermine the nitrogen concentration and carbon isotope ratio(see below)
For CO2-response curves phylloclades were allowed toequilibrate at 400 ppm to induce stomatal opening and the netCO2 assimilation per leaf area was determined at Ci steps of400 300 200 100 50 400 400 500 600 800 1200 14001600 ppm with 3ndash4min equilibration time at each step Wedetermined maximum rate of carboxylation and maximum rateof electron transport per leaf area (Vcmax and Jmax) from plots ofCi vs A corrected to 25C (Farquhar and von Caemmerer 1980)
Cuticular conductance (ie minimumepidermal conductancegmin sensu Kerstiens 1996) was determined for 3ndash4 maturephylloclades and 10 cm lengths of stems from each of threeindividuals of each species Phylloclade and stem sampleswere hydrated and then the cut ends were sealed with waxSamples were dried for at least 30min on a laboratory bench atPAR of lt10mmol photons mndash2 sndash1 to induce stomatal closurethen samples were weighed for at least eight intervals of 30minduring which the slope of water loss versus time was highlylinear (R2 gt 0995) and therefore taken to represent transpirationafter stomata had closed fully The gmin was calculated as thetranspiration rate divided by the mole fraction vapour pressuredeficit (VPD determined from a weather station HOBO MicroStation with Smart Sensors Onset Bourne MA USA)
Pressurendashvolume curve parametersPressurendashvolume curve parameters were determined for matureshoots using the bench-drying method (Koide et al 2000 Sacket al 2003a) Shoots were ~10ndash15 cm long with about fourphylloclades per shoot for R microglossum and 15ndash20phylloclades for R aculeatus Shoots were progressively driedon a laboratory bench and measured at intervals by equilibratingfor 10min in a Whirlpak bag (Whirl-Pak Nasco Fort AtkinsonWIUSA) beforeweighing andmeasuring for leafwater potential(Yleaf) with a pressure chamber (Model 1000 Plant MoistureStress Instruments Albany OR USA) Subsequently dry masswas determined after more than 48 h in an oven at 70C Wedetermined the leaf dry matter content (LDMC) saturated watercontent (SWC) turgor loss point (Ytlp) relative water content atturgor loss point (RWCtlp) and osmotic potential at full turgor
(po) relative capacitance at full turgor (Cft DRWCDYleaf) andrelative capacitance at turgor loss point (Ctlp DRWCDYleaf) Wedetermined the modulus of elasticity (e) as the linear slope of theline fitted for pressure potential versus relative water contentabove and including turgor loss point (Sack et al 2013b)
Shoot hydraulic conductance
We measured the hydraulic conductance of mature shoots(Kshoot) ~10ndash15 cm long bearing about four phylloclades pershoot for R microglossum and 15ndash20 phylloclades forR aculeatus using the evaporative flux method (Sack et al2002) Samples were harvested and the ends were re-cut underdistilled degassed water with a fresh razor blade then hydratedovernight Shoots were connected to tubing containing distilleddegassed water running to a graduated cylinder on a balanceinterfaced to a computer logging data every minute to calculatethe flow rate of water from the balance into the sample Sampleswere held in place above a fan on wood frames strung withfishing line Lights were arranged above a plexiglass containerof water that acted as a heat trap producing PAR ofgt1200mmolmndash2 sndash1 at shoot level When steady-statetranspiration was achieved samples were covered with aplastic bag and removed from the tubing and the leaf waterpotential was determined with a pressure chamber (Model 1000Plant Moisture Stress Instruments) Kshoot was calculated as thesteady-state transpirational flow rate (E mmolmndash2 sndash1) dividedby the water potential driving force (DYleaF= ndashYleaf MPa)further normalised by total phylloclade area Kshoot valueswere standardised to 25C to correct for the temperaturedependence of the viscosity of water (Sack et al 2003a)
Foliar nitrogen concentration and carbon isotope ratio
To analyse total tissue nitrogen concentration and carbon isotopecomposition (d13C) for three replicates from each of threeindividuals for each species phylloclade samples were oven-dried at gt70C for more than 48 h and ground by mortar andpestle The nitrogen concentration and d13C were determined atthe UCDavis Stable Isotope Facility using an elemental analyser(PDZEuropaANCA-GSL SerconLtd Cheshire UK) interfacedto an isotope ratiomass spectrometer (PDZEuropa20ndash20 SerconLtd) at the UC Davis Stable Isotope Facility Final d13C contentvalues are expressed relative to international standardVienna PeeDee belemnite (V-PDB) for carbon
d13CethpermilTHORN frac14 etheth13C=12C of sampleTHORN=eth13C=12C of standardTHORN 1THORN 1000
eth4THORN
d13C of plant tissues can provide a measure of intrinsic WUE(Ags) at the time that carbon was assimilated giving long-termwater-use efficiency
Comparative data compilation and trait comparison
To consider Ruscus trait values in a global context we compileddata from the literature for (1) temperate and tropical broadleafevergreen species (2) Mediterranean species and (3) woodyangiosperms in general When available we considered data forplants grown in the shade and for species that are shade tolerant
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology I
however eco-physiological trait data is generally collected forsun leaves or plants making a comparison solely for shade grownplants not possible Formore commonly studied traits (egAmax)comparative data was taken from studies with large databasesand multiple traits (eg GLOPNET Wright et al 2004) Wedetermined minimum mean and maximum values for traitsfrom comparative studies or the mean and standard error ifraw species values were not available Studies that wereincluded for comparative data are referenced in the captionsfor Table 1ndash5 in association with the specific traits wecompared Differences between Ruscus trait values andcomparative data from the literature were determined for bothR aculeatus and R microglossum Hypotheses were deemedsupported for a Ruscus species if the trait value was higher orlower than the mean comparator value in the way predicted
Results
Both Ruscus species studied had leaf morphology consistentwith adaptation to shade and drought relative to comparator
species (Tables 1ndash5)We constructed a radar graph to encapsulatethe key traits that would confer adaptation to shade drought andthe combination for R aculeatus which had the more extremeadaptation of the two Ruscus species considered (Fig 3) Thisfigure summarises the major results of our study with values forcomparator species appearing as the inner circle and R aculeatustrait values displayed as the bold outer line
Gross morphology of phylloclades
Both R aculeatus and R microglossum had LMA valueslower than comparator species and R microglossum hadphylloclades larger than the mean leaf area for comparatorspecies Both Ruscus species had lower bulk tissue density thancomparator species consistent with the water storage in theRuscus phylloclades Notably despite its water storage tissuethe LDMC and SWC values of R aculeatus were typical ofthose for comparator species whereas R microglossum had alower LDMC and a higher SWC than for comparator speciessets (Table 1)
Fig 3 Radar graph illustrating percent difference between selected traits of Ruscus aculeatuswithcomparative data (see Tables 1ndash5 for symbols and sources of comparative data) Values forR aculeatus outside the circle indicate shade andor drought tolerance Traits are arrangedaccording to whether they would contribute shade tolerance drought tolerance or both The innerdisc represents the mean for comparative species for given traits and the values for R aculeatus isscaled relative to the magnitude of that value () with a value outside the disc representing greatershade andor drought tolerance (+) and (ndash) indicate if the axis is scaled such that a value outside thecircle represents percent higher or lower than comparative values respectively For traits expressed asnegative values (+) and (ndash) indicate more negative or less negative respectively
J Functional Plant Biology A Pivovaroff et al
Anatomy of phylloclades
The Ruscus species possessed numerous anatomical traitsconsistent with benefits for both shade and drought tolerance(Table 2) The phylloclade cross-sections were symmetrical(Fig 2) and thus the cross-sectional anatomy of the adaxialand abaxial halves were not different for all traits (paired t-testP gt 005) and values were averaged within species The twospecies were similar in their substantial leaf thickness and inthe thickness of cuticle epidermis cell walls and water storagecompartment (Table 2) The fractions of the lamina occupied bythe epidermis mesophyll and water storage tissues were 1552ndash56 and 29ndash32 respectively
Both species had parallel longitudinal veins of three sizes(midrib intermediate and small veins) Consistent with droughtadaptation the two species had low Dm R aculeatus having thelower value andRmicroglossumhadahigher IVD (Table 2)Thetwo species had on average the same maximum conduitdiameters in their midribs intermediate veins and small veinsand the maximum conduit diameter decreased ~35 from themidrib to the small veins The average theoretical hydraulicconductivity of xylem conduits did not differ between thespecies and decreased by up to 86 from the midrib to theminor vein R aculeatus had on average walls that were 85thicker in the fibrous bundle sheath (Table 2)
Gas-exchange measurements
Consistent with adaptation to simultaneous shade and droughtthe Ruscus species had very low values for Rarea Rmass and gs(Table 3) relative to comparator temperate broadleaf evergreenspecies (Fig 3) The Aarea Amass LCP Vcmax and Jmax were alsovery low relative to comparator species (Fig 3 Table 3)consistent with shade adaptation The two Ruscus species hadvery high values for Ags (Table 3) consistent with excellentWUE Consistent with strong drought tolerance via retention ofstored water both species had very low values for leaf and stemgmin especially relative to comparator vascular plant species(Fig 3 Table 3)
Hydraulic conductance pressure volume curveparameters and leaf water storage
Consistent with expectations for combined drought and shadetolerance both Ruscus species had low Kshoot relative tocomparator tropical and temperate woody angiosperms (Fig 3Table 4) The pressurendashvolume curve parameters of Ruscuswereconsistent with achieving drought tolerance through tissue waterstorage Both species had less negative po and ytlp than meanvalues for comparative evergreen woody species (Fig 3Table 4) Notably both species had lower e values thancomparative evergreen woody species and higher RWCtlp andCft values (Fig 3 Table 4) consistent with drought tolerance
Nitrogen concentration and carbon isotope composition
Both species had high values for phyllyclade Narea and Nmass
relative to comparative temperate broadleaf evergreen speciesconsistent with drought adaptation (Fig 3) The d13C valueswerevery negative and typical of values often observed for understoryplants (da Silveira et al 1989) consistent with shade tolerance
Indeed the d13C values were notably strongly negative given thehigh WUE found for these species
Testing hypotheses for shade and drought tolerancewith trait survey data
Overall we quantified 57 traits for the twoRuscus species and for21 traits we had a priori hypotheses for a benefit for shadetolerance and for 24 traits we had a priori hypotheses for abenefit for drought tolerance Using comparative data we foundthat 16 of 21 hypotheses were supported for shade tolerance and22 of 24 were supported for drought tolerance Of the ninehypotheses for traits that would contribute to both shade anddrought tolerance simultaneously (ie expectations were both forhigher or lower values than comparative species) eight weresupported by trait data All these proportions were significantlyhigher than the 50 support that would have been expected toarise only from chance (P = 0001ndash0058 proportion tests)Notably in the seven cases when shade tolerance traits wouldconflict with drought tolerance traits four indicated a benefit fordrought tolerance rather than shade tolerance for both species(Tables 1ndash5)
Discussion
Both R aculeatus (Fig 3) and R microglossum showed traitvalues consistent with combined shade and drought toleranceNumerous traits were consistent with a combined shade anddrought tolerance through improving carbon balance enablinga conservative resource use ie via slow respiration and long-lived parts Other traits would contribute specifically to droughttolerance via reduced demand for water during activephotosynthesis and the ability to survive strong drought afterstomatal closure Notably many such traits were related to waterstorage providingnew insights into effective formsof succulencein shaded habitats This suite of traits would contributeimportantly to the ability of Ruscus to occupy shaded sitesprone to strong drought across a wide geographical range
Traits contributing to simultaneous drought and shadetolerance
Ruscus species showed specialisation associated withconservative resource use consistent with tolerance of shadeand drought These specialised traits included thick lamina andcomponent tissues that contribute to long tissue life-spans (shootslastgt5 years Sack et al 2003bWright et al 2004) AdditionallyRuscus species had very low gas-exchange rates including lowgs and lowKshoot whichwould correspond to a low investment invascular tissue (Tyree andZimmermann1983 Sack et al 2003b)and low Rarea Rmass Aarea and Amass all representing an ability tomaintain photosynthesis and growth with low requirements forlight and water
Traits contributing to drought tolerance
According to Jones et al (1992) drought tolerance can beachieved through avoidance of plant water deficits toleranceof plant water deficits or efficiency mechanisms Ruscus speciesshowed traits associated with drought tolerance either byproviding the ability to maintain photosynthesis and growth indrying soil andor the ability to survive chronic drought as
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology K
previously shown experimentally for R aculeatus (Sack 2004)Traits that would contribute to the ability to maintain gasexchange in drying soil include small leaf size high WUE andlow IVD High WUE achieved in part with high Narea meansthese Ruscus species can attain positive carbon balance evenwith extremely low gs Low IVD and water storage capacitanceallow the phylloclades to maintain water supply to the mesophylland tolerate transiently high evaporation rates for example dueto sunflecks without desiccating the leaf (Sack et al 2003a)Traits contributing to the ability of the phylloclades to surviveextended drought included those enabling a low evaporationrate per leaf area once stomata have shut such as low gmin inleaf and stem and those related to specialised water storagetissue linked with low leaf density low e and po values thatwere low in magnitude
Ruscus water storage
The water storage tissue of Ruscus that occupied a third of theleaf thickness although contributing most directly to droughttolerance is also consistent with shade tolerance given itscontribution to reduced tissue costs for the phylloclade as awhole The water storage tissue had thin cell walls reflected inthe low bulk e and low solute concentration contributing tobulk po values that were low in magnitude
This water storage tissue would also contribute to both typesof drought tolerance ndash the ability to maintain photosynthesis indrying soil and to survive after stomata have shut duringextended drought The lsquosucculencersquo of Ruscus phylloclades isdistinctive relative to more typical succulent-leafed andsucculent-stemmed species which tend to have high leaf watercontent and capacitance values (Vendramini et al 2002 Ogburnand Edwards 2012) In contrast in Ruscus species the SWCwas low relative to typical leaf succulent species and forR aculeatus Cft fell within the range of typical evergreenleaves We note that the strong tissue differentiation in Ruscus(ie separation of mesophyll cell and water storage in spaceand their distinctions in anatomy) would contribute to higheffectiveness of water storage even if the bulk tissue overallhad low SWC and capacitance Indeed across species theretends to be no relationship between the magnitude of SWC orcapacitance and the degree of within-leaf tissue differentiation(Ogburn and Edwards 2012) Notably such differentiationwould contribute special advantages for supply of waterwhether stomata are open or closed as the large thin-walledwater storage cells with low solute concentration can yield theirwater to supply the evaporative load while the photosynthetictissues can maintain their volume according to their thickerwalls and stronger solute concentration
Although the capacitance and SWC values were low forRuscus species these values would be more substantial ifconsidered relative to water demand Across several speciesCft has been found to correlate with Kshoot and with gs (Sacket al 2003a Blackman et al 2010) indicating that leaves tendto be built with capacitance to match their maximum flux ratesand thus to buffer the leaf water potential against surges intranspiration Thus because Ruscus has low gs when stomataare open and low Kshoot the capacitance would be expectedto supply transpiration transiently during sunflecks or high
VPD Likewise when stomata close the capacitance suppliesongoing water loss via cuticular conductance Given theextremely low gmin of Ruscus even its moderate Ctlp canenable survival for weeks (Sack et al 2003b Sack 2004)Further at turgor loss the water content would equalSWCRWCtlp and the relatively high RWCtlp wouldcontribute to the high water content once turgor is lost Thusthe lsquosucculencersquo of Ruscus is moderate in absolute terms butcombined with its other very strong mechanisms to reducetranspiration when stomata are open or closed ie low gs andgmin even this moderate capacitance would provide strongfunctionality
Water-use efficiency and carbon isotope composition
It was noteworthy that despite extremely high WUE valuesphylloclade d13C values of the Ruscus species were verynegative This presents a strong anomaly worthy of furtherinvestigation as species with high WUE typically have higherd13C (less negative ie closer to zero) The d13C can beinfluenced by a host of processes including source CO2 storedplant carbon and time-integrated CO2 concentration at the siteof carboxylation (Farquhar et al 1989) For Ruscus althoughd13C values were typical of an understory plant they did notappear to be drivenby internalCO2 concentration because the leafisotopic values were depleted in 13C whereas the high WUEdetermined by gas exchange would likely promote enrichedisotopic values It is more likely that d13C in this species wasdetermined by source CO2 or stored carbon or recycling ofrespired CO2 (da Silveira et al 1989) Our study individualsof Ruscus were cultivated in a shaded understory similar totheir natural habitat Previous studies have shown that there canbe higher concentrations of respired CO2 in the forest understorythan in the canopy resulting in more negative carbon isotoperatios in understory plant tissue than canopy plant tissue (daSilveira et al 1989) This is a function of decomposing leavesand litter cover slow air mixing as well as plant environmentalresponses
A second possible explanation for the d13C values ofRuscus is related to its growth form and phenology asRuscus has extensive rhizomes (Sack et al 2003b) whichact as a carbon store for the plant Using this recycled carbonduring the growth season when the plant may not be able tomeet its carbon requirement by photosynthesis alone becauseof low maximum rates under light limitation may alsocontribute to more negative d13C values (Vizzini 2003)Notably Ruscus stems are hollow and thus can also storerelatively large amounts of CO2 for use during a growth periodwhen carbon is otherwise limiting especially given very lowgs Some of this stored carbon might be photosyntheticallyfixed by the stem (Nilsen and Sharifi 1997) In each of thesecases respired stored or recycled CO2 would supply carbonthat was previously fixed by Rubisco with more negative d13Cvalues (ndash27o) than air (ndash8o)
There was also a dissonance between the d13C value and CiCa The fully mature phylloclades which were selected andused for gas-exchange and d13C measurements were producedduring late winter and early spring with mild temperatures(average at midday 190ndash220C) and low atmospheric VPD
L Functional Plant Biology A Pivovaroff et al
(average at midday 06ndash15 kPa) However the gas-exchangemeasurements were taken during summer with hightemperature (average at midday 300ndash355C) and high VPD(average at midday 25ndash35 kPa) We harvested thephylloclades that we used for gas exchange to determine thecarbon isotope ratio Although the low value of instantaneousCiCa may be caused by high temperature and high atmosphericVPD the very low value of d13C may represent the time whenthe phyllocladersquos carbon was assimilated (during mildtemperature and low VPD)
Implications for drought and shade tolerance Ruscusas a model
We found strong support for a large number of hypotheses fortrait-based shade and drought tolerance providing a strong traitbasis for combined tolerance Although the detailed functionaltrait survey conducted here is relatively novel in its breadth(see also Pasquet-Kok et al 2010) this is a logical extensionof the traditional approach for understanding the basis forplant adaptation to environment ie testing expectations forindividual traits established by previous studies of thefunctional significance of these traits in other species Weacknowledge there is some degree of uncertainty ininterpreting a large number of traits simultaneously based onstudies of other species First the interpretation of the value oftraits based on other species may not be in all cases equally validfor Ruscus Some trait variation may relate to other functionsFurther studies for example using mutants would be necessaryas conclusive evidence of the value of specific traits in a givenspecies However one advantage of testing numerousexpectations for each hypothesis is that the key finding will berobust to the removal of some traits from the analysis if thoseare later found to be inappropriate Ideally when a model forestimating plant performance from leaf traits becomes availableone could determine how the specific quantitative combinationsof traits presented here scales up to plant shade and droughttolerance
The shade and drought tolerance of Ruscus consistent withthe suite of traits examined here is one case demonstratinghow plants can avoid a general trade-off between shade anddrought tolerance Further Ruscus is noteworthy as one of onlya few stem photosynthetic plants that occupy a shaded habitatHistorically it is likely that shade tolerance preceded droughttolerance given this speciesrsquo ancestors were species of moisttropical forests (Kim et al 2010) and thus Ruscus or itsancestor apparently evolved drought tolerance whileexpanding its range into drier habitat or during past climatechange Considering its unique adaptations and trait valuesRuscus can serve as an excellent model for the basis ofcombined shade and drought tolerance
Acknowledgements
We thank the Mildred E Mathias Botanical Garden for access to garden andplants Andy Truong for assistance with measurements Howard GriffithsLouis Santiago and Cheryl Swift for helpful discussions and comments onthe manuscript This work was supported by National Science FoundationGrant IOB-0546784
References
Bartlett MK Scoffoni C Sack L (2012) The determinants of leaf turgor losspoint and prediction of drought tolerance of species and biomes a globalmeta-analysis Ecology Letters 15 393ndash405 doi101111j1461-0248201201751x
Blackman CJ Brodribb TJ Jordan GJ (2010) Leaf hydraulic vulnerability isrelated to conduit dimensions and drought resistance across a diverserange of woody angiosperms New Phytologist 188 1113ndash1123doi101111j1469-8137201003439x
Brodribb TJ Feild TS Jordan GJ (2007) Leaf maximum photosynthetic rateand venation are linked by hydraulicsPlant Physiology 144 1890ndash1898doi101104pp107101352
Caspersen JP (2001) Interspecific variation in sapling mortality in relationto growth and soil moisture Oikos 92 160ndash168 doi101034j1600-07062001920119x
Cochard H Nardini A Coll L (2004) Hydraulic architecture of leaf bladeswhere is the main resistance Plant Cell amp Environment 27 1257ndash1267doi101111j1365-3040200401233x
Cooney-Sovetts C Sattler R (1987) Phylloclade development in theAsparagaceae an example of homoeosis Botanical Journal of theLinnean Society 94 327ndash371 doi101111j1095-83391986tb01053x
Cowling R Rundel P Lamont BB ArroyoMK ArianoutsouM (1996) Plantdiversity in Mediterranean-climate regions Trends in Ecology ampEvolution 11 362ndash366 doi1010160169-5347(96)10044-6
da Silveira L Sternberg L Mulkey SS Wright SJ (1989) Ecologicalinterpretation of leaf carbon isotope ratios influence of respired carbondioxide Ecology 70 1317ndash1324 doi1023071938191
de Lillis M Fontanella A (1992) Comparative phenology and growth indifferent species of the Mediterranean maquis of central Italy PlantEcology 99-100 83ndash96 doi101007BF00118213
Dracup JA (1991)DroughtmonitoringStochasticHydrologyandHydraulics5 261ndash266 doi101007BF01543134
Engelbrecht BMJ Kursar TA (2003) Comparative drought-resistance ofseedlings of 28 species of co-occurring tropical woody plantsOecologia 136 383ndash393 doi101007s00442-003-1290-8
Farquhar GD von Caemmerer S (1980) A biochemical model ofphotosynthetic CO2 assimilation in leaves of C3 species Planta 14978ndash90 doi101007BF00386231
FarquharGD Ehleringer JR HubickK (1989) Carbon isotope discriminationand photosynthesis Annual Review of Plant Physiology and PlantMolecular Biology 40 503ndash537 doi101146annurevpp40060189002443
Givnish TJ (1988) Adaptation to sun and shade a whole-plant perspectiveAustralian Journal of Plant Physiology 15 63ndash92 doi101071PP9880063
Gulias J Flexas J Mus M Cifre J Lefi E Medrano H (2003) Relationshipbetweenmaximum leaf photosynthesis nitrogen content and specific leafarea inBalearic endemic and non-endemicMediterranean speciesAnnalsof Botany 92 215ndash222 doi101093aobmcg123
Haberlandt G (1914) lsquoPhysiological plant anatomyrsquo (MacMillan London)Hallik L Niinemets U Wright J (2009) Are species shade and drought
tolerance reflected in leaf-level structural and functional differentiationin Northern Hemisphere temperate woody flora New Phytologist 184257ndash274 doi101111j1469-8137200902918x
Jones HG (1992) lsquoPlants and microclimate a quantitative approach toenvironmental plant physiologyrsquo (Cambridge University PressCambridge)
Kerstiens G (1996) Cuticular water permeability and its physiologicalsignificance Journal of Experimental Botany 47 1813ndash1832doi101093jxb47121813
Kim JH Kim DK Forest F Fay MF Chase MW (2010) Molecularphylogenetics of Ruscaceae sensu lato and related families(Asparagales) based on plastid and nuclear DNA sequences Annals ofBotany 106 775ndash790 doi101093aobmcq167
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology M
Koide RT Robichaux RH Morse SR Smith CM (2000) Plant water statushydraulic resistance and capacitance In lsquoPlant physiological ecologyfield methods and instrumentationrsquo (Eds R Pearcy JR Ehleringer HMooney P Rundel) pp 161ndash183 (Kluwer Dordrecht The Netherlands)
Koumlrner C Farquhar GD Wong SC (1991) Carbon isotope discrimination byplants follows latitudinal and altitudinal trends Oecologia 88 30ndash40doi101007BF00328400
Lewis AM Boose ER (1995) Estimating volume flow rates through xylemconduits American Journal of Botany 82 1112ndash1116 doi1023072446063
Martiacutenez-Palleacute E Aronne G (1999) Flower development and reproductivecontinuity inMediterraneanRuscusaculeatusL (Liliaceae)Protoplasma208 58ndash64 doi101007BF01279075
Martiacutenez-Tilleriacutea K Loayza AP Sandquist DR Squeo FA (2012) Noevidence of a trade-off between drought and shade tolerance inseedlings of six coastal desert shrub species in north-central ChileJournal of Vegetation Science 23 1051ndash1061 doi101111j1654-1103201201427x
Matalas NC (1963) Probability distribution of low flows USGS ProfessionalPapers 434-A US Government Printing Office Washington DC
Niinemets U (1999) Research review components of leaf dry mass per area ndashthickness and density ndash alter leaf photosynthetic capacity in reversedirections in woody plants New Phytologist 144 35ndash47 doi101046j1469-8137199900466x
Niinemets U Valladares F (2006) Tolerance to shade drought andwaterlogging of temperate northern hemisphere trees and shrubsEcological Monographs 76 521ndash547 doi1018900012-9615(2006)076[0521TTSDAW]20CO2
Nilsen ET Sharifi MR (1997) Carbon isotope composition of legumes withphotosynthetic stems from Mediterranean and desert habitats AmericanJournal of Botany 84 1707ndash1713 doi1023072446469
Ogburn RM Edwards EJ (2012) Quantifying succulence a rapidphysiologically meaningful metric of plant water storage Plant Cell ampEnvironment 35 1533ndash1542 doi101111j1365-3040201202503x
Pasquet-Kok J Creese C Sack L (2010) Turning over a new lsquoleafrsquo multiplefunctional significances of leaves versus phyllodes in Hawaiian Acaciakoa Plant Cell amp Environment 33 2084ndash2100 doi101111j1365-3040201002207x
SackL (2004)Responses of temperatewoody seedlings to shade and droughtdo trade-offs limit potential niche differentiation Oikos 107 110ndash127doi101111j0030-1299200413184x
SackLFroleK (2006)Leaf structural diversity is related to hydraulic capacityin tropical rain forest trees Ecology 87 483ndash491 doi10189005-0710
Sack L Holbrook NM (2006) Leaf hydraulics Annual Review of PlantBiology 57 361ndash381 doi101146annurevarplant56032604144141
Sack L Melcher PJ Zwieniecki MA Holbrook NM (2002) The hydraulicconductance of the angiosperm leaf lamina a comparison of threemeasurement methods Journal of Experimental Botany 532177ndash2184 doi101093jxberf069
Sack L Cowan P Jaikumar N Holbrook NM (2003a) The ldquohydrologyrdquo ofleaves co-ordination of structure and function in temperate woodyspecies Plant Cell amp Environment 26 1343ndash1356 doi101046j0016-8025200301058x
Sack L Grubb P Maronon T (2003b) The functional morphology of juvenileplants tolerant of strong summer drought in shaded forest understoriesin southern Spain Plant Ecology 168 139ndash163 doi101023A1024423820136
SackLScoffoniCMcKownADFroleKRawlsMHavran JCTranHTranT (2012) Developmentally based scaling of leaf venation architectureexplains global ecological patterns Nature Communications 3 837doi101038ncomms1835
Sack L Chatelet D Scoffoni C PrometheusWiki contributors (2013a)ldquoEstimating the mesophyll surface area per leaf area from leaf celland tissue dimensions measured from transverse cross-sectionsrdquoPrometheusWiki Available at httpwwwpublishcsiroauprometheuswikitiki-pagehistoryphppage=Estimating the mesophyllsurface area per leaf area from leaf cell and tissue dimensionsmeasured from transverse cross-sectionsamppreview=16 [accessed June18 2013]
SackL Pasquet-Kok JPrometheusWiki contributors (2013b) ldquoLeaf pressure-volume curve parametersrdquo PrometheusWiki Available at httpprometheuswikipublishcsiroautiki-indexphppage=Leaf+pressure-volume+curve+parameters
Scoffoni C Pou A Aasamaa K Sack L (2008) The rapid light response ofleaf hydraulic conductance new evidence from two experimentalmethods Plant Cell amp Environment 31 1803ndash1812 doi101111j1365-3040200801884x
Smith T Huston M (1989) A theory of the spatial and temporal dynamics ofplant communities Vegetatio 83 49ndash69 doi101007BF00031680
Sterck F Markesteijn L Schieving F Poorter L (2011) Functional traitsdetermine trade-offs and niches in a tropical forest communityProceedings of the National Academy of Sciences of the United Statesof America 108 20 627ndash20 632 doi101073pnas1106950108
Thomas GS (1992) lsquoOrnamental shrubs climbers and bamboosrsquo (JohnMurray Publishers Great Britain)
TyreeMT ZimmermannMH (1983) lsquoXylem structure and the ascent of saprsquo(Springer-Verlag Berlin)
USDA ARS National Genetic Resources Program (2009) GermplasmResources Information Network (GRIN) online database NationalGermplasm Resources Laboratory Beltsville Maryland Availableathttpwwwars-gringovcgi-binnpgshtmltaxonpl410577[Accessed 20 July 2009]
Vendramini F Diacuteaz S Gurvich DE Wilson PJ Thompson K Hodgson JG(2002) Leaf traits as indicators of resource-use strategy in floras withsucculent species New Phytologist 154 147ndash157 doi101046j1469-8137200200357x
Vile D (2005) Specific leaf area and dry matter content estimate thicknessin laminar leaves Annals of Botany 96 1129ndash1136 doi101093aobmci264
Vizzini S (2003) d13C and d15N variability in Posidonia oceanica associatedwith seasonality and plant fraction Aquatic Botany 76 195ndash202doi101016S0304-3770(03)00052-4
Walters M Reich PB (1999) Research review low-light carbon balance andshade tolerance in the seedlings of woody plants dowinter deciduous andbroad-leaved evergreen species differ New Phytologist 143 143ndash154doi101046j1469-8137199900425x
Witkowski E Lamont BB (1991) Leaf specific mass confounds leaf densityand thickness Oecologia 88 486ndash493
Wright IJWestobyM (2003)Nutrient concentration resorption and lifespanleaf traits of Australian sclerophyll species Functional Ecology 1710ndash19 doi101046j1365-2435200300694x
Wright IJ Reich PB Westoby M Ackerly DD Baruch Z Bongers FCavender-Bares J Chapin T Cornelissen JHC Diemer M Flexas JGarnier E Groom PK Gulias J Hikosaka K Lamont BB Lee T LeeWLusk C Midgley JJ Navas M-L Niinemets U Oleksyn J Osada NPoorter H Poot P Prior L Pyankov VI Roumet C Thomas SC TjoelkerMG Veneklaas EJ Villar R (2004) The worldwide leaf economicsspectrum Nature 428 821ndash827 doi101038nature02403
Wullschleger SD (1993) Biochemical limitations to carbon assimilation inC3 plants ndash a retrospective analysis of the ACi curves from 109 speciesJournal of Experimental Botany 44 907ndash920 doi101093jxb445907
N Functional Plant Biology A Pivovaroff et al
wwwpublishcsiroaujournalsfpb
with those hypothesised to confer shade tolerance droughtavoidance or both Overall for 21 traits we had a priorihypotheses of a benefit for shade tolerance and for 24 traits wehad a priori hypotheses of a benefit for drought avoidance Wethen compared the traits for Ruscus with values compiled fromthe literature for (1) temperate and tropical broadleaf evergreenspecies (2) Mediterranean species and (3) woody angiospermsin general in order to put Ruscus trait values in a global contextThis approach involved measuring a large number of key aspectsof structure and function and when possible compiling specifichypotheses for traits potentially involved in the shade anddrought tolerance Ruscus species relative to comparatorspecies (Tables 1ndash5) According to the previous literature onshade and drought tolerance (for example Givnish 1988 Jones1992) these suites of traits in Ruscus are expected to directly orindirectly contribute to mechanisms operating across cell typesand levels of leaf organisation conferring combined shade anddrought adaptation
Thus on the general understanding of shade and droughttolerance traits we hypothesised that Ruscus species wouldhave mechanisms of drought adaptation including traitsenabling the delay of tissue dehydration and traits enablingmaintained function even as tissue dehydrates Such traitsinclude a high water-use efficiency (Wright and Westoby2003) as well as water storage tissue with high water storagecapacitance associated with low bulk leaf modulus of elasticityhigh relative water content at turgor loss point and low cuticularconductance (Sack et al 2003a Pasquet-Kok et al 2010Ogburnand Edwards 2012) Traits potentially contributing to shadetolerance include low rates of maximum photosynthetic CO2
assimilation per leaf area and per leaf mass low lightcompensation point low maximum rate of carboxylation lowmaximum rate of electron transport and more negative carbon
isotope ratios (Walters and Reich 1999) Traits that potentiallyconfer a combined drought and shade tolerance through a generalconservative and cost-efficient resource use include thick laminawith thick epidermis low respiration rates per area andmass lowstomatal conductance and low shoot hydraulic conductance(Sack et al 2003b) Given the exceptional biology of thesespecies ndash their extreme tolerance and their possession ofphylloclades ndash we also qualified additional traits in particularthe detailed anatomical traits such as cell sizes for which wecould not compile hypotheses due to the paucity of comparativedata in the published literature However such anatomical traitshave been argued to be strongly associated with environmentaladaptation in principle (Haberlandt 1914) and thus these datafor Ruscus are likely to be important as future studies providecomparative data for many species
Materials and methodsStudy species and site
Ruscus (Asparagaceae) is a genus of six species of evergreensclerophyllous woody shrubs native to western and southernEurope (including north to southern England) Macaronesianorth-west Africa and south-western Asia ranging east to theCaucasus (de Lillis and Fontanella 1992 Martiacutenez-Palleacute andAronne 1999) Ruscus is thus found in a wide range oftemperate forests as well as in Mediterranean-type climatescharacterised by wet cool winters and dry warm summersthat result in an annual seasonal period of low wateravailability or drought (Matalas 1963 Dracup 1991 Cowlinget al 1996) Ruscus exhibits phylloclades which are flattenedphotosynthetic stems that resemble leaves and are consideredintermediate organs that combine stem and leaf features (Fig 1Cooney-Sovetts and Sattler 1987) Ruscus aculeatus L is a
Table 1 Mean values for morphological traits se for Ruscus aculeatus and R microglossum with units and replicationFor given traits expectations are given forwhetherRuscus should have a higher or lower value relative to comparator species according to the hypotheses of shadeor drought adaptation Comparator species data were taken according to availability in the previously published literature and number of species and minimummean andmaximum trait values are provided ormean se if only thesewere available Sources of comparative data leaf area (Sack et al 2012) LMA (Wright
et al 2004) density (Niinemets 1999) LDMC (Vile 2005) and SWC (Vendramini et al 2002 Ogburn and Edwards 2012)
Morphological traits Units R aculeatus R microglossum Hypotheses Comparator speciesMean plusmn se N Meanplusmn se N Shade
adaptedDroughtadapted
(N) (min mean max)
Leaf area cm2 180 plusmn 006 92 193 plusmn 034 84 LowerA Dicots (485) 010 178 280Leaf mass per area (LMA) gmndash2 1228 plusmn 89 92 910 plusmn 09 84 Higher LowerABC Temperate broadleaf
evergreen (132)580 153 429
Tropical broadleafevergreen (72)
406 145 370
Density g cmndash3 039 plusmn 003 10 027 plusmn 001 10 Higher LowerABC Woody trees andshrubs (38)
009 041 133
Leaf dry mattercontent (LDMC)
g∙gndash1 0389plusmn 0016 6 0310plusmn 0005 6 HigherA LowerBC Shrubs (gt100) 0384plusmn 00084
Saturated watercontent (SWC)
g∙gndash1 159 plusmn 012 6 223 plusmn 005 6 HigherBC Evergreen trees andshrubs (6)
067 182 60
Succulents (45) 170 117 520
AAn expectation according to a hypothesis was confirmed for R aculeatusBAn expectation according to a hypothesis was confirmed for R microglossumCExpectations for these traits are based on drought tolerance conferred by water storage tissue (lsquosucculencersquo) the opposite expectations would arise for droughttolerance conferred by the ability to maintain turgor with dehydration
B Functional Plant Biology A Pivovaroff et al
Tab
le2
Meanva
lues
foran
atom
ical
traitsseforRuscusaculeatusan
dRm
icroglossumw
ithun
itsan
dreplication
Forgiventraitsexpectatio
nsaregivenforw
hetherRuscusshouldhave
ahigherorlowervaluerelativ
etocomparatorspeciesaccordingtothehypothesesofshadeordroughtadaptationCom
paratorspeciesdata
weretakenaccordingtoavailabilityinthepreviouslypublishedliteratureandnumbero
fspeciesand
minim
umm
eanandmaxim
umtraitvaluesareprovidedSources
ofcomparativ
edatatissuethicknessand
airspace
(SackandFrole20
06)Dm(Brodribbetal2
007)N
Sn
otstatistically
significant
atPlt005
Anatomicaltraits
Units
Ra
culeatus
Rm
icroglossum
Hypotheses
Com
paratorspecies
Meanplusmnse
NMeanplusmnse
NShade
adapted
Drought
adapted
(N)
(minm
ean
max)
Tissuethicknessesan
dairspace
Lam
ina
mm27
8plusmn334
529
6plusmn857
5HigherA
BHigherA
BTropicalevergreen
trees(10)
156
267
512
Cuticle
mm352
plusmn022
5329
plusmn023
5Higher
Higher
Tropicalevergreen
trees(10)
1254
60
105
Epiderm
ismm
202plusmn0513
523
plusmn0657
5HigherA
BHigherA
BTropicalevergreen
trees(10)
9751
40
173
Epiderm
iscellwall
mm341
plusmn011
5369
plusmn025
5Mesop
hyll
mm72
6plusmn179
583
0plusmn272
5Water
storage
mm88
7plusmn705
586
2plusmn587
5Present
AB
totalleaf
airspace
10
5plusmn138
520
8plusmn173
5
Celldimension
sEpiderm
iscellarea
mm2
447plusmn27
05
599plusmn35
35
Epiderm
iscellperimeter
mm78
9plusmn261
594
1plusmn278
5Mesop
hyllcellarea
mm2
657plusmn21
55
846plusmn42
65
Mesophyllcellperimeter
mm94
5plusmn172
511
0plusmn241
5
chloroplastarea
inmesop
hyll
cell
21
2plusmn149
521
1plusmn307
5
Mesop
hyllareatotalleaf
area
(AmesA)
247plusmn13
520
5plusmn045
5
Water
storagecellarea
mm2
3414
plusmn19
65
5972
plusmn84
75
Water
storagecellperimeter
mm22
1plusmn572
529
1plusmn19
15
Vasculartraits
Minim
umdistance
from
vein
toepidermis(D
m)
mm20
3plusmn18
05
370plusmn49
35
HigherB
Low
erA
Dicotyledons
(11)
129
233
428
Inter-veinaldistance
(IVD)
mm27
2plusmn27
55
509plusmn71
55
Fibrous
bundlesheath
cellheight
mm18
4plusmn127
517
5plusmn149
5Fibrous
bundlesheath
cellwidth
mm17
4plusmn066
516
3plusmn092
5Fibrous
bundlesheath
cellwall
thickness
mm457
plusmn041
5248
plusmn023
5
Average
theoreticalxylemconduit
cond
uctiv
ityin
themidrib
10
5mmolm
ndash2
sndash1MPandash
1124
plusmn025
5198
plusmn055
5
Average
theoreticalxylemconduit
conductiv
ityin
aninterm
ediary
vein
10
5mmolm
ndash2
sndash1MPa
ndash1
063
plusmn014
5119
plusmn038
5
Average
theoreticalxylemconduit
cond
uctiv
ityin
aminor
vein
10
5mmolm
ndash2
sndash1MPandash
1037
plusmn012
5028
plusmn006
5
(contin
uednextpa
ge)
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology C
typical understory species in forests in the Mediterranean basin(Fig 1 de Lillis and Fontanella 1992Martiacutenez-Palleacute andAronne1999 Sack et al 2003b)RuscusmicroglossumBertol is a hybridproduced by crossing species Ruscus hypoglossum L from theBlack Sea region andRuscus hypophyllumL fromNorthAfrica(Fig 1 Thomas 1992 USDA ARS National Genetic ResourcesProgram 2009)
Experiments were conducted on R aculeatus andR microglossum plants in the Mildred E Mathias BotanicalGarden at the University California Los Angeles from Juneto August 2009 Measurements were made in shaded understorysites on at least three individuals of each species Diurnal lightmeasurements were made on sunny days above the Ruscus plantcanopies with a quantum sensor (Li-190S Li-Cor BiosciencesLincoln NE USA) Individuals received full sunlight averaging~1500mmolmndash2 sndash1 photosynthetically active radiation (PAR)for ~1 h each day and otherwise experienced PAR of~50mmolmndash2 sndash1 interspersed with sunflecks averaging~190mmolmndash2 sndash1 Plants were irrigated as needed and allRuscus individuals were watered before the start of this studyto reduce any differences in water availability betweenindividuals
Phylloclade morphology
We determined leaf morphological traits on recently formedmature phylloclades Although methods were applied tophylloclades for convenience we retained the names ofmethods and traits as applied to leaves (eg leaf mass perarea) Phylloclade area was measured on excised samples withan areameter (Li-3100 Li-Cor Biosciences) Samples were driedin an oven at gt70C for more than 48 h to determine drymass andcalculate leaf mass per area (LMA) as dry mass divided by areaPhylloclade thickness was measured with electronic digitalcalipers (Fisher Scientific Pittsburgh PA USA) and densitywas calculated as mass per area divided by thickness (Witkowskiand Lamont 1991)
Phylloclade anatomy
We sampled phylloclades of each species and prepared cross-sections for anatomical measurements Phylloclades werepreserved in formalin acetic acid (37 formaldehyde glacialacidic acid 95 ethanol and deionised water in a 10 5 50 35mixture) We measured the transverse cross-sectional anatomyusing sections cut halfway along the phylloclade lengthembedded in LR White (London Resin Co London UK) cut05mm thick using a microtome (Ultracut E Reichert-JungUltracut E Leica Microsystems Arcadia CA USA) stainedwith 001toluidine blue in 1sodiumborate andviewedunderthe light microscope using a 20ndash40 objective (DMRB LeicaMicrosystems Wetzlar Germany)
We measured tissue thicknesses and cell dimensions in thelamina and dimensions of vascular bundles and of xylemconduits using Image J software (ver 142 q NationalInstitutes of Health Bethesda MD USA) on microscopeimages We measured thickness for the lamina cuticleepidermis epidermis cell wall mesophyll and water storagecompartment We averaged three measurements of each type foreach cross-section The phylloclade tissues were arranged
Tab
le2
(contin
ued)
Anatomicaltraits
Units
Ra
culeatus
Rm
icroglossum
Hyp
otheses
Com
paratorspecies
Meanplusmnse
NMeanplusmnse
NShade
adapted
Droug
htadapted
(N)
(minm
ean
max)
Meanmaxim
umxy
lem
cond
uit
diam
eter
inthemidrib
mm966
plusmn044
511
0plusmn078
5
Meanmaxim
umxy
lem
cond
uit
diam
eterinan
interm
ediaryvein
mm831
plusmn055
5949
plusmn072
5
Meanmaxim
umxy
lem
cond
uit
diam
eter
inaminor
vein
mm715
plusmn072
5719
plusmn048
5
AAnexpectationaccordingto
ahypothesiswas
confi
rmed
forRa
culeatus
BAnexpectationaccordingto
ahypothesiswas
confi
rmed
forRm
icroglossum
D Functional Plant Biology A Pivovaroff et al
Tab
le3
Meanva
lues
forga
sexchan
getraitsseforRuscusaculeatusan
dRm
icroglossumw
ithun
itsan
dreplication
Forgiventraitsexpectatio
nsaregivenforw
hetherRuscusshouldhave
ahigherorlowervaluerelativ
etocomparatorspeciesaccordingtothehypothesesofshadeordroughtadaptationCom
paratorspeciesdata
weretakenaccordingtoavailabilityinthepreviouslypu
blishedliteratureandnu
mberofspeciesand
minim
umm
eanandmaxim
umtraitvaluesareprov
idedSourcesofcomparativ
edataA
areaA
massRareaR
mass
g s(W
rightetal2
004)LCP(W
altersandReich
1999)Ag
s(G
uliasetal2
003)VcmaxJ
max(W
ullschleger19
93)g m
in(K
erstiens
1996)NSn
otstatistically
significant
atPlt005
Gas-exchang
etraits
Abb
reviation
Units
Ra
culeatus
Rm
icroglossum
Hypotheses
Com
paratorspecies
Meanplusmnse
NMeanplusmnse
NShade
adapted
Droug
htadapted
(N)
(minm
ean
max)
Light-saturated
rateof
photosyn
thesisperarea
Aarea
mmolm
ndash2sndash
1522
plusmn133
451
plusmn047
6Low
erAB
Tem
peratebroadleaf
evergreens
(78)
2629
42
2265
Light-saturated
rateof
photosyn
thesisperm
ass
Amass
nmolCO2gndash
1sndash
1004
3plusmn001
14
0050plusmn0005
6Low
erAB
Tem
peratebroadleaf
evergreens
(78)
2287
46
209
Respiratio
nrateperarea
Rarea
mmolm
ndash2sndash
1004
4plusmn000
98
0156plusmn0015
10Low
erAB
Low
erAB
Tem
peratebroadleaf
evergreens
(50)
0320
93
170
Respiratio
npermass
Rmass
nmolCO2gndash
1sndash
1356
E-04plusmn702
E-05
8171E-03plusmn166E-04
10Low
erAB
Low
erAB
Tem
peratebroadleaf
evergreens
(50)
3337
06
157
Maxim
umstom
atal
conductanceperarea
g smmolm
ndash2sndash
133
plusmn000
74
35plusmn0006
6Low
erAB
Low
erAB
Tem
peratebroadleaf
evergreens
(48)
491
423
09
Light
compensationpo
int
LCP
mmol
photonsm
ndash2sndash
1485
plusmn0
4361
plusmn0
6Low
erAB
Tropicalevergreenshade-
tolerators(15)
16582
4
Intrinsicwater
use
efficiency
Ag
smm
olmol
ndash1
154plusmn8
414
2plusmn19
06
HigherA
BMediterraneanspecies
(78)
2335
94
142
Ratio
ofintercellularto
ambient[CO2]
CiC
a032
7plusmn010
64
0431plusmn0064
6
Maxim
umrateof
carbox
ylation
Vcmax
mmolm
ndash2sndash
126
6plusmn318
424
7plusmn542
5Low
erAB
Tem
peratehardwood
species(19)
114
711
9
Maxim
umrateof
electron
transport
J max
601plusmn635
550
6plusmn786
5Low
erAB
Tem
peratehardwood
species(19)
291
042
37
Leafcuticularcond
uctance
g min
mmolm
ndash2sndash
1037
9plusmn008
210
0295plusmn0082
12Low
erAB
Vascularplants(201)
01
1321
07Stem
cuticular
conductance
g min
mmolm
ndash2sndash
1009
5plusmn002
56
0030plusmn0003
4
AAnexpectationaccordingto
ahypothesiswas
confi
rmed
forRa
culeatus
BAnexpectationaccordingto
ahy
pothesiswas
confi
rmed
forRm
icroglossum
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology E
Tab
le4
Meanva
lues
forhy
drau
licstraitsseforRuscusaculeatusan
dRm
icroglossumw
ithun
itsan
dreplication
Forgiventraitsexp
ectatio
nsaregivenforw
hetherRuscusshouldhave
ahigh
erorlowervaluerelativ
etocomparatorspeciesaccordingtothehy
pothesesofshadeordrough
tadaptationCom
paratorspeciesdata
weretakenaccordingtoavailabilityinthepreviouslypu
blishedliteratureandnu
mberofspeciesand
minim
umm
eanandmaxim
umtraitvaluesareprov
idedorm
eanstandarderrorifon
lythesewereavailable
Sources
ofcomparativ
edataK
shoot(SackandHolbroo
k20
06)Y
tlpR
WCtlpP
oe
(Bartlettetal2
012)Cft(Scoffon
ietal2
008)NSnotstatistically
significant
atPlt005
Hydraulicstraits
Abbreviation
Units
Ra
culeatus
Rm
icroglossum
Hyp
otheses
Com
paratorspecies
Meanplusmnse
NMeanplusmnse
NShade
adapted
Drought
adapted
(N)
(minm
ean
max)
Sho
othy
draulic
cond
uctance
Kshoot
mmolm
ndash2sndash
1
MPandash
1216
plusmn010
10269
plusmn013
11Low
erAB
Low
erAB
Tem
peratewoo
dyangiosperm
s(38)
8plusmn1
Tropicalwoody
angiosperm
s(49)
13plusmn15
Osm
oticpotentialatfull
turgor
p oMPa
ndash128
plusmn010
6ndash065
plusmn003
6HigherA
BHigherA
BC
Evergreen
woo
dyspecies
(182)
-34ndash
183ndash
049
Turgo
rloss
point
Ytlp
MPa
ndash184
plusmn010
6ndash113
plusmn007
6HigherA
BHigherA
BC
Evergreen
woo
dyspecies
(158)
-425
ndash220ndash
054
Modulus
ofelasticity
eMPa
110plusmn141
6588
plusmn053
6Low
erABC
Evergreen
woo
dyspecies(139)
3561
71
734
Relativewater
contentat
turgor
loss
point
RWCtlp
88
8plusmn109
689
3plusmn085
6HigherA
BC
Evergreen
woo
dyspecies(19)
718
12
911
Relativecapacitanceatfull
turgor
Cft
MPandash
10067plusmn0008
60102plusmn0010
6HigherA
BC
Evergreen
species(6)
0040
0066
0113
Relativecapacitanceat
turgor
loss
Ctlp
MPandash
10104plusmn0015
60089plusmn0003
6
Predawnwater
potential
Ypre
MPa
ndash045
plusmn008
4ndash088
plusmn008
4Middaywater
potential
Ymid
MPa
ndash141
plusmn008
3ndash129
plusmn028
4
AAnexpectationaccordingto
ahy
pothesiswas
confi
rmed
forRa
culeatus
BAnexpectationaccordingto
ahypothesiswas
confi
rmed
forRm
icroglossum
CExpectatio
nsforthesetraitsarebasedon
drou
ghttolerance
conferredby
waterstoragetissue(lsquosucculencersquo)the
oppositeexpectations
wou
ldarisefordroughttoleranceconferredby
theabilitytomaintainturgor
with
dehydration
F Functional Plant Biology A Pivovaroff et al
symmetrically with adaxial and abaxial layers of mesophyllepidermis and cuticle above and below a central achlorophyllouswater storage tissue (Fig 2) The per cent air space in each of theadaxial and abaxial mesophyll and in the water storagecompartment was estimated to the nearest 5 and the totalphylloclade airspace was calculated
PAir space in each tissue
fraction of leaf cross section occupied by that tissue100
eth1THORN
As indices of cell size the cross-sectional areas andperimeters were measured for three cells in the epidermis(adaxial and abaxial) the mesophyll (adaxial and abaxialie above and below the water storage tissue) and thewater storage tissue The area occupied by chloroplastswithin a mesophyll cell was measured for three cells(adaxial and abaxial) and the percent cross-sectionalchloroplast area was calculated as the ratio of chlorophyllarea divided by mesophyll cell area
We calculated the surface area of mesophyll cells per leafarea (AmesA) as described by Sack et al (2013a) a measure ofthe area available for CO2 uptake formesophyll cell layers Giventhe lack of palisade-form cells we modelled all mesophyll cellsas spheres for these calculations
We also measured vascular anatomy to quantify traits relatedto the efficiency of water transport within and outside the xylemWe measured the inter-veinal distance (IVD) and also theminimum distance from edge of bundle sheath to epidermis(Dm) as the hypotenuse between the distance between veinsand the distance to the epidermis (Brodribb Feild and Jordan2007)
Dm frac14ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
IVD2
2thorn ethdistance from bundle sheath edge to epidermisTHORN2
s
eth2THORN
We averaged IVD and Dm from three values for each cross-section
For all anatomical traits except those relating to the centralwater storage tissue measurements were made both adaxial andabaxial halves and values for the two halveswere averagedwhennot significantly different (at P lt 005 in paired t-tests) exceptthey were summed for total AmesA
To characterise the midrib for three typical fibrous bundlesheath cells we measured the cross-sectional heights widthsand cell wall thicknesses calculating mean values for each traitfrom three measurements per cross-section To characterise thexylem anatomy and theoretical conductivity of xylem conduitsfor a typical conduit within the midrib an intermediary veinand a minor vein of each sampled phylloclade we treated theconduit as an ellipse and determined the major and minor axisdiameters We calculated the theoretical hydraulic conductivityof the xylem conduit using Poiseuillersquos equation for ellipsesbased on conduit dimensions (Lewis and Boose 1995 CochardNardini and Coll 2004)
Tab
le5
Meanvalues
fortissue
compo
sition
traitsseforRuscusaculeatusan
dRm
icroglossumw
ithun
itsan
dreplication
andsign
ificanceof
differencesbetw
eenspecies(t-tests)
Forgiventraitsexpectatio
nsaregivenforw
hetherRaculeatus
shouldhave
ahigherorlowervaluerelativ
etocomparatorspeciesaccordingtothehypothesesofshadeordroughtadaptationCom
paratorspecies
dataweretakenaccordingtoavailabilityinthepreviouslypublishedliteratureandnumberofspeciesand
minim
umm
eanandmaxim
umtraitvaluesareprovidedSourcesofcomparativ
edatad
13C(K
oumlrneretal
1991)NmassNarea(W
righ
tetal2
004)N
Sn
otstatistically
significant
atPlt005
Tissuecompositio
ntraits
Abbreviation
Units
Ra
culeatus
Rm
icroglossum
Hypotheses
Com
paratorspecies
Meanplusmnse
NMeanplusmnse
NShade
adapted
Drought
adapted
(N)
(minm
ean
max)
Npermass
Nmass
194
plusmn006
9192
plusmn007
9Low
erHigherA
BTem
peratebroadleaf
evergreen(129)
0581
19
231
Nperarea
Narea
238
plusmn007
9175
plusmn006
9Low
erHigherA
BTem
peratebroadleaf
evergreen(129)
0727
1733
63
Cpermass
Cmass
44
4plusmn048
943
6plusmn052
9Carbonto
nitrogen
ratio
CN
231plusmn068
922
9plusmn058
9Carbonisotoperatio
d13C
permilndash33
32plusmn029
9ndash33
05plusmn020
9Low
erAB
Higher
Tem
peratespecies(35)
ndash27
64
ndash26
03
ndash24
09
AAnexpectationaccordingto
ahypothesiswas
confi
rmed
forRa
culeatus
BAnexpectationaccordingto
ahy
pothesiswas
confi
rmed
forRm
icroglossum
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology G
K t frac14 pa3b3
64hetha2 thorn b2THORN eth3THORN
where a and b are the major and minor axes of the ellipse and his water viscosity at 25C
Gas-exchange measurements responses to light and CO2and cuticular conductance
In July and August 2009 photosynthetic light response curvesandCO2 response curvesweremeasuredusing aLi-6400portablephotosynthesis system (Li-Cor Biosciences ) with light provided
Ruscusaculeatus
Ruscusmicroglossum
01 mm
Fig 2 Lamina and midrib cross-sections of Ruscus aculeatus and R microglossum phylloclades (05mm thick) Note that bothspecies exhibit shade tolerance features such as absence of palisade tissue as well as drought tolerance features such as large waterstorage compartment and thick-walled epidermis andfibrousbundle sheath cells surrounding thexylemandphloemfor bothmajor andminor veins especially prominent in R aculeatus which is native to drier habitats
(a) (b)
Fig 1 (a)Ruscus aculeatus and (b)Rmicroglossumgrowing at theMildredEMathiasBotanicalGardenNote thatwhat atfirst glance appear to be leaves are infact phylloclades
H Functional Plant Biology A Pivovaroff et al
by a red-blue light source (6400ndash02B no SI-710 Li-CorBiosciences ) Gas-exchange measurements were made on atleast 1ndash2 phylloclades per individual
For light-response curves phylloclades were acclimatedfor at least 5min at 1500mmolmndash2 sndash1 PAR at temperatures of2527C with RH maintained at ~50 and CO2 concentrationof 400mmolmolndash1 Then phylloclades were measured for netCO2 assimilation per leaf area at PAR steps of 1600 1400 12001000 800 600 500 400 300 200 100 50 and 0mmolmndash2 sndash1with 180ndash240 s stabilisation at each irradiance step Wedetermined light-saturated photosynthetic rate per area andmass (Aarea and Amass) dark respiration rate per area and mass(Rarea and Rmass) ie the negative A at zero PAR maximumstomatal conductance per area (gs) light compensation point(LCP) as the x-intercept intrinsic water use efficiency (WUEAareags) and the ratio of intercellular to ambient CO2
concentration (CiCa) at 100mmolmndash2 sndash1 PAR We thenharvested the phylloclades measured for gas exchange todetermine the nitrogen concentration and carbon isotope ratio(see below)
For CO2-response curves phylloclades were allowed toequilibrate at 400 ppm to induce stomatal opening and the netCO2 assimilation per leaf area was determined at Ci steps of400 300 200 100 50 400 400 500 600 800 1200 14001600 ppm with 3ndash4min equilibration time at each step Wedetermined maximum rate of carboxylation and maximum rateof electron transport per leaf area (Vcmax and Jmax) from plots ofCi vs A corrected to 25C (Farquhar and von Caemmerer 1980)
Cuticular conductance (ie minimumepidermal conductancegmin sensu Kerstiens 1996) was determined for 3ndash4 maturephylloclades and 10 cm lengths of stems from each of threeindividuals of each species Phylloclade and stem sampleswere hydrated and then the cut ends were sealed with waxSamples were dried for at least 30min on a laboratory bench atPAR of lt10mmol photons mndash2 sndash1 to induce stomatal closurethen samples were weighed for at least eight intervals of 30minduring which the slope of water loss versus time was highlylinear (R2 gt 0995) and therefore taken to represent transpirationafter stomata had closed fully The gmin was calculated as thetranspiration rate divided by the mole fraction vapour pressuredeficit (VPD determined from a weather station HOBO MicroStation with Smart Sensors Onset Bourne MA USA)
Pressurendashvolume curve parametersPressurendashvolume curve parameters were determined for matureshoots using the bench-drying method (Koide et al 2000 Sacket al 2003a) Shoots were ~10ndash15 cm long with about fourphylloclades per shoot for R microglossum and 15ndash20phylloclades for R aculeatus Shoots were progressively driedon a laboratory bench and measured at intervals by equilibratingfor 10min in a Whirlpak bag (Whirl-Pak Nasco Fort AtkinsonWIUSA) beforeweighing andmeasuring for leafwater potential(Yleaf) with a pressure chamber (Model 1000 Plant MoistureStress Instruments Albany OR USA) Subsequently dry masswas determined after more than 48 h in an oven at 70C Wedetermined the leaf dry matter content (LDMC) saturated watercontent (SWC) turgor loss point (Ytlp) relative water content atturgor loss point (RWCtlp) and osmotic potential at full turgor
(po) relative capacitance at full turgor (Cft DRWCDYleaf) andrelative capacitance at turgor loss point (Ctlp DRWCDYleaf) Wedetermined the modulus of elasticity (e) as the linear slope of theline fitted for pressure potential versus relative water contentabove and including turgor loss point (Sack et al 2013b)
Shoot hydraulic conductance
We measured the hydraulic conductance of mature shoots(Kshoot) ~10ndash15 cm long bearing about four phylloclades pershoot for R microglossum and 15ndash20 phylloclades forR aculeatus using the evaporative flux method (Sack et al2002) Samples were harvested and the ends were re-cut underdistilled degassed water with a fresh razor blade then hydratedovernight Shoots were connected to tubing containing distilleddegassed water running to a graduated cylinder on a balanceinterfaced to a computer logging data every minute to calculatethe flow rate of water from the balance into the sample Sampleswere held in place above a fan on wood frames strung withfishing line Lights were arranged above a plexiglass containerof water that acted as a heat trap producing PAR ofgt1200mmolmndash2 sndash1 at shoot level When steady-statetranspiration was achieved samples were covered with aplastic bag and removed from the tubing and the leaf waterpotential was determined with a pressure chamber (Model 1000Plant Moisture Stress Instruments) Kshoot was calculated as thesteady-state transpirational flow rate (E mmolmndash2 sndash1) dividedby the water potential driving force (DYleaF= ndashYleaf MPa)further normalised by total phylloclade area Kshoot valueswere standardised to 25C to correct for the temperaturedependence of the viscosity of water (Sack et al 2003a)
Foliar nitrogen concentration and carbon isotope ratio
To analyse total tissue nitrogen concentration and carbon isotopecomposition (d13C) for three replicates from each of threeindividuals for each species phylloclade samples were oven-dried at gt70C for more than 48 h and ground by mortar andpestle The nitrogen concentration and d13C were determined atthe UCDavis Stable Isotope Facility using an elemental analyser(PDZEuropaANCA-GSL SerconLtd Cheshire UK) interfacedto an isotope ratiomass spectrometer (PDZEuropa20ndash20 SerconLtd) at the UC Davis Stable Isotope Facility Final d13C contentvalues are expressed relative to international standardVienna PeeDee belemnite (V-PDB) for carbon
d13CethpermilTHORN frac14 etheth13C=12C of sampleTHORN=eth13C=12C of standardTHORN 1THORN 1000
eth4THORN
d13C of plant tissues can provide a measure of intrinsic WUE(Ags) at the time that carbon was assimilated giving long-termwater-use efficiency
Comparative data compilation and trait comparison
To consider Ruscus trait values in a global context we compileddata from the literature for (1) temperate and tropical broadleafevergreen species (2) Mediterranean species and (3) woodyangiosperms in general When available we considered data forplants grown in the shade and for species that are shade tolerant
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology I
however eco-physiological trait data is generally collected forsun leaves or plants making a comparison solely for shade grownplants not possible Formore commonly studied traits (egAmax)comparative data was taken from studies with large databasesand multiple traits (eg GLOPNET Wright et al 2004) Wedetermined minimum mean and maximum values for traitsfrom comparative studies or the mean and standard error ifraw species values were not available Studies that wereincluded for comparative data are referenced in the captionsfor Table 1ndash5 in association with the specific traits wecompared Differences between Ruscus trait values andcomparative data from the literature were determined for bothR aculeatus and R microglossum Hypotheses were deemedsupported for a Ruscus species if the trait value was higher orlower than the mean comparator value in the way predicted
Results
Both Ruscus species studied had leaf morphology consistentwith adaptation to shade and drought relative to comparator
species (Tables 1ndash5)We constructed a radar graph to encapsulatethe key traits that would confer adaptation to shade drought andthe combination for R aculeatus which had the more extremeadaptation of the two Ruscus species considered (Fig 3) Thisfigure summarises the major results of our study with values forcomparator species appearing as the inner circle and R aculeatustrait values displayed as the bold outer line
Gross morphology of phylloclades
Both R aculeatus and R microglossum had LMA valueslower than comparator species and R microglossum hadphylloclades larger than the mean leaf area for comparatorspecies Both Ruscus species had lower bulk tissue density thancomparator species consistent with the water storage in theRuscus phylloclades Notably despite its water storage tissuethe LDMC and SWC values of R aculeatus were typical ofthose for comparator species whereas R microglossum had alower LDMC and a higher SWC than for comparator speciessets (Table 1)
Fig 3 Radar graph illustrating percent difference between selected traits of Ruscus aculeatuswithcomparative data (see Tables 1ndash5 for symbols and sources of comparative data) Values forR aculeatus outside the circle indicate shade andor drought tolerance Traits are arrangedaccording to whether they would contribute shade tolerance drought tolerance or both The innerdisc represents the mean for comparative species for given traits and the values for R aculeatus isscaled relative to the magnitude of that value () with a value outside the disc representing greatershade andor drought tolerance (+) and (ndash) indicate if the axis is scaled such that a value outside thecircle represents percent higher or lower than comparative values respectively For traits expressed asnegative values (+) and (ndash) indicate more negative or less negative respectively
J Functional Plant Biology A Pivovaroff et al
Anatomy of phylloclades
The Ruscus species possessed numerous anatomical traitsconsistent with benefits for both shade and drought tolerance(Table 2) The phylloclade cross-sections were symmetrical(Fig 2) and thus the cross-sectional anatomy of the adaxialand abaxial halves were not different for all traits (paired t-testP gt 005) and values were averaged within species The twospecies were similar in their substantial leaf thickness and inthe thickness of cuticle epidermis cell walls and water storagecompartment (Table 2) The fractions of the lamina occupied bythe epidermis mesophyll and water storage tissues were 1552ndash56 and 29ndash32 respectively
Both species had parallel longitudinal veins of three sizes(midrib intermediate and small veins) Consistent with droughtadaptation the two species had low Dm R aculeatus having thelower value andRmicroglossumhadahigher IVD (Table 2)Thetwo species had on average the same maximum conduitdiameters in their midribs intermediate veins and small veinsand the maximum conduit diameter decreased ~35 from themidrib to the small veins The average theoretical hydraulicconductivity of xylem conduits did not differ between thespecies and decreased by up to 86 from the midrib to theminor vein R aculeatus had on average walls that were 85thicker in the fibrous bundle sheath (Table 2)
Gas-exchange measurements
Consistent with adaptation to simultaneous shade and droughtthe Ruscus species had very low values for Rarea Rmass and gs(Table 3) relative to comparator temperate broadleaf evergreenspecies (Fig 3) The Aarea Amass LCP Vcmax and Jmax were alsovery low relative to comparator species (Fig 3 Table 3)consistent with shade adaptation The two Ruscus species hadvery high values for Ags (Table 3) consistent with excellentWUE Consistent with strong drought tolerance via retention ofstored water both species had very low values for leaf and stemgmin especially relative to comparator vascular plant species(Fig 3 Table 3)
Hydraulic conductance pressure volume curveparameters and leaf water storage
Consistent with expectations for combined drought and shadetolerance both Ruscus species had low Kshoot relative tocomparator tropical and temperate woody angiosperms (Fig 3Table 4) The pressurendashvolume curve parameters of Ruscuswereconsistent with achieving drought tolerance through tissue waterstorage Both species had less negative po and ytlp than meanvalues for comparative evergreen woody species (Fig 3Table 4) Notably both species had lower e values thancomparative evergreen woody species and higher RWCtlp andCft values (Fig 3 Table 4) consistent with drought tolerance
Nitrogen concentration and carbon isotope composition
Both species had high values for phyllyclade Narea and Nmass
relative to comparative temperate broadleaf evergreen speciesconsistent with drought adaptation (Fig 3) The d13C valueswerevery negative and typical of values often observed for understoryplants (da Silveira et al 1989) consistent with shade tolerance
Indeed the d13C values were notably strongly negative given thehigh WUE found for these species
Testing hypotheses for shade and drought tolerancewith trait survey data
Overall we quantified 57 traits for the twoRuscus species and for21 traits we had a priori hypotheses for a benefit for shadetolerance and for 24 traits we had a priori hypotheses for abenefit for drought tolerance Using comparative data we foundthat 16 of 21 hypotheses were supported for shade tolerance and22 of 24 were supported for drought tolerance Of the ninehypotheses for traits that would contribute to both shade anddrought tolerance simultaneously (ie expectations were both forhigher or lower values than comparative species) eight weresupported by trait data All these proportions were significantlyhigher than the 50 support that would have been expected toarise only from chance (P = 0001ndash0058 proportion tests)Notably in the seven cases when shade tolerance traits wouldconflict with drought tolerance traits four indicated a benefit fordrought tolerance rather than shade tolerance for both species(Tables 1ndash5)
Discussion
Both R aculeatus (Fig 3) and R microglossum showed traitvalues consistent with combined shade and drought toleranceNumerous traits were consistent with a combined shade anddrought tolerance through improving carbon balance enablinga conservative resource use ie via slow respiration and long-lived parts Other traits would contribute specifically to droughttolerance via reduced demand for water during activephotosynthesis and the ability to survive strong drought afterstomatal closure Notably many such traits were related to waterstorage providingnew insights into effective formsof succulencein shaded habitats This suite of traits would contributeimportantly to the ability of Ruscus to occupy shaded sitesprone to strong drought across a wide geographical range
Traits contributing to simultaneous drought and shadetolerance
Ruscus species showed specialisation associated withconservative resource use consistent with tolerance of shadeand drought These specialised traits included thick lamina andcomponent tissues that contribute to long tissue life-spans (shootslastgt5 years Sack et al 2003bWright et al 2004) AdditionallyRuscus species had very low gas-exchange rates including lowgs and lowKshoot whichwould correspond to a low investment invascular tissue (Tyree andZimmermann1983 Sack et al 2003b)and low Rarea Rmass Aarea and Amass all representing an ability tomaintain photosynthesis and growth with low requirements forlight and water
Traits contributing to drought tolerance
According to Jones et al (1992) drought tolerance can beachieved through avoidance of plant water deficits toleranceof plant water deficits or efficiency mechanisms Ruscus speciesshowed traits associated with drought tolerance either byproviding the ability to maintain photosynthesis and growth indrying soil andor the ability to survive chronic drought as
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology K
previously shown experimentally for R aculeatus (Sack 2004)Traits that would contribute to the ability to maintain gasexchange in drying soil include small leaf size high WUE andlow IVD High WUE achieved in part with high Narea meansthese Ruscus species can attain positive carbon balance evenwith extremely low gs Low IVD and water storage capacitanceallow the phylloclades to maintain water supply to the mesophylland tolerate transiently high evaporation rates for example dueto sunflecks without desiccating the leaf (Sack et al 2003a)Traits contributing to the ability of the phylloclades to surviveextended drought included those enabling a low evaporationrate per leaf area once stomata have shut such as low gmin inleaf and stem and those related to specialised water storagetissue linked with low leaf density low e and po values thatwere low in magnitude
Ruscus water storage
The water storage tissue of Ruscus that occupied a third of theleaf thickness although contributing most directly to droughttolerance is also consistent with shade tolerance given itscontribution to reduced tissue costs for the phylloclade as awhole The water storage tissue had thin cell walls reflected inthe low bulk e and low solute concentration contributing tobulk po values that were low in magnitude
This water storage tissue would also contribute to both typesof drought tolerance ndash the ability to maintain photosynthesis indrying soil and to survive after stomata have shut duringextended drought The lsquosucculencersquo of Ruscus phylloclades isdistinctive relative to more typical succulent-leafed andsucculent-stemmed species which tend to have high leaf watercontent and capacitance values (Vendramini et al 2002 Ogburnand Edwards 2012) In contrast in Ruscus species the SWCwas low relative to typical leaf succulent species and forR aculeatus Cft fell within the range of typical evergreenleaves We note that the strong tissue differentiation in Ruscus(ie separation of mesophyll cell and water storage in spaceand their distinctions in anatomy) would contribute to higheffectiveness of water storage even if the bulk tissue overallhad low SWC and capacitance Indeed across species theretends to be no relationship between the magnitude of SWC orcapacitance and the degree of within-leaf tissue differentiation(Ogburn and Edwards 2012) Notably such differentiationwould contribute special advantages for supply of waterwhether stomata are open or closed as the large thin-walledwater storage cells with low solute concentration can yield theirwater to supply the evaporative load while the photosynthetictissues can maintain their volume according to their thickerwalls and stronger solute concentration
Although the capacitance and SWC values were low forRuscus species these values would be more substantial ifconsidered relative to water demand Across several speciesCft has been found to correlate with Kshoot and with gs (Sacket al 2003a Blackman et al 2010) indicating that leaves tendto be built with capacitance to match their maximum flux ratesand thus to buffer the leaf water potential against surges intranspiration Thus because Ruscus has low gs when stomataare open and low Kshoot the capacitance would be expectedto supply transpiration transiently during sunflecks or high
VPD Likewise when stomata close the capacitance suppliesongoing water loss via cuticular conductance Given theextremely low gmin of Ruscus even its moderate Ctlp canenable survival for weeks (Sack et al 2003b Sack 2004)Further at turgor loss the water content would equalSWCRWCtlp and the relatively high RWCtlp wouldcontribute to the high water content once turgor is lost Thusthe lsquosucculencersquo of Ruscus is moderate in absolute terms butcombined with its other very strong mechanisms to reducetranspiration when stomata are open or closed ie low gs andgmin even this moderate capacitance would provide strongfunctionality
Water-use efficiency and carbon isotope composition
It was noteworthy that despite extremely high WUE valuesphylloclade d13C values of the Ruscus species were verynegative This presents a strong anomaly worthy of furtherinvestigation as species with high WUE typically have higherd13C (less negative ie closer to zero) The d13C can beinfluenced by a host of processes including source CO2 storedplant carbon and time-integrated CO2 concentration at the siteof carboxylation (Farquhar et al 1989) For Ruscus althoughd13C values were typical of an understory plant they did notappear to be drivenby internalCO2 concentration because the leafisotopic values were depleted in 13C whereas the high WUEdetermined by gas exchange would likely promote enrichedisotopic values It is more likely that d13C in this species wasdetermined by source CO2 or stored carbon or recycling ofrespired CO2 (da Silveira et al 1989) Our study individualsof Ruscus were cultivated in a shaded understory similar totheir natural habitat Previous studies have shown that there canbe higher concentrations of respired CO2 in the forest understorythan in the canopy resulting in more negative carbon isotoperatios in understory plant tissue than canopy plant tissue (daSilveira et al 1989) This is a function of decomposing leavesand litter cover slow air mixing as well as plant environmentalresponses
A second possible explanation for the d13C values ofRuscus is related to its growth form and phenology asRuscus has extensive rhizomes (Sack et al 2003b) whichact as a carbon store for the plant Using this recycled carbonduring the growth season when the plant may not be able tomeet its carbon requirement by photosynthesis alone becauseof low maximum rates under light limitation may alsocontribute to more negative d13C values (Vizzini 2003)Notably Ruscus stems are hollow and thus can also storerelatively large amounts of CO2 for use during a growth periodwhen carbon is otherwise limiting especially given very lowgs Some of this stored carbon might be photosyntheticallyfixed by the stem (Nilsen and Sharifi 1997) In each of thesecases respired stored or recycled CO2 would supply carbonthat was previously fixed by Rubisco with more negative d13Cvalues (ndash27o) than air (ndash8o)
There was also a dissonance between the d13C value and CiCa The fully mature phylloclades which were selected andused for gas-exchange and d13C measurements were producedduring late winter and early spring with mild temperatures(average at midday 190ndash220C) and low atmospheric VPD
L Functional Plant Biology A Pivovaroff et al
(average at midday 06ndash15 kPa) However the gas-exchangemeasurements were taken during summer with hightemperature (average at midday 300ndash355C) and high VPD(average at midday 25ndash35 kPa) We harvested thephylloclades that we used for gas exchange to determine thecarbon isotope ratio Although the low value of instantaneousCiCa may be caused by high temperature and high atmosphericVPD the very low value of d13C may represent the time whenthe phyllocladersquos carbon was assimilated (during mildtemperature and low VPD)
Implications for drought and shade tolerance Ruscusas a model
We found strong support for a large number of hypotheses fortrait-based shade and drought tolerance providing a strong traitbasis for combined tolerance Although the detailed functionaltrait survey conducted here is relatively novel in its breadth(see also Pasquet-Kok et al 2010) this is a logical extensionof the traditional approach for understanding the basis forplant adaptation to environment ie testing expectations forindividual traits established by previous studies of thefunctional significance of these traits in other species Weacknowledge there is some degree of uncertainty ininterpreting a large number of traits simultaneously based onstudies of other species First the interpretation of the value oftraits based on other species may not be in all cases equally validfor Ruscus Some trait variation may relate to other functionsFurther studies for example using mutants would be necessaryas conclusive evidence of the value of specific traits in a givenspecies However one advantage of testing numerousexpectations for each hypothesis is that the key finding will berobust to the removal of some traits from the analysis if thoseare later found to be inappropriate Ideally when a model forestimating plant performance from leaf traits becomes availableone could determine how the specific quantitative combinationsof traits presented here scales up to plant shade and droughttolerance
The shade and drought tolerance of Ruscus consistent withthe suite of traits examined here is one case demonstratinghow plants can avoid a general trade-off between shade anddrought tolerance Further Ruscus is noteworthy as one of onlya few stem photosynthetic plants that occupy a shaded habitatHistorically it is likely that shade tolerance preceded droughttolerance given this speciesrsquo ancestors were species of moisttropical forests (Kim et al 2010) and thus Ruscus or itsancestor apparently evolved drought tolerance whileexpanding its range into drier habitat or during past climatechange Considering its unique adaptations and trait valuesRuscus can serve as an excellent model for the basis ofcombined shade and drought tolerance
Acknowledgements
We thank the Mildred E Mathias Botanical Garden for access to garden andplants Andy Truong for assistance with measurements Howard GriffithsLouis Santiago and Cheryl Swift for helpful discussions and comments onthe manuscript This work was supported by National Science FoundationGrant IOB-0546784
References
Bartlett MK Scoffoni C Sack L (2012) The determinants of leaf turgor losspoint and prediction of drought tolerance of species and biomes a globalmeta-analysis Ecology Letters 15 393ndash405 doi101111j1461-0248201201751x
Blackman CJ Brodribb TJ Jordan GJ (2010) Leaf hydraulic vulnerability isrelated to conduit dimensions and drought resistance across a diverserange of woody angiosperms New Phytologist 188 1113ndash1123doi101111j1469-8137201003439x
Brodribb TJ Feild TS Jordan GJ (2007) Leaf maximum photosynthetic rateand venation are linked by hydraulicsPlant Physiology 144 1890ndash1898doi101104pp107101352
Caspersen JP (2001) Interspecific variation in sapling mortality in relationto growth and soil moisture Oikos 92 160ndash168 doi101034j1600-07062001920119x
Cochard H Nardini A Coll L (2004) Hydraulic architecture of leaf bladeswhere is the main resistance Plant Cell amp Environment 27 1257ndash1267doi101111j1365-3040200401233x
Cooney-Sovetts C Sattler R (1987) Phylloclade development in theAsparagaceae an example of homoeosis Botanical Journal of theLinnean Society 94 327ndash371 doi101111j1095-83391986tb01053x
Cowling R Rundel P Lamont BB ArroyoMK ArianoutsouM (1996) Plantdiversity in Mediterranean-climate regions Trends in Ecology ampEvolution 11 362ndash366 doi1010160169-5347(96)10044-6
da Silveira L Sternberg L Mulkey SS Wright SJ (1989) Ecologicalinterpretation of leaf carbon isotope ratios influence of respired carbondioxide Ecology 70 1317ndash1324 doi1023071938191
de Lillis M Fontanella A (1992) Comparative phenology and growth indifferent species of the Mediterranean maquis of central Italy PlantEcology 99-100 83ndash96 doi101007BF00118213
Dracup JA (1991)DroughtmonitoringStochasticHydrologyandHydraulics5 261ndash266 doi101007BF01543134
Engelbrecht BMJ Kursar TA (2003) Comparative drought-resistance ofseedlings of 28 species of co-occurring tropical woody plantsOecologia 136 383ndash393 doi101007s00442-003-1290-8
Farquhar GD von Caemmerer S (1980) A biochemical model ofphotosynthetic CO2 assimilation in leaves of C3 species Planta 14978ndash90 doi101007BF00386231
FarquharGD Ehleringer JR HubickK (1989) Carbon isotope discriminationand photosynthesis Annual Review of Plant Physiology and PlantMolecular Biology 40 503ndash537 doi101146annurevpp40060189002443
Givnish TJ (1988) Adaptation to sun and shade a whole-plant perspectiveAustralian Journal of Plant Physiology 15 63ndash92 doi101071PP9880063
Gulias J Flexas J Mus M Cifre J Lefi E Medrano H (2003) Relationshipbetweenmaximum leaf photosynthesis nitrogen content and specific leafarea inBalearic endemic and non-endemicMediterranean speciesAnnalsof Botany 92 215ndash222 doi101093aobmcg123
Haberlandt G (1914) lsquoPhysiological plant anatomyrsquo (MacMillan London)Hallik L Niinemets U Wright J (2009) Are species shade and drought
tolerance reflected in leaf-level structural and functional differentiationin Northern Hemisphere temperate woody flora New Phytologist 184257ndash274 doi101111j1469-8137200902918x
Jones HG (1992) lsquoPlants and microclimate a quantitative approach toenvironmental plant physiologyrsquo (Cambridge University PressCambridge)
Kerstiens G (1996) Cuticular water permeability and its physiologicalsignificance Journal of Experimental Botany 47 1813ndash1832doi101093jxb47121813
Kim JH Kim DK Forest F Fay MF Chase MW (2010) Molecularphylogenetics of Ruscaceae sensu lato and related families(Asparagales) based on plastid and nuclear DNA sequences Annals ofBotany 106 775ndash790 doi101093aobmcq167
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology M
Koide RT Robichaux RH Morse SR Smith CM (2000) Plant water statushydraulic resistance and capacitance In lsquoPlant physiological ecologyfield methods and instrumentationrsquo (Eds R Pearcy JR Ehleringer HMooney P Rundel) pp 161ndash183 (Kluwer Dordrecht The Netherlands)
Koumlrner C Farquhar GD Wong SC (1991) Carbon isotope discrimination byplants follows latitudinal and altitudinal trends Oecologia 88 30ndash40doi101007BF00328400
Lewis AM Boose ER (1995) Estimating volume flow rates through xylemconduits American Journal of Botany 82 1112ndash1116 doi1023072446063
Martiacutenez-Palleacute E Aronne G (1999) Flower development and reproductivecontinuity inMediterraneanRuscusaculeatusL (Liliaceae)Protoplasma208 58ndash64 doi101007BF01279075
Martiacutenez-Tilleriacutea K Loayza AP Sandquist DR Squeo FA (2012) Noevidence of a trade-off between drought and shade tolerance inseedlings of six coastal desert shrub species in north-central ChileJournal of Vegetation Science 23 1051ndash1061 doi101111j1654-1103201201427x
Matalas NC (1963) Probability distribution of low flows USGS ProfessionalPapers 434-A US Government Printing Office Washington DC
Niinemets U (1999) Research review components of leaf dry mass per area ndashthickness and density ndash alter leaf photosynthetic capacity in reversedirections in woody plants New Phytologist 144 35ndash47 doi101046j1469-8137199900466x
Niinemets U Valladares F (2006) Tolerance to shade drought andwaterlogging of temperate northern hemisphere trees and shrubsEcological Monographs 76 521ndash547 doi1018900012-9615(2006)076[0521TTSDAW]20CO2
Nilsen ET Sharifi MR (1997) Carbon isotope composition of legumes withphotosynthetic stems from Mediterranean and desert habitats AmericanJournal of Botany 84 1707ndash1713 doi1023072446469
Ogburn RM Edwards EJ (2012) Quantifying succulence a rapidphysiologically meaningful metric of plant water storage Plant Cell ampEnvironment 35 1533ndash1542 doi101111j1365-3040201202503x
Pasquet-Kok J Creese C Sack L (2010) Turning over a new lsquoleafrsquo multiplefunctional significances of leaves versus phyllodes in Hawaiian Acaciakoa Plant Cell amp Environment 33 2084ndash2100 doi101111j1365-3040201002207x
SackL (2004)Responses of temperatewoody seedlings to shade and droughtdo trade-offs limit potential niche differentiation Oikos 107 110ndash127doi101111j0030-1299200413184x
SackLFroleK (2006)Leaf structural diversity is related to hydraulic capacityin tropical rain forest trees Ecology 87 483ndash491 doi10189005-0710
Sack L Holbrook NM (2006) Leaf hydraulics Annual Review of PlantBiology 57 361ndash381 doi101146annurevarplant56032604144141
Sack L Melcher PJ Zwieniecki MA Holbrook NM (2002) The hydraulicconductance of the angiosperm leaf lamina a comparison of threemeasurement methods Journal of Experimental Botany 532177ndash2184 doi101093jxberf069
Sack L Cowan P Jaikumar N Holbrook NM (2003a) The ldquohydrologyrdquo ofleaves co-ordination of structure and function in temperate woodyspecies Plant Cell amp Environment 26 1343ndash1356 doi101046j0016-8025200301058x
Sack L Grubb P Maronon T (2003b) The functional morphology of juvenileplants tolerant of strong summer drought in shaded forest understoriesin southern Spain Plant Ecology 168 139ndash163 doi101023A1024423820136
SackLScoffoniCMcKownADFroleKRawlsMHavran JCTranHTranT (2012) Developmentally based scaling of leaf venation architectureexplains global ecological patterns Nature Communications 3 837doi101038ncomms1835
Sack L Chatelet D Scoffoni C PrometheusWiki contributors (2013a)ldquoEstimating the mesophyll surface area per leaf area from leaf celland tissue dimensions measured from transverse cross-sectionsrdquoPrometheusWiki Available at httpwwwpublishcsiroauprometheuswikitiki-pagehistoryphppage=Estimating the mesophyllsurface area per leaf area from leaf cell and tissue dimensionsmeasured from transverse cross-sectionsamppreview=16 [accessed June18 2013]
SackL Pasquet-Kok JPrometheusWiki contributors (2013b) ldquoLeaf pressure-volume curve parametersrdquo PrometheusWiki Available at httpprometheuswikipublishcsiroautiki-indexphppage=Leaf+pressure-volume+curve+parameters
Scoffoni C Pou A Aasamaa K Sack L (2008) The rapid light response ofleaf hydraulic conductance new evidence from two experimentalmethods Plant Cell amp Environment 31 1803ndash1812 doi101111j1365-3040200801884x
Smith T Huston M (1989) A theory of the spatial and temporal dynamics ofplant communities Vegetatio 83 49ndash69 doi101007BF00031680
Sterck F Markesteijn L Schieving F Poorter L (2011) Functional traitsdetermine trade-offs and niches in a tropical forest communityProceedings of the National Academy of Sciences of the United Statesof America 108 20 627ndash20 632 doi101073pnas1106950108
Thomas GS (1992) lsquoOrnamental shrubs climbers and bamboosrsquo (JohnMurray Publishers Great Britain)
TyreeMT ZimmermannMH (1983) lsquoXylem structure and the ascent of saprsquo(Springer-Verlag Berlin)
USDA ARS National Genetic Resources Program (2009) GermplasmResources Information Network (GRIN) online database NationalGermplasm Resources Laboratory Beltsville Maryland Availableathttpwwwars-gringovcgi-binnpgshtmltaxonpl410577[Accessed 20 July 2009]
Vendramini F Diacuteaz S Gurvich DE Wilson PJ Thompson K Hodgson JG(2002) Leaf traits as indicators of resource-use strategy in floras withsucculent species New Phytologist 154 147ndash157 doi101046j1469-8137200200357x
Vile D (2005) Specific leaf area and dry matter content estimate thicknessin laminar leaves Annals of Botany 96 1129ndash1136 doi101093aobmci264
Vizzini S (2003) d13C and d15N variability in Posidonia oceanica associatedwith seasonality and plant fraction Aquatic Botany 76 195ndash202doi101016S0304-3770(03)00052-4
Walters M Reich PB (1999) Research review low-light carbon balance andshade tolerance in the seedlings of woody plants dowinter deciduous andbroad-leaved evergreen species differ New Phytologist 143 143ndash154doi101046j1469-8137199900425x
Witkowski E Lamont BB (1991) Leaf specific mass confounds leaf densityand thickness Oecologia 88 486ndash493
Wright IJWestobyM (2003)Nutrient concentration resorption and lifespanleaf traits of Australian sclerophyll species Functional Ecology 1710ndash19 doi101046j1365-2435200300694x
Wright IJ Reich PB Westoby M Ackerly DD Baruch Z Bongers FCavender-Bares J Chapin T Cornelissen JHC Diemer M Flexas JGarnier E Groom PK Gulias J Hikosaka K Lamont BB Lee T LeeWLusk C Midgley JJ Navas M-L Niinemets U Oleksyn J Osada NPoorter H Poot P Prior L Pyankov VI Roumet C Thomas SC TjoelkerMG Veneklaas EJ Villar R (2004) The worldwide leaf economicsspectrum Nature 428 821ndash827 doi101038nature02403
Wullschleger SD (1993) Biochemical limitations to carbon assimilation inC3 plants ndash a retrospective analysis of the ACi curves from 109 speciesJournal of Experimental Botany 44 907ndash920 doi101093jxb445907
N Functional Plant Biology A Pivovaroff et al
wwwpublishcsiroaujournalsfpb
Tab
le2
Meanva
lues
foran
atom
ical
traitsseforRuscusaculeatusan
dRm
icroglossumw
ithun
itsan
dreplication
Forgiventraitsexpectatio
nsaregivenforw
hetherRuscusshouldhave
ahigherorlowervaluerelativ
etocomparatorspeciesaccordingtothehypothesesofshadeordroughtadaptationCom
paratorspeciesdata
weretakenaccordingtoavailabilityinthepreviouslypublishedliteratureandnumbero
fspeciesand
minim
umm
eanandmaxim
umtraitvaluesareprovidedSources
ofcomparativ
edatatissuethicknessand
airspace
(SackandFrole20
06)Dm(Brodribbetal2
007)N
Sn
otstatistically
significant
atPlt005
Anatomicaltraits
Units
Ra
culeatus
Rm
icroglossum
Hypotheses
Com
paratorspecies
Meanplusmnse
NMeanplusmnse
NShade
adapted
Drought
adapted
(N)
(minm
ean
max)
Tissuethicknessesan
dairspace
Lam
ina
mm27
8plusmn334
529
6plusmn857
5HigherA
BHigherA
BTropicalevergreen
trees(10)
156
267
512
Cuticle
mm352
plusmn022
5329
plusmn023
5Higher
Higher
Tropicalevergreen
trees(10)
1254
60
105
Epiderm
ismm
202plusmn0513
523
plusmn0657
5HigherA
BHigherA
BTropicalevergreen
trees(10)
9751
40
173
Epiderm
iscellwall
mm341
plusmn011
5369
plusmn025
5Mesop
hyll
mm72
6plusmn179
583
0plusmn272
5Water
storage
mm88
7plusmn705
586
2plusmn587
5Present
AB
totalleaf
airspace
10
5plusmn138
520
8plusmn173
5
Celldimension
sEpiderm
iscellarea
mm2
447plusmn27
05
599plusmn35
35
Epiderm
iscellperimeter
mm78
9plusmn261
594
1plusmn278
5Mesop
hyllcellarea
mm2
657plusmn21
55
846plusmn42
65
Mesophyllcellperimeter
mm94
5plusmn172
511
0plusmn241
5
chloroplastarea
inmesop
hyll
cell
21
2plusmn149
521
1plusmn307
5
Mesop
hyllareatotalleaf
area
(AmesA)
247plusmn13
520
5plusmn045
5
Water
storagecellarea
mm2
3414
plusmn19
65
5972
plusmn84
75
Water
storagecellperimeter
mm22
1plusmn572
529
1plusmn19
15
Vasculartraits
Minim
umdistance
from
vein
toepidermis(D
m)
mm20
3plusmn18
05
370plusmn49
35
HigherB
Low
erA
Dicotyledons
(11)
129
233
428
Inter-veinaldistance
(IVD)
mm27
2plusmn27
55
509plusmn71
55
Fibrous
bundlesheath
cellheight
mm18
4plusmn127
517
5plusmn149
5Fibrous
bundlesheath
cellwidth
mm17
4plusmn066
516
3plusmn092
5Fibrous
bundlesheath
cellwall
thickness
mm457
plusmn041
5248
plusmn023
5
Average
theoreticalxylemconduit
cond
uctiv
ityin
themidrib
10
5mmolm
ndash2
sndash1MPandash
1124
plusmn025
5198
plusmn055
5
Average
theoreticalxylemconduit
conductiv
ityin
aninterm
ediary
vein
10
5mmolm
ndash2
sndash1MPa
ndash1
063
plusmn014
5119
plusmn038
5
Average
theoreticalxylemconduit
cond
uctiv
ityin
aminor
vein
10
5mmolm
ndash2
sndash1MPandash
1037
plusmn012
5028
plusmn006
5
(contin
uednextpa
ge)
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology C
typical understory species in forests in the Mediterranean basin(Fig 1 de Lillis and Fontanella 1992Martiacutenez-Palleacute andAronne1999 Sack et al 2003b)RuscusmicroglossumBertol is a hybridproduced by crossing species Ruscus hypoglossum L from theBlack Sea region andRuscus hypophyllumL fromNorthAfrica(Fig 1 Thomas 1992 USDA ARS National Genetic ResourcesProgram 2009)
Experiments were conducted on R aculeatus andR microglossum plants in the Mildred E Mathias BotanicalGarden at the University California Los Angeles from Juneto August 2009 Measurements were made in shaded understorysites on at least three individuals of each species Diurnal lightmeasurements were made on sunny days above the Ruscus plantcanopies with a quantum sensor (Li-190S Li-Cor BiosciencesLincoln NE USA) Individuals received full sunlight averaging~1500mmolmndash2 sndash1 photosynthetically active radiation (PAR)for ~1 h each day and otherwise experienced PAR of~50mmolmndash2 sndash1 interspersed with sunflecks averaging~190mmolmndash2 sndash1 Plants were irrigated as needed and allRuscus individuals were watered before the start of this studyto reduce any differences in water availability betweenindividuals
Phylloclade morphology
We determined leaf morphological traits on recently formedmature phylloclades Although methods were applied tophylloclades for convenience we retained the names ofmethods and traits as applied to leaves (eg leaf mass perarea) Phylloclade area was measured on excised samples withan areameter (Li-3100 Li-Cor Biosciences) Samples were driedin an oven at gt70C for more than 48 h to determine drymass andcalculate leaf mass per area (LMA) as dry mass divided by areaPhylloclade thickness was measured with electronic digitalcalipers (Fisher Scientific Pittsburgh PA USA) and densitywas calculated as mass per area divided by thickness (Witkowskiand Lamont 1991)
Phylloclade anatomy
We sampled phylloclades of each species and prepared cross-sections for anatomical measurements Phylloclades werepreserved in formalin acetic acid (37 formaldehyde glacialacidic acid 95 ethanol and deionised water in a 10 5 50 35mixture) We measured the transverse cross-sectional anatomyusing sections cut halfway along the phylloclade lengthembedded in LR White (London Resin Co London UK) cut05mm thick using a microtome (Ultracut E Reichert-JungUltracut E Leica Microsystems Arcadia CA USA) stainedwith 001toluidine blue in 1sodiumborate andviewedunderthe light microscope using a 20ndash40 objective (DMRB LeicaMicrosystems Wetzlar Germany)
We measured tissue thicknesses and cell dimensions in thelamina and dimensions of vascular bundles and of xylemconduits using Image J software (ver 142 q NationalInstitutes of Health Bethesda MD USA) on microscopeimages We measured thickness for the lamina cuticleepidermis epidermis cell wall mesophyll and water storagecompartment We averaged three measurements of each type foreach cross-section The phylloclade tissues were arranged
Tab
le2
(contin
ued)
Anatomicaltraits
Units
Ra
culeatus
Rm
icroglossum
Hyp
otheses
Com
paratorspecies
Meanplusmnse
NMeanplusmnse
NShade
adapted
Droug
htadapted
(N)
(minm
ean
max)
Meanmaxim
umxy
lem
cond
uit
diam
eter
inthemidrib
mm966
plusmn044
511
0plusmn078
5
Meanmaxim
umxy
lem
cond
uit
diam
eterinan
interm
ediaryvein
mm831
plusmn055
5949
plusmn072
5
Meanmaxim
umxy
lem
cond
uit
diam
eter
inaminor
vein
mm715
plusmn072
5719
plusmn048
5
AAnexpectationaccordingto
ahypothesiswas
confi
rmed
forRa
culeatus
BAnexpectationaccordingto
ahypothesiswas
confi
rmed
forRm
icroglossum
D Functional Plant Biology A Pivovaroff et al
Tab
le3
Meanva
lues
forga
sexchan
getraitsseforRuscusaculeatusan
dRm
icroglossumw
ithun
itsan
dreplication
Forgiventraitsexpectatio
nsaregivenforw
hetherRuscusshouldhave
ahigherorlowervaluerelativ
etocomparatorspeciesaccordingtothehypothesesofshadeordroughtadaptationCom
paratorspeciesdata
weretakenaccordingtoavailabilityinthepreviouslypu
blishedliteratureandnu
mberofspeciesand
minim
umm
eanandmaxim
umtraitvaluesareprov
idedSourcesofcomparativ
edataA
areaA
massRareaR
mass
g s(W
rightetal2
004)LCP(W
altersandReich
1999)Ag
s(G
uliasetal2
003)VcmaxJ
max(W
ullschleger19
93)g m
in(K
erstiens
1996)NSn
otstatistically
significant
atPlt005
Gas-exchang
etraits
Abb
reviation
Units
Ra
culeatus
Rm
icroglossum
Hypotheses
Com
paratorspecies
Meanplusmnse
NMeanplusmnse
NShade
adapted
Droug
htadapted
(N)
(minm
ean
max)
Light-saturated
rateof
photosyn
thesisperarea
Aarea
mmolm
ndash2sndash
1522
plusmn133
451
plusmn047
6Low
erAB
Tem
peratebroadleaf
evergreens
(78)
2629
42
2265
Light-saturated
rateof
photosyn
thesisperm
ass
Amass
nmolCO2gndash
1sndash
1004
3plusmn001
14
0050plusmn0005
6Low
erAB
Tem
peratebroadleaf
evergreens
(78)
2287
46
209
Respiratio
nrateperarea
Rarea
mmolm
ndash2sndash
1004
4plusmn000
98
0156plusmn0015
10Low
erAB
Low
erAB
Tem
peratebroadleaf
evergreens
(50)
0320
93
170
Respiratio
npermass
Rmass
nmolCO2gndash
1sndash
1356
E-04plusmn702
E-05
8171E-03plusmn166E-04
10Low
erAB
Low
erAB
Tem
peratebroadleaf
evergreens
(50)
3337
06
157
Maxim
umstom
atal
conductanceperarea
g smmolm
ndash2sndash
133
plusmn000
74
35plusmn0006
6Low
erAB
Low
erAB
Tem
peratebroadleaf
evergreens
(48)
491
423
09
Light
compensationpo
int
LCP
mmol
photonsm
ndash2sndash
1485
plusmn0
4361
plusmn0
6Low
erAB
Tropicalevergreenshade-
tolerators(15)
16582
4
Intrinsicwater
use
efficiency
Ag
smm
olmol
ndash1
154plusmn8
414
2plusmn19
06
HigherA
BMediterraneanspecies
(78)
2335
94
142
Ratio
ofintercellularto
ambient[CO2]
CiC
a032
7plusmn010
64
0431plusmn0064
6
Maxim
umrateof
carbox
ylation
Vcmax
mmolm
ndash2sndash
126
6plusmn318
424
7plusmn542
5Low
erAB
Tem
peratehardwood
species(19)
114
711
9
Maxim
umrateof
electron
transport
J max
601plusmn635
550
6plusmn786
5Low
erAB
Tem
peratehardwood
species(19)
291
042
37
Leafcuticularcond
uctance
g min
mmolm
ndash2sndash
1037
9plusmn008
210
0295plusmn0082
12Low
erAB
Vascularplants(201)
01
1321
07Stem
cuticular
conductance
g min
mmolm
ndash2sndash
1009
5plusmn002
56
0030plusmn0003
4
AAnexpectationaccordingto
ahypothesiswas
confi
rmed
forRa
culeatus
BAnexpectationaccordingto
ahy
pothesiswas
confi
rmed
forRm
icroglossum
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology E
Tab
le4
Meanva
lues
forhy
drau
licstraitsseforRuscusaculeatusan
dRm
icroglossumw
ithun
itsan
dreplication
Forgiventraitsexp
ectatio
nsaregivenforw
hetherRuscusshouldhave
ahigh
erorlowervaluerelativ
etocomparatorspeciesaccordingtothehy
pothesesofshadeordrough
tadaptationCom
paratorspeciesdata
weretakenaccordingtoavailabilityinthepreviouslypu
blishedliteratureandnu
mberofspeciesand
minim
umm
eanandmaxim
umtraitvaluesareprov
idedorm
eanstandarderrorifon
lythesewereavailable
Sources
ofcomparativ
edataK
shoot(SackandHolbroo
k20
06)Y
tlpR
WCtlpP
oe
(Bartlettetal2
012)Cft(Scoffon
ietal2
008)NSnotstatistically
significant
atPlt005
Hydraulicstraits
Abbreviation
Units
Ra
culeatus
Rm
icroglossum
Hyp
otheses
Com
paratorspecies
Meanplusmnse
NMeanplusmnse
NShade
adapted
Drought
adapted
(N)
(minm
ean
max)
Sho
othy
draulic
cond
uctance
Kshoot
mmolm
ndash2sndash
1
MPandash
1216
plusmn010
10269
plusmn013
11Low
erAB
Low
erAB
Tem
peratewoo
dyangiosperm
s(38)
8plusmn1
Tropicalwoody
angiosperm
s(49)
13plusmn15
Osm
oticpotentialatfull
turgor
p oMPa
ndash128
plusmn010
6ndash065
plusmn003
6HigherA
BHigherA
BC
Evergreen
woo
dyspecies
(182)
-34ndash
183ndash
049
Turgo
rloss
point
Ytlp
MPa
ndash184
plusmn010
6ndash113
plusmn007
6HigherA
BHigherA
BC
Evergreen
woo
dyspecies
(158)
-425
ndash220ndash
054
Modulus
ofelasticity
eMPa
110plusmn141
6588
plusmn053
6Low
erABC
Evergreen
woo
dyspecies(139)
3561
71
734
Relativewater
contentat
turgor
loss
point
RWCtlp
88
8plusmn109
689
3plusmn085
6HigherA
BC
Evergreen
woo
dyspecies(19)
718
12
911
Relativecapacitanceatfull
turgor
Cft
MPandash
10067plusmn0008
60102plusmn0010
6HigherA
BC
Evergreen
species(6)
0040
0066
0113
Relativecapacitanceat
turgor
loss
Ctlp
MPandash
10104plusmn0015
60089plusmn0003
6
Predawnwater
potential
Ypre
MPa
ndash045
plusmn008
4ndash088
plusmn008
4Middaywater
potential
Ymid
MPa
ndash141
plusmn008
3ndash129
plusmn028
4
AAnexpectationaccordingto
ahy
pothesiswas
confi
rmed
forRa
culeatus
BAnexpectationaccordingto
ahypothesiswas
confi
rmed
forRm
icroglossum
CExpectatio
nsforthesetraitsarebasedon
drou
ghttolerance
conferredby
waterstoragetissue(lsquosucculencersquo)the
oppositeexpectations
wou
ldarisefordroughttoleranceconferredby
theabilitytomaintainturgor
with
dehydration
F Functional Plant Biology A Pivovaroff et al
symmetrically with adaxial and abaxial layers of mesophyllepidermis and cuticle above and below a central achlorophyllouswater storage tissue (Fig 2) The per cent air space in each of theadaxial and abaxial mesophyll and in the water storagecompartment was estimated to the nearest 5 and the totalphylloclade airspace was calculated
PAir space in each tissue
fraction of leaf cross section occupied by that tissue100
eth1THORN
As indices of cell size the cross-sectional areas andperimeters were measured for three cells in the epidermis(adaxial and abaxial) the mesophyll (adaxial and abaxialie above and below the water storage tissue) and thewater storage tissue The area occupied by chloroplastswithin a mesophyll cell was measured for three cells(adaxial and abaxial) and the percent cross-sectionalchloroplast area was calculated as the ratio of chlorophyllarea divided by mesophyll cell area
We calculated the surface area of mesophyll cells per leafarea (AmesA) as described by Sack et al (2013a) a measure ofthe area available for CO2 uptake formesophyll cell layers Giventhe lack of palisade-form cells we modelled all mesophyll cellsas spheres for these calculations
We also measured vascular anatomy to quantify traits relatedto the efficiency of water transport within and outside the xylemWe measured the inter-veinal distance (IVD) and also theminimum distance from edge of bundle sheath to epidermis(Dm) as the hypotenuse between the distance between veinsand the distance to the epidermis (Brodribb Feild and Jordan2007)
Dm frac14ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
IVD2
2thorn ethdistance from bundle sheath edge to epidermisTHORN2
s
eth2THORN
We averaged IVD and Dm from three values for each cross-section
For all anatomical traits except those relating to the centralwater storage tissue measurements were made both adaxial andabaxial halves and values for the two halveswere averagedwhennot significantly different (at P lt 005 in paired t-tests) exceptthey were summed for total AmesA
To characterise the midrib for three typical fibrous bundlesheath cells we measured the cross-sectional heights widthsand cell wall thicknesses calculating mean values for each traitfrom three measurements per cross-section To characterise thexylem anatomy and theoretical conductivity of xylem conduitsfor a typical conduit within the midrib an intermediary veinand a minor vein of each sampled phylloclade we treated theconduit as an ellipse and determined the major and minor axisdiameters We calculated the theoretical hydraulic conductivityof the xylem conduit using Poiseuillersquos equation for ellipsesbased on conduit dimensions (Lewis and Boose 1995 CochardNardini and Coll 2004)
Tab
le5
Meanvalues
fortissue
compo
sition
traitsseforRuscusaculeatusan
dRm
icroglossumw
ithun
itsan
dreplication
andsign
ificanceof
differencesbetw
eenspecies(t-tests)
Forgiventraitsexpectatio
nsaregivenforw
hetherRaculeatus
shouldhave
ahigherorlowervaluerelativ
etocomparatorspeciesaccordingtothehypothesesofshadeordroughtadaptationCom
paratorspecies
dataweretakenaccordingtoavailabilityinthepreviouslypublishedliteratureandnumberofspeciesand
minim
umm
eanandmaxim
umtraitvaluesareprovidedSourcesofcomparativ
edatad
13C(K
oumlrneretal
1991)NmassNarea(W
righ
tetal2
004)N
Sn
otstatistically
significant
atPlt005
Tissuecompositio
ntraits
Abbreviation
Units
Ra
culeatus
Rm
icroglossum
Hypotheses
Com
paratorspecies
Meanplusmnse
NMeanplusmnse
NShade
adapted
Drought
adapted
(N)
(minm
ean
max)
Npermass
Nmass
194
plusmn006
9192
plusmn007
9Low
erHigherA
BTem
peratebroadleaf
evergreen(129)
0581
19
231
Nperarea
Narea
238
plusmn007
9175
plusmn006
9Low
erHigherA
BTem
peratebroadleaf
evergreen(129)
0727
1733
63
Cpermass
Cmass
44
4plusmn048
943
6plusmn052
9Carbonto
nitrogen
ratio
CN
231plusmn068
922
9plusmn058
9Carbonisotoperatio
d13C
permilndash33
32plusmn029
9ndash33
05plusmn020
9Low
erAB
Higher
Tem
peratespecies(35)
ndash27
64
ndash26
03
ndash24
09
AAnexpectationaccordingto
ahypothesiswas
confi
rmed
forRa
culeatus
BAnexpectationaccordingto
ahy
pothesiswas
confi
rmed
forRm
icroglossum
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology G
K t frac14 pa3b3
64hetha2 thorn b2THORN eth3THORN
where a and b are the major and minor axes of the ellipse and his water viscosity at 25C
Gas-exchange measurements responses to light and CO2and cuticular conductance
In July and August 2009 photosynthetic light response curvesandCO2 response curvesweremeasuredusing aLi-6400portablephotosynthesis system (Li-Cor Biosciences ) with light provided
Ruscusaculeatus
Ruscusmicroglossum
01 mm
Fig 2 Lamina and midrib cross-sections of Ruscus aculeatus and R microglossum phylloclades (05mm thick) Note that bothspecies exhibit shade tolerance features such as absence of palisade tissue as well as drought tolerance features such as large waterstorage compartment and thick-walled epidermis andfibrousbundle sheath cells surrounding thexylemandphloemfor bothmajor andminor veins especially prominent in R aculeatus which is native to drier habitats
(a) (b)
Fig 1 (a)Ruscus aculeatus and (b)Rmicroglossumgrowing at theMildredEMathiasBotanicalGardenNote thatwhat atfirst glance appear to be leaves are infact phylloclades
H Functional Plant Biology A Pivovaroff et al
by a red-blue light source (6400ndash02B no SI-710 Li-CorBiosciences ) Gas-exchange measurements were made on atleast 1ndash2 phylloclades per individual
For light-response curves phylloclades were acclimatedfor at least 5min at 1500mmolmndash2 sndash1 PAR at temperatures of2527C with RH maintained at ~50 and CO2 concentrationof 400mmolmolndash1 Then phylloclades were measured for netCO2 assimilation per leaf area at PAR steps of 1600 1400 12001000 800 600 500 400 300 200 100 50 and 0mmolmndash2 sndash1with 180ndash240 s stabilisation at each irradiance step Wedetermined light-saturated photosynthetic rate per area andmass (Aarea and Amass) dark respiration rate per area and mass(Rarea and Rmass) ie the negative A at zero PAR maximumstomatal conductance per area (gs) light compensation point(LCP) as the x-intercept intrinsic water use efficiency (WUEAareags) and the ratio of intercellular to ambient CO2
concentration (CiCa) at 100mmolmndash2 sndash1 PAR We thenharvested the phylloclades measured for gas exchange todetermine the nitrogen concentration and carbon isotope ratio(see below)
For CO2-response curves phylloclades were allowed toequilibrate at 400 ppm to induce stomatal opening and the netCO2 assimilation per leaf area was determined at Ci steps of400 300 200 100 50 400 400 500 600 800 1200 14001600 ppm with 3ndash4min equilibration time at each step Wedetermined maximum rate of carboxylation and maximum rateof electron transport per leaf area (Vcmax and Jmax) from plots ofCi vs A corrected to 25C (Farquhar and von Caemmerer 1980)
Cuticular conductance (ie minimumepidermal conductancegmin sensu Kerstiens 1996) was determined for 3ndash4 maturephylloclades and 10 cm lengths of stems from each of threeindividuals of each species Phylloclade and stem sampleswere hydrated and then the cut ends were sealed with waxSamples were dried for at least 30min on a laboratory bench atPAR of lt10mmol photons mndash2 sndash1 to induce stomatal closurethen samples were weighed for at least eight intervals of 30minduring which the slope of water loss versus time was highlylinear (R2 gt 0995) and therefore taken to represent transpirationafter stomata had closed fully The gmin was calculated as thetranspiration rate divided by the mole fraction vapour pressuredeficit (VPD determined from a weather station HOBO MicroStation with Smart Sensors Onset Bourne MA USA)
Pressurendashvolume curve parametersPressurendashvolume curve parameters were determined for matureshoots using the bench-drying method (Koide et al 2000 Sacket al 2003a) Shoots were ~10ndash15 cm long with about fourphylloclades per shoot for R microglossum and 15ndash20phylloclades for R aculeatus Shoots were progressively driedon a laboratory bench and measured at intervals by equilibratingfor 10min in a Whirlpak bag (Whirl-Pak Nasco Fort AtkinsonWIUSA) beforeweighing andmeasuring for leafwater potential(Yleaf) with a pressure chamber (Model 1000 Plant MoistureStress Instruments Albany OR USA) Subsequently dry masswas determined after more than 48 h in an oven at 70C Wedetermined the leaf dry matter content (LDMC) saturated watercontent (SWC) turgor loss point (Ytlp) relative water content atturgor loss point (RWCtlp) and osmotic potential at full turgor
(po) relative capacitance at full turgor (Cft DRWCDYleaf) andrelative capacitance at turgor loss point (Ctlp DRWCDYleaf) Wedetermined the modulus of elasticity (e) as the linear slope of theline fitted for pressure potential versus relative water contentabove and including turgor loss point (Sack et al 2013b)
Shoot hydraulic conductance
We measured the hydraulic conductance of mature shoots(Kshoot) ~10ndash15 cm long bearing about four phylloclades pershoot for R microglossum and 15ndash20 phylloclades forR aculeatus using the evaporative flux method (Sack et al2002) Samples were harvested and the ends were re-cut underdistilled degassed water with a fresh razor blade then hydratedovernight Shoots were connected to tubing containing distilleddegassed water running to a graduated cylinder on a balanceinterfaced to a computer logging data every minute to calculatethe flow rate of water from the balance into the sample Sampleswere held in place above a fan on wood frames strung withfishing line Lights were arranged above a plexiglass containerof water that acted as a heat trap producing PAR ofgt1200mmolmndash2 sndash1 at shoot level When steady-statetranspiration was achieved samples were covered with aplastic bag and removed from the tubing and the leaf waterpotential was determined with a pressure chamber (Model 1000Plant Moisture Stress Instruments) Kshoot was calculated as thesteady-state transpirational flow rate (E mmolmndash2 sndash1) dividedby the water potential driving force (DYleaF= ndashYleaf MPa)further normalised by total phylloclade area Kshoot valueswere standardised to 25C to correct for the temperaturedependence of the viscosity of water (Sack et al 2003a)
Foliar nitrogen concentration and carbon isotope ratio
To analyse total tissue nitrogen concentration and carbon isotopecomposition (d13C) for three replicates from each of threeindividuals for each species phylloclade samples were oven-dried at gt70C for more than 48 h and ground by mortar andpestle The nitrogen concentration and d13C were determined atthe UCDavis Stable Isotope Facility using an elemental analyser(PDZEuropaANCA-GSL SerconLtd Cheshire UK) interfacedto an isotope ratiomass spectrometer (PDZEuropa20ndash20 SerconLtd) at the UC Davis Stable Isotope Facility Final d13C contentvalues are expressed relative to international standardVienna PeeDee belemnite (V-PDB) for carbon
d13CethpermilTHORN frac14 etheth13C=12C of sampleTHORN=eth13C=12C of standardTHORN 1THORN 1000
eth4THORN
d13C of plant tissues can provide a measure of intrinsic WUE(Ags) at the time that carbon was assimilated giving long-termwater-use efficiency
Comparative data compilation and trait comparison
To consider Ruscus trait values in a global context we compileddata from the literature for (1) temperate and tropical broadleafevergreen species (2) Mediterranean species and (3) woodyangiosperms in general When available we considered data forplants grown in the shade and for species that are shade tolerant
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology I
however eco-physiological trait data is generally collected forsun leaves or plants making a comparison solely for shade grownplants not possible Formore commonly studied traits (egAmax)comparative data was taken from studies with large databasesand multiple traits (eg GLOPNET Wright et al 2004) Wedetermined minimum mean and maximum values for traitsfrom comparative studies or the mean and standard error ifraw species values were not available Studies that wereincluded for comparative data are referenced in the captionsfor Table 1ndash5 in association with the specific traits wecompared Differences between Ruscus trait values andcomparative data from the literature were determined for bothR aculeatus and R microglossum Hypotheses were deemedsupported for a Ruscus species if the trait value was higher orlower than the mean comparator value in the way predicted
Results
Both Ruscus species studied had leaf morphology consistentwith adaptation to shade and drought relative to comparator
species (Tables 1ndash5)We constructed a radar graph to encapsulatethe key traits that would confer adaptation to shade drought andthe combination for R aculeatus which had the more extremeadaptation of the two Ruscus species considered (Fig 3) Thisfigure summarises the major results of our study with values forcomparator species appearing as the inner circle and R aculeatustrait values displayed as the bold outer line
Gross morphology of phylloclades
Both R aculeatus and R microglossum had LMA valueslower than comparator species and R microglossum hadphylloclades larger than the mean leaf area for comparatorspecies Both Ruscus species had lower bulk tissue density thancomparator species consistent with the water storage in theRuscus phylloclades Notably despite its water storage tissuethe LDMC and SWC values of R aculeatus were typical ofthose for comparator species whereas R microglossum had alower LDMC and a higher SWC than for comparator speciessets (Table 1)
Fig 3 Radar graph illustrating percent difference between selected traits of Ruscus aculeatuswithcomparative data (see Tables 1ndash5 for symbols and sources of comparative data) Values forR aculeatus outside the circle indicate shade andor drought tolerance Traits are arrangedaccording to whether they would contribute shade tolerance drought tolerance or both The innerdisc represents the mean for comparative species for given traits and the values for R aculeatus isscaled relative to the magnitude of that value () with a value outside the disc representing greatershade andor drought tolerance (+) and (ndash) indicate if the axis is scaled such that a value outside thecircle represents percent higher or lower than comparative values respectively For traits expressed asnegative values (+) and (ndash) indicate more negative or less negative respectively
J Functional Plant Biology A Pivovaroff et al
Anatomy of phylloclades
The Ruscus species possessed numerous anatomical traitsconsistent with benefits for both shade and drought tolerance(Table 2) The phylloclade cross-sections were symmetrical(Fig 2) and thus the cross-sectional anatomy of the adaxialand abaxial halves were not different for all traits (paired t-testP gt 005) and values were averaged within species The twospecies were similar in their substantial leaf thickness and inthe thickness of cuticle epidermis cell walls and water storagecompartment (Table 2) The fractions of the lamina occupied bythe epidermis mesophyll and water storage tissues were 1552ndash56 and 29ndash32 respectively
Both species had parallel longitudinal veins of three sizes(midrib intermediate and small veins) Consistent with droughtadaptation the two species had low Dm R aculeatus having thelower value andRmicroglossumhadahigher IVD (Table 2)Thetwo species had on average the same maximum conduitdiameters in their midribs intermediate veins and small veinsand the maximum conduit diameter decreased ~35 from themidrib to the small veins The average theoretical hydraulicconductivity of xylem conduits did not differ between thespecies and decreased by up to 86 from the midrib to theminor vein R aculeatus had on average walls that were 85thicker in the fibrous bundle sheath (Table 2)
Gas-exchange measurements
Consistent with adaptation to simultaneous shade and droughtthe Ruscus species had very low values for Rarea Rmass and gs(Table 3) relative to comparator temperate broadleaf evergreenspecies (Fig 3) The Aarea Amass LCP Vcmax and Jmax were alsovery low relative to comparator species (Fig 3 Table 3)consistent with shade adaptation The two Ruscus species hadvery high values for Ags (Table 3) consistent with excellentWUE Consistent with strong drought tolerance via retention ofstored water both species had very low values for leaf and stemgmin especially relative to comparator vascular plant species(Fig 3 Table 3)
Hydraulic conductance pressure volume curveparameters and leaf water storage
Consistent with expectations for combined drought and shadetolerance both Ruscus species had low Kshoot relative tocomparator tropical and temperate woody angiosperms (Fig 3Table 4) The pressurendashvolume curve parameters of Ruscuswereconsistent with achieving drought tolerance through tissue waterstorage Both species had less negative po and ytlp than meanvalues for comparative evergreen woody species (Fig 3Table 4) Notably both species had lower e values thancomparative evergreen woody species and higher RWCtlp andCft values (Fig 3 Table 4) consistent with drought tolerance
Nitrogen concentration and carbon isotope composition
Both species had high values for phyllyclade Narea and Nmass
relative to comparative temperate broadleaf evergreen speciesconsistent with drought adaptation (Fig 3) The d13C valueswerevery negative and typical of values often observed for understoryplants (da Silveira et al 1989) consistent with shade tolerance
Indeed the d13C values were notably strongly negative given thehigh WUE found for these species
Testing hypotheses for shade and drought tolerancewith trait survey data
Overall we quantified 57 traits for the twoRuscus species and for21 traits we had a priori hypotheses for a benefit for shadetolerance and for 24 traits we had a priori hypotheses for abenefit for drought tolerance Using comparative data we foundthat 16 of 21 hypotheses were supported for shade tolerance and22 of 24 were supported for drought tolerance Of the ninehypotheses for traits that would contribute to both shade anddrought tolerance simultaneously (ie expectations were both forhigher or lower values than comparative species) eight weresupported by trait data All these proportions were significantlyhigher than the 50 support that would have been expected toarise only from chance (P = 0001ndash0058 proportion tests)Notably in the seven cases when shade tolerance traits wouldconflict with drought tolerance traits four indicated a benefit fordrought tolerance rather than shade tolerance for both species(Tables 1ndash5)
Discussion
Both R aculeatus (Fig 3) and R microglossum showed traitvalues consistent with combined shade and drought toleranceNumerous traits were consistent with a combined shade anddrought tolerance through improving carbon balance enablinga conservative resource use ie via slow respiration and long-lived parts Other traits would contribute specifically to droughttolerance via reduced demand for water during activephotosynthesis and the ability to survive strong drought afterstomatal closure Notably many such traits were related to waterstorage providingnew insights into effective formsof succulencein shaded habitats This suite of traits would contributeimportantly to the ability of Ruscus to occupy shaded sitesprone to strong drought across a wide geographical range
Traits contributing to simultaneous drought and shadetolerance
Ruscus species showed specialisation associated withconservative resource use consistent with tolerance of shadeand drought These specialised traits included thick lamina andcomponent tissues that contribute to long tissue life-spans (shootslastgt5 years Sack et al 2003bWright et al 2004) AdditionallyRuscus species had very low gas-exchange rates including lowgs and lowKshoot whichwould correspond to a low investment invascular tissue (Tyree andZimmermann1983 Sack et al 2003b)and low Rarea Rmass Aarea and Amass all representing an ability tomaintain photosynthesis and growth with low requirements forlight and water
Traits contributing to drought tolerance
According to Jones et al (1992) drought tolerance can beachieved through avoidance of plant water deficits toleranceof plant water deficits or efficiency mechanisms Ruscus speciesshowed traits associated with drought tolerance either byproviding the ability to maintain photosynthesis and growth indrying soil andor the ability to survive chronic drought as
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology K
previously shown experimentally for R aculeatus (Sack 2004)Traits that would contribute to the ability to maintain gasexchange in drying soil include small leaf size high WUE andlow IVD High WUE achieved in part with high Narea meansthese Ruscus species can attain positive carbon balance evenwith extremely low gs Low IVD and water storage capacitanceallow the phylloclades to maintain water supply to the mesophylland tolerate transiently high evaporation rates for example dueto sunflecks without desiccating the leaf (Sack et al 2003a)Traits contributing to the ability of the phylloclades to surviveextended drought included those enabling a low evaporationrate per leaf area once stomata have shut such as low gmin inleaf and stem and those related to specialised water storagetissue linked with low leaf density low e and po values thatwere low in magnitude
Ruscus water storage
The water storage tissue of Ruscus that occupied a third of theleaf thickness although contributing most directly to droughttolerance is also consistent with shade tolerance given itscontribution to reduced tissue costs for the phylloclade as awhole The water storage tissue had thin cell walls reflected inthe low bulk e and low solute concentration contributing tobulk po values that were low in magnitude
This water storage tissue would also contribute to both typesof drought tolerance ndash the ability to maintain photosynthesis indrying soil and to survive after stomata have shut duringextended drought The lsquosucculencersquo of Ruscus phylloclades isdistinctive relative to more typical succulent-leafed andsucculent-stemmed species which tend to have high leaf watercontent and capacitance values (Vendramini et al 2002 Ogburnand Edwards 2012) In contrast in Ruscus species the SWCwas low relative to typical leaf succulent species and forR aculeatus Cft fell within the range of typical evergreenleaves We note that the strong tissue differentiation in Ruscus(ie separation of mesophyll cell and water storage in spaceand their distinctions in anatomy) would contribute to higheffectiveness of water storage even if the bulk tissue overallhad low SWC and capacitance Indeed across species theretends to be no relationship between the magnitude of SWC orcapacitance and the degree of within-leaf tissue differentiation(Ogburn and Edwards 2012) Notably such differentiationwould contribute special advantages for supply of waterwhether stomata are open or closed as the large thin-walledwater storage cells with low solute concentration can yield theirwater to supply the evaporative load while the photosynthetictissues can maintain their volume according to their thickerwalls and stronger solute concentration
Although the capacitance and SWC values were low forRuscus species these values would be more substantial ifconsidered relative to water demand Across several speciesCft has been found to correlate with Kshoot and with gs (Sacket al 2003a Blackman et al 2010) indicating that leaves tendto be built with capacitance to match their maximum flux ratesand thus to buffer the leaf water potential against surges intranspiration Thus because Ruscus has low gs when stomataare open and low Kshoot the capacitance would be expectedto supply transpiration transiently during sunflecks or high
VPD Likewise when stomata close the capacitance suppliesongoing water loss via cuticular conductance Given theextremely low gmin of Ruscus even its moderate Ctlp canenable survival for weeks (Sack et al 2003b Sack 2004)Further at turgor loss the water content would equalSWCRWCtlp and the relatively high RWCtlp wouldcontribute to the high water content once turgor is lost Thusthe lsquosucculencersquo of Ruscus is moderate in absolute terms butcombined with its other very strong mechanisms to reducetranspiration when stomata are open or closed ie low gs andgmin even this moderate capacitance would provide strongfunctionality
Water-use efficiency and carbon isotope composition
It was noteworthy that despite extremely high WUE valuesphylloclade d13C values of the Ruscus species were verynegative This presents a strong anomaly worthy of furtherinvestigation as species with high WUE typically have higherd13C (less negative ie closer to zero) The d13C can beinfluenced by a host of processes including source CO2 storedplant carbon and time-integrated CO2 concentration at the siteof carboxylation (Farquhar et al 1989) For Ruscus althoughd13C values were typical of an understory plant they did notappear to be drivenby internalCO2 concentration because the leafisotopic values were depleted in 13C whereas the high WUEdetermined by gas exchange would likely promote enrichedisotopic values It is more likely that d13C in this species wasdetermined by source CO2 or stored carbon or recycling ofrespired CO2 (da Silveira et al 1989) Our study individualsof Ruscus were cultivated in a shaded understory similar totheir natural habitat Previous studies have shown that there canbe higher concentrations of respired CO2 in the forest understorythan in the canopy resulting in more negative carbon isotoperatios in understory plant tissue than canopy plant tissue (daSilveira et al 1989) This is a function of decomposing leavesand litter cover slow air mixing as well as plant environmentalresponses
A second possible explanation for the d13C values ofRuscus is related to its growth form and phenology asRuscus has extensive rhizomes (Sack et al 2003b) whichact as a carbon store for the plant Using this recycled carbonduring the growth season when the plant may not be able tomeet its carbon requirement by photosynthesis alone becauseof low maximum rates under light limitation may alsocontribute to more negative d13C values (Vizzini 2003)Notably Ruscus stems are hollow and thus can also storerelatively large amounts of CO2 for use during a growth periodwhen carbon is otherwise limiting especially given very lowgs Some of this stored carbon might be photosyntheticallyfixed by the stem (Nilsen and Sharifi 1997) In each of thesecases respired stored or recycled CO2 would supply carbonthat was previously fixed by Rubisco with more negative d13Cvalues (ndash27o) than air (ndash8o)
There was also a dissonance between the d13C value and CiCa The fully mature phylloclades which were selected andused for gas-exchange and d13C measurements were producedduring late winter and early spring with mild temperatures(average at midday 190ndash220C) and low atmospheric VPD
L Functional Plant Biology A Pivovaroff et al
(average at midday 06ndash15 kPa) However the gas-exchangemeasurements were taken during summer with hightemperature (average at midday 300ndash355C) and high VPD(average at midday 25ndash35 kPa) We harvested thephylloclades that we used for gas exchange to determine thecarbon isotope ratio Although the low value of instantaneousCiCa may be caused by high temperature and high atmosphericVPD the very low value of d13C may represent the time whenthe phyllocladersquos carbon was assimilated (during mildtemperature and low VPD)
Implications for drought and shade tolerance Ruscusas a model
We found strong support for a large number of hypotheses fortrait-based shade and drought tolerance providing a strong traitbasis for combined tolerance Although the detailed functionaltrait survey conducted here is relatively novel in its breadth(see also Pasquet-Kok et al 2010) this is a logical extensionof the traditional approach for understanding the basis forplant adaptation to environment ie testing expectations forindividual traits established by previous studies of thefunctional significance of these traits in other species Weacknowledge there is some degree of uncertainty ininterpreting a large number of traits simultaneously based onstudies of other species First the interpretation of the value oftraits based on other species may not be in all cases equally validfor Ruscus Some trait variation may relate to other functionsFurther studies for example using mutants would be necessaryas conclusive evidence of the value of specific traits in a givenspecies However one advantage of testing numerousexpectations for each hypothesis is that the key finding will berobust to the removal of some traits from the analysis if thoseare later found to be inappropriate Ideally when a model forestimating plant performance from leaf traits becomes availableone could determine how the specific quantitative combinationsof traits presented here scales up to plant shade and droughttolerance
The shade and drought tolerance of Ruscus consistent withthe suite of traits examined here is one case demonstratinghow plants can avoid a general trade-off between shade anddrought tolerance Further Ruscus is noteworthy as one of onlya few stem photosynthetic plants that occupy a shaded habitatHistorically it is likely that shade tolerance preceded droughttolerance given this speciesrsquo ancestors were species of moisttropical forests (Kim et al 2010) and thus Ruscus or itsancestor apparently evolved drought tolerance whileexpanding its range into drier habitat or during past climatechange Considering its unique adaptations and trait valuesRuscus can serve as an excellent model for the basis ofcombined shade and drought tolerance
Acknowledgements
We thank the Mildred E Mathias Botanical Garden for access to garden andplants Andy Truong for assistance with measurements Howard GriffithsLouis Santiago and Cheryl Swift for helpful discussions and comments onthe manuscript This work was supported by National Science FoundationGrant IOB-0546784
References
Bartlett MK Scoffoni C Sack L (2012) The determinants of leaf turgor losspoint and prediction of drought tolerance of species and biomes a globalmeta-analysis Ecology Letters 15 393ndash405 doi101111j1461-0248201201751x
Blackman CJ Brodribb TJ Jordan GJ (2010) Leaf hydraulic vulnerability isrelated to conduit dimensions and drought resistance across a diverserange of woody angiosperms New Phytologist 188 1113ndash1123doi101111j1469-8137201003439x
Brodribb TJ Feild TS Jordan GJ (2007) Leaf maximum photosynthetic rateand venation are linked by hydraulicsPlant Physiology 144 1890ndash1898doi101104pp107101352
Caspersen JP (2001) Interspecific variation in sapling mortality in relationto growth and soil moisture Oikos 92 160ndash168 doi101034j1600-07062001920119x
Cochard H Nardini A Coll L (2004) Hydraulic architecture of leaf bladeswhere is the main resistance Plant Cell amp Environment 27 1257ndash1267doi101111j1365-3040200401233x
Cooney-Sovetts C Sattler R (1987) Phylloclade development in theAsparagaceae an example of homoeosis Botanical Journal of theLinnean Society 94 327ndash371 doi101111j1095-83391986tb01053x
Cowling R Rundel P Lamont BB ArroyoMK ArianoutsouM (1996) Plantdiversity in Mediterranean-climate regions Trends in Ecology ampEvolution 11 362ndash366 doi1010160169-5347(96)10044-6
da Silveira L Sternberg L Mulkey SS Wright SJ (1989) Ecologicalinterpretation of leaf carbon isotope ratios influence of respired carbondioxide Ecology 70 1317ndash1324 doi1023071938191
de Lillis M Fontanella A (1992) Comparative phenology and growth indifferent species of the Mediterranean maquis of central Italy PlantEcology 99-100 83ndash96 doi101007BF00118213
Dracup JA (1991)DroughtmonitoringStochasticHydrologyandHydraulics5 261ndash266 doi101007BF01543134
Engelbrecht BMJ Kursar TA (2003) Comparative drought-resistance ofseedlings of 28 species of co-occurring tropical woody plantsOecologia 136 383ndash393 doi101007s00442-003-1290-8
Farquhar GD von Caemmerer S (1980) A biochemical model ofphotosynthetic CO2 assimilation in leaves of C3 species Planta 14978ndash90 doi101007BF00386231
FarquharGD Ehleringer JR HubickK (1989) Carbon isotope discriminationand photosynthesis Annual Review of Plant Physiology and PlantMolecular Biology 40 503ndash537 doi101146annurevpp40060189002443
Givnish TJ (1988) Adaptation to sun and shade a whole-plant perspectiveAustralian Journal of Plant Physiology 15 63ndash92 doi101071PP9880063
Gulias J Flexas J Mus M Cifre J Lefi E Medrano H (2003) Relationshipbetweenmaximum leaf photosynthesis nitrogen content and specific leafarea inBalearic endemic and non-endemicMediterranean speciesAnnalsof Botany 92 215ndash222 doi101093aobmcg123
Haberlandt G (1914) lsquoPhysiological plant anatomyrsquo (MacMillan London)Hallik L Niinemets U Wright J (2009) Are species shade and drought
tolerance reflected in leaf-level structural and functional differentiationin Northern Hemisphere temperate woody flora New Phytologist 184257ndash274 doi101111j1469-8137200902918x
Jones HG (1992) lsquoPlants and microclimate a quantitative approach toenvironmental plant physiologyrsquo (Cambridge University PressCambridge)
Kerstiens G (1996) Cuticular water permeability and its physiologicalsignificance Journal of Experimental Botany 47 1813ndash1832doi101093jxb47121813
Kim JH Kim DK Forest F Fay MF Chase MW (2010) Molecularphylogenetics of Ruscaceae sensu lato and related families(Asparagales) based on plastid and nuclear DNA sequences Annals ofBotany 106 775ndash790 doi101093aobmcq167
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology M
Koide RT Robichaux RH Morse SR Smith CM (2000) Plant water statushydraulic resistance and capacitance In lsquoPlant physiological ecologyfield methods and instrumentationrsquo (Eds R Pearcy JR Ehleringer HMooney P Rundel) pp 161ndash183 (Kluwer Dordrecht The Netherlands)
Koumlrner C Farquhar GD Wong SC (1991) Carbon isotope discrimination byplants follows latitudinal and altitudinal trends Oecologia 88 30ndash40doi101007BF00328400
Lewis AM Boose ER (1995) Estimating volume flow rates through xylemconduits American Journal of Botany 82 1112ndash1116 doi1023072446063
Martiacutenez-Palleacute E Aronne G (1999) Flower development and reproductivecontinuity inMediterraneanRuscusaculeatusL (Liliaceae)Protoplasma208 58ndash64 doi101007BF01279075
Martiacutenez-Tilleriacutea K Loayza AP Sandquist DR Squeo FA (2012) Noevidence of a trade-off between drought and shade tolerance inseedlings of six coastal desert shrub species in north-central ChileJournal of Vegetation Science 23 1051ndash1061 doi101111j1654-1103201201427x
Matalas NC (1963) Probability distribution of low flows USGS ProfessionalPapers 434-A US Government Printing Office Washington DC
Niinemets U (1999) Research review components of leaf dry mass per area ndashthickness and density ndash alter leaf photosynthetic capacity in reversedirections in woody plants New Phytologist 144 35ndash47 doi101046j1469-8137199900466x
Niinemets U Valladares F (2006) Tolerance to shade drought andwaterlogging of temperate northern hemisphere trees and shrubsEcological Monographs 76 521ndash547 doi1018900012-9615(2006)076[0521TTSDAW]20CO2
Nilsen ET Sharifi MR (1997) Carbon isotope composition of legumes withphotosynthetic stems from Mediterranean and desert habitats AmericanJournal of Botany 84 1707ndash1713 doi1023072446469
Ogburn RM Edwards EJ (2012) Quantifying succulence a rapidphysiologically meaningful metric of plant water storage Plant Cell ampEnvironment 35 1533ndash1542 doi101111j1365-3040201202503x
Pasquet-Kok J Creese C Sack L (2010) Turning over a new lsquoleafrsquo multiplefunctional significances of leaves versus phyllodes in Hawaiian Acaciakoa Plant Cell amp Environment 33 2084ndash2100 doi101111j1365-3040201002207x
SackL (2004)Responses of temperatewoody seedlings to shade and droughtdo trade-offs limit potential niche differentiation Oikos 107 110ndash127doi101111j0030-1299200413184x
SackLFroleK (2006)Leaf structural diversity is related to hydraulic capacityin tropical rain forest trees Ecology 87 483ndash491 doi10189005-0710
Sack L Holbrook NM (2006) Leaf hydraulics Annual Review of PlantBiology 57 361ndash381 doi101146annurevarplant56032604144141
Sack L Melcher PJ Zwieniecki MA Holbrook NM (2002) The hydraulicconductance of the angiosperm leaf lamina a comparison of threemeasurement methods Journal of Experimental Botany 532177ndash2184 doi101093jxberf069
Sack L Cowan P Jaikumar N Holbrook NM (2003a) The ldquohydrologyrdquo ofleaves co-ordination of structure and function in temperate woodyspecies Plant Cell amp Environment 26 1343ndash1356 doi101046j0016-8025200301058x
Sack L Grubb P Maronon T (2003b) The functional morphology of juvenileplants tolerant of strong summer drought in shaded forest understoriesin southern Spain Plant Ecology 168 139ndash163 doi101023A1024423820136
SackLScoffoniCMcKownADFroleKRawlsMHavran JCTranHTranT (2012) Developmentally based scaling of leaf venation architectureexplains global ecological patterns Nature Communications 3 837doi101038ncomms1835
Sack L Chatelet D Scoffoni C PrometheusWiki contributors (2013a)ldquoEstimating the mesophyll surface area per leaf area from leaf celland tissue dimensions measured from transverse cross-sectionsrdquoPrometheusWiki Available at httpwwwpublishcsiroauprometheuswikitiki-pagehistoryphppage=Estimating the mesophyllsurface area per leaf area from leaf cell and tissue dimensionsmeasured from transverse cross-sectionsamppreview=16 [accessed June18 2013]
SackL Pasquet-Kok JPrometheusWiki contributors (2013b) ldquoLeaf pressure-volume curve parametersrdquo PrometheusWiki Available at httpprometheuswikipublishcsiroautiki-indexphppage=Leaf+pressure-volume+curve+parameters
Scoffoni C Pou A Aasamaa K Sack L (2008) The rapid light response ofleaf hydraulic conductance new evidence from two experimentalmethods Plant Cell amp Environment 31 1803ndash1812 doi101111j1365-3040200801884x
Smith T Huston M (1989) A theory of the spatial and temporal dynamics ofplant communities Vegetatio 83 49ndash69 doi101007BF00031680
Sterck F Markesteijn L Schieving F Poorter L (2011) Functional traitsdetermine trade-offs and niches in a tropical forest communityProceedings of the National Academy of Sciences of the United Statesof America 108 20 627ndash20 632 doi101073pnas1106950108
Thomas GS (1992) lsquoOrnamental shrubs climbers and bamboosrsquo (JohnMurray Publishers Great Britain)
TyreeMT ZimmermannMH (1983) lsquoXylem structure and the ascent of saprsquo(Springer-Verlag Berlin)
USDA ARS National Genetic Resources Program (2009) GermplasmResources Information Network (GRIN) online database NationalGermplasm Resources Laboratory Beltsville Maryland Availableathttpwwwars-gringovcgi-binnpgshtmltaxonpl410577[Accessed 20 July 2009]
Vendramini F Diacuteaz S Gurvich DE Wilson PJ Thompson K Hodgson JG(2002) Leaf traits as indicators of resource-use strategy in floras withsucculent species New Phytologist 154 147ndash157 doi101046j1469-8137200200357x
Vile D (2005) Specific leaf area and dry matter content estimate thicknessin laminar leaves Annals of Botany 96 1129ndash1136 doi101093aobmci264
Vizzini S (2003) d13C and d15N variability in Posidonia oceanica associatedwith seasonality and plant fraction Aquatic Botany 76 195ndash202doi101016S0304-3770(03)00052-4
Walters M Reich PB (1999) Research review low-light carbon balance andshade tolerance in the seedlings of woody plants dowinter deciduous andbroad-leaved evergreen species differ New Phytologist 143 143ndash154doi101046j1469-8137199900425x
Witkowski E Lamont BB (1991) Leaf specific mass confounds leaf densityand thickness Oecologia 88 486ndash493
Wright IJWestobyM (2003)Nutrient concentration resorption and lifespanleaf traits of Australian sclerophyll species Functional Ecology 1710ndash19 doi101046j1365-2435200300694x
Wright IJ Reich PB Westoby M Ackerly DD Baruch Z Bongers FCavender-Bares J Chapin T Cornelissen JHC Diemer M Flexas JGarnier E Groom PK Gulias J Hikosaka K Lamont BB Lee T LeeWLusk C Midgley JJ Navas M-L Niinemets U Oleksyn J Osada NPoorter H Poot P Prior L Pyankov VI Roumet C Thomas SC TjoelkerMG Veneklaas EJ Villar R (2004) The worldwide leaf economicsspectrum Nature 428 821ndash827 doi101038nature02403
Wullschleger SD (1993) Biochemical limitations to carbon assimilation inC3 plants ndash a retrospective analysis of the ACi curves from 109 speciesJournal of Experimental Botany 44 907ndash920 doi101093jxb445907
N Functional Plant Biology A Pivovaroff et al
wwwpublishcsiroaujournalsfpb
typical understory species in forests in the Mediterranean basin(Fig 1 de Lillis and Fontanella 1992Martiacutenez-Palleacute andAronne1999 Sack et al 2003b)RuscusmicroglossumBertol is a hybridproduced by crossing species Ruscus hypoglossum L from theBlack Sea region andRuscus hypophyllumL fromNorthAfrica(Fig 1 Thomas 1992 USDA ARS National Genetic ResourcesProgram 2009)
Experiments were conducted on R aculeatus andR microglossum plants in the Mildred E Mathias BotanicalGarden at the University California Los Angeles from Juneto August 2009 Measurements were made in shaded understorysites on at least three individuals of each species Diurnal lightmeasurements were made on sunny days above the Ruscus plantcanopies with a quantum sensor (Li-190S Li-Cor BiosciencesLincoln NE USA) Individuals received full sunlight averaging~1500mmolmndash2 sndash1 photosynthetically active radiation (PAR)for ~1 h each day and otherwise experienced PAR of~50mmolmndash2 sndash1 interspersed with sunflecks averaging~190mmolmndash2 sndash1 Plants were irrigated as needed and allRuscus individuals were watered before the start of this studyto reduce any differences in water availability betweenindividuals
Phylloclade morphology
We determined leaf morphological traits on recently formedmature phylloclades Although methods were applied tophylloclades for convenience we retained the names ofmethods and traits as applied to leaves (eg leaf mass perarea) Phylloclade area was measured on excised samples withan areameter (Li-3100 Li-Cor Biosciences) Samples were driedin an oven at gt70C for more than 48 h to determine drymass andcalculate leaf mass per area (LMA) as dry mass divided by areaPhylloclade thickness was measured with electronic digitalcalipers (Fisher Scientific Pittsburgh PA USA) and densitywas calculated as mass per area divided by thickness (Witkowskiand Lamont 1991)
Phylloclade anatomy
We sampled phylloclades of each species and prepared cross-sections for anatomical measurements Phylloclades werepreserved in formalin acetic acid (37 formaldehyde glacialacidic acid 95 ethanol and deionised water in a 10 5 50 35mixture) We measured the transverse cross-sectional anatomyusing sections cut halfway along the phylloclade lengthembedded in LR White (London Resin Co London UK) cut05mm thick using a microtome (Ultracut E Reichert-JungUltracut E Leica Microsystems Arcadia CA USA) stainedwith 001toluidine blue in 1sodiumborate andviewedunderthe light microscope using a 20ndash40 objective (DMRB LeicaMicrosystems Wetzlar Germany)
We measured tissue thicknesses and cell dimensions in thelamina and dimensions of vascular bundles and of xylemconduits using Image J software (ver 142 q NationalInstitutes of Health Bethesda MD USA) on microscopeimages We measured thickness for the lamina cuticleepidermis epidermis cell wall mesophyll and water storagecompartment We averaged three measurements of each type foreach cross-section The phylloclade tissues were arranged
Tab
le2
(contin
ued)
Anatomicaltraits
Units
Ra
culeatus
Rm
icroglossum
Hyp
otheses
Com
paratorspecies
Meanplusmnse
NMeanplusmnse
NShade
adapted
Droug
htadapted
(N)
(minm
ean
max)
Meanmaxim
umxy
lem
cond
uit
diam
eter
inthemidrib
mm966
plusmn044
511
0plusmn078
5
Meanmaxim
umxy
lem
cond
uit
diam
eterinan
interm
ediaryvein
mm831
plusmn055
5949
plusmn072
5
Meanmaxim
umxy
lem
cond
uit
diam
eter
inaminor
vein
mm715
plusmn072
5719
plusmn048
5
AAnexpectationaccordingto
ahypothesiswas
confi
rmed
forRa
culeatus
BAnexpectationaccordingto
ahypothesiswas
confi
rmed
forRm
icroglossum
D Functional Plant Biology A Pivovaroff et al
Tab
le3
Meanva
lues
forga
sexchan
getraitsseforRuscusaculeatusan
dRm
icroglossumw
ithun
itsan
dreplication
Forgiventraitsexpectatio
nsaregivenforw
hetherRuscusshouldhave
ahigherorlowervaluerelativ
etocomparatorspeciesaccordingtothehypothesesofshadeordroughtadaptationCom
paratorspeciesdata
weretakenaccordingtoavailabilityinthepreviouslypu
blishedliteratureandnu
mberofspeciesand
minim
umm
eanandmaxim
umtraitvaluesareprov
idedSourcesofcomparativ
edataA
areaA
massRareaR
mass
g s(W
rightetal2
004)LCP(W
altersandReich
1999)Ag
s(G
uliasetal2
003)VcmaxJ
max(W
ullschleger19
93)g m
in(K
erstiens
1996)NSn
otstatistically
significant
atPlt005
Gas-exchang
etraits
Abb
reviation
Units
Ra
culeatus
Rm
icroglossum
Hypotheses
Com
paratorspecies
Meanplusmnse
NMeanplusmnse
NShade
adapted
Droug
htadapted
(N)
(minm
ean
max)
Light-saturated
rateof
photosyn
thesisperarea
Aarea
mmolm
ndash2sndash
1522
plusmn133
451
plusmn047
6Low
erAB
Tem
peratebroadleaf
evergreens
(78)
2629
42
2265
Light-saturated
rateof
photosyn
thesisperm
ass
Amass
nmolCO2gndash
1sndash
1004
3plusmn001
14
0050plusmn0005
6Low
erAB
Tem
peratebroadleaf
evergreens
(78)
2287
46
209
Respiratio
nrateperarea
Rarea
mmolm
ndash2sndash
1004
4plusmn000
98
0156plusmn0015
10Low
erAB
Low
erAB
Tem
peratebroadleaf
evergreens
(50)
0320
93
170
Respiratio
npermass
Rmass
nmolCO2gndash
1sndash
1356
E-04plusmn702
E-05
8171E-03plusmn166E-04
10Low
erAB
Low
erAB
Tem
peratebroadleaf
evergreens
(50)
3337
06
157
Maxim
umstom
atal
conductanceperarea
g smmolm
ndash2sndash
133
plusmn000
74
35plusmn0006
6Low
erAB
Low
erAB
Tem
peratebroadleaf
evergreens
(48)
491
423
09
Light
compensationpo
int
LCP
mmol
photonsm
ndash2sndash
1485
plusmn0
4361
plusmn0
6Low
erAB
Tropicalevergreenshade-
tolerators(15)
16582
4
Intrinsicwater
use
efficiency
Ag
smm
olmol
ndash1
154plusmn8
414
2plusmn19
06
HigherA
BMediterraneanspecies
(78)
2335
94
142
Ratio
ofintercellularto
ambient[CO2]
CiC
a032
7plusmn010
64
0431plusmn0064
6
Maxim
umrateof
carbox
ylation
Vcmax
mmolm
ndash2sndash
126
6plusmn318
424
7plusmn542
5Low
erAB
Tem
peratehardwood
species(19)
114
711
9
Maxim
umrateof
electron
transport
J max
601plusmn635
550
6plusmn786
5Low
erAB
Tem
peratehardwood
species(19)
291
042
37
Leafcuticularcond
uctance
g min
mmolm
ndash2sndash
1037
9plusmn008
210
0295plusmn0082
12Low
erAB
Vascularplants(201)
01
1321
07Stem
cuticular
conductance
g min
mmolm
ndash2sndash
1009
5plusmn002
56
0030plusmn0003
4
AAnexpectationaccordingto
ahypothesiswas
confi
rmed
forRa
culeatus
BAnexpectationaccordingto
ahy
pothesiswas
confi
rmed
forRm
icroglossum
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology E
Tab
le4
Meanva
lues
forhy
drau
licstraitsseforRuscusaculeatusan
dRm
icroglossumw
ithun
itsan
dreplication
Forgiventraitsexp
ectatio
nsaregivenforw
hetherRuscusshouldhave
ahigh
erorlowervaluerelativ
etocomparatorspeciesaccordingtothehy
pothesesofshadeordrough
tadaptationCom
paratorspeciesdata
weretakenaccordingtoavailabilityinthepreviouslypu
blishedliteratureandnu
mberofspeciesand
minim
umm
eanandmaxim
umtraitvaluesareprov
idedorm
eanstandarderrorifon
lythesewereavailable
Sources
ofcomparativ
edataK
shoot(SackandHolbroo
k20
06)Y
tlpR
WCtlpP
oe
(Bartlettetal2
012)Cft(Scoffon
ietal2
008)NSnotstatistically
significant
atPlt005
Hydraulicstraits
Abbreviation
Units
Ra
culeatus
Rm
icroglossum
Hyp
otheses
Com
paratorspecies
Meanplusmnse
NMeanplusmnse
NShade
adapted
Drought
adapted
(N)
(minm
ean
max)
Sho
othy
draulic
cond
uctance
Kshoot
mmolm
ndash2sndash
1
MPandash
1216
plusmn010
10269
plusmn013
11Low
erAB
Low
erAB
Tem
peratewoo
dyangiosperm
s(38)
8plusmn1
Tropicalwoody
angiosperm
s(49)
13plusmn15
Osm
oticpotentialatfull
turgor
p oMPa
ndash128
plusmn010
6ndash065
plusmn003
6HigherA
BHigherA
BC
Evergreen
woo
dyspecies
(182)
-34ndash
183ndash
049
Turgo
rloss
point
Ytlp
MPa
ndash184
plusmn010
6ndash113
plusmn007
6HigherA
BHigherA
BC
Evergreen
woo
dyspecies
(158)
-425
ndash220ndash
054
Modulus
ofelasticity
eMPa
110plusmn141
6588
plusmn053
6Low
erABC
Evergreen
woo
dyspecies(139)
3561
71
734
Relativewater
contentat
turgor
loss
point
RWCtlp
88
8plusmn109
689
3plusmn085
6HigherA
BC
Evergreen
woo
dyspecies(19)
718
12
911
Relativecapacitanceatfull
turgor
Cft
MPandash
10067plusmn0008
60102plusmn0010
6HigherA
BC
Evergreen
species(6)
0040
0066
0113
Relativecapacitanceat
turgor
loss
Ctlp
MPandash
10104plusmn0015
60089plusmn0003
6
Predawnwater
potential
Ypre
MPa
ndash045
plusmn008
4ndash088
plusmn008
4Middaywater
potential
Ymid
MPa
ndash141
plusmn008
3ndash129
plusmn028
4
AAnexpectationaccordingto
ahy
pothesiswas
confi
rmed
forRa
culeatus
BAnexpectationaccordingto
ahypothesiswas
confi
rmed
forRm
icroglossum
CExpectatio
nsforthesetraitsarebasedon
drou
ghttolerance
conferredby
waterstoragetissue(lsquosucculencersquo)the
oppositeexpectations
wou
ldarisefordroughttoleranceconferredby
theabilitytomaintainturgor
with
dehydration
F Functional Plant Biology A Pivovaroff et al
symmetrically with adaxial and abaxial layers of mesophyllepidermis and cuticle above and below a central achlorophyllouswater storage tissue (Fig 2) The per cent air space in each of theadaxial and abaxial mesophyll and in the water storagecompartment was estimated to the nearest 5 and the totalphylloclade airspace was calculated
PAir space in each tissue
fraction of leaf cross section occupied by that tissue100
eth1THORN
As indices of cell size the cross-sectional areas andperimeters were measured for three cells in the epidermis(adaxial and abaxial) the mesophyll (adaxial and abaxialie above and below the water storage tissue) and thewater storage tissue The area occupied by chloroplastswithin a mesophyll cell was measured for three cells(adaxial and abaxial) and the percent cross-sectionalchloroplast area was calculated as the ratio of chlorophyllarea divided by mesophyll cell area
We calculated the surface area of mesophyll cells per leafarea (AmesA) as described by Sack et al (2013a) a measure ofthe area available for CO2 uptake formesophyll cell layers Giventhe lack of palisade-form cells we modelled all mesophyll cellsas spheres for these calculations
We also measured vascular anatomy to quantify traits relatedto the efficiency of water transport within and outside the xylemWe measured the inter-veinal distance (IVD) and also theminimum distance from edge of bundle sheath to epidermis(Dm) as the hypotenuse between the distance between veinsand the distance to the epidermis (Brodribb Feild and Jordan2007)
Dm frac14ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
IVD2
2thorn ethdistance from bundle sheath edge to epidermisTHORN2
s
eth2THORN
We averaged IVD and Dm from three values for each cross-section
For all anatomical traits except those relating to the centralwater storage tissue measurements were made both adaxial andabaxial halves and values for the two halveswere averagedwhennot significantly different (at P lt 005 in paired t-tests) exceptthey were summed for total AmesA
To characterise the midrib for three typical fibrous bundlesheath cells we measured the cross-sectional heights widthsand cell wall thicknesses calculating mean values for each traitfrom three measurements per cross-section To characterise thexylem anatomy and theoretical conductivity of xylem conduitsfor a typical conduit within the midrib an intermediary veinand a minor vein of each sampled phylloclade we treated theconduit as an ellipse and determined the major and minor axisdiameters We calculated the theoretical hydraulic conductivityof the xylem conduit using Poiseuillersquos equation for ellipsesbased on conduit dimensions (Lewis and Boose 1995 CochardNardini and Coll 2004)
Tab
le5
Meanvalues
fortissue
compo
sition
traitsseforRuscusaculeatusan
dRm
icroglossumw
ithun
itsan
dreplication
andsign
ificanceof
differencesbetw
eenspecies(t-tests)
Forgiventraitsexpectatio
nsaregivenforw
hetherRaculeatus
shouldhave
ahigherorlowervaluerelativ
etocomparatorspeciesaccordingtothehypothesesofshadeordroughtadaptationCom
paratorspecies
dataweretakenaccordingtoavailabilityinthepreviouslypublishedliteratureandnumberofspeciesand
minim
umm
eanandmaxim
umtraitvaluesareprovidedSourcesofcomparativ
edatad
13C(K
oumlrneretal
1991)NmassNarea(W
righ
tetal2
004)N
Sn
otstatistically
significant
atPlt005
Tissuecompositio
ntraits
Abbreviation
Units
Ra
culeatus
Rm
icroglossum
Hypotheses
Com
paratorspecies
Meanplusmnse
NMeanplusmnse
NShade
adapted
Drought
adapted
(N)
(minm
ean
max)
Npermass
Nmass
194
plusmn006
9192
plusmn007
9Low
erHigherA
BTem
peratebroadleaf
evergreen(129)
0581
19
231
Nperarea
Narea
238
plusmn007
9175
plusmn006
9Low
erHigherA
BTem
peratebroadleaf
evergreen(129)
0727
1733
63
Cpermass
Cmass
44
4plusmn048
943
6plusmn052
9Carbonto
nitrogen
ratio
CN
231plusmn068
922
9plusmn058
9Carbonisotoperatio
d13C
permilndash33
32plusmn029
9ndash33
05plusmn020
9Low
erAB
Higher
Tem
peratespecies(35)
ndash27
64
ndash26
03
ndash24
09
AAnexpectationaccordingto
ahypothesiswas
confi
rmed
forRa
culeatus
BAnexpectationaccordingto
ahy
pothesiswas
confi
rmed
forRm
icroglossum
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology G
K t frac14 pa3b3
64hetha2 thorn b2THORN eth3THORN
where a and b are the major and minor axes of the ellipse and his water viscosity at 25C
Gas-exchange measurements responses to light and CO2and cuticular conductance
In July and August 2009 photosynthetic light response curvesandCO2 response curvesweremeasuredusing aLi-6400portablephotosynthesis system (Li-Cor Biosciences ) with light provided
Ruscusaculeatus
Ruscusmicroglossum
01 mm
Fig 2 Lamina and midrib cross-sections of Ruscus aculeatus and R microglossum phylloclades (05mm thick) Note that bothspecies exhibit shade tolerance features such as absence of palisade tissue as well as drought tolerance features such as large waterstorage compartment and thick-walled epidermis andfibrousbundle sheath cells surrounding thexylemandphloemfor bothmajor andminor veins especially prominent in R aculeatus which is native to drier habitats
(a) (b)
Fig 1 (a)Ruscus aculeatus and (b)Rmicroglossumgrowing at theMildredEMathiasBotanicalGardenNote thatwhat atfirst glance appear to be leaves are infact phylloclades
H Functional Plant Biology A Pivovaroff et al
by a red-blue light source (6400ndash02B no SI-710 Li-CorBiosciences ) Gas-exchange measurements were made on atleast 1ndash2 phylloclades per individual
For light-response curves phylloclades were acclimatedfor at least 5min at 1500mmolmndash2 sndash1 PAR at temperatures of2527C with RH maintained at ~50 and CO2 concentrationof 400mmolmolndash1 Then phylloclades were measured for netCO2 assimilation per leaf area at PAR steps of 1600 1400 12001000 800 600 500 400 300 200 100 50 and 0mmolmndash2 sndash1with 180ndash240 s stabilisation at each irradiance step Wedetermined light-saturated photosynthetic rate per area andmass (Aarea and Amass) dark respiration rate per area and mass(Rarea and Rmass) ie the negative A at zero PAR maximumstomatal conductance per area (gs) light compensation point(LCP) as the x-intercept intrinsic water use efficiency (WUEAareags) and the ratio of intercellular to ambient CO2
concentration (CiCa) at 100mmolmndash2 sndash1 PAR We thenharvested the phylloclades measured for gas exchange todetermine the nitrogen concentration and carbon isotope ratio(see below)
For CO2-response curves phylloclades were allowed toequilibrate at 400 ppm to induce stomatal opening and the netCO2 assimilation per leaf area was determined at Ci steps of400 300 200 100 50 400 400 500 600 800 1200 14001600 ppm with 3ndash4min equilibration time at each step Wedetermined maximum rate of carboxylation and maximum rateof electron transport per leaf area (Vcmax and Jmax) from plots ofCi vs A corrected to 25C (Farquhar and von Caemmerer 1980)
Cuticular conductance (ie minimumepidermal conductancegmin sensu Kerstiens 1996) was determined for 3ndash4 maturephylloclades and 10 cm lengths of stems from each of threeindividuals of each species Phylloclade and stem sampleswere hydrated and then the cut ends were sealed with waxSamples were dried for at least 30min on a laboratory bench atPAR of lt10mmol photons mndash2 sndash1 to induce stomatal closurethen samples were weighed for at least eight intervals of 30minduring which the slope of water loss versus time was highlylinear (R2 gt 0995) and therefore taken to represent transpirationafter stomata had closed fully The gmin was calculated as thetranspiration rate divided by the mole fraction vapour pressuredeficit (VPD determined from a weather station HOBO MicroStation with Smart Sensors Onset Bourne MA USA)
Pressurendashvolume curve parametersPressurendashvolume curve parameters were determined for matureshoots using the bench-drying method (Koide et al 2000 Sacket al 2003a) Shoots were ~10ndash15 cm long with about fourphylloclades per shoot for R microglossum and 15ndash20phylloclades for R aculeatus Shoots were progressively driedon a laboratory bench and measured at intervals by equilibratingfor 10min in a Whirlpak bag (Whirl-Pak Nasco Fort AtkinsonWIUSA) beforeweighing andmeasuring for leafwater potential(Yleaf) with a pressure chamber (Model 1000 Plant MoistureStress Instruments Albany OR USA) Subsequently dry masswas determined after more than 48 h in an oven at 70C Wedetermined the leaf dry matter content (LDMC) saturated watercontent (SWC) turgor loss point (Ytlp) relative water content atturgor loss point (RWCtlp) and osmotic potential at full turgor
(po) relative capacitance at full turgor (Cft DRWCDYleaf) andrelative capacitance at turgor loss point (Ctlp DRWCDYleaf) Wedetermined the modulus of elasticity (e) as the linear slope of theline fitted for pressure potential versus relative water contentabove and including turgor loss point (Sack et al 2013b)
Shoot hydraulic conductance
We measured the hydraulic conductance of mature shoots(Kshoot) ~10ndash15 cm long bearing about four phylloclades pershoot for R microglossum and 15ndash20 phylloclades forR aculeatus using the evaporative flux method (Sack et al2002) Samples were harvested and the ends were re-cut underdistilled degassed water with a fresh razor blade then hydratedovernight Shoots were connected to tubing containing distilleddegassed water running to a graduated cylinder on a balanceinterfaced to a computer logging data every minute to calculatethe flow rate of water from the balance into the sample Sampleswere held in place above a fan on wood frames strung withfishing line Lights were arranged above a plexiglass containerof water that acted as a heat trap producing PAR ofgt1200mmolmndash2 sndash1 at shoot level When steady-statetranspiration was achieved samples were covered with aplastic bag and removed from the tubing and the leaf waterpotential was determined with a pressure chamber (Model 1000Plant Moisture Stress Instruments) Kshoot was calculated as thesteady-state transpirational flow rate (E mmolmndash2 sndash1) dividedby the water potential driving force (DYleaF= ndashYleaf MPa)further normalised by total phylloclade area Kshoot valueswere standardised to 25C to correct for the temperaturedependence of the viscosity of water (Sack et al 2003a)
Foliar nitrogen concentration and carbon isotope ratio
To analyse total tissue nitrogen concentration and carbon isotopecomposition (d13C) for three replicates from each of threeindividuals for each species phylloclade samples were oven-dried at gt70C for more than 48 h and ground by mortar andpestle The nitrogen concentration and d13C were determined atthe UCDavis Stable Isotope Facility using an elemental analyser(PDZEuropaANCA-GSL SerconLtd Cheshire UK) interfacedto an isotope ratiomass spectrometer (PDZEuropa20ndash20 SerconLtd) at the UC Davis Stable Isotope Facility Final d13C contentvalues are expressed relative to international standardVienna PeeDee belemnite (V-PDB) for carbon
d13CethpermilTHORN frac14 etheth13C=12C of sampleTHORN=eth13C=12C of standardTHORN 1THORN 1000
eth4THORN
d13C of plant tissues can provide a measure of intrinsic WUE(Ags) at the time that carbon was assimilated giving long-termwater-use efficiency
Comparative data compilation and trait comparison
To consider Ruscus trait values in a global context we compileddata from the literature for (1) temperate and tropical broadleafevergreen species (2) Mediterranean species and (3) woodyangiosperms in general When available we considered data forplants grown in the shade and for species that are shade tolerant
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology I
however eco-physiological trait data is generally collected forsun leaves or plants making a comparison solely for shade grownplants not possible Formore commonly studied traits (egAmax)comparative data was taken from studies with large databasesand multiple traits (eg GLOPNET Wright et al 2004) Wedetermined minimum mean and maximum values for traitsfrom comparative studies or the mean and standard error ifraw species values were not available Studies that wereincluded for comparative data are referenced in the captionsfor Table 1ndash5 in association with the specific traits wecompared Differences between Ruscus trait values andcomparative data from the literature were determined for bothR aculeatus and R microglossum Hypotheses were deemedsupported for a Ruscus species if the trait value was higher orlower than the mean comparator value in the way predicted
Results
Both Ruscus species studied had leaf morphology consistentwith adaptation to shade and drought relative to comparator
species (Tables 1ndash5)We constructed a radar graph to encapsulatethe key traits that would confer adaptation to shade drought andthe combination for R aculeatus which had the more extremeadaptation of the two Ruscus species considered (Fig 3) Thisfigure summarises the major results of our study with values forcomparator species appearing as the inner circle and R aculeatustrait values displayed as the bold outer line
Gross morphology of phylloclades
Both R aculeatus and R microglossum had LMA valueslower than comparator species and R microglossum hadphylloclades larger than the mean leaf area for comparatorspecies Both Ruscus species had lower bulk tissue density thancomparator species consistent with the water storage in theRuscus phylloclades Notably despite its water storage tissuethe LDMC and SWC values of R aculeatus were typical ofthose for comparator species whereas R microglossum had alower LDMC and a higher SWC than for comparator speciessets (Table 1)
Fig 3 Radar graph illustrating percent difference between selected traits of Ruscus aculeatuswithcomparative data (see Tables 1ndash5 for symbols and sources of comparative data) Values forR aculeatus outside the circle indicate shade andor drought tolerance Traits are arrangedaccording to whether they would contribute shade tolerance drought tolerance or both The innerdisc represents the mean for comparative species for given traits and the values for R aculeatus isscaled relative to the magnitude of that value () with a value outside the disc representing greatershade andor drought tolerance (+) and (ndash) indicate if the axis is scaled such that a value outside thecircle represents percent higher or lower than comparative values respectively For traits expressed asnegative values (+) and (ndash) indicate more negative or less negative respectively
J Functional Plant Biology A Pivovaroff et al
Anatomy of phylloclades
The Ruscus species possessed numerous anatomical traitsconsistent with benefits for both shade and drought tolerance(Table 2) The phylloclade cross-sections were symmetrical(Fig 2) and thus the cross-sectional anatomy of the adaxialand abaxial halves were not different for all traits (paired t-testP gt 005) and values were averaged within species The twospecies were similar in their substantial leaf thickness and inthe thickness of cuticle epidermis cell walls and water storagecompartment (Table 2) The fractions of the lamina occupied bythe epidermis mesophyll and water storage tissues were 1552ndash56 and 29ndash32 respectively
Both species had parallel longitudinal veins of three sizes(midrib intermediate and small veins) Consistent with droughtadaptation the two species had low Dm R aculeatus having thelower value andRmicroglossumhadahigher IVD (Table 2)Thetwo species had on average the same maximum conduitdiameters in their midribs intermediate veins and small veinsand the maximum conduit diameter decreased ~35 from themidrib to the small veins The average theoretical hydraulicconductivity of xylem conduits did not differ between thespecies and decreased by up to 86 from the midrib to theminor vein R aculeatus had on average walls that were 85thicker in the fibrous bundle sheath (Table 2)
Gas-exchange measurements
Consistent with adaptation to simultaneous shade and droughtthe Ruscus species had very low values for Rarea Rmass and gs(Table 3) relative to comparator temperate broadleaf evergreenspecies (Fig 3) The Aarea Amass LCP Vcmax and Jmax were alsovery low relative to comparator species (Fig 3 Table 3)consistent with shade adaptation The two Ruscus species hadvery high values for Ags (Table 3) consistent with excellentWUE Consistent with strong drought tolerance via retention ofstored water both species had very low values for leaf and stemgmin especially relative to comparator vascular plant species(Fig 3 Table 3)
Hydraulic conductance pressure volume curveparameters and leaf water storage
Consistent with expectations for combined drought and shadetolerance both Ruscus species had low Kshoot relative tocomparator tropical and temperate woody angiosperms (Fig 3Table 4) The pressurendashvolume curve parameters of Ruscuswereconsistent with achieving drought tolerance through tissue waterstorage Both species had less negative po and ytlp than meanvalues for comparative evergreen woody species (Fig 3Table 4) Notably both species had lower e values thancomparative evergreen woody species and higher RWCtlp andCft values (Fig 3 Table 4) consistent with drought tolerance
Nitrogen concentration and carbon isotope composition
Both species had high values for phyllyclade Narea and Nmass
relative to comparative temperate broadleaf evergreen speciesconsistent with drought adaptation (Fig 3) The d13C valueswerevery negative and typical of values often observed for understoryplants (da Silveira et al 1989) consistent with shade tolerance
Indeed the d13C values were notably strongly negative given thehigh WUE found for these species
Testing hypotheses for shade and drought tolerancewith trait survey data
Overall we quantified 57 traits for the twoRuscus species and for21 traits we had a priori hypotheses for a benefit for shadetolerance and for 24 traits we had a priori hypotheses for abenefit for drought tolerance Using comparative data we foundthat 16 of 21 hypotheses were supported for shade tolerance and22 of 24 were supported for drought tolerance Of the ninehypotheses for traits that would contribute to both shade anddrought tolerance simultaneously (ie expectations were both forhigher or lower values than comparative species) eight weresupported by trait data All these proportions were significantlyhigher than the 50 support that would have been expected toarise only from chance (P = 0001ndash0058 proportion tests)Notably in the seven cases when shade tolerance traits wouldconflict with drought tolerance traits four indicated a benefit fordrought tolerance rather than shade tolerance for both species(Tables 1ndash5)
Discussion
Both R aculeatus (Fig 3) and R microglossum showed traitvalues consistent with combined shade and drought toleranceNumerous traits were consistent with a combined shade anddrought tolerance through improving carbon balance enablinga conservative resource use ie via slow respiration and long-lived parts Other traits would contribute specifically to droughttolerance via reduced demand for water during activephotosynthesis and the ability to survive strong drought afterstomatal closure Notably many such traits were related to waterstorage providingnew insights into effective formsof succulencein shaded habitats This suite of traits would contributeimportantly to the ability of Ruscus to occupy shaded sitesprone to strong drought across a wide geographical range
Traits contributing to simultaneous drought and shadetolerance
Ruscus species showed specialisation associated withconservative resource use consistent with tolerance of shadeand drought These specialised traits included thick lamina andcomponent tissues that contribute to long tissue life-spans (shootslastgt5 years Sack et al 2003bWright et al 2004) AdditionallyRuscus species had very low gas-exchange rates including lowgs and lowKshoot whichwould correspond to a low investment invascular tissue (Tyree andZimmermann1983 Sack et al 2003b)and low Rarea Rmass Aarea and Amass all representing an ability tomaintain photosynthesis and growth with low requirements forlight and water
Traits contributing to drought tolerance
According to Jones et al (1992) drought tolerance can beachieved through avoidance of plant water deficits toleranceof plant water deficits or efficiency mechanisms Ruscus speciesshowed traits associated with drought tolerance either byproviding the ability to maintain photosynthesis and growth indrying soil andor the ability to survive chronic drought as
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology K
previously shown experimentally for R aculeatus (Sack 2004)Traits that would contribute to the ability to maintain gasexchange in drying soil include small leaf size high WUE andlow IVD High WUE achieved in part with high Narea meansthese Ruscus species can attain positive carbon balance evenwith extremely low gs Low IVD and water storage capacitanceallow the phylloclades to maintain water supply to the mesophylland tolerate transiently high evaporation rates for example dueto sunflecks without desiccating the leaf (Sack et al 2003a)Traits contributing to the ability of the phylloclades to surviveextended drought included those enabling a low evaporationrate per leaf area once stomata have shut such as low gmin inleaf and stem and those related to specialised water storagetissue linked with low leaf density low e and po values thatwere low in magnitude
Ruscus water storage
The water storage tissue of Ruscus that occupied a third of theleaf thickness although contributing most directly to droughttolerance is also consistent with shade tolerance given itscontribution to reduced tissue costs for the phylloclade as awhole The water storage tissue had thin cell walls reflected inthe low bulk e and low solute concentration contributing tobulk po values that were low in magnitude
This water storage tissue would also contribute to both typesof drought tolerance ndash the ability to maintain photosynthesis indrying soil and to survive after stomata have shut duringextended drought The lsquosucculencersquo of Ruscus phylloclades isdistinctive relative to more typical succulent-leafed andsucculent-stemmed species which tend to have high leaf watercontent and capacitance values (Vendramini et al 2002 Ogburnand Edwards 2012) In contrast in Ruscus species the SWCwas low relative to typical leaf succulent species and forR aculeatus Cft fell within the range of typical evergreenleaves We note that the strong tissue differentiation in Ruscus(ie separation of mesophyll cell and water storage in spaceand their distinctions in anatomy) would contribute to higheffectiveness of water storage even if the bulk tissue overallhad low SWC and capacitance Indeed across species theretends to be no relationship between the magnitude of SWC orcapacitance and the degree of within-leaf tissue differentiation(Ogburn and Edwards 2012) Notably such differentiationwould contribute special advantages for supply of waterwhether stomata are open or closed as the large thin-walledwater storage cells with low solute concentration can yield theirwater to supply the evaporative load while the photosynthetictissues can maintain their volume according to their thickerwalls and stronger solute concentration
Although the capacitance and SWC values were low forRuscus species these values would be more substantial ifconsidered relative to water demand Across several speciesCft has been found to correlate with Kshoot and with gs (Sacket al 2003a Blackman et al 2010) indicating that leaves tendto be built with capacitance to match their maximum flux ratesand thus to buffer the leaf water potential against surges intranspiration Thus because Ruscus has low gs when stomataare open and low Kshoot the capacitance would be expectedto supply transpiration transiently during sunflecks or high
VPD Likewise when stomata close the capacitance suppliesongoing water loss via cuticular conductance Given theextremely low gmin of Ruscus even its moderate Ctlp canenable survival for weeks (Sack et al 2003b Sack 2004)Further at turgor loss the water content would equalSWCRWCtlp and the relatively high RWCtlp wouldcontribute to the high water content once turgor is lost Thusthe lsquosucculencersquo of Ruscus is moderate in absolute terms butcombined with its other very strong mechanisms to reducetranspiration when stomata are open or closed ie low gs andgmin even this moderate capacitance would provide strongfunctionality
Water-use efficiency and carbon isotope composition
It was noteworthy that despite extremely high WUE valuesphylloclade d13C values of the Ruscus species were verynegative This presents a strong anomaly worthy of furtherinvestigation as species with high WUE typically have higherd13C (less negative ie closer to zero) The d13C can beinfluenced by a host of processes including source CO2 storedplant carbon and time-integrated CO2 concentration at the siteof carboxylation (Farquhar et al 1989) For Ruscus althoughd13C values were typical of an understory plant they did notappear to be drivenby internalCO2 concentration because the leafisotopic values were depleted in 13C whereas the high WUEdetermined by gas exchange would likely promote enrichedisotopic values It is more likely that d13C in this species wasdetermined by source CO2 or stored carbon or recycling ofrespired CO2 (da Silveira et al 1989) Our study individualsof Ruscus were cultivated in a shaded understory similar totheir natural habitat Previous studies have shown that there canbe higher concentrations of respired CO2 in the forest understorythan in the canopy resulting in more negative carbon isotoperatios in understory plant tissue than canopy plant tissue (daSilveira et al 1989) This is a function of decomposing leavesand litter cover slow air mixing as well as plant environmentalresponses
A second possible explanation for the d13C values ofRuscus is related to its growth form and phenology asRuscus has extensive rhizomes (Sack et al 2003b) whichact as a carbon store for the plant Using this recycled carbonduring the growth season when the plant may not be able tomeet its carbon requirement by photosynthesis alone becauseof low maximum rates under light limitation may alsocontribute to more negative d13C values (Vizzini 2003)Notably Ruscus stems are hollow and thus can also storerelatively large amounts of CO2 for use during a growth periodwhen carbon is otherwise limiting especially given very lowgs Some of this stored carbon might be photosyntheticallyfixed by the stem (Nilsen and Sharifi 1997) In each of thesecases respired stored or recycled CO2 would supply carbonthat was previously fixed by Rubisco with more negative d13Cvalues (ndash27o) than air (ndash8o)
There was also a dissonance between the d13C value and CiCa The fully mature phylloclades which were selected andused for gas-exchange and d13C measurements were producedduring late winter and early spring with mild temperatures(average at midday 190ndash220C) and low atmospheric VPD
L Functional Plant Biology A Pivovaroff et al
(average at midday 06ndash15 kPa) However the gas-exchangemeasurements were taken during summer with hightemperature (average at midday 300ndash355C) and high VPD(average at midday 25ndash35 kPa) We harvested thephylloclades that we used for gas exchange to determine thecarbon isotope ratio Although the low value of instantaneousCiCa may be caused by high temperature and high atmosphericVPD the very low value of d13C may represent the time whenthe phyllocladersquos carbon was assimilated (during mildtemperature and low VPD)
Implications for drought and shade tolerance Ruscusas a model
We found strong support for a large number of hypotheses fortrait-based shade and drought tolerance providing a strong traitbasis for combined tolerance Although the detailed functionaltrait survey conducted here is relatively novel in its breadth(see also Pasquet-Kok et al 2010) this is a logical extensionof the traditional approach for understanding the basis forplant adaptation to environment ie testing expectations forindividual traits established by previous studies of thefunctional significance of these traits in other species Weacknowledge there is some degree of uncertainty ininterpreting a large number of traits simultaneously based onstudies of other species First the interpretation of the value oftraits based on other species may not be in all cases equally validfor Ruscus Some trait variation may relate to other functionsFurther studies for example using mutants would be necessaryas conclusive evidence of the value of specific traits in a givenspecies However one advantage of testing numerousexpectations for each hypothesis is that the key finding will berobust to the removal of some traits from the analysis if thoseare later found to be inappropriate Ideally when a model forestimating plant performance from leaf traits becomes availableone could determine how the specific quantitative combinationsof traits presented here scales up to plant shade and droughttolerance
The shade and drought tolerance of Ruscus consistent withthe suite of traits examined here is one case demonstratinghow plants can avoid a general trade-off between shade anddrought tolerance Further Ruscus is noteworthy as one of onlya few stem photosynthetic plants that occupy a shaded habitatHistorically it is likely that shade tolerance preceded droughttolerance given this speciesrsquo ancestors were species of moisttropical forests (Kim et al 2010) and thus Ruscus or itsancestor apparently evolved drought tolerance whileexpanding its range into drier habitat or during past climatechange Considering its unique adaptations and trait valuesRuscus can serve as an excellent model for the basis ofcombined shade and drought tolerance
Acknowledgements
We thank the Mildred E Mathias Botanical Garden for access to garden andplants Andy Truong for assistance with measurements Howard GriffithsLouis Santiago and Cheryl Swift for helpful discussions and comments onthe manuscript This work was supported by National Science FoundationGrant IOB-0546784
References
Bartlett MK Scoffoni C Sack L (2012) The determinants of leaf turgor losspoint and prediction of drought tolerance of species and biomes a globalmeta-analysis Ecology Letters 15 393ndash405 doi101111j1461-0248201201751x
Blackman CJ Brodribb TJ Jordan GJ (2010) Leaf hydraulic vulnerability isrelated to conduit dimensions and drought resistance across a diverserange of woody angiosperms New Phytologist 188 1113ndash1123doi101111j1469-8137201003439x
Brodribb TJ Feild TS Jordan GJ (2007) Leaf maximum photosynthetic rateand venation are linked by hydraulicsPlant Physiology 144 1890ndash1898doi101104pp107101352
Caspersen JP (2001) Interspecific variation in sapling mortality in relationto growth and soil moisture Oikos 92 160ndash168 doi101034j1600-07062001920119x
Cochard H Nardini A Coll L (2004) Hydraulic architecture of leaf bladeswhere is the main resistance Plant Cell amp Environment 27 1257ndash1267doi101111j1365-3040200401233x
Cooney-Sovetts C Sattler R (1987) Phylloclade development in theAsparagaceae an example of homoeosis Botanical Journal of theLinnean Society 94 327ndash371 doi101111j1095-83391986tb01053x
Cowling R Rundel P Lamont BB ArroyoMK ArianoutsouM (1996) Plantdiversity in Mediterranean-climate regions Trends in Ecology ampEvolution 11 362ndash366 doi1010160169-5347(96)10044-6
da Silveira L Sternberg L Mulkey SS Wright SJ (1989) Ecologicalinterpretation of leaf carbon isotope ratios influence of respired carbondioxide Ecology 70 1317ndash1324 doi1023071938191
de Lillis M Fontanella A (1992) Comparative phenology and growth indifferent species of the Mediterranean maquis of central Italy PlantEcology 99-100 83ndash96 doi101007BF00118213
Dracup JA (1991)DroughtmonitoringStochasticHydrologyandHydraulics5 261ndash266 doi101007BF01543134
Engelbrecht BMJ Kursar TA (2003) Comparative drought-resistance ofseedlings of 28 species of co-occurring tropical woody plantsOecologia 136 383ndash393 doi101007s00442-003-1290-8
Farquhar GD von Caemmerer S (1980) A biochemical model ofphotosynthetic CO2 assimilation in leaves of C3 species Planta 14978ndash90 doi101007BF00386231
FarquharGD Ehleringer JR HubickK (1989) Carbon isotope discriminationand photosynthesis Annual Review of Plant Physiology and PlantMolecular Biology 40 503ndash537 doi101146annurevpp40060189002443
Givnish TJ (1988) Adaptation to sun and shade a whole-plant perspectiveAustralian Journal of Plant Physiology 15 63ndash92 doi101071PP9880063
Gulias J Flexas J Mus M Cifre J Lefi E Medrano H (2003) Relationshipbetweenmaximum leaf photosynthesis nitrogen content and specific leafarea inBalearic endemic and non-endemicMediterranean speciesAnnalsof Botany 92 215ndash222 doi101093aobmcg123
Haberlandt G (1914) lsquoPhysiological plant anatomyrsquo (MacMillan London)Hallik L Niinemets U Wright J (2009) Are species shade and drought
tolerance reflected in leaf-level structural and functional differentiationin Northern Hemisphere temperate woody flora New Phytologist 184257ndash274 doi101111j1469-8137200902918x
Jones HG (1992) lsquoPlants and microclimate a quantitative approach toenvironmental plant physiologyrsquo (Cambridge University PressCambridge)
Kerstiens G (1996) Cuticular water permeability and its physiologicalsignificance Journal of Experimental Botany 47 1813ndash1832doi101093jxb47121813
Kim JH Kim DK Forest F Fay MF Chase MW (2010) Molecularphylogenetics of Ruscaceae sensu lato and related families(Asparagales) based on plastid and nuclear DNA sequences Annals ofBotany 106 775ndash790 doi101093aobmcq167
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology M
Koide RT Robichaux RH Morse SR Smith CM (2000) Plant water statushydraulic resistance and capacitance In lsquoPlant physiological ecologyfield methods and instrumentationrsquo (Eds R Pearcy JR Ehleringer HMooney P Rundel) pp 161ndash183 (Kluwer Dordrecht The Netherlands)
Koumlrner C Farquhar GD Wong SC (1991) Carbon isotope discrimination byplants follows latitudinal and altitudinal trends Oecologia 88 30ndash40doi101007BF00328400
Lewis AM Boose ER (1995) Estimating volume flow rates through xylemconduits American Journal of Botany 82 1112ndash1116 doi1023072446063
Martiacutenez-Palleacute E Aronne G (1999) Flower development and reproductivecontinuity inMediterraneanRuscusaculeatusL (Liliaceae)Protoplasma208 58ndash64 doi101007BF01279075
Martiacutenez-Tilleriacutea K Loayza AP Sandquist DR Squeo FA (2012) Noevidence of a trade-off between drought and shade tolerance inseedlings of six coastal desert shrub species in north-central ChileJournal of Vegetation Science 23 1051ndash1061 doi101111j1654-1103201201427x
Matalas NC (1963) Probability distribution of low flows USGS ProfessionalPapers 434-A US Government Printing Office Washington DC
Niinemets U (1999) Research review components of leaf dry mass per area ndashthickness and density ndash alter leaf photosynthetic capacity in reversedirections in woody plants New Phytologist 144 35ndash47 doi101046j1469-8137199900466x
Niinemets U Valladares F (2006) Tolerance to shade drought andwaterlogging of temperate northern hemisphere trees and shrubsEcological Monographs 76 521ndash547 doi1018900012-9615(2006)076[0521TTSDAW]20CO2
Nilsen ET Sharifi MR (1997) Carbon isotope composition of legumes withphotosynthetic stems from Mediterranean and desert habitats AmericanJournal of Botany 84 1707ndash1713 doi1023072446469
Ogburn RM Edwards EJ (2012) Quantifying succulence a rapidphysiologically meaningful metric of plant water storage Plant Cell ampEnvironment 35 1533ndash1542 doi101111j1365-3040201202503x
Pasquet-Kok J Creese C Sack L (2010) Turning over a new lsquoleafrsquo multiplefunctional significances of leaves versus phyllodes in Hawaiian Acaciakoa Plant Cell amp Environment 33 2084ndash2100 doi101111j1365-3040201002207x
SackL (2004)Responses of temperatewoody seedlings to shade and droughtdo trade-offs limit potential niche differentiation Oikos 107 110ndash127doi101111j0030-1299200413184x
SackLFroleK (2006)Leaf structural diversity is related to hydraulic capacityin tropical rain forest trees Ecology 87 483ndash491 doi10189005-0710
Sack L Holbrook NM (2006) Leaf hydraulics Annual Review of PlantBiology 57 361ndash381 doi101146annurevarplant56032604144141
Sack L Melcher PJ Zwieniecki MA Holbrook NM (2002) The hydraulicconductance of the angiosperm leaf lamina a comparison of threemeasurement methods Journal of Experimental Botany 532177ndash2184 doi101093jxberf069
Sack L Cowan P Jaikumar N Holbrook NM (2003a) The ldquohydrologyrdquo ofleaves co-ordination of structure and function in temperate woodyspecies Plant Cell amp Environment 26 1343ndash1356 doi101046j0016-8025200301058x
Sack L Grubb P Maronon T (2003b) The functional morphology of juvenileplants tolerant of strong summer drought in shaded forest understoriesin southern Spain Plant Ecology 168 139ndash163 doi101023A1024423820136
SackLScoffoniCMcKownADFroleKRawlsMHavran JCTranHTranT (2012) Developmentally based scaling of leaf venation architectureexplains global ecological patterns Nature Communications 3 837doi101038ncomms1835
Sack L Chatelet D Scoffoni C PrometheusWiki contributors (2013a)ldquoEstimating the mesophyll surface area per leaf area from leaf celland tissue dimensions measured from transverse cross-sectionsrdquoPrometheusWiki Available at httpwwwpublishcsiroauprometheuswikitiki-pagehistoryphppage=Estimating the mesophyllsurface area per leaf area from leaf cell and tissue dimensionsmeasured from transverse cross-sectionsamppreview=16 [accessed June18 2013]
SackL Pasquet-Kok JPrometheusWiki contributors (2013b) ldquoLeaf pressure-volume curve parametersrdquo PrometheusWiki Available at httpprometheuswikipublishcsiroautiki-indexphppage=Leaf+pressure-volume+curve+parameters
Scoffoni C Pou A Aasamaa K Sack L (2008) The rapid light response ofleaf hydraulic conductance new evidence from two experimentalmethods Plant Cell amp Environment 31 1803ndash1812 doi101111j1365-3040200801884x
Smith T Huston M (1989) A theory of the spatial and temporal dynamics ofplant communities Vegetatio 83 49ndash69 doi101007BF00031680
Sterck F Markesteijn L Schieving F Poorter L (2011) Functional traitsdetermine trade-offs and niches in a tropical forest communityProceedings of the National Academy of Sciences of the United Statesof America 108 20 627ndash20 632 doi101073pnas1106950108
Thomas GS (1992) lsquoOrnamental shrubs climbers and bamboosrsquo (JohnMurray Publishers Great Britain)
TyreeMT ZimmermannMH (1983) lsquoXylem structure and the ascent of saprsquo(Springer-Verlag Berlin)
USDA ARS National Genetic Resources Program (2009) GermplasmResources Information Network (GRIN) online database NationalGermplasm Resources Laboratory Beltsville Maryland Availableathttpwwwars-gringovcgi-binnpgshtmltaxonpl410577[Accessed 20 July 2009]
Vendramini F Diacuteaz S Gurvich DE Wilson PJ Thompson K Hodgson JG(2002) Leaf traits as indicators of resource-use strategy in floras withsucculent species New Phytologist 154 147ndash157 doi101046j1469-8137200200357x
Vile D (2005) Specific leaf area and dry matter content estimate thicknessin laminar leaves Annals of Botany 96 1129ndash1136 doi101093aobmci264
Vizzini S (2003) d13C and d15N variability in Posidonia oceanica associatedwith seasonality and plant fraction Aquatic Botany 76 195ndash202doi101016S0304-3770(03)00052-4
Walters M Reich PB (1999) Research review low-light carbon balance andshade tolerance in the seedlings of woody plants dowinter deciduous andbroad-leaved evergreen species differ New Phytologist 143 143ndash154doi101046j1469-8137199900425x
Witkowski E Lamont BB (1991) Leaf specific mass confounds leaf densityand thickness Oecologia 88 486ndash493
Wright IJWestobyM (2003)Nutrient concentration resorption and lifespanleaf traits of Australian sclerophyll species Functional Ecology 1710ndash19 doi101046j1365-2435200300694x
Wright IJ Reich PB Westoby M Ackerly DD Baruch Z Bongers FCavender-Bares J Chapin T Cornelissen JHC Diemer M Flexas JGarnier E Groom PK Gulias J Hikosaka K Lamont BB Lee T LeeWLusk C Midgley JJ Navas M-L Niinemets U Oleksyn J Osada NPoorter H Poot P Prior L Pyankov VI Roumet C Thomas SC TjoelkerMG Veneklaas EJ Villar R (2004) The worldwide leaf economicsspectrum Nature 428 821ndash827 doi101038nature02403
Wullschleger SD (1993) Biochemical limitations to carbon assimilation inC3 plants ndash a retrospective analysis of the ACi curves from 109 speciesJournal of Experimental Botany 44 907ndash920 doi101093jxb445907
N Functional Plant Biology A Pivovaroff et al
wwwpublishcsiroaujournalsfpb
Tab
le3
Meanva
lues
forga
sexchan
getraitsseforRuscusaculeatusan
dRm
icroglossumw
ithun
itsan
dreplication
Forgiventraitsexpectatio
nsaregivenforw
hetherRuscusshouldhave
ahigherorlowervaluerelativ
etocomparatorspeciesaccordingtothehypothesesofshadeordroughtadaptationCom
paratorspeciesdata
weretakenaccordingtoavailabilityinthepreviouslypu
blishedliteratureandnu
mberofspeciesand
minim
umm
eanandmaxim
umtraitvaluesareprov
idedSourcesofcomparativ
edataA
areaA
massRareaR
mass
g s(W
rightetal2
004)LCP(W
altersandReich
1999)Ag
s(G
uliasetal2
003)VcmaxJ
max(W
ullschleger19
93)g m
in(K
erstiens
1996)NSn
otstatistically
significant
atPlt005
Gas-exchang
etraits
Abb
reviation
Units
Ra
culeatus
Rm
icroglossum
Hypotheses
Com
paratorspecies
Meanplusmnse
NMeanplusmnse
NShade
adapted
Droug
htadapted
(N)
(minm
ean
max)
Light-saturated
rateof
photosyn
thesisperarea
Aarea
mmolm
ndash2sndash
1522
plusmn133
451
plusmn047
6Low
erAB
Tem
peratebroadleaf
evergreens
(78)
2629
42
2265
Light-saturated
rateof
photosyn
thesisperm
ass
Amass
nmolCO2gndash
1sndash
1004
3plusmn001
14
0050plusmn0005
6Low
erAB
Tem
peratebroadleaf
evergreens
(78)
2287
46
209
Respiratio
nrateperarea
Rarea
mmolm
ndash2sndash
1004
4plusmn000
98
0156plusmn0015
10Low
erAB
Low
erAB
Tem
peratebroadleaf
evergreens
(50)
0320
93
170
Respiratio
npermass
Rmass
nmolCO2gndash
1sndash
1356
E-04plusmn702
E-05
8171E-03plusmn166E-04
10Low
erAB
Low
erAB
Tem
peratebroadleaf
evergreens
(50)
3337
06
157
Maxim
umstom
atal
conductanceperarea
g smmolm
ndash2sndash
133
plusmn000
74
35plusmn0006
6Low
erAB
Low
erAB
Tem
peratebroadleaf
evergreens
(48)
491
423
09
Light
compensationpo
int
LCP
mmol
photonsm
ndash2sndash
1485
plusmn0
4361
plusmn0
6Low
erAB
Tropicalevergreenshade-
tolerators(15)
16582
4
Intrinsicwater
use
efficiency
Ag
smm
olmol
ndash1
154plusmn8
414
2plusmn19
06
HigherA
BMediterraneanspecies
(78)
2335
94
142
Ratio
ofintercellularto
ambient[CO2]
CiC
a032
7plusmn010
64
0431plusmn0064
6
Maxim
umrateof
carbox
ylation
Vcmax
mmolm
ndash2sndash
126
6plusmn318
424
7plusmn542
5Low
erAB
Tem
peratehardwood
species(19)
114
711
9
Maxim
umrateof
electron
transport
J max
601plusmn635
550
6plusmn786
5Low
erAB
Tem
peratehardwood
species(19)
291
042
37
Leafcuticularcond
uctance
g min
mmolm
ndash2sndash
1037
9plusmn008
210
0295plusmn0082
12Low
erAB
Vascularplants(201)
01
1321
07Stem
cuticular
conductance
g min
mmolm
ndash2sndash
1009
5plusmn002
56
0030plusmn0003
4
AAnexpectationaccordingto
ahypothesiswas
confi
rmed
forRa
culeatus
BAnexpectationaccordingto
ahy
pothesiswas
confi
rmed
forRm
icroglossum
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology E
Tab
le4
Meanva
lues
forhy
drau
licstraitsseforRuscusaculeatusan
dRm
icroglossumw
ithun
itsan
dreplication
Forgiventraitsexp
ectatio
nsaregivenforw
hetherRuscusshouldhave
ahigh
erorlowervaluerelativ
etocomparatorspeciesaccordingtothehy
pothesesofshadeordrough
tadaptationCom
paratorspeciesdata
weretakenaccordingtoavailabilityinthepreviouslypu
blishedliteratureandnu
mberofspeciesand
minim
umm
eanandmaxim
umtraitvaluesareprov
idedorm
eanstandarderrorifon
lythesewereavailable
Sources
ofcomparativ
edataK
shoot(SackandHolbroo
k20
06)Y
tlpR
WCtlpP
oe
(Bartlettetal2
012)Cft(Scoffon
ietal2
008)NSnotstatistically
significant
atPlt005
Hydraulicstraits
Abbreviation
Units
Ra
culeatus
Rm
icroglossum
Hyp
otheses
Com
paratorspecies
Meanplusmnse
NMeanplusmnse
NShade
adapted
Drought
adapted
(N)
(minm
ean
max)
Sho
othy
draulic
cond
uctance
Kshoot
mmolm
ndash2sndash
1
MPandash
1216
plusmn010
10269
plusmn013
11Low
erAB
Low
erAB
Tem
peratewoo
dyangiosperm
s(38)
8plusmn1
Tropicalwoody
angiosperm
s(49)
13plusmn15
Osm
oticpotentialatfull
turgor
p oMPa
ndash128
plusmn010
6ndash065
plusmn003
6HigherA
BHigherA
BC
Evergreen
woo
dyspecies
(182)
-34ndash
183ndash
049
Turgo
rloss
point
Ytlp
MPa
ndash184
plusmn010
6ndash113
plusmn007
6HigherA
BHigherA
BC
Evergreen
woo
dyspecies
(158)
-425
ndash220ndash
054
Modulus
ofelasticity
eMPa
110plusmn141
6588
plusmn053
6Low
erABC
Evergreen
woo
dyspecies(139)
3561
71
734
Relativewater
contentat
turgor
loss
point
RWCtlp
88
8plusmn109
689
3plusmn085
6HigherA
BC
Evergreen
woo
dyspecies(19)
718
12
911
Relativecapacitanceatfull
turgor
Cft
MPandash
10067plusmn0008
60102plusmn0010
6HigherA
BC
Evergreen
species(6)
0040
0066
0113
Relativecapacitanceat
turgor
loss
Ctlp
MPandash
10104plusmn0015
60089plusmn0003
6
Predawnwater
potential
Ypre
MPa
ndash045
plusmn008
4ndash088
plusmn008
4Middaywater
potential
Ymid
MPa
ndash141
plusmn008
3ndash129
plusmn028
4
AAnexpectationaccordingto
ahy
pothesiswas
confi
rmed
forRa
culeatus
BAnexpectationaccordingto
ahypothesiswas
confi
rmed
forRm
icroglossum
CExpectatio
nsforthesetraitsarebasedon
drou
ghttolerance
conferredby
waterstoragetissue(lsquosucculencersquo)the
oppositeexpectations
wou
ldarisefordroughttoleranceconferredby
theabilitytomaintainturgor
with
dehydration
F Functional Plant Biology A Pivovaroff et al
symmetrically with adaxial and abaxial layers of mesophyllepidermis and cuticle above and below a central achlorophyllouswater storage tissue (Fig 2) The per cent air space in each of theadaxial and abaxial mesophyll and in the water storagecompartment was estimated to the nearest 5 and the totalphylloclade airspace was calculated
PAir space in each tissue
fraction of leaf cross section occupied by that tissue100
eth1THORN
As indices of cell size the cross-sectional areas andperimeters were measured for three cells in the epidermis(adaxial and abaxial) the mesophyll (adaxial and abaxialie above and below the water storage tissue) and thewater storage tissue The area occupied by chloroplastswithin a mesophyll cell was measured for three cells(adaxial and abaxial) and the percent cross-sectionalchloroplast area was calculated as the ratio of chlorophyllarea divided by mesophyll cell area
We calculated the surface area of mesophyll cells per leafarea (AmesA) as described by Sack et al (2013a) a measure ofthe area available for CO2 uptake formesophyll cell layers Giventhe lack of palisade-form cells we modelled all mesophyll cellsas spheres for these calculations
We also measured vascular anatomy to quantify traits relatedto the efficiency of water transport within and outside the xylemWe measured the inter-veinal distance (IVD) and also theminimum distance from edge of bundle sheath to epidermis(Dm) as the hypotenuse between the distance between veinsand the distance to the epidermis (Brodribb Feild and Jordan2007)
Dm frac14ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
IVD2
2thorn ethdistance from bundle sheath edge to epidermisTHORN2
s
eth2THORN
We averaged IVD and Dm from three values for each cross-section
For all anatomical traits except those relating to the centralwater storage tissue measurements were made both adaxial andabaxial halves and values for the two halveswere averagedwhennot significantly different (at P lt 005 in paired t-tests) exceptthey were summed for total AmesA
To characterise the midrib for three typical fibrous bundlesheath cells we measured the cross-sectional heights widthsand cell wall thicknesses calculating mean values for each traitfrom three measurements per cross-section To characterise thexylem anatomy and theoretical conductivity of xylem conduitsfor a typical conduit within the midrib an intermediary veinand a minor vein of each sampled phylloclade we treated theconduit as an ellipse and determined the major and minor axisdiameters We calculated the theoretical hydraulic conductivityof the xylem conduit using Poiseuillersquos equation for ellipsesbased on conduit dimensions (Lewis and Boose 1995 CochardNardini and Coll 2004)
Tab
le5
Meanvalues
fortissue
compo
sition
traitsseforRuscusaculeatusan
dRm
icroglossumw
ithun
itsan
dreplication
andsign
ificanceof
differencesbetw
eenspecies(t-tests)
Forgiventraitsexpectatio
nsaregivenforw
hetherRaculeatus
shouldhave
ahigherorlowervaluerelativ
etocomparatorspeciesaccordingtothehypothesesofshadeordroughtadaptationCom
paratorspecies
dataweretakenaccordingtoavailabilityinthepreviouslypublishedliteratureandnumberofspeciesand
minim
umm
eanandmaxim
umtraitvaluesareprovidedSourcesofcomparativ
edatad
13C(K
oumlrneretal
1991)NmassNarea(W
righ
tetal2
004)N
Sn
otstatistically
significant
atPlt005
Tissuecompositio
ntraits
Abbreviation
Units
Ra
culeatus
Rm
icroglossum
Hypotheses
Com
paratorspecies
Meanplusmnse
NMeanplusmnse
NShade
adapted
Drought
adapted
(N)
(minm
ean
max)
Npermass
Nmass
194
plusmn006
9192
plusmn007
9Low
erHigherA
BTem
peratebroadleaf
evergreen(129)
0581
19
231
Nperarea
Narea
238
plusmn007
9175
plusmn006
9Low
erHigherA
BTem
peratebroadleaf
evergreen(129)
0727
1733
63
Cpermass
Cmass
44
4plusmn048
943
6plusmn052
9Carbonto
nitrogen
ratio
CN
231plusmn068
922
9plusmn058
9Carbonisotoperatio
d13C
permilndash33
32plusmn029
9ndash33
05plusmn020
9Low
erAB
Higher
Tem
peratespecies(35)
ndash27
64
ndash26
03
ndash24
09
AAnexpectationaccordingto
ahypothesiswas
confi
rmed
forRa
culeatus
BAnexpectationaccordingto
ahy
pothesiswas
confi
rmed
forRm
icroglossum
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology G
K t frac14 pa3b3
64hetha2 thorn b2THORN eth3THORN
where a and b are the major and minor axes of the ellipse and his water viscosity at 25C
Gas-exchange measurements responses to light and CO2and cuticular conductance
In July and August 2009 photosynthetic light response curvesandCO2 response curvesweremeasuredusing aLi-6400portablephotosynthesis system (Li-Cor Biosciences ) with light provided
Ruscusaculeatus
Ruscusmicroglossum
01 mm
Fig 2 Lamina and midrib cross-sections of Ruscus aculeatus and R microglossum phylloclades (05mm thick) Note that bothspecies exhibit shade tolerance features such as absence of palisade tissue as well as drought tolerance features such as large waterstorage compartment and thick-walled epidermis andfibrousbundle sheath cells surrounding thexylemandphloemfor bothmajor andminor veins especially prominent in R aculeatus which is native to drier habitats
(a) (b)
Fig 1 (a)Ruscus aculeatus and (b)Rmicroglossumgrowing at theMildredEMathiasBotanicalGardenNote thatwhat atfirst glance appear to be leaves are infact phylloclades
H Functional Plant Biology A Pivovaroff et al
by a red-blue light source (6400ndash02B no SI-710 Li-CorBiosciences ) Gas-exchange measurements were made on atleast 1ndash2 phylloclades per individual
For light-response curves phylloclades were acclimatedfor at least 5min at 1500mmolmndash2 sndash1 PAR at temperatures of2527C with RH maintained at ~50 and CO2 concentrationof 400mmolmolndash1 Then phylloclades were measured for netCO2 assimilation per leaf area at PAR steps of 1600 1400 12001000 800 600 500 400 300 200 100 50 and 0mmolmndash2 sndash1with 180ndash240 s stabilisation at each irradiance step Wedetermined light-saturated photosynthetic rate per area andmass (Aarea and Amass) dark respiration rate per area and mass(Rarea and Rmass) ie the negative A at zero PAR maximumstomatal conductance per area (gs) light compensation point(LCP) as the x-intercept intrinsic water use efficiency (WUEAareags) and the ratio of intercellular to ambient CO2
concentration (CiCa) at 100mmolmndash2 sndash1 PAR We thenharvested the phylloclades measured for gas exchange todetermine the nitrogen concentration and carbon isotope ratio(see below)
For CO2-response curves phylloclades were allowed toequilibrate at 400 ppm to induce stomatal opening and the netCO2 assimilation per leaf area was determined at Ci steps of400 300 200 100 50 400 400 500 600 800 1200 14001600 ppm with 3ndash4min equilibration time at each step Wedetermined maximum rate of carboxylation and maximum rateof electron transport per leaf area (Vcmax and Jmax) from plots ofCi vs A corrected to 25C (Farquhar and von Caemmerer 1980)
Cuticular conductance (ie minimumepidermal conductancegmin sensu Kerstiens 1996) was determined for 3ndash4 maturephylloclades and 10 cm lengths of stems from each of threeindividuals of each species Phylloclade and stem sampleswere hydrated and then the cut ends were sealed with waxSamples were dried for at least 30min on a laboratory bench atPAR of lt10mmol photons mndash2 sndash1 to induce stomatal closurethen samples were weighed for at least eight intervals of 30minduring which the slope of water loss versus time was highlylinear (R2 gt 0995) and therefore taken to represent transpirationafter stomata had closed fully The gmin was calculated as thetranspiration rate divided by the mole fraction vapour pressuredeficit (VPD determined from a weather station HOBO MicroStation with Smart Sensors Onset Bourne MA USA)
Pressurendashvolume curve parametersPressurendashvolume curve parameters were determined for matureshoots using the bench-drying method (Koide et al 2000 Sacket al 2003a) Shoots were ~10ndash15 cm long with about fourphylloclades per shoot for R microglossum and 15ndash20phylloclades for R aculeatus Shoots were progressively driedon a laboratory bench and measured at intervals by equilibratingfor 10min in a Whirlpak bag (Whirl-Pak Nasco Fort AtkinsonWIUSA) beforeweighing andmeasuring for leafwater potential(Yleaf) with a pressure chamber (Model 1000 Plant MoistureStress Instruments Albany OR USA) Subsequently dry masswas determined after more than 48 h in an oven at 70C Wedetermined the leaf dry matter content (LDMC) saturated watercontent (SWC) turgor loss point (Ytlp) relative water content atturgor loss point (RWCtlp) and osmotic potential at full turgor
(po) relative capacitance at full turgor (Cft DRWCDYleaf) andrelative capacitance at turgor loss point (Ctlp DRWCDYleaf) Wedetermined the modulus of elasticity (e) as the linear slope of theline fitted for pressure potential versus relative water contentabove and including turgor loss point (Sack et al 2013b)
Shoot hydraulic conductance
We measured the hydraulic conductance of mature shoots(Kshoot) ~10ndash15 cm long bearing about four phylloclades pershoot for R microglossum and 15ndash20 phylloclades forR aculeatus using the evaporative flux method (Sack et al2002) Samples were harvested and the ends were re-cut underdistilled degassed water with a fresh razor blade then hydratedovernight Shoots were connected to tubing containing distilleddegassed water running to a graduated cylinder on a balanceinterfaced to a computer logging data every minute to calculatethe flow rate of water from the balance into the sample Sampleswere held in place above a fan on wood frames strung withfishing line Lights were arranged above a plexiglass containerof water that acted as a heat trap producing PAR ofgt1200mmolmndash2 sndash1 at shoot level When steady-statetranspiration was achieved samples were covered with aplastic bag and removed from the tubing and the leaf waterpotential was determined with a pressure chamber (Model 1000Plant Moisture Stress Instruments) Kshoot was calculated as thesteady-state transpirational flow rate (E mmolmndash2 sndash1) dividedby the water potential driving force (DYleaF= ndashYleaf MPa)further normalised by total phylloclade area Kshoot valueswere standardised to 25C to correct for the temperaturedependence of the viscosity of water (Sack et al 2003a)
Foliar nitrogen concentration and carbon isotope ratio
To analyse total tissue nitrogen concentration and carbon isotopecomposition (d13C) for three replicates from each of threeindividuals for each species phylloclade samples were oven-dried at gt70C for more than 48 h and ground by mortar andpestle The nitrogen concentration and d13C were determined atthe UCDavis Stable Isotope Facility using an elemental analyser(PDZEuropaANCA-GSL SerconLtd Cheshire UK) interfacedto an isotope ratiomass spectrometer (PDZEuropa20ndash20 SerconLtd) at the UC Davis Stable Isotope Facility Final d13C contentvalues are expressed relative to international standardVienna PeeDee belemnite (V-PDB) for carbon
d13CethpermilTHORN frac14 etheth13C=12C of sampleTHORN=eth13C=12C of standardTHORN 1THORN 1000
eth4THORN
d13C of plant tissues can provide a measure of intrinsic WUE(Ags) at the time that carbon was assimilated giving long-termwater-use efficiency
Comparative data compilation and trait comparison
To consider Ruscus trait values in a global context we compileddata from the literature for (1) temperate and tropical broadleafevergreen species (2) Mediterranean species and (3) woodyangiosperms in general When available we considered data forplants grown in the shade and for species that are shade tolerant
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology I
however eco-physiological trait data is generally collected forsun leaves or plants making a comparison solely for shade grownplants not possible Formore commonly studied traits (egAmax)comparative data was taken from studies with large databasesand multiple traits (eg GLOPNET Wright et al 2004) Wedetermined minimum mean and maximum values for traitsfrom comparative studies or the mean and standard error ifraw species values were not available Studies that wereincluded for comparative data are referenced in the captionsfor Table 1ndash5 in association with the specific traits wecompared Differences between Ruscus trait values andcomparative data from the literature were determined for bothR aculeatus and R microglossum Hypotheses were deemedsupported for a Ruscus species if the trait value was higher orlower than the mean comparator value in the way predicted
Results
Both Ruscus species studied had leaf morphology consistentwith adaptation to shade and drought relative to comparator
species (Tables 1ndash5)We constructed a radar graph to encapsulatethe key traits that would confer adaptation to shade drought andthe combination for R aculeatus which had the more extremeadaptation of the two Ruscus species considered (Fig 3) Thisfigure summarises the major results of our study with values forcomparator species appearing as the inner circle and R aculeatustrait values displayed as the bold outer line
Gross morphology of phylloclades
Both R aculeatus and R microglossum had LMA valueslower than comparator species and R microglossum hadphylloclades larger than the mean leaf area for comparatorspecies Both Ruscus species had lower bulk tissue density thancomparator species consistent with the water storage in theRuscus phylloclades Notably despite its water storage tissuethe LDMC and SWC values of R aculeatus were typical ofthose for comparator species whereas R microglossum had alower LDMC and a higher SWC than for comparator speciessets (Table 1)
Fig 3 Radar graph illustrating percent difference between selected traits of Ruscus aculeatuswithcomparative data (see Tables 1ndash5 for symbols and sources of comparative data) Values forR aculeatus outside the circle indicate shade andor drought tolerance Traits are arrangedaccording to whether they would contribute shade tolerance drought tolerance or both The innerdisc represents the mean for comparative species for given traits and the values for R aculeatus isscaled relative to the magnitude of that value () with a value outside the disc representing greatershade andor drought tolerance (+) and (ndash) indicate if the axis is scaled such that a value outside thecircle represents percent higher or lower than comparative values respectively For traits expressed asnegative values (+) and (ndash) indicate more negative or less negative respectively
J Functional Plant Biology A Pivovaroff et al
Anatomy of phylloclades
The Ruscus species possessed numerous anatomical traitsconsistent with benefits for both shade and drought tolerance(Table 2) The phylloclade cross-sections were symmetrical(Fig 2) and thus the cross-sectional anatomy of the adaxialand abaxial halves were not different for all traits (paired t-testP gt 005) and values were averaged within species The twospecies were similar in their substantial leaf thickness and inthe thickness of cuticle epidermis cell walls and water storagecompartment (Table 2) The fractions of the lamina occupied bythe epidermis mesophyll and water storage tissues were 1552ndash56 and 29ndash32 respectively
Both species had parallel longitudinal veins of three sizes(midrib intermediate and small veins) Consistent with droughtadaptation the two species had low Dm R aculeatus having thelower value andRmicroglossumhadahigher IVD (Table 2)Thetwo species had on average the same maximum conduitdiameters in their midribs intermediate veins and small veinsand the maximum conduit diameter decreased ~35 from themidrib to the small veins The average theoretical hydraulicconductivity of xylem conduits did not differ between thespecies and decreased by up to 86 from the midrib to theminor vein R aculeatus had on average walls that were 85thicker in the fibrous bundle sheath (Table 2)
Gas-exchange measurements
Consistent with adaptation to simultaneous shade and droughtthe Ruscus species had very low values for Rarea Rmass and gs(Table 3) relative to comparator temperate broadleaf evergreenspecies (Fig 3) The Aarea Amass LCP Vcmax and Jmax were alsovery low relative to comparator species (Fig 3 Table 3)consistent with shade adaptation The two Ruscus species hadvery high values for Ags (Table 3) consistent with excellentWUE Consistent with strong drought tolerance via retention ofstored water both species had very low values for leaf and stemgmin especially relative to comparator vascular plant species(Fig 3 Table 3)
Hydraulic conductance pressure volume curveparameters and leaf water storage
Consistent with expectations for combined drought and shadetolerance both Ruscus species had low Kshoot relative tocomparator tropical and temperate woody angiosperms (Fig 3Table 4) The pressurendashvolume curve parameters of Ruscuswereconsistent with achieving drought tolerance through tissue waterstorage Both species had less negative po and ytlp than meanvalues for comparative evergreen woody species (Fig 3Table 4) Notably both species had lower e values thancomparative evergreen woody species and higher RWCtlp andCft values (Fig 3 Table 4) consistent with drought tolerance
Nitrogen concentration and carbon isotope composition
Both species had high values for phyllyclade Narea and Nmass
relative to comparative temperate broadleaf evergreen speciesconsistent with drought adaptation (Fig 3) The d13C valueswerevery negative and typical of values often observed for understoryplants (da Silveira et al 1989) consistent with shade tolerance
Indeed the d13C values were notably strongly negative given thehigh WUE found for these species
Testing hypotheses for shade and drought tolerancewith trait survey data
Overall we quantified 57 traits for the twoRuscus species and for21 traits we had a priori hypotheses for a benefit for shadetolerance and for 24 traits we had a priori hypotheses for abenefit for drought tolerance Using comparative data we foundthat 16 of 21 hypotheses were supported for shade tolerance and22 of 24 were supported for drought tolerance Of the ninehypotheses for traits that would contribute to both shade anddrought tolerance simultaneously (ie expectations were both forhigher or lower values than comparative species) eight weresupported by trait data All these proportions were significantlyhigher than the 50 support that would have been expected toarise only from chance (P = 0001ndash0058 proportion tests)Notably in the seven cases when shade tolerance traits wouldconflict with drought tolerance traits four indicated a benefit fordrought tolerance rather than shade tolerance for both species(Tables 1ndash5)
Discussion
Both R aculeatus (Fig 3) and R microglossum showed traitvalues consistent with combined shade and drought toleranceNumerous traits were consistent with a combined shade anddrought tolerance through improving carbon balance enablinga conservative resource use ie via slow respiration and long-lived parts Other traits would contribute specifically to droughttolerance via reduced demand for water during activephotosynthesis and the ability to survive strong drought afterstomatal closure Notably many such traits were related to waterstorage providingnew insights into effective formsof succulencein shaded habitats This suite of traits would contributeimportantly to the ability of Ruscus to occupy shaded sitesprone to strong drought across a wide geographical range
Traits contributing to simultaneous drought and shadetolerance
Ruscus species showed specialisation associated withconservative resource use consistent with tolerance of shadeand drought These specialised traits included thick lamina andcomponent tissues that contribute to long tissue life-spans (shootslastgt5 years Sack et al 2003bWright et al 2004) AdditionallyRuscus species had very low gas-exchange rates including lowgs and lowKshoot whichwould correspond to a low investment invascular tissue (Tyree andZimmermann1983 Sack et al 2003b)and low Rarea Rmass Aarea and Amass all representing an ability tomaintain photosynthesis and growth with low requirements forlight and water
Traits contributing to drought tolerance
According to Jones et al (1992) drought tolerance can beachieved through avoidance of plant water deficits toleranceof plant water deficits or efficiency mechanisms Ruscus speciesshowed traits associated with drought tolerance either byproviding the ability to maintain photosynthesis and growth indrying soil andor the ability to survive chronic drought as
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology K
previously shown experimentally for R aculeatus (Sack 2004)Traits that would contribute to the ability to maintain gasexchange in drying soil include small leaf size high WUE andlow IVD High WUE achieved in part with high Narea meansthese Ruscus species can attain positive carbon balance evenwith extremely low gs Low IVD and water storage capacitanceallow the phylloclades to maintain water supply to the mesophylland tolerate transiently high evaporation rates for example dueto sunflecks without desiccating the leaf (Sack et al 2003a)Traits contributing to the ability of the phylloclades to surviveextended drought included those enabling a low evaporationrate per leaf area once stomata have shut such as low gmin inleaf and stem and those related to specialised water storagetissue linked with low leaf density low e and po values thatwere low in magnitude
Ruscus water storage
The water storage tissue of Ruscus that occupied a third of theleaf thickness although contributing most directly to droughttolerance is also consistent with shade tolerance given itscontribution to reduced tissue costs for the phylloclade as awhole The water storage tissue had thin cell walls reflected inthe low bulk e and low solute concentration contributing tobulk po values that were low in magnitude
This water storage tissue would also contribute to both typesof drought tolerance ndash the ability to maintain photosynthesis indrying soil and to survive after stomata have shut duringextended drought The lsquosucculencersquo of Ruscus phylloclades isdistinctive relative to more typical succulent-leafed andsucculent-stemmed species which tend to have high leaf watercontent and capacitance values (Vendramini et al 2002 Ogburnand Edwards 2012) In contrast in Ruscus species the SWCwas low relative to typical leaf succulent species and forR aculeatus Cft fell within the range of typical evergreenleaves We note that the strong tissue differentiation in Ruscus(ie separation of mesophyll cell and water storage in spaceand their distinctions in anatomy) would contribute to higheffectiveness of water storage even if the bulk tissue overallhad low SWC and capacitance Indeed across species theretends to be no relationship between the magnitude of SWC orcapacitance and the degree of within-leaf tissue differentiation(Ogburn and Edwards 2012) Notably such differentiationwould contribute special advantages for supply of waterwhether stomata are open or closed as the large thin-walledwater storage cells with low solute concentration can yield theirwater to supply the evaporative load while the photosynthetictissues can maintain their volume according to their thickerwalls and stronger solute concentration
Although the capacitance and SWC values were low forRuscus species these values would be more substantial ifconsidered relative to water demand Across several speciesCft has been found to correlate with Kshoot and with gs (Sacket al 2003a Blackman et al 2010) indicating that leaves tendto be built with capacitance to match their maximum flux ratesand thus to buffer the leaf water potential against surges intranspiration Thus because Ruscus has low gs when stomataare open and low Kshoot the capacitance would be expectedto supply transpiration transiently during sunflecks or high
VPD Likewise when stomata close the capacitance suppliesongoing water loss via cuticular conductance Given theextremely low gmin of Ruscus even its moderate Ctlp canenable survival for weeks (Sack et al 2003b Sack 2004)Further at turgor loss the water content would equalSWCRWCtlp and the relatively high RWCtlp wouldcontribute to the high water content once turgor is lost Thusthe lsquosucculencersquo of Ruscus is moderate in absolute terms butcombined with its other very strong mechanisms to reducetranspiration when stomata are open or closed ie low gs andgmin even this moderate capacitance would provide strongfunctionality
Water-use efficiency and carbon isotope composition
It was noteworthy that despite extremely high WUE valuesphylloclade d13C values of the Ruscus species were verynegative This presents a strong anomaly worthy of furtherinvestigation as species with high WUE typically have higherd13C (less negative ie closer to zero) The d13C can beinfluenced by a host of processes including source CO2 storedplant carbon and time-integrated CO2 concentration at the siteof carboxylation (Farquhar et al 1989) For Ruscus althoughd13C values were typical of an understory plant they did notappear to be drivenby internalCO2 concentration because the leafisotopic values were depleted in 13C whereas the high WUEdetermined by gas exchange would likely promote enrichedisotopic values It is more likely that d13C in this species wasdetermined by source CO2 or stored carbon or recycling ofrespired CO2 (da Silveira et al 1989) Our study individualsof Ruscus were cultivated in a shaded understory similar totheir natural habitat Previous studies have shown that there canbe higher concentrations of respired CO2 in the forest understorythan in the canopy resulting in more negative carbon isotoperatios in understory plant tissue than canopy plant tissue (daSilveira et al 1989) This is a function of decomposing leavesand litter cover slow air mixing as well as plant environmentalresponses
A second possible explanation for the d13C values ofRuscus is related to its growth form and phenology asRuscus has extensive rhizomes (Sack et al 2003b) whichact as a carbon store for the plant Using this recycled carbonduring the growth season when the plant may not be able tomeet its carbon requirement by photosynthesis alone becauseof low maximum rates under light limitation may alsocontribute to more negative d13C values (Vizzini 2003)Notably Ruscus stems are hollow and thus can also storerelatively large amounts of CO2 for use during a growth periodwhen carbon is otherwise limiting especially given very lowgs Some of this stored carbon might be photosyntheticallyfixed by the stem (Nilsen and Sharifi 1997) In each of thesecases respired stored or recycled CO2 would supply carbonthat was previously fixed by Rubisco with more negative d13Cvalues (ndash27o) than air (ndash8o)
There was also a dissonance between the d13C value and CiCa The fully mature phylloclades which were selected andused for gas-exchange and d13C measurements were producedduring late winter and early spring with mild temperatures(average at midday 190ndash220C) and low atmospheric VPD
L Functional Plant Biology A Pivovaroff et al
(average at midday 06ndash15 kPa) However the gas-exchangemeasurements were taken during summer with hightemperature (average at midday 300ndash355C) and high VPD(average at midday 25ndash35 kPa) We harvested thephylloclades that we used for gas exchange to determine thecarbon isotope ratio Although the low value of instantaneousCiCa may be caused by high temperature and high atmosphericVPD the very low value of d13C may represent the time whenthe phyllocladersquos carbon was assimilated (during mildtemperature and low VPD)
Implications for drought and shade tolerance Ruscusas a model
We found strong support for a large number of hypotheses fortrait-based shade and drought tolerance providing a strong traitbasis for combined tolerance Although the detailed functionaltrait survey conducted here is relatively novel in its breadth(see also Pasquet-Kok et al 2010) this is a logical extensionof the traditional approach for understanding the basis forplant adaptation to environment ie testing expectations forindividual traits established by previous studies of thefunctional significance of these traits in other species Weacknowledge there is some degree of uncertainty ininterpreting a large number of traits simultaneously based onstudies of other species First the interpretation of the value oftraits based on other species may not be in all cases equally validfor Ruscus Some trait variation may relate to other functionsFurther studies for example using mutants would be necessaryas conclusive evidence of the value of specific traits in a givenspecies However one advantage of testing numerousexpectations for each hypothesis is that the key finding will berobust to the removal of some traits from the analysis if thoseare later found to be inappropriate Ideally when a model forestimating plant performance from leaf traits becomes availableone could determine how the specific quantitative combinationsof traits presented here scales up to plant shade and droughttolerance
The shade and drought tolerance of Ruscus consistent withthe suite of traits examined here is one case demonstratinghow plants can avoid a general trade-off between shade anddrought tolerance Further Ruscus is noteworthy as one of onlya few stem photosynthetic plants that occupy a shaded habitatHistorically it is likely that shade tolerance preceded droughttolerance given this speciesrsquo ancestors were species of moisttropical forests (Kim et al 2010) and thus Ruscus or itsancestor apparently evolved drought tolerance whileexpanding its range into drier habitat or during past climatechange Considering its unique adaptations and trait valuesRuscus can serve as an excellent model for the basis ofcombined shade and drought tolerance
Acknowledgements
We thank the Mildred E Mathias Botanical Garden for access to garden andplants Andy Truong for assistance with measurements Howard GriffithsLouis Santiago and Cheryl Swift for helpful discussions and comments onthe manuscript This work was supported by National Science FoundationGrant IOB-0546784
References
Bartlett MK Scoffoni C Sack L (2012) The determinants of leaf turgor losspoint and prediction of drought tolerance of species and biomes a globalmeta-analysis Ecology Letters 15 393ndash405 doi101111j1461-0248201201751x
Blackman CJ Brodribb TJ Jordan GJ (2010) Leaf hydraulic vulnerability isrelated to conduit dimensions and drought resistance across a diverserange of woody angiosperms New Phytologist 188 1113ndash1123doi101111j1469-8137201003439x
Brodribb TJ Feild TS Jordan GJ (2007) Leaf maximum photosynthetic rateand venation are linked by hydraulicsPlant Physiology 144 1890ndash1898doi101104pp107101352
Caspersen JP (2001) Interspecific variation in sapling mortality in relationto growth and soil moisture Oikos 92 160ndash168 doi101034j1600-07062001920119x
Cochard H Nardini A Coll L (2004) Hydraulic architecture of leaf bladeswhere is the main resistance Plant Cell amp Environment 27 1257ndash1267doi101111j1365-3040200401233x
Cooney-Sovetts C Sattler R (1987) Phylloclade development in theAsparagaceae an example of homoeosis Botanical Journal of theLinnean Society 94 327ndash371 doi101111j1095-83391986tb01053x
Cowling R Rundel P Lamont BB ArroyoMK ArianoutsouM (1996) Plantdiversity in Mediterranean-climate regions Trends in Ecology ampEvolution 11 362ndash366 doi1010160169-5347(96)10044-6
da Silveira L Sternberg L Mulkey SS Wright SJ (1989) Ecologicalinterpretation of leaf carbon isotope ratios influence of respired carbondioxide Ecology 70 1317ndash1324 doi1023071938191
de Lillis M Fontanella A (1992) Comparative phenology and growth indifferent species of the Mediterranean maquis of central Italy PlantEcology 99-100 83ndash96 doi101007BF00118213
Dracup JA (1991)DroughtmonitoringStochasticHydrologyandHydraulics5 261ndash266 doi101007BF01543134
Engelbrecht BMJ Kursar TA (2003) Comparative drought-resistance ofseedlings of 28 species of co-occurring tropical woody plantsOecologia 136 383ndash393 doi101007s00442-003-1290-8
Farquhar GD von Caemmerer S (1980) A biochemical model ofphotosynthetic CO2 assimilation in leaves of C3 species Planta 14978ndash90 doi101007BF00386231
FarquharGD Ehleringer JR HubickK (1989) Carbon isotope discriminationand photosynthesis Annual Review of Plant Physiology and PlantMolecular Biology 40 503ndash537 doi101146annurevpp40060189002443
Givnish TJ (1988) Adaptation to sun and shade a whole-plant perspectiveAustralian Journal of Plant Physiology 15 63ndash92 doi101071PP9880063
Gulias J Flexas J Mus M Cifre J Lefi E Medrano H (2003) Relationshipbetweenmaximum leaf photosynthesis nitrogen content and specific leafarea inBalearic endemic and non-endemicMediterranean speciesAnnalsof Botany 92 215ndash222 doi101093aobmcg123
Haberlandt G (1914) lsquoPhysiological plant anatomyrsquo (MacMillan London)Hallik L Niinemets U Wright J (2009) Are species shade and drought
tolerance reflected in leaf-level structural and functional differentiationin Northern Hemisphere temperate woody flora New Phytologist 184257ndash274 doi101111j1469-8137200902918x
Jones HG (1992) lsquoPlants and microclimate a quantitative approach toenvironmental plant physiologyrsquo (Cambridge University PressCambridge)
Kerstiens G (1996) Cuticular water permeability and its physiologicalsignificance Journal of Experimental Botany 47 1813ndash1832doi101093jxb47121813
Kim JH Kim DK Forest F Fay MF Chase MW (2010) Molecularphylogenetics of Ruscaceae sensu lato and related families(Asparagales) based on plastid and nuclear DNA sequences Annals ofBotany 106 775ndash790 doi101093aobmcq167
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology M
Koide RT Robichaux RH Morse SR Smith CM (2000) Plant water statushydraulic resistance and capacitance In lsquoPlant physiological ecologyfield methods and instrumentationrsquo (Eds R Pearcy JR Ehleringer HMooney P Rundel) pp 161ndash183 (Kluwer Dordrecht The Netherlands)
Koumlrner C Farquhar GD Wong SC (1991) Carbon isotope discrimination byplants follows latitudinal and altitudinal trends Oecologia 88 30ndash40doi101007BF00328400
Lewis AM Boose ER (1995) Estimating volume flow rates through xylemconduits American Journal of Botany 82 1112ndash1116 doi1023072446063
Martiacutenez-Palleacute E Aronne G (1999) Flower development and reproductivecontinuity inMediterraneanRuscusaculeatusL (Liliaceae)Protoplasma208 58ndash64 doi101007BF01279075
Martiacutenez-Tilleriacutea K Loayza AP Sandquist DR Squeo FA (2012) Noevidence of a trade-off between drought and shade tolerance inseedlings of six coastal desert shrub species in north-central ChileJournal of Vegetation Science 23 1051ndash1061 doi101111j1654-1103201201427x
Matalas NC (1963) Probability distribution of low flows USGS ProfessionalPapers 434-A US Government Printing Office Washington DC
Niinemets U (1999) Research review components of leaf dry mass per area ndashthickness and density ndash alter leaf photosynthetic capacity in reversedirections in woody plants New Phytologist 144 35ndash47 doi101046j1469-8137199900466x
Niinemets U Valladares F (2006) Tolerance to shade drought andwaterlogging of temperate northern hemisphere trees and shrubsEcological Monographs 76 521ndash547 doi1018900012-9615(2006)076[0521TTSDAW]20CO2
Nilsen ET Sharifi MR (1997) Carbon isotope composition of legumes withphotosynthetic stems from Mediterranean and desert habitats AmericanJournal of Botany 84 1707ndash1713 doi1023072446469
Ogburn RM Edwards EJ (2012) Quantifying succulence a rapidphysiologically meaningful metric of plant water storage Plant Cell ampEnvironment 35 1533ndash1542 doi101111j1365-3040201202503x
Pasquet-Kok J Creese C Sack L (2010) Turning over a new lsquoleafrsquo multiplefunctional significances of leaves versus phyllodes in Hawaiian Acaciakoa Plant Cell amp Environment 33 2084ndash2100 doi101111j1365-3040201002207x
SackL (2004)Responses of temperatewoody seedlings to shade and droughtdo trade-offs limit potential niche differentiation Oikos 107 110ndash127doi101111j0030-1299200413184x
SackLFroleK (2006)Leaf structural diversity is related to hydraulic capacityin tropical rain forest trees Ecology 87 483ndash491 doi10189005-0710
Sack L Holbrook NM (2006) Leaf hydraulics Annual Review of PlantBiology 57 361ndash381 doi101146annurevarplant56032604144141
Sack L Melcher PJ Zwieniecki MA Holbrook NM (2002) The hydraulicconductance of the angiosperm leaf lamina a comparison of threemeasurement methods Journal of Experimental Botany 532177ndash2184 doi101093jxberf069
Sack L Cowan P Jaikumar N Holbrook NM (2003a) The ldquohydrologyrdquo ofleaves co-ordination of structure and function in temperate woodyspecies Plant Cell amp Environment 26 1343ndash1356 doi101046j0016-8025200301058x
Sack L Grubb P Maronon T (2003b) The functional morphology of juvenileplants tolerant of strong summer drought in shaded forest understoriesin southern Spain Plant Ecology 168 139ndash163 doi101023A1024423820136
SackLScoffoniCMcKownADFroleKRawlsMHavran JCTranHTranT (2012) Developmentally based scaling of leaf venation architectureexplains global ecological patterns Nature Communications 3 837doi101038ncomms1835
Sack L Chatelet D Scoffoni C PrometheusWiki contributors (2013a)ldquoEstimating the mesophyll surface area per leaf area from leaf celland tissue dimensions measured from transverse cross-sectionsrdquoPrometheusWiki Available at httpwwwpublishcsiroauprometheuswikitiki-pagehistoryphppage=Estimating the mesophyllsurface area per leaf area from leaf cell and tissue dimensionsmeasured from transverse cross-sectionsamppreview=16 [accessed June18 2013]
SackL Pasquet-Kok JPrometheusWiki contributors (2013b) ldquoLeaf pressure-volume curve parametersrdquo PrometheusWiki Available at httpprometheuswikipublishcsiroautiki-indexphppage=Leaf+pressure-volume+curve+parameters
Scoffoni C Pou A Aasamaa K Sack L (2008) The rapid light response ofleaf hydraulic conductance new evidence from two experimentalmethods Plant Cell amp Environment 31 1803ndash1812 doi101111j1365-3040200801884x
Smith T Huston M (1989) A theory of the spatial and temporal dynamics ofplant communities Vegetatio 83 49ndash69 doi101007BF00031680
Sterck F Markesteijn L Schieving F Poorter L (2011) Functional traitsdetermine trade-offs and niches in a tropical forest communityProceedings of the National Academy of Sciences of the United Statesof America 108 20 627ndash20 632 doi101073pnas1106950108
Thomas GS (1992) lsquoOrnamental shrubs climbers and bamboosrsquo (JohnMurray Publishers Great Britain)
TyreeMT ZimmermannMH (1983) lsquoXylem structure and the ascent of saprsquo(Springer-Verlag Berlin)
USDA ARS National Genetic Resources Program (2009) GermplasmResources Information Network (GRIN) online database NationalGermplasm Resources Laboratory Beltsville Maryland Availableathttpwwwars-gringovcgi-binnpgshtmltaxonpl410577[Accessed 20 July 2009]
Vendramini F Diacuteaz S Gurvich DE Wilson PJ Thompson K Hodgson JG(2002) Leaf traits as indicators of resource-use strategy in floras withsucculent species New Phytologist 154 147ndash157 doi101046j1469-8137200200357x
Vile D (2005) Specific leaf area and dry matter content estimate thicknessin laminar leaves Annals of Botany 96 1129ndash1136 doi101093aobmci264
Vizzini S (2003) d13C and d15N variability in Posidonia oceanica associatedwith seasonality and plant fraction Aquatic Botany 76 195ndash202doi101016S0304-3770(03)00052-4
Walters M Reich PB (1999) Research review low-light carbon balance andshade tolerance in the seedlings of woody plants dowinter deciduous andbroad-leaved evergreen species differ New Phytologist 143 143ndash154doi101046j1469-8137199900425x
Witkowski E Lamont BB (1991) Leaf specific mass confounds leaf densityand thickness Oecologia 88 486ndash493
Wright IJWestobyM (2003)Nutrient concentration resorption and lifespanleaf traits of Australian sclerophyll species Functional Ecology 1710ndash19 doi101046j1365-2435200300694x
Wright IJ Reich PB Westoby M Ackerly DD Baruch Z Bongers FCavender-Bares J Chapin T Cornelissen JHC Diemer M Flexas JGarnier E Groom PK Gulias J Hikosaka K Lamont BB Lee T LeeWLusk C Midgley JJ Navas M-L Niinemets U Oleksyn J Osada NPoorter H Poot P Prior L Pyankov VI Roumet C Thomas SC TjoelkerMG Veneklaas EJ Villar R (2004) The worldwide leaf economicsspectrum Nature 428 821ndash827 doi101038nature02403
Wullschleger SD (1993) Biochemical limitations to carbon assimilation inC3 plants ndash a retrospective analysis of the ACi curves from 109 speciesJournal of Experimental Botany 44 907ndash920 doi101093jxb445907
N Functional Plant Biology A Pivovaroff et al
wwwpublishcsiroaujournalsfpb
Tab
le4
Meanva
lues
forhy
drau
licstraitsseforRuscusaculeatusan
dRm
icroglossumw
ithun
itsan
dreplication
Forgiventraitsexp
ectatio
nsaregivenforw
hetherRuscusshouldhave
ahigh
erorlowervaluerelativ
etocomparatorspeciesaccordingtothehy
pothesesofshadeordrough
tadaptationCom
paratorspeciesdata
weretakenaccordingtoavailabilityinthepreviouslypu
blishedliteratureandnu
mberofspeciesand
minim
umm
eanandmaxim
umtraitvaluesareprov
idedorm
eanstandarderrorifon
lythesewereavailable
Sources
ofcomparativ
edataK
shoot(SackandHolbroo
k20
06)Y
tlpR
WCtlpP
oe
(Bartlettetal2
012)Cft(Scoffon
ietal2
008)NSnotstatistically
significant
atPlt005
Hydraulicstraits
Abbreviation
Units
Ra
culeatus
Rm
icroglossum
Hyp
otheses
Com
paratorspecies
Meanplusmnse
NMeanplusmnse
NShade
adapted
Drought
adapted
(N)
(minm
ean
max)
Sho
othy
draulic
cond
uctance
Kshoot
mmolm
ndash2sndash
1
MPandash
1216
plusmn010
10269
plusmn013
11Low
erAB
Low
erAB
Tem
peratewoo
dyangiosperm
s(38)
8plusmn1
Tropicalwoody
angiosperm
s(49)
13plusmn15
Osm
oticpotentialatfull
turgor
p oMPa
ndash128
plusmn010
6ndash065
plusmn003
6HigherA
BHigherA
BC
Evergreen
woo
dyspecies
(182)
-34ndash
183ndash
049
Turgo
rloss
point
Ytlp
MPa
ndash184
plusmn010
6ndash113
plusmn007
6HigherA
BHigherA
BC
Evergreen
woo
dyspecies
(158)
-425
ndash220ndash
054
Modulus
ofelasticity
eMPa
110plusmn141
6588
plusmn053
6Low
erABC
Evergreen
woo
dyspecies(139)
3561
71
734
Relativewater
contentat
turgor
loss
point
RWCtlp
88
8plusmn109
689
3plusmn085
6HigherA
BC
Evergreen
woo
dyspecies(19)
718
12
911
Relativecapacitanceatfull
turgor
Cft
MPandash
10067plusmn0008
60102plusmn0010
6HigherA
BC
Evergreen
species(6)
0040
0066
0113
Relativecapacitanceat
turgor
loss
Ctlp
MPandash
10104plusmn0015
60089plusmn0003
6
Predawnwater
potential
Ypre
MPa
ndash045
plusmn008
4ndash088
plusmn008
4Middaywater
potential
Ymid
MPa
ndash141
plusmn008
3ndash129
plusmn028
4
AAnexpectationaccordingto
ahy
pothesiswas
confi
rmed
forRa
culeatus
BAnexpectationaccordingto
ahypothesiswas
confi
rmed
forRm
icroglossum
CExpectatio
nsforthesetraitsarebasedon
drou
ghttolerance
conferredby
waterstoragetissue(lsquosucculencersquo)the
oppositeexpectations
wou
ldarisefordroughttoleranceconferredby
theabilitytomaintainturgor
with
dehydration
F Functional Plant Biology A Pivovaroff et al
symmetrically with adaxial and abaxial layers of mesophyllepidermis and cuticle above and below a central achlorophyllouswater storage tissue (Fig 2) The per cent air space in each of theadaxial and abaxial mesophyll and in the water storagecompartment was estimated to the nearest 5 and the totalphylloclade airspace was calculated
PAir space in each tissue
fraction of leaf cross section occupied by that tissue100
eth1THORN
As indices of cell size the cross-sectional areas andperimeters were measured for three cells in the epidermis(adaxial and abaxial) the mesophyll (adaxial and abaxialie above and below the water storage tissue) and thewater storage tissue The area occupied by chloroplastswithin a mesophyll cell was measured for three cells(adaxial and abaxial) and the percent cross-sectionalchloroplast area was calculated as the ratio of chlorophyllarea divided by mesophyll cell area
We calculated the surface area of mesophyll cells per leafarea (AmesA) as described by Sack et al (2013a) a measure ofthe area available for CO2 uptake formesophyll cell layers Giventhe lack of palisade-form cells we modelled all mesophyll cellsas spheres for these calculations
We also measured vascular anatomy to quantify traits relatedto the efficiency of water transport within and outside the xylemWe measured the inter-veinal distance (IVD) and also theminimum distance from edge of bundle sheath to epidermis(Dm) as the hypotenuse between the distance between veinsand the distance to the epidermis (Brodribb Feild and Jordan2007)
Dm frac14ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
IVD2
2thorn ethdistance from bundle sheath edge to epidermisTHORN2
s
eth2THORN
We averaged IVD and Dm from three values for each cross-section
For all anatomical traits except those relating to the centralwater storage tissue measurements were made both adaxial andabaxial halves and values for the two halveswere averagedwhennot significantly different (at P lt 005 in paired t-tests) exceptthey were summed for total AmesA
To characterise the midrib for three typical fibrous bundlesheath cells we measured the cross-sectional heights widthsand cell wall thicknesses calculating mean values for each traitfrom three measurements per cross-section To characterise thexylem anatomy and theoretical conductivity of xylem conduitsfor a typical conduit within the midrib an intermediary veinand a minor vein of each sampled phylloclade we treated theconduit as an ellipse and determined the major and minor axisdiameters We calculated the theoretical hydraulic conductivityof the xylem conduit using Poiseuillersquos equation for ellipsesbased on conduit dimensions (Lewis and Boose 1995 CochardNardini and Coll 2004)
Tab
le5
Meanvalues
fortissue
compo
sition
traitsseforRuscusaculeatusan
dRm
icroglossumw
ithun
itsan
dreplication
andsign
ificanceof
differencesbetw
eenspecies(t-tests)
Forgiventraitsexpectatio
nsaregivenforw
hetherRaculeatus
shouldhave
ahigherorlowervaluerelativ
etocomparatorspeciesaccordingtothehypothesesofshadeordroughtadaptationCom
paratorspecies
dataweretakenaccordingtoavailabilityinthepreviouslypublishedliteratureandnumberofspeciesand
minim
umm
eanandmaxim
umtraitvaluesareprovidedSourcesofcomparativ
edatad
13C(K
oumlrneretal
1991)NmassNarea(W
righ
tetal2
004)N
Sn
otstatistically
significant
atPlt005
Tissuecompositio
ntraits
Abbreviation
Units
Ra
culeatus
Rm
icroglossum
Hypotheses
Com
paratorspecies
Meanplusmnse
NMeanplusmnse
NShade
adapted
Drought
adapted
(N)
(minm
ean
max)
Npermass
Nmass
194
plusmn006
9192
plusmn007
9Low
erHigherA
BTem
peratebroadleaf
evergreen(129)
0581
19
231
Nperarea
Narea
238
plusmn007
9175
plusmn006
9Low
erHigherA
BTem
peratebroadleaf
evergreen(129)
0727
1733
63
Cpermass
Cmass
44
4plusmn048
943
6plusmn052
9Carbonto
nitrogen
ratio
CN
231plusmn068
922
9plusmn058
9Carbonisotoperatio
d13C
permilndash33
32plusmn029
9ndash33
05plusmn020
9Low
erAB
Higher
Tem
peratespecies(35)
ndash27
64
ndash26
03
ndash24
09
AAnexpectationaccordingto
ahypothesiswas
confi
rmed
forRa
culeatus
BAnexpectationaccordingto
ahy
pothesiswas
confi
rmed
forRm
icroglossum
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology G
K t frac14 pa3b3
64hetha2 thorn b2THORN eth3THORN
where a and b are the major and minor axes of the ellipse and his water viscosity at 25C
Gas-exchange measurements responses to light and CO2and cuticular conductance
In July and August 2009 photosynthetic light response curvesandCO2 response curvesweremeasuredusing aLi-6400portablephotosynthesis system (Li-Cor Biosciences ) with light provided
Ruscusaculeatus
Ruscusmicroglossum
01 mm
Fig 2 Lamina and midrib cross-sections of Ruscus aculeatus and R microglossum phylloclades (05mm thick) Note that bothspecies exhibit shade tolerance features such as absence of palisade tissue as well as drought tolerance features such as large waterstorage compartment and thick-walled epidermis andfibrousbundle sheath cells surrounding thexylemandphloemfor bothmajor andminor veins especially prominent in R aculeatus which is native to drier habitats
(a) (b)
Fig 1 (a)Ruscus aculeatus and (b)Rmicroglossumgrowing at theMildredEMathiasBotanicalGardenNote thatwhat atfirst glance appear to be leaves are infact phylloclades
H Functional Plant Biology A Pivovaroff et al
by a red-blue light source (6400ndash02B no SI-710 Li-CorBiosciences ) Gas-exchange measurements were made on atleast 1ndash2 phylloclades per individual
For light-response curves phylloclades were acclimatedfor at least 5min at 1500mmolmndash2 sndash1 PAR at temperatures of2527C with RH maintained at ~50 and CO2 concentrationof 400mmolmolndash1 Then phylloclades were measured for netCO2 assimilation per leaf area at PAR steps of 1600 1400 12001000 800 600 500 400 300 200 100 50 and 0mmolmndash2 sndash1with 180ndash240 s stabilisation at each irradiance step Wedetermined light-saturated photosynthetic rate per area andmass (Aarea and Amass) dark respiration rate per area and mass(Rarea and Rmass) ie the negative A at zero PAR maximumstomatal conductance per area (gs) light compensation point(LCP) as the x-intercept intrinsic water use efficiency (WUEAareags) and the ratio of intercellular to ambient CO2
concentration (CiCa) at 100mmolmndash2 sndash1 PAR We thenharvested the phylloclades measured for gas exchange todetermine the nitrogen concentration and carbon isotope ratio(see below)
For CO2-response curves phylloclades were allowed toequilibrate at 400 ppm to induce stomatal opening and the netCO2 assimilation per leaf area was determined at Ci steps of400 300 200 100 50 400 400 500 600 800 1200 14001600 ppm with 3ndash4min equilibration time at each step Wedetermined maximum rate of carboxylation and maximum rateof electron transport per leaf area (Vcmax and Jmax) from plots ofCi vs A corrected to 25C (Farquhar and von Caemmerer 1980)
Cuticular conductance (ie minimumepidermal conductancegmin sensu Kerstiens 1996) was determined for 3ndash4 maturephylloclades and 10 cm lengths of stems from each of threeindividuals of each species Phylloclade and stem sampleswere hydrated and then the cut ends were sealed with waxSamples were dried for at least 30min on a laboratory bench atPAR of lt10mmol photons mndash2 sndash1 to induce stomatal closurethen samples were weighed for at least eight intervals of 30minduring which the slope of water loss versus time was highlylinear (R2 gt 0995) and therefore taken to represent transpirationafter stomata had closed fully The gmin was calculated as thetranspiration rate divided by the mole fraction vapour pressuredeficit (VPD determined from a weather station HOBO MicroStation with Smart Sensors Onset Bourne MA USA)
Pressurendashvolume curve parametersPressurendashvolume curve parameters were determined for matureshoots using the bench-drying method (Koide et al 2000 Sacket al 2003a) Shoots were ~10ndash15 cm long with about fourphylloclades per shoot for R microglossum and 15ndash20phylloclades for R aculeatus Shoots were progressively driedon a laboratory bench and measured at intervals by equilibratingfor 10min in a Whirlpak bag (Whirl-Pak Nasco Fort AtkinsonWIUSA) beforeweighing andmeasuring for leafwater potential(Yleaf) with a pressure chamber (Model 1000 Plant MoistureStress Instruments Albany OR USA) Subsequently dry masswas determined after more than 48 h in an oven at 70C Wedetermined the leaf dry matter content (LDMC) saturated watercontent (SWC) turgor loss point (Ytlp) relative water content atturgor loss point (RWCtlp) and osmotic potential at full turgor
(po) relative capacitance at full turgor (Cft DRWCDYleaf) andrelative capacitance at turgor loss point (Ctlp DRWCDYleaf) Wedetermined the modulus of elasticity (e) as the linear slope of theline fitted for pressure potential versus relative water contentabove and including turgor loss point (Sack et al 2013b)
Shoot hydraulic conductance
We measured the hydraulic conductance of mature shoots(Kshoot) ~10ndash15 cm long bearing about four phylloclades pershoot for R microglossum and 15ndash20 phylloclades forR aculeatus using the evaporative flux method (Sack et al2002) Samples were harvested and the ends were re-cut underdistilled degassed water with a fresh razor blade then hydratedovernight Shoots were connected to tubing containing distilleddegassed water running to a graduated cylinder on a balanceinterfaced to a computer logging data every minute to calculatethe flow rate of water from the balance into the sample Sampleswere held in place above a fan on wood frames strung withfishing line Lights were arranged above a plexiglass containerof water that acted as a heat trap producing PAR ofgt1200mmolmndash2 sndash1 at shoot level When steady-statetranspiration was achieved samples were covered with aplastic bag and removed from the tubing and the leaf waterpotential was determined with a pressure chamber (Model 1000Plant Moisture Stress Instruments) Kshoot was calculated as thesteady-state transpirational flow rate (E mmolmndash2 sndash1) dividedby the water potential driving force (DYleaF= ndashYleaf MPa)further normalised by total phylloclade area Kshoot valueswere standardised to 25C to correct for the temperaturedependence of the viscosity of water (Sack et al 2003a)
Foliar nitrogen concentration and carbon isotope ratio
To analyse total tissue nitrogen concentration and carbon isotopecomposition (d13C) for three replicates from each of threeindividuals for each species phylloclade samples were oven-dried at gt70C for more than 48 h and ground by mortar andpestle The nitrogen concentration and d13C were determined atthe UCDavis Stable Isotope Facility using an elemental analyser(PDZEuropaANCA-GSL SerconLtd Cheshire UK) interfacedto an isotope ratiomass spectrometer (PDZEuropa20ndash20 SerconLtd) at the UC Davis Stable Isotope Facility Final d13C contentvalues are expressed relative to international standardVienna PeeDee belemnite (V-PDB) for carbon
d13CethpermilTHORN frac14 etheth13C=12C of sampleTHORN=eth13C=12C of standardTHORN 1THORN 1000
eth4THORN
d13C of plant tissues can provide a measure of intrinsic WUE(Ags) at the time that carbon was assimilated giving long-termwater-use efficiency
Comparative data compilation and trait comparison
To consider Ruscus trait values in a global context we compileddata from the literature for (1) temperate and tropical broadleafevergreen species (2) Mediterranean species and (3) woodyangiosperms in general When available we considered data forplants grown in the shade and for species that are shade tolerant
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology I
however eco-physiological trait data is generally collected forsun leaves or plants making a comparison solely for shade grownplants not possible Formore commonly studied traits (egAmax)comparative data was taken from studies with large databasesand multiple traits (eg GLOPNET Wright et al 2004) Wedetermined minimum mean and maximum values for traitsfrom comparative studies or the mean and standard error ifraw species values were not available Studies that wereincluded for comparative data are referenced in the captionsfor Table 1ndash5 in association with the specific traits wecompared Differences between Ruscus trait values andcomparative data from the literature were determined for bothR aculeatus and R microglossum Hypotheses were deemedsupported for a Ruscus species if the trait value was higher orlower than the mean comparator value in the way predicted
Results
Both Ruscus species studied had leaf morphology consistentwith adaptation to shade and drought relative to comparator
species (Tables 1ndash5)We constructed a radar graph to encapsulatethe key traits that would confer adaptation to shade drought andthe combination for R aculeatus which had the more extremeadaptation of the two Ruscus species considered (Fig 3) Thisfigure summarises the major results of our study with values forcomparator species appearing as the inner circle and R aculeatustrait values displayed as the bold outer line
Gross morphology of phylloclades
Both R aculeatus and R microglossum had LMA valueslower than comparator species and R microglossum hadphylloclades larger than the mean leaf area for comparatorspecies Both Ruscus species had lower bulk tissue density thancomparator species consistent with the water storage in theRuscus phylloclades Notably despite its water storage tissuethe LDMC and SWC values of R aculeatus were typical ofthose for comparator species whereas R microglossum had alower LDMC and a higher SWC than for comparator speciessets (Table 1)
Fig 3 Radar graph illustrating percent difference between selected traits of Ruscus aculeatuswithcomparative data (see Tables 1ndash5 for symbols and sources of comparative data) Values forR aculeatus outside the circle indicate shade andor drought tolerance Traits are arrangedaccording to whether they would contribute shade tolerance drought tolerance or both The innerdisc represents the mean for comparative species for given traits and the values for R aculeatus isscaled relative to the magnitude of that value () with a value outside the disc representing greatershade andor drought tolerance (+) and (ndash) indicate if the axis is scaled such that a value outside thecircle represents percent higher or lower than comparative values respectively For traits expressed asnegative values (+) and (ndash) indicate more negative or less negative respectively
J Functional Plant Biology A Pivovaroff et al
Anatomy of phylloclades
The Ruscus species possessed numerous anatomical traitsconsistent with benefits for both shade and drought tolerance(Table 2) The phylloclade cross-sections were symmetrical(Fig 2) and thus the cross-sectional anatomy of the adaxialand abaxial halves were not different for all traits (paired t-testP gt 005) and values were averaged within species The twospecies were similar in their substantial leaf thickness and inthe thickness of cuticle epidermis cell walls and water storagecompartment (Table 2) The fractions of the lamina occupied bythe epidermis mesophyll and water storage tissues were 1552ndash56 and 29ndash32 respectively
Both species had parallel longitudinal veins of three sizes(midrib intermediate and small veins) Consistent with droughtadaptation the two species had low Dm R aculeatus having thelower value andRmicroglossumhadahigher IVD (Table 2)Thetwo species had on average the same maximum conduitdiameters in their midribs intermediate veins and small veinsand the maximum conduit diameter decreased ~35 from themidrib to the small veins The average theoretical hydraulicconductivity of xylem conduits did not differ between thespecies and decreased by up to 86 from the midrib to theminor vein R aculeatus had on average walls that were 85thicker in the fibrous bundle sheath (Table 2)
Gas-exchange measurements
Consistent with adaptation to simultaneous shade and droughtthe Ruscus species had very low values for Rarea Rmass and gs(Table 3) relative to comparator temperate broadleaf evergreenspecies (Fig 3) The Aarea Amass LCP Vcmax and Jmax were alsovery low relative to comparator species (Fig 3 Table 3)consistent with shade adaptation The two Ruscus species hadvery high values for Ags (Table 3) consistent with excellentWUE Consistent with strong drought tolerance via retention ofstored water both species had very low values for leaf and stemgmin especially relative to comparator vascular plant species(Fig 3 Table 3)
Hydraulic conductance pressure volume curveparameters and leaf water storage
Consistent with expectations for combined drought and shadetolerance both Ruscus species had low Kshoot relative tocomparator tropical and temperate woody angiosperms (Fig 3Table 4) The pressurendashvolume curve parameters of Ruscuswereconsistent with achieving drought tolerance through tissue waterstorage Both species had less negative po and ytlp than meanvalues for comparative evergreen woody species (Fig 3Table 4) Notably both species had lower e values thancomparative evergreen woody species and higher RWCtlp andCft values (Fig 3 Table 4) consistent with drought tolerance
Nitrogen concentration and carbon isotope composition
Both species had high values for phyllyclade Narea and Nmass
relative to comparative temperate broadleaf evergreen speciesconsistent with drought adaptation (Fig 3) The d13C valueswerevery negative and typical of values often observed for understoryplants (da Silveira et al 1989) consistent with shade tolerance
Indeed the d13C values were notably strongly negative given thehigh WUE found for these species
Testing hypotheses for shade and drought tolerancewith trait survey data
Overall we quantified 57 traits for the twoRuscus species and for21 traits we had a priori hypotheses for a benefit for shadetolerance and for 24 traits we had a priori hypotheses for abenefit for drought tolerance Using comparative data we foundthat 16 of 21 hypotheses were supported for shade tolerance and22 of 24 were supported for drought tolerance Of the ninehypotheses for traits that would contribute to both shade anddrought tolerance simultaneously (ie expectations were both forhigher or lower values than comparative species) eight weresupported by trait data All these proportions were significantlyhigher than the 50 support that would have been expected toarise only from chance (P = 0001ndash0058 proportion tests)Notably in the seven cases when shade tolerance traits wouldconflict with drought tolerance traits four indicated a benefit fordrought tolerance rather than shade tolerance for both species(Tables 1ndash5)
Discussion
Both R aculeatus (Fig 3) and R microglossum showed traitvalues consistent with combined shade and drought toleranceNumerous traits were consistent with a combined shade anddrought tolerance through improving carbon balance enablinga conservative resource use ie via slow respiration and long-lived parts Other traits would contribute specifically to droughttolerance via reduced demand for water during activephotosynthesis and the ability to survive strong drought afterstomatal closure Notably many such traits were related to waterstorage providingnew insights into effective formsof succulencein shaded habitats This suite of traits would contributeimportantly to the ability of Ruscus to occupy shaded sitesprone to strong drought across a wide geographical range
Traits contributing to simultaneous drought and shadetolerance
Ruscus species showed specialisation associated withconservative resource use consistent with tolerance of shadeand drought These specialised traits included thick lamina andcomponent tissues that contribute to long tissue life-spans (shootslastgt5 years Sack et al 2003bWright et al 2004) AdditionallyRuscus species had very low gas-exchange rates including lowgs and lowKshoot whichwould correspond to a low investment invascular tissue (Tyree andZimmermann1983 Sack et al 2003b)and low Rarea Rmass Aarea and Amass all representing an ability tomaintain photosynthesis and growth with low requirements forlight and water
Traits contributing to drought tolerance
According to Jones et al (1992) drought tolerance can beachieved through avoidance of plant water deficits toleranceof plant water deficits or efficiency mechanisms Ruscus speciesshowed traits associated with drought tolerance either byproviding the ability to maintain photosynthesis and growth indrying soil andor the ability to survive chronic drought as
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology K
previously shown experimentally for R aculeatus (Sack 2004)Traits that would contribute to the ability to maintain gasexchange in drying soil include small leaf size high WUE andlow IVD High WUE achieved in part with high Narea meansthese Ruscus species can attain positive carbon balance evenwith extremely low gs Low IVD and water storage capacitanceallow the phylloclades to maintain water supply to the mesophylland tolerate transiently high evaporation rates for example dueto sunflecks without desiccating the leaf (Sack et al 2003a)Traits contributing to the ability of the phylloclades to surviveextended drought included those enabling a low evaporationrate per leaf area once stomata have shut such as low gmin inleaf and stem and those related to specialised water storagetissue linked with low leaf density low e and po values thatwere low in magnitude
Ruscus water storage
The water storage tissue of Ruscus that occupied a third of theleaf thickness although contributing most directly to droughttolerance is also consistent with shade tolerance given itscontribution to reduced tissue costs for the phylloclade as awhole The water storage tissue had thin cell walls reflected inthe low bulk e and low solute concentration contributing tobulk po values that were low in magnitude
This water storage tissue would also contribute to both typesof drought tolerance ndash the ability to maintain photosynthesis indrying soil and to survive after stomata have shut duringextended drought The lsquosucculencersquo of Ruscus phylloclades isdistinctive relative to more typical succulent-leafed andsucculent-stemmed species which tend to have high leaf watercontent and capacitance values (Vendramini et al 2002 Ogburnand Edwards 2012) In contrast in Ruscus species the SWCwas low relative to typical leaf succulent species and forR aculeatus Cft fell within the range of typical evergreenleaves We note that the strong tissue differentiation in Ruscus(ie separation of mesophyll cell and water storage in spaceand their distinctions in anatomy) would contribute to higheffectiveness of water storage even if the bulk tissue overallhad low SWC and capacitance Indeed across species theretends to be no relationship between the magnitude of SWC orcapacitance and the degree of within-leaf tissue differentiation(Ogburn and Edwards 2012) Notably such differentiationwould contribute special advantages for supply of waterwhether stomata are open or closed as the large thin-walledwater storage cells with low solute concentration can yield theirwater to supply the evaporative load while the photosynthetictissues can maintain their volume according to their thickerwalls and stronger solute concentration
Although the capacitance and SWC values were low forRuscus species these values would be more substantial ifconsidered relative to water demand Across several speciesCft has been found to correlate with Kshoot and with gs (Sacket al 2003a Blackman et al 2010) indicating that leaves tendto be built with capacitance to match their maximum flux ratesand thus to buffer the leaf water potential against surges intranspiration Thus because Ruscus has low gs when stomataare open and low Kshoot the capacitance would be expectedto supply transpiration transiently during sunflecks or high
VPD Likewise when stomata close the capacitance suppliesongoing water loss via cuticular conductance Given theextremely low gmin of Ruscus even its moderate Ctlp canenable survival for weeks (Sack et al 2003b Sack 2004)Further at turgor loss the water content would equalSWCRWCtlp and the relatively high RWCtlp wouldcontribute to the high water content once turgor is lost Thusthe lsquosucculencersquo of Ruscus is moderate in absolute terms butcombined with its other very strong mechanisms to reducetranspiration when stomata are open or closed ie low gs andgmin even this moderate capacitance would provide strongfunctionality
Water-use efficiency and carbon isotope composition
It was noteworthy that despite extremely high WUE valuesphylloclade d13C values of the Ruscus species were verynegative This presents a strong anomaly worthy of furtherinvestigation as species with high WUE typically have higherd13C (less negative ie closer to zero) The d13C can beinfluenced by a host of processes including source CO2 storedplant carbon and time-integrated CO2 concentration at the siteof carboxylation (Farquhar et al 1989) For Ruscus althoughd13C values were typical of an understory plant they did notappear to be drivenby internalCO2 concentration because the leafisotopic values were depleted in 13C whereas the high WUEdetermined by gas exchange would likely promote enrichedisotopic values It is more likely that d13C in this species wasdetermined by source CO2 or stored carbon or recycling ofrespired CO2 (da Silveira et al 1989) Our study individualsof Ruscus were cultivated in a shaded understory similar totheir natural habitat Previous studies have shown that there canbe higher concentrations of respired CO2 in the forest understorythan in the canopy resulting in more negative carbon isotoperatios in understory plant tissue than canopy plant tissue (daSilveira et al 1989) This is a function of decomposing leavesand litter cover slow air mixing as well as plant environmentalresponses
A second possible explanation for the d13C values ofRuscus is related to its growth form and phenology asRuscus has extensive rhizomes (Sack et al 2003b) whichact as a carbon store for the plant Using this recycled carbonduring the growth season when the plant may not be able tomeet its carbon requirement by photosynthesis alone becauseof low maximum rates under light limitation may alsocontribute to more negative d13C values (Vizzini 2003)Notably Ruscus stems are hollow and thus can also storerelatively large amounts of CO2 for use during a growth periodwhen carbon is otherwise limiting especially given very lowgs Some of this stored carbon might be photosyntheticallyfixed by the stem (Nilsen and Sharifi 1997) In each of thesecases respired stored or recycled CO2 would supply carbonthat was previously fixed by Rubisco with more negative d13Cvalues (ndash27o) than air (ndash8o)
There was also a dissonance between the d13C value and CiCa The fully mature phylloclades which were selected andused for gas-exchange and d13C measurements were producedduring late winter and early spring with mild temperatures(average at midday 190ndash220C) and low atmospheric VPD
L Functional Plant Biology A Pivovaroff et al
(average at midday 06ndash15 kPa) However the gas-exchangemeasurements were taken during summer with hightemperature (average at midday 300ndash355C) and high VPD(average at midday 25ndash35 kPa) We harvested thephylloclades that we used for gas exchange to determine thecarbon isotope ratio Although the low value of instantaneousCiCa may be caused by high temperature and high atmosphericVPD the very low value of d13C may represent the time whenthe phyllocladersquos carbon was assimilated (during mildtemperature and low VPD)
Implications for drought and shade tolerance Ruscusas a model
We found strong support for a large number of hypotheses fortrait-based shade and drought tolerance providing a strong traitbasis for combined tolerance Although the detailed functionaltrait survey conducted here is relatively novel in its breadth(see also Pasquet-Kok et al 2010) this is a logical extensionof the traditional approach for understanding the basis forplant adaptation to environment ie testing expectations forindividual traits established by previous studies of thefunctional significance of these traits in other species Weacknowledge there is some degree of uncertainty ininterpreting a large number of traits simultaneously based onstudies of other species First the interpretation of the value oftraits based on other species may not be in all cases equally validfor Ruscus Some trait variation may relate to other functionsFurther studies for example using mutants would be necessaryas conclusive evidence of the value of specific traits in a givenspecies However one advantage of testing numerousexpectations for each hypothesis is that the key finding will berobust to the removal of some traits from the analysis if thoseare later found to be inappropriate Ideally when a model forestimating plant performance from leaf traits becomes availableone could determine how the specific quantitative combinationsof traits presented here scales up to plant shade and droughttolerance
The shade and drought tolerance of Ruscus consistent withthe suite of traits examined here is one case demonstratinghow plants can avoid a general trade-off between shade anddrought tolerance Further Ruscus is noteworthy as one of onlya few stem photosynthetic plants that occupy a shaded habitatHistorically it is likely that shade tolerance preceded droughttolerance given this speciesrsquo ancestors were species of moisttropical forests (Kim et al 2010) and thus Ruscus or itsancestor apparently evolved drought tolerance whileexpanding its range into drier habitat or during past climatechange Considering its unique adaptations and trait valuesRuscus can serve as an excellent model for the basis ofcombined shade and drought tolerance
Acknowledgements
We thank the Mildred E Mathias Botanical Garden for access to garden andplants Andy Truong for assistance with measurements Howard GriffithsLouis Santiago and Cheryl Swift for helpful discussions and comments onthe manuscript This work was supported by National Science FoundationGrant IOB-0546784
References
Bartlett MK Scoffoni C Sack L (2012) The determinants of leaf turgor losspoint and prediction of drought tolerance of species and biomes a globalmeta-analysis Ecology Letters 15 393ndash405 doi101111j1461-0248201201751x
Blackman CJ Brodribb TJ Jordan GJ (2010) Leaf hydraulic vulnerability isrelated to conduit dimensions and drought resistance across a diverserange of woody angiosperms New Phytologist 188 1113ndash1123doi101111j1469-8137201003439x
Brodribb TJ Feild TS Jordan GJ (2007) Leaf maximum photosynthetic rateand venation are linked by hydraulicsPlant Physiology 144 1890ndash1898doi101104pp107101352
Caspersen JP (2001) Interspecific variation in sapling mortality in relationto growth and soil moisture Oikos 92 160ndash168 doi101034j1600-07062001920119x
Cochard H Nardini A Coll L (2004) Hydraulic architecture of leaf bladeswhere is the main resistance Plant Cell amp Environment 27 1257ndash1267doi101111j1365-3040200401233x
Cooney-Sovetts C Sattler R (1987) Phylloclade development in theAsparagaceae an example of homoeosis Botanical Journal of theLinnean Society 94 327ndash371 doi101111j1095-83391986tb01053x
Cowling R Rundel P Lamont BB ArroyoMK ArianoutsouM (1996) Plantdiversity in Mediterranean-climate regions Trends in Ecology ampEvolution 11 362ndash366 doi1010160169-5347(96)10044-6
da Silveira L Sternberg L Mulkey SS Wright SJ (1989) Ecologicalinterpretation of leaf carbon isotope ratios influence of respired carbondioxide Ecology 70 1317ndash1324 doi1023071938191
de Lillis M Fontanella A (1992) Comparative phenology and growth indifferent species of the Mediterranean maquis of central Italy PlantEcology 99-100 83ndash96 doi101007BF00118213
Dracup JA (1991)DroughtmonitoringStochasticHydrologyandHydraulics5 261ndash266 doi101007BF01543134
Engelbrecht BMJ Kursar TA (2003) Comparative drought-resistance ofseedlings of 28 species of co-occurring tropical woody plantsOecologia 136 383ndash393 doi101007s00442-003-1290-8
Farquhar GD von Caemmerer S (1980) A biochemical model ofphotosynthetic CO2 assimilation in leaves of C3 species Planta 14978ndash90 doi101007BF00386231
FarquharGD Ehleringer JR HubickK (1989) Carbon isotope discriminationand photosynthesis Annual Review of Plant Physiology and PlantMolecular Biology 40 503ndash537 doi101146annurevpp40060189002443
Givnish TJ (1988) Adaptation to sun and shade a whole-plant perspectiveAustralian Journal of Plant Physiology 15 63ndash92 doi101071PP9880063
Gulias J Flexas J Mus M Cifre J Lefi E Medrano H (2003) Relationshipbetweenmaximum leaf photosynthesis nitrogen content and specific leafarea inBalearic endemic and non-endemicMediterranean speciesAnnalsof Botany 92 215ndash222 doi101093aobmcg123
Haberlandt G (1914) lsquoPhysiological plant anatomyrsquo (MacMillan London)Hallik L Niinemets U Wright J (2009) Are species shade and drought
tolerance reflected in leaf-level structural and functional differentiationin Northern Hemisphere temperate woody flora New Phytologist 184257ndash274 doi101111j1469-8137200902918x
Jones HG (1992) lsquoPlants and microclimate a quantitative approach toenvironmental plant physiologyrsquo (Cambridge University PressCambridge)
Kerstiens G (1996) Cuticular water permeability and its physiologicalsignificance Journal of Experimental Botany 47 1813ndash1832doi101093jxb47121813
Kim JH Kim DK Forest F Fay MF Chase MW (2010) Molecularphylogenetics of Ruscaceae sensu lato and related families(Asparagales) based on plastid and nuclear DNA sequences Annals ofBotany 106 775ndash790 doi101093aobmcq167
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology M
Koide RT Robichaux RH Morse SR Smith CM (2000) Plant water statushydraulic resistance and capacitance In lsquoPlant physiological ecologyfield methods and instrumentationrsquo (Eds R Pearcy JR Ehleringer HMooney P Rundel) pp 161ndash183 (Kluwer Dordrecht The Netherlands)
Koumlrner C Farquhar GD Wong SC (1991) Carbon isotope discrimination byplants follows latitudinal and altitudinal trends Oecologia 88 30ndash40doi101007BF00328400
Lewis AM Boose ER (1995) Estimating volume flow rates through xylemconduits American Journal of Botany 82 1112ndash1116 doi1023072446063
Martiacutenez-Palleacute E Aronne G (1999) Flower development and reproductivecontinuity inMediterraneanRuscusaculeatusL (Liliaceae)Protoplasma208 58ndash64 doi101007BF01279075
Martiacutenez-Tilleriacutea K Loayza AP Sandquist DR Squeo FA (2012) Noevidence of a trade-off between drought and shade tolerance inseedlings of six coastal desert shrub species in north-central ChileJournal of Vegetation Science 23 1051ndash1061 doi101111j1654-1103201201427x
Matalas NC (1963) Probability distribution of low flows USGS ProfessionalPapers 434-A US Government Printing Office Washington DC
Niinemets U (1999) Research review components of leaf dry mass per area ndashthickness and density ndash alter leaf photosynthetic capacity in reversedirections in woody plants New Phytologist 144 35ndash47 doi101046j1469-8137199900466x
Niinemets U Valladares F (2006) Tolerance to shade drought andwaterlogging of temperate northern hemisphere trees and shrubsEcological Monographs 76 521ndash547 doi1018900012-9615(2006)076[0521TTSDAW]20CO2
Nilsen ET Sharifi MR (1997) Carbon isotope composition of legumes withphotosynthetic stems from Mediterranean and desert habitats AmericanJournal of Botany 84 1707ndash1713 doi1023072446469
Ogburn RM Edwards EJ (2012) Quantifying succulence a rapidphysiologically meaningful metric of plant water storage Plant Cell ampEnvironment 35 1533ndash1542 doi101111j1365-3040201202503x
Pasquet-Kok J Creese C Sack L (2010) Turning over a new lsquoleafrsquo multiplefunctional significances of leaves versus phyllodes in Hawaiian Acaciakoa Plant Cell amp Environment 33 2084ndash2100 doi101111j1365-3040201002207x
SackL (2004)Responses of temperatewoody seedlings to shade and droughtdo trade-offs limit potential niche differentiation Oikos 107 110ndash127doi101111j0030-1299200413184x
SackLFroleK (2006)Leaf structural diversity is related to hydraulic capacityin tropical rain forest trees Ecology 87 483ndash491 doi10189005-0710
Sack L Holbrook NM (2006) Leaf hydraulics Annual Review of PlantBiology 57 361ndash381 doi101146annurevarplant56032604144141
Sack L Melcher PJ Zwieniecki MA Holbrook NM (2002) The hydraulicconductance of the angiosperm leaf lamina a comparison of threemeasurement methods Journal of Experimental Botany 532177ndash2184 doi101093jxberf069
Sack L Cowan P Jaikumar N Holbrook NM (2003a) The ldquohydrologyrdquo ofleaves co-ordination of structure and function in temperate woodyspecies Plant Cell amp Environment 26 1343ndash1356 doi101046j0016-8025200301058x
Sack L Grubb P Maronon T (2003b) The functional morphology of juvenileplants tolerant of strong summer drought in shaded forest understoriesin southern Spain Plant Ecology 168 139ndash163 doi101023A1024423820136
SackLScoffoniCMcKownADFroleKRawlsMHavran JCTranHTranT (2012) Developmentally based scaling of leaf venation architectureexplains global ecological patterns Nature Communications 3 837doi101038ncomms1835
Sack L Chatelet D Scoffoni C PrometheusWiki contributors (2013a)ldquoEstimating the mesophyll surface area per leaf area from leaf celland tissue dimensions measured from transverse cross-sectionsrdquoPrometheusWiki Available at httpwwwpublishcsiroauprometheuswikitiki-pagehistoryphppage=Estimating the mesophyllsurface area per leaf area from leaf cell and tissue dimensionsmeasured from transverse cross-sectionsamppreview=16 [accessed June18 2013]
SackL Pasquet-Kok JPrometheusWiki contributors (2013b) ldquoLeaf pressure-volume curve parametersrdquo PrometheusWiki Available at httpprometheuswikipublishcsiroautiki-indexphppage=Leaf+pressure-volume+curve+parameters
Scoffoni C Pou A Aasamaa K Sack L (2008) The rapid light response ofleaf hydraulic conductance new evidence from two experimentalmethods Plant Cell amp Environment 31 1803ndash1812 doi101111j1365-3040200801884x
Smith T Huston M (1989) A theory of the spatial and temporal dynamics ofplant communities Vegetatio 83 49ndash69 doi101007BF00031680
Sterck F Markesteijn L Schieving F Poorter L (2011) Functional traitsdetermine trade-offs and niches in a tropical forest communityProceedings of the National Academy of Sciences of the United Statesof America 108 20 627ndash20 632 doi101073pnas1106950108
Thomas GS (1992) lsquoOrnamental shrubs climbers and bamboosrsquo (JohnMurray Publishers Great Britain)
TyreeMT ZimmermannMH (1983) lsquoXylem structure and the ascent of saprsquo(Springer-Verlag Berlin)
USDA ARS National Genetic Resources Program (2009) GermplasmResources Information Network (GRIN) online database NationalGermplasm Resources Laboratory Beltsville Maryland Availableathttpwwwars-gringovcgi-binnpgshtmltaxonpl410577[Accessed 20 July 2009]
Vendramini F Diacuteaz S Gurvich DE Wilson PJ Thompson K Hodgson JG(2002) Leaf traits as indicators of resource-use strategy in floras withsucculent species New Phytologist 154 147ndash157 doi101046j1469-8137200200357x
Vile D (2005) Specific leaf area and dry matter content estimate thicknessin laminar leaves Annals of Botany 96 1129ndash1136 doi101093aobmci264
Vizzini S (2003) d13C and d15N variability in Posidonia oceanica associatedwith seasonality and plant fraction Aquatic Botany 76 195ndash202doi101016S0304-3770(03)00052-4
Walters M Reich PB (1999) Research review low-light carbon balance andshade tolerance in the seedlings of woody plants dowinter deciduous andbroad-leaved evergreen species differ New Phytologist 143 143ndash154doi101046j1469-8137199900425x
Witkowski E Lamont BB (1991) Leaf specific mass confounds leaf densityand thickness Oecologia 88 486ndash493
Wright IJWestobyM (2003)Nutrient concentration resorption and lifespanleaf traits of Australian sclerophyll species Functional Ecology 1710ndash19 doi101046j1365-2435200300694x
Wright IJ Reich PB Westoby M Ackerly DD Baruch Z Bongers FCavender-Bares J Chapin T Cornelissen JHC Diemer M Flexas JGarnier E Groom PK Gulias J Hikosaka K Lamont BB Lee T LeeWLusk C Midgley JJ Navas M-L Niinemets U Oleksyn J Osada NPoorter H Poot P Prior L Pyankov VI Roumet C Thomas SC TjoelkerMG Veneklaas EJ Villar R (2004) The worldwide leaf economicsspectrum Nature 428 821ndash827 doi101038nature02403
Wullschleger SD (1993) Biochemical limitations to carbon assimilation inC3 plants ndash a retrospective analysis of the ACi curves from 109 speciesJournal of Experimental Botany 44 907ndash920 doi101093jxb445907
N Functional Plant Biology A Pivovaroff et al
wwwpublishcsiroaujournalsfpb
symmetrically with adaxial and abaxial layers of mesophyllepidermis and cuticle above and below a central achlorophyllouswater storage tissue (Fig 2) The per cent air space in each of theadaxial and abaxial mesophyll and in the water storagecompartment was estimated to the nearest 5 and the totalphylloclade airspace was calculated
PAir space in each tissue
fraction of leaf cross section occupied by that tissue100
eth1THORN
As indices of cell size the cross-sectional areas andperimeters were measured for three cells in the epidermis(adaxial and abaxial) the mesophyll (adaxial and abaxialie above and below the water storage tissue) and thewater storage tissue The area occupied by chloroplastswithin a mesophyll cell was measured for three cells(adaxial and abaxial) and the percent cross-sectionalchloroplast area was calculated as the ratio of chlorophyllarea divided by mesophyll cell area
We calculated the surface area of mesophyll cells per leafarea (AmesA) as described by Sack et al (2013a) a measure ofthe area available for CO2 uptake formesophyll cell layers Giventhe lack of palisade-form cells we modelled all mesophyll cellsas spheres for these calculations
We also measured vascular anatomy to quantify traits relatedto the efficiency of water transport within and outside the xylemWe measured the inter-veinal distance (IVD) and also theminimum distance from edge of bundle sheath to epidermis(Dm) as the hypotenuse between the distance between veinsand the distance to the epidermis (Brodribb Feild and Jordan2007)
Dm frac14ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
IVD2
2thorn ethdistance from bundle sheath edge to epidermisTHORN2
s
eth2THORN
We averaged IVD and Dm from three values for each cross-section
For all anatomical traits except those relating to the centralwater storage tissue measurements were made both adaxial andabaxial halves and values for the two halveswere averagedwhennot significantly different (at P lt 005 in paired t-tests) exceptthey were summed for total AmesA
To characterise the midrib for three typical fibrous bundlesheath cells we measured the cross-sectional heights widthsand cell wall thicknesses calculating mean values for each traitfrom three measurements per cross-section To characterise thexylem anatomy and theoretical conductivity of xylem conduitsfor a typical conduit within the midrib an intermediary veinand a minor vein of each sampled phylloclade we treated theconduit as an ellipse and determined the major and minor axisdiameters We calculated the theoretical hydraulic conductivityof the xylem conduit using Poiseuillersquos equation for ellipsesbased on conduit dimensions (Lewis and Boose 1995 CochardNardini and Coll 2004)
Tab
le5
Meanvalues
fortissue
compo
sition
traitsseforRuscusaculeatusan
dRm
icroglossumw
ithun
itsan
dreplication
andsign
ificanceof
differencesbetw
eenspecies(t-tests)
Forgiventraitsexpectatio
nsaregivenforw
hetherRaculeatus
shouldhave
ahigherorlowervaluerelativ
etocomparatorspeciesaccordingtothehypothesesofshadeordroughtadaptationCom
paratorspecies
dataweretakenaccordingtoavailabilityinthepreviouslypublishedliteratureandnumberofspeciesand
minim
umm
eanandmaxim
umtraitvaluesareprovidedSourcesofcomparativ
edatad
13C(K
oumlrneretal
1991)NmassNarea(W
righ
tetal2
004)N
Sn
otstatistically
significant
atPlt005
Tissuecompositio
ntraits
Abbreviation
Units
Ra
culeatus
Rm
icroglossum
Hypotheses
Com
paratorspecies
Meanplusmnse
NMeanplusmnse
NShade
adapted
Drought
adapted
(N)
(minm
ean
max)
Npermass
Nmass
194
plusmn006
9192
plusmn007
9Low
erHigherA
BTem
peratebroadleaf
evergreen(129)
0581
19
231
Nperarea
Narea
238
plusmn007
9175
plusmn006
9Low
erHigherA
BTem
peratebroadleaf
evergreen(129)
0727
1733
63
Cpermass
Cmass
44
4plusmn048
943
6plusmn052
9Carbonto
nitrogen
ratio
CN
231plusmn068
922
9plusmn058
9Carbonisotoperatio
d13C
permilndash33
32plusmn029
9ndash33
05plusmn020
9Low
erAB
Higher
Tem
peratespecies(35)
ndash27
64
ndash26
03
ndash24
09
AAnexpectationaccordingto
ahypothesiswas
confi
rmed
forRa
culeatus
BAnexpectationaccordingto
ahy
pothesiswas
confi
rmed
forRm
icroglossum
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology G
K t frac14 pa3b3
64hetha2 thorn b2THORN eth3THORN
where a and b are the major and minor axes of the ellipse and his water viscosity at 25C
Gas-exchange measurements responses to light and CO2and cuticular conductance
In July and August 2009 photosynthetic light response curvesandCO2 response curvesweremeasuredusing aLi-6400portablephotosynthesis system (Li-Cor Biosciences ) with light provided
Ruscusaculeatus
Ruscusmicroglossum
01 mm
Fig 2 Lamina and midrib cross-sections of Ruscus aculeatus and R microglossum phylloclades (05mm thick) Note that bothspecies exhibit shade tolerance features such as absence of palisade tissue as well as drought tolerance features such as large waterstorage compartment and thick-walled epidermis andfibrousbundle sheath cells surrounding thexylemandphloemfor bothmajor andminor veins especially prominent in R aculeatus which is native to drier habitats
(a) (b)
Fig 1 (a)Ruscus aculeatus and (b)Rmicroglossumgrowing at theMildredEMathiasBotanicalGardenNote thatwhat atfirst glance appear to be leaves are infact phylloclades
H Functional Plant Biology A Pivovaroff et al
by a red-blue light source (6400ndash02B no SI-710 Li-CorBiosciences ) Gas-exchange measurements were made on atleast 1ndash2 phylloclades per individual
For light-response curves phylloclades were acclimatedfor at least 5min at 1500mmolmndash2 sndash1 PAR at temperatures of2527C with RH maintained at ~50 and CO2 concentrationof 400mmolmolndash1 Then phylloclades were measured for netCO2 assimilation per leaf area at PAR steps of 1600 1400 12001000 800 600 500 400 300 200 100 50 and 0mmolmndash2 sndash1with 180ndash240 s stabilisation at each irradiance step Wedetermined light-saturated photosynthetic rate per area andmass (Aarea and Amass) dark respiration rate per area and mass(Rarea and Rmass) ie the negative A at zero PAR maximumstomatal conductance per area (gs) light compensation point(LCP) as the x-intercept intrinsic water use efficiency (WUEAareags) and the ratio of intercellular to ambient CO2
concentration (CiCa) at 100mmolmndash2 sndash1 PAR We thenharvested the phylloclades measured for gas exchange todetermine the nitrogen concentration and carbon isotope ratio(see below)
For CO2-response curves phylloclades were allowed toequilibrate at 400 ppm to induce stomatal opening and the netCO2 assimilation per leaf area was determined at Ci steps of400 300 200 100 50 400 400 500 600 800 1200 14001600 ppm with 3ndash4min equilibration time at each step Wedetermined maximum rate of carboxylation and maximum rateof electron transport per leaf area (Vcmax and Jmax) from plots ofCi vs A corrected to 25C (Farquhar and von Caemmerer 1980)
Cuticular conductance (ie minimumepidermal conductancegmin sensu Kerstiens 1996) was determined for 3ndash4 maturephylloclades and 10 cm lengths of stems from each of threeindividuals of each species Phylloclade and stem sampleswere hydrated and then the cut ends were sealed with waxSamples were dried for at least 30min on a laboratory bench atPAR of lt10mmol photons mndash2 sndash1 to induce stomatal closurethen samples were weighed for at least eight intervals of 30minduring which the slope of water loss versus time was highlylinear (R2 gt 0995) and therefore taken to represent transpirationafter stomata had closed fully The gmin was calculated as thetranspiration rate divided by the mole fraction vapour pressuredeficit (VPD determined from a weather station HOBO MicroStation with Smart Sensors Onset Bourne MA USA)
Pressurendashvolume curve parametersPressurendashvolume curve parameters were determined for matureshoots using the bench-drying method (Koide et al 2000 Sacket al 2003a) Shoots were ~10ndash15 cm long with about fourphylloclades per shoot for R microglossum and 15ndash20phylloclades for R aculeatus Shoots were progressively driedon a laboratory bench and measured at intervals by equilibratingfor 10min in a Whirlpak bag (Whirl-Pak Nasco Fort AtkinsonWIUSA) beforeweighing andmeasuring for leafwater potential(Yleaf) with a pressure chamber (Model 1000 Plant MoistureStress Instruments Albany OR USA) Subsequently dry masswas determined after more than 48 h in an oven at 70C Wedetermined the leaf dry matter content (LDMC) saturated watercontent (SWC) turgor loss point (Ytlp) relative water content atturgor loss point (RWCtlp) and osmotic potential at full turgor
(po) relative capacitance at full turgor (Cft DRWCDYleaf) andrelative capacitance at turgor loss point (Ctlp DRWCDYleaf) Wedetermined the modulus of elasticity (e) as the linear slope of theline fitted for pressure potential versus relative water contentabove and including turgor loss point (Sack et al 2013b)
Shoot hydraulic conductance
We measured the hydraulic conductance of mature shoots(Kshoot) ~10ndash15 cm long bearing about four phylloclades pershoot for R microglossum and 15ndash20 phylloclades forR aculeatus using the evaporative flux method (Sack et al2002) Samples were harvested and the ends were re-cut underdistilled degassed water with a fresh razor blade then hydratedovernight Shoots were connected to tubing containing distilleddegassed water running to a graduated cylinder on a balanceinterfaced to a computer logging data every minute to calculatethe flow rate of water from the balance into the sample Sampleswere held in place above a fan on wood frames strung withfishing line Lights were arranged above a plexiglass containerof water that acted as a heat trap producing PAR ofgt1200mmolmndash2 sndash1 at shoot level When steady-statetranspiration was achieved samples were covered with aplastic bag and removed from the tubing and the leaf waterpotential was determined with a pressure chamber (Model 1000Plant Moisture Stress Instruments) Kshoot was calculated as thesteady-state transpirational flow rate (E mmolmndash2 sndash1) dividedby the water potential driving force (DYleaF= ndashYleaf MPa)further normalised by total phylloclade area Kshoot valueswere standardised to 25C to correct for the temperaturedependence of the viscosity of water (Sack et al 2003a)
Foliar nitrogen concentration and carbon isotope ratio
To analyse total tissue nitrogen concentration and carbon isotopecomposition (d13C) for three replicates from each of threeindividuals for each species phylloclade samples were oven-dried at gt70C for more than 48 h and ground by mortar andpestle The nitrogen concentration and d13C were determined atthe UCDavis Stable Isotope Facility using an elemental analyser(PDZEuropaANCA-GSL SerconLtd Cheshire UK) interfacedto an isotope ratiomass spectrometer (PDZEuropa20ndash20 SerconLtd) at the UC Davis Stable Isotope Facility Final d13C contentvalues are expressed relative to international standardVienna PeeDee belemnite (V-PDB) for carbon
d13CethpermilTHORN frac14 etheth13C=12C of sampleTHORN=eth13C=12C of standardTHORN 1THORN 1000
eth4THORN
d13C of plant tissues can provide a measure of intrinsic WUE(Ags) at the time that carbon was assimilated giving long-termwater-use efficiency
Comparative data compilation and trait comparison
To consider Ruscus trait values in a global context we compileddata from the literature for (1) temperate and tropical broadleafevergreen species (2) Mediterranean species and (3) woodyangiosperms in general When available we considered data forplants grown in the shade and for species that are shade tolerant
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology I
however eco-physiological trait data is generally collected forsun leaves or plants making a comparison solely for shade grownplants not possible Formore commonly studied traits (egAmax)comparative data was taken from studies with large databasesand multiple traits (eg GLOPNET Wright et al 2004) Wedetermined minimum mean and maximum values for traitsfrom comparative studies or the mean and standard error ifraw species values were not available Studies that wereincluded for comparative data are referenced in the captionsfor Table 1ndash5 in association with the specific traits wecompared Differences between Ruscus trait values andcomparative data from the literature were determined for bothR aculeatus and R microglossum Hypotheses were deemedsupported for a Ruscus species if the trait value was higher orlower than the mean comparator value in the way predicted
Results
Both Ruscus species studied had leaf morphology consistentwith adaptation to shade and drought relative to comparator
species (Tables 1ndash5)We constructed a radar graph to encapsulatethe key traits that would confer adaptation to shade drought andthe combination for R aculeatus which had the more extremeadaptation of the two Ruscus species considered (Fig 3) Thisfigure summarises the major results of our study with values forcomparator species appearing as the inner circle and R aculeatustrait values displayed as the bold outer line
Gross morphology of phylloclades
Both R aculeatus and R microglossum had LMA valueslower than comparator species and R microglossum hadphylloclades larger than the mean leaf area for comparatorspecies Both Ruscus species had lower bulk tissue density thancomparator species consistent with the water storage in theRuscus phylloclades Notably despite its water storage tissuethe LDMC and SWC values of R aculeatus were typical ofthose for comparator species whereas R microglossum had alower LDMC and a higher SWC than for comparator speciessets (Table 1)
Fig 3 Radar graph illustrating percent difference between selected traits of Ruscus aculeatuswithcomparative data (see Tables 1ndash5 for symbols and sources of comparative data) Values forR aculeatus outside the circle indicate shade andor drought tolerance Traits are arrangedaccording to whether they would contribute shade tolerance drought tolerance or both The innerdisc represents the mean for comparative species for given traits and the values for R aculeatus isscaled relative to the magnitude of that value () with a value outside the disc representing greatershade andor drought tolerance (+) and (ndash) indicate if the axis is scaled such that a value outside thecircle represents percent higher or lower than comparative values respectively For traits expressed asnegative values (+) and (ndash) indicate more negative or less negative respectively
J Functional Plant Biology A Pivovaroff et al
Anatomy of phylloclades
The Ruscus species possessed numerous anatomical traitsconsistent with benefits for both shade and drought tolerance(Table 2) The phylloclade cross-sections were symmetrical(Fig 2) and thus the cross-sectional anatomy of the adaxialand abaxial halves were not different for all traits (paired t-testP gt 005) and values were averaged within species The twospecies were similar in their substantial leaf thickness and inthe thickness of cuticle epidermis cell walls and water storagecompartment (Table 2) The fractions of the lamina occupied bythe epidermis mesophyll and water storage tissues were 1552ndash56 and 29ndash32 respectively
Both species had parallel longitudinal veins of three sizes(midrib intermediate and small veins) Consistent with droughtadaptation the two species had low Dm R aculeatus having thelower value andRmicroglossumhadahigher IVD (Table 2)Thetwo species had on average the same maximum conduitdiameters in their midribs intermediate veins and small veinsand the maximum conduit diameter decreased ~35 from themidrib to the small veins The average theoretical hydraulicconductivity of xylem conduits did not differ between thespecies and decreased by up to 86 from the midrib to theminor vein R aculeatus had on average walls that were 85thicker in the fibrous bundle sheath (Table 2)
Gas-exchange measurements
Consistent with adaptation to simultaneous shade and droughtthe Ruscus species had very low values for Rarea Rmass and gs(Table 3) relative to comparator temperate broadleaf evergreenspecies (Fig 3) The Aarea Amass LCP Vcmax and Jmax were alsovery low relative to comparator species (Fig 3 Table 3)consistent with shade adaptation The two Ruscus species hadvery high values for Ags (Table 3) consistent with excellentWUE Consistent with strong drought tolerance via retention ofstored water both species had very low values for leaf and stemgmin especially relative to comparator vascular plant species(Fig 3 Table 3)
Hydraulic conductance pressure volume curveparameters and leaf water storage
Consistent with expectations for combined drought and shadetolerance both Ruscus species had low Kshoot relative tocomparator tropical and temperate woody angiosperms (Fig 3Table 4) The pressurendashvolume curve parameters of Ruscuswereconsistent with achieving drought tolerance through tissue waterstorage Both species had less negative po and ytlp than meanvalues for comparative evergreen woody species (Fig 3Table 4) Notably both species had lower e values thancomparative evergreen woody species and higher RWCtlp andCft values (Fig 3 Table 4) consistent with drought tolerance
Nitrogen concentration and carbon isotope composition
Both species had high values for phyllyclade Narea and Nmass
relative to comparative temperate broadleaf evergreen speciesconsistent with drought adaptation (Fig 3) The d13C valueswerevery negative and typical of values often observed for understoryplants (da Silveira et al 1989) consistent with shade tolerance
Indeed the d13C values were notably strongly negative given thehigh WUE found for these species
Testing hypotheses for shade and drought tolerancewith trait survey data
Overall we quantified 57 traits for the twoRuscus species and for21 traits we had a priori hypotheses for a benefit for shadetolerance and for 24 traits we had a priori hypotheses for abenefit for drought tolerance Using comparative data we foundthat 16 of 21 hypotheses were supported for shade tolerance and22 of 24 were supported for drought tolerance Of the ninehypotheses for traits that would contribute to both shade anddrought tolerance simultaneously (ie expectations were both forhigher or lower values than comparative species) eight weresupported by trait data All these proportions were significantlyhigher than the 50 support that would have been expected toarise only from chance (P = 0001ndash0058 proportion tests)Notably in the seven cases when shade tolerance traits wouldconflict with drought tolerance traits four indicated a benefit fordrought tolerance rather than shade tolerance for both species(Tables 1ndash5)
Discussion
Both R aculeatus (Fig 3) and R microglossum showed traitvalues consistent with combined shade and drought toleranceNumerous traits were consistent with a combined shade anddrought tolerance through improving carbon balance enablinga conservative resource use ie via slow respiration and long-lived parts Other traits would contribute specifically to droughttolerance via reduced demand for water during activephotosynthesis and the ability to survive strong drought afterstomatal closure Notably many such traits were related to waterstorage providingnew insights into effective formsof succulencein shaded habitats This suite of traits would contributeimportantly to the ability of Ruscus to occupy shaded sitesprone to strong drought across a wide geographical range
Traits contributing to simultaneous drought and shadetolerance
Ruscus species showed specialisation associated withconservative resource use consistent with tolerance of shadeand drought These specialised traits included thick lamina andcomponent tissues that contribute to long tissue life-spans (shootslastgt5 years Sack et al 2003bWright et al 2004) AdditionallyRuscus species had very low gas-exchange rates including lowgs and lowKshoot whichwould correspond to a low investment invascular tissue (Tyree andZimmermann1983 Sack et al 2003b)and low Rarea Rmass Aarea and Amass all representing an ability tomaintain photosynthesis and growth with low requirements forlight and water
Traits contributing to drought tolerance
According to Jones et al (1992) drought tolerance can beachieved through avoidance of plant water deficits toleranceof plant water deficits or efficiency mechanisms Ruscus speciesshowed traits associated with drought tolerance either byproviding the ability to maintain photosynthesis and growth indrying soil andor the ability to survive chronic drought as
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology K
previously shown experimentally for R aculeatus (Sack 2004)Traits that would contribute to the ability to maintain gasexchange in drying soil include small leaf size high WUE andlow IVD High WUE achieved in part with high Narea meansthese Ruscus species can attain positive carbon balance evenwith extremely low gs Low IVD and water storage capacitanceallow the phylloclades to maintain water supply to the mesophylland tolerate transiently high evaporation rates for example dueto sunflecks without desiccating the leaf (Sack et al 2003a)Traits contributing to the ability of the phylloclades to surviveextended drought included those enabling a low evaporationrate per leaf area once stomata have shut such as low gmin inleaf and stem and those related to specialised water storagetissue linked with low leaf density low e and po values thatwere low in magnitude
Ruscus water storage
The water storage tissue of Ruscus that occupied a third of theleaf thickness although contributing most directly to droughttolerance is also consistent with shade tolerance given itscontribution to reduced tissue costs for the phylloclade as awhole The water storage tissue had thin cell walls reflected inthe low bulk e and low solute concentration contributing tobulk po values that were low in magnitude
This water storage tissue would also contribute to both typesof drought tolerance ndash the ability to maintain photosynthesis indrying soil and to survive after stomata have shut duringextended drought The lsquosucculencersquo of Ruscus phylloclades isdistinctive relative to more typical succulent-leafed andsucculent-stemmed species which tend to have high leaf watercontent and capacitance values (Vendramini et al 2002 Ogburnand Edwards 2012) In contrast in Ruscus species the SWCwas low relative to typical leaf succulent species and forR aculeatus Cft fell within the range of typical evergreenleaves We note that the strong tissue differentiation in Ruscus(ie separation of mesophyll cell and water storage in spaceand their distinctions in anatomy) would contribute to higheffectiveness of water storage even if the bulk tissue overallhad low SWC and capacitance Indeed across species theretends to be no relationship between the magnitude of SWC orcapacitance and the degree of within-leaf tissue differentiation(Ogburn and Edwards 2012) Notably such differentiationwould contribute special advantages for supply of waterwhether stomata are open or closed as the large thin-walledwater storage cells with low solute concentration can yield theirwater to supply the evaporative load while the photosynthetictissues can maintain their volume according to their thickerwalls and stronger solute concentration
Although the capacitance and SWC values were low forRuscus species these values would be more substantial ifconsidered relative to water demand Across several speciesCft has been found to correlate with Kshoot and with gs (Sacket al 2003a Blackman et al 2010) indicating that leaves tendto be built with capacitance to match their maximum flux ratesand thus to buffer the leaf water potential against surges intranspiration Thus because Ruscus has low gs when stomataare open and low Kshoot the capacitance would be expectedto supply transpiration transiently during sunflecks or high
VPD Likewise when stomata close the capacitance suppliesongoing water loss via cuticular conductance Given theextremely low gmin of Ruscus even its moderate Ctlp canenable survival for weeks (Sack et al 2003b Sack 2004)Further at turgor loss the water content would equalSWCRWCtlp and the relatively high RWCtlp wouldcontribute to the high water content once turgor is lost Thusthe lsquosucculencersquo of Ruscus is moderate in absolute terms butcombined with its other very strong mechanisms to reducetranspiration when stomata are open or closed ie low gs andgmin even this moderate capacitance would provide strongfunctionality
Water-use efficiency and carbon isotope composition
It was noteworthy that despite extremely high WUE valuesphylloclade d13C values of the Ruscus species were verynegative This presents a strong anomaly worthy of furtherinvestigation as species with high WUE typically have higherd13C (less negative ie closer to zero) The d13C can beinfluenced by a host of processes including source CO2 storedplant carbon and time-integrated CO2 concentration at the siteof carboxylation (Farquhar et al 1989) For Ruscus althoughd13C values were typical of an understory plant they did notappear to be drivenby internalCO2 concentration because the leafisotopic values were depleted in 13C whereas the high WUEdetermined by gas exchange would likely promote enrichedisotopic values It is more likely that d13C in this species wasdetermined by source CO2 or stored carbon or recycling ofrespired CO2 (da Silveira et al 1989) Our study individualsof Ruscus were cultivated in a shaded understory similar totheir natural habitat Previous studies have shown that there canbe higher concentrations of respired CO2 in the forest understorythan in the canopy resulting in more negative carbon isotoperatios in understory plant tissue than canopy plant tissue (daSilveira et al 1989) This is a function of decomposing leavesand litter cover slow air mixing as well as plant environmentalresponses
A second possible explanation for the d13C values ofRuscus is related to its growth form and phenology asRuscus has extensive rhizomes (Sack et al 2003b) whichact as a carbon store for the plant Using this recycled carbonduring the growth season when the plant may not be able tomeet its carbon requirement by photosynthesis alone becauseof low maximum rates under light limitation may alsocontribute to more negative d13C values (Vizzini 2003)Notably Ruscus stems are hollow and thus can also storerelatively large amounts of CO2 for use during a growth periodwhen carbon is otherwise limiting especially given very lowgs Some of this stored carbon might be photosyntheticallyfixed by the stem (Nilsen and Sharifi 1997) In each of thesecases respired stored or recycled CO2 would supply carbonthat was previously fixed by Rubisco with more negative d13Cvalues (ndash27o) than air (ndash8o)
There was also a dissonance between the d13C value and CiCa The fully mature phylloclades which were selected andused for gas-exchange and d13C measurements were producedduring late winter and early spring with mild temperatures(average at midday 190ndash220C) and low atmospheric VPD
L Functional Plant Biology A Pivovaroff et al
(average at midday 06ndash15 kPa) However the gas-exchangemeasurements were taken during summer with hightemperature (average at midday 300ndash355C) and high VPD(average at midday 25ndash35 kPa) We harvested thephylloclades that we used for gas exchange to determine thecarbon isotope ratio Although the low value of instantaneousCiCa may be caused by high temperature and high atmosphericVPD the very low value of d13C may represent the time whenthe phyllocladersquos carbon was assimilated (during mildtemperature and low VPD)
Implications for drought and shade tolerance Ruscusas a model
We found strong support for a large number of hypotheses fortrait-based shade and drought tolerance providing a strong traitbasis for combined tolerance Although the detailed functionaltrait survey conducted here is relatively novel in its breadth(see also Pasquet-Kok et al 2010) this is a logical extensionof the traditional approach for understanding the basis forplant adaptation to environment ie testing expectations forindividual traits established by previous studies of thefunctional significance of these traits in other species Weacknowledge there is some degree of uncertainty ininterpreting a large number of traits simultaneously based onstudies of other species First the interpretation of the value oftraits based on other species may not be in all cases equally validfor Ruscus Some trait variation may relate to other functionsFurther studies for example using mutants would be necessaryas conclusive evidence of the value of specific traits in a givenspecies However one advantage of testing numerousexpectations for each hypothesis is that the key finding will berobust to the removal of some traits from the analysis if thoseare later found to be inappropriate Ideally when a model forestimating plant performance from leaf traits becomes availableone could determine how the specific quantitative combinationsof traits presented here scales up to plant shade and droughttolerance
The shade and drought tolerance of Ruscus consistent withthe suite of traits examined here is one case demonstratinghow plants can avoid a general trade-off between shade anddrought tolerance Further Ruscus is noteworthy as one of onlya few stem photosynthetic plants that occupy a shaded habitatHistorically it is likely that shade tolerance preceded droughttolerance given this speciesrsquo ancestors were species of moisttropical forests (Kim et al 2010) and thus Ruscus or itsancestor apparently evolved drought tolerance whileexpanding its range into drier habitat or during past climatechange Considering its unique adaptations and trait valuesRuscus can serve as an excellent model for the basis ofcombined shade and drought tolerance
Acknowledgements
We thank the Mildred E Mathias Botanical Garden for access to garden andplants Andy Truong for assistance with measurements Howard GriffithsLouis Santiago and Cheryl Swift for helpful discussions and comments onthe manuscript This work was supported by National Science FoundationGrant IOB-0546784
References
Bartlett MK Scoffoni C Sack L (2012) The determinants of leaf turgor losspoint and prediction of drought tolerance of species and biomes a globalmeta-analysis Ecology Letters 15 393ndash405 doi101111j1461-0248201201751x
Blackman CJ Brodribb TJ Jordan GJ (2010) Leaf hydraulic vulnerability isrelated to conduit dimensions and drought resistance across a diverserange of woody angiosperms New Phytologist 188 1113ndash1123doi101111j1469-8137201003439x
Brodribb TJ Feild TS Jordan GJ (2007) Leaf maximum photosynthetic rateand venation are linked by hydraulicsPlant Physiology 144 1890ndash1898doi101104pp107101352
Caspersen JP (2001) Interspecific variation in sapling mortality in relationto growth and soil moisture Oikos 92 160ndash168 doi101034j1600-07062001920119x
Cochard H Nardini A Coll L (2004) Hydraulic architecture of leaf bladeswhere is the main resistance Plant Cell amp Environment 27 1257ndash1267doi101111j1365-3040200401233x
Cooney-Sovetts C Sattler R (1987) Phylloclade development in theAsparagaceae an example of homoeosis Botanical Journal of theLinnean Society 94 327ndash371 doi101111j1095-83391986tb01053x
Cowling R Rundel P Lamont BB ArroyoMK ArianoutsouM (1996) Plantdiversity in Mediterranean-climate regions Trends in Ecology ampEvolution 11 362ndash366 doi1010160169-5347(96)10044-6
da Silveira L Sternberg L Mulkey SS Wright SJ (1989) Ecologicalinterpretation of leaf carbon isotope ratios influence of respired carbondioxide Ecology 70 1317ndash1324 doi1023071938191
de Lillis M Fontanella A (1992) Comparative phenology and growth indifferent species of the Mediterranean maquis of central Italy PlantEcology 99-100 83ndash96 doi101007BF00118213
Dracup JA (1991)DroughtmonitoringStochasticHydrologyandHydraulics5 261ndash266 doi101007BF01543134
Engelbrecht BMJ Kursar TA (2003) Comparative drought-resistance ofseedlings of 28 species of co-occurring tropical woody plantsOecologia 136 383ndash393 doi101007s00442-003-1290-8
Farquhar GD von Caemmerer S (1980) A biochemical model ofphotosynthetic CO2 assimilation in leaves of C3 species Planta 14978ndash90 doi101007BF00386231
FarquharGD Ehleringer JR HubickK (1989) Carbon isotope discriminationand photosynthesis Annual Review of Plant Physiology and PlantMolecular Biology 40 503ndash537 doi101146annurevpp40060189002443
Givnish TJ (1988) Adaptation to sun and shade a whole-plant perspectiveAustralian Journal of Plant Physiology 15 63ndash92 doi101071PP9880063
Gulias J Flexas J Mus M Cifre J Lefi E Medrano H (2003) Relationshipbetweenmaximum leaf photosynthesis nitrogen content and specific leafarea inBalearic endemic and non-endemicMediterranean speciesAnnalsof Botany 92 215ndash222 doi101093aobmcg123
Haberlandt G (1914) lsquoPhysiological plant anatomyrsquo (MacMillan London)Hallik L Niinemets U Wright J (2009) Are species shade and drought
tolerance reflected in leaf-level structural and functional differentiationin Northern Hemisphere temperate woody flora New Phytologist 184257ndash274 doi101111j1469-8137200902918x
Jones HG (1992) lsquoPlants and microclimate a quantitative approach toenvironmental plant physiologyrsquo (Cambridge University PressCambridge)
Kerstiens G (1996) Cuticular water permeability and its physiologicalsignificance Journal of Experimental Botany 47 1813ndash1832doi101093jxb47121813
Kim JH Kim DK Forest F Fay MF Chase MW (2010) Molecularphylogenetics of Ruscaceae sensu lato and related families(Asparagales) based on plastid and nuclear DNA sequences Annals ofBotany 106 775ndash790 doi101093aobmcq167
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology M
Koide RT Robichaux RH Morse SR Smith CM (2000) Plant water statushydraulic resistance and capacitance In lsquoPlant physiological ecologyfield methods and instrumentationrsquo (Eds R Pearcy JR Ehleringer HMooney P Rundel) pp 161ndash183 (Kluwer Dordrecht The Netherlands)
Koumlrner C Farquhar GD Wong SC (1991) Carbon isotope discrimination byplants follows latitudinal and altitudinal trends Oecologia 88 30ndash40doi101007BF00328400
Lewis AM Boose ER (1995) Estimating volume flow rates through xylemconduits American Journal of Botany 82 1112ndash1116 doi1023072446063
Martiacutenez-Palleacute E Aronne G (1999) Flower development and reproductivecontinuity inMediterraneanRuscusaculeatusL (Liliaceae)Protoplasma208 58ndash64 doi101007BF01279075
Martiacutenez-Tilleriacutea K Loayza AP Sandquist DR Squeo FA (2012) Noevidence of a trade-off between drought and shade tolerance inseedlings of six coastal desert shrub species in north-central ChileJournal of Vegetation Science 23 1051ndash1061 doi101111j1654-1103201201427x
Matalas NC (1963) Probability distribution of low flows USGS ProfessionalPapers 434-A US Government Printing Office Washington DC
Niinemets U (1999) Research review components of leaf dry mass per area ndashthickness and density ndash alter leaf photosynthetic capacity in reversedirections in woody plants New Phytologist 144 35ndash47 doi101046j1469-8137199900466x
Niinemets U Valladares F (2006) Tolerance to shade drought andwaterlogging of temperate northern hemisphere trees and shrubsEcological Monographs 76 521ndash547 doi1018900012-9615(2006)076[0521TTSDAW]20CO2
Nilsen ET Sharifi MR (1997) Carbon isotope composition of legumes withphotosynthetic stems from Mediterranean and desert habitats AmericanJournal of Botany 84 1707ndash1713 doi1023072446469
Ogburn RM Edwards EJ (2012) Quantifying succulence a rapidphysiologically meaningful metric of plant water storage Plant Cell ampEnvironment 35 1533ndash1542 doi101111j1365-3040201202503x
Pasquet-Kok J Creese C Sack L (2010) Turning over a new lsquoleafrsquo multiplefunctional significances of leaves versus phyllodes in Hawaiian Acaciakoa Plant Cell amp Environment 33 2084ndash2100 doi101111j1365-3040201002207x
SackL (2004)Responses of temperatewoody seedlings to shade and droughtdo trade-offs limit potential niche differentiation Oikos 107 110ndash127doi101111j0030-1299200413184x
SackLFroleK (2006)Leaf structural diversity is related to hydraulic capacityin tropical rain forest trees Ecology 87 483ndash491 doi10189005-0710
Sack L Holbrook NM (2006) Leaf hydraulics Annual Review of PlantBiology 57 361ndash381 doi101146annurevarplant56032604144141
Sack L Melcher PJ Zwieniecki MA Holbrook NM (2002) The hydraulicconductance of the angiosperm leaf lamina a comparison of threemeasurement methods Journal of Experimental Botany 532177ndash2184 doi101093jxberf069
Sack L Cowan P Jaikumar N Holbrook NM (2003a) The ldquohydrologyrdquo ofleaves co-ordination of structure and function in temperate woodyspecies Plant Cell amp Environment 26 1343ndash1356 doi101046j0016-8025200301058x
Sack L Grubb P Maronon T (2003b) The functional morphology of juvenileplants tolerant of strong summer drought in shaded forest understoriesin southern Spain Plant Ecology 168 139ndash163 doi101023A1024423820136
SackLScoffoniCMcKownADFroleKRawlsMHavran JCTranHTranT (2012) Developmentally based scaling of leaf venation architectureexplains global ecological patterns Nature Communications 3 837doi101038ncomms1835
Sack L Chatelet D Scoffoni C PrometheusWiki contributors (2013a)ldquoEstimating the mesophyll surface area per leaf area from leaf celland tissue dimensions measured from transverse cross-sectionsrdquoPrometheusWiki Available at httpwwwpublishcsiroauprometheuswikitiki-pagehistoryphppage=Estimating the mesophyllsurface area per leaf area from leaf cell and tissue dimensionsmeasured from transverse cross-sectionsamppreview=16 [accessed June18 2013]
SackL Pasquet-Kok JPrometheusWiki contributors (2013b) ldquoLeaf pressure-volume curve parametersrdquo PrometheusWiki Available at httpprometheuswikipublishcsiroautiki-indexphppage=Leaf+pressure-volume+curve+parameters
Scoffoni C Pou A Aasamaa K Sack L (2008) The rapid light response ofleaf hydraulic conductance new evidence from two experimentalmethods Plant Cell amp Environment 31 1803ndash1812 doi101111j1365-3040200801884x
Smith T Huston M (1989) A theory of the spatial and temporal dynamics ofplant communities Vegetatio 83 49ndash69 doi101007BF00031680
Sterck F Markesteijn L Schieving F Poorter L (2011) Functional traitsdetermine trade-offs and niches in a tropical forest communityProceedings of the National Academy of Sciences of the United Statesof America 108 20 627ndash20 632 doi101073pnas1106950108
Thomas GS (1992) lsquoOrnamental shrubs climbers and bamboosrsquo (JohnMurray Publishers Great Britain)
TyreeMT ZimmermannMH (1983) lsquoXylem structure and the ascent of saprsquo(Springer-Verlag Berlin)
USDA ARS National Genetic Resources Program (2009) GermplasmResources Information Network (GRIN) online database NationalGermplasm Resources Laboratory Beltsville Maryland Availableathttpwwwars-gringovcgi-binnpgshtmltaxonpl410577[Accessed 20 July 2009]
Vendramini F Diacuteaz S Gurvich DE Wilson PJ Thompson K Hodgson JG(2002) Leaf traits as indicators of resource-use strategy in floras withsucculent species New Phytologist 154 147ndash157 doi101046j1469-8137200200357x
Vile D (2005) Specific leaf area and dry matter content estimate thicknessin laminar leaves Annals of Botany 96 1129ndash1136 doi101093aobmci264
Vizzini S (2003) d13C and d15N variability in Posidonia oceanica associatedwith seasonality and plant fraction Aquatic Botany 76 195ndash202doi101016S0304-3770(03)00052-4
Walters M Reich PB (1999) Research review low-light carbon balance andshade tolerance in the seedlings of woody plants dowinter deciduous andbroad-leaved evergreen species differ New Phytologist 143 143ndash154doi101046j1469-8137199900425x
Witkowski E Lamont BB (1991) Leaf specific mass confounds leaf densityand thickness Oecologia 88 486ndash493
Wright IJWestobyM (2003)Nutrient concentration resorption and lifespanleaf traits of Australian sclerophyll species Functional Ecology 1710ndash19 doi101046j1365-2435200300694x
Wright IJ Reich PB Westoby M Ackerly DD Baruch Z Bongers FCavender-Bares J Chapin T Cornelissen JHC Diemer M Flexas JGarnier E Groom PK Gulias J Hikosaka K Lamont BB Lee T LeeWLusk C Midgley JJ Navas M-L Niinemets U Oleksyn J Osada NPoorter H Poot P Prior L Pyankov VI Roumet C Thomas SC TjoelkerMG Veneklaas EJ Villar R (2004) The worldwide leaf economicsspectrum Nature 428 821ndash827 doi101038nature02403
Wullschleger SD (1993) Biochemical limitations to carbon assimilation inC3 plants ndash a retrospective analysis of the ACi curves from 109 speciesJournal of Experimental Botany 44 907ndash920 doi101093jxb445907
N Functional Plant Biology A Pivovaroff et al
wwwpublishcsiroaujournalsfpb
K t frac14 pa3b3
64hetha2 thorn b2THORN eth3THORN
where a and b are the major and minor axes of the ellipse and his water viscosity at 25C
Gas-exchange measurements responses to light and CO2and cuticular conductance
In July and August 2009 photosynthetic light response curvesandCO2 response curvesweremeasuredusing aLi-6400portablephotosynthesis system (Li-Cor Biosciences ) with light provided
Ruscusaculeatus
Ruscusmicroglossum
01 mm
Fig 2 Lamina and midrib cross-sections of Ruscus aculeatus and R microglossum phylloclades (05mm thick) Note that bothspecies exhibit shade tolerance features such as absence of palisade tissue as well as drought tolerance features such as large waterstorage compartment and thick-walled epidermis andfibrousbundle sheath cells surrounding thexylemandphloemfor bothmajor andminor veins especially prominent in R aculeatus which is native to drier habitats
(a) (b)
Fig 1 (a)Ruscus aculeatus and (b)Rmicroglossumgrowing at theMildredEMathiasBotanicalGardenNote thatwhat atfirst glance appear to be leaves are infact phylloclades
H Functional Plant Biology A Pivovaroff et al
by a red-blue light source (6400ndash02B no SI-710 Li-CorBiosciences ) Gas-exchange measurements were made on atleast 1ndash2 phylloclades per individual
For light-response curves phylloclades were acclimatedfor at least 5min at 1500mmolmndash2 sndash1 PAR at temperatures of2527C with RH maintained at ~50 and CO2 concentrationof 400mmolmolndash1 Then phylloclades were measured for netCO2 assimilation per leaf area at PAR steps of 1600 1400 12001000 800 600 500 400 300 200 100 50 and 0mmolmndash2 sndash1with 180ndash240 s stabilisation at each irradiance step Wedetermined light-saturated photosynthetic rate per area andmass (Aarea and Amass) dark respiration rate per area and mass(Rarea and Rmass) ie the negative A at zero PAR maximumstomatal conductance per area (gs) light compensation point(LCP) as the x-intercept intrinsic water use efficiency (WUEAareags) and the ratio of intercellular to ambient CO2
concentration (CiCa) at 100mmolmndash2 sndash1 PAR We thenharvested the phylloclades measured for gas exchange todetermine the nitrogen concentration and carbon isotope ratio(see below)
For CO2-response curves phylloclades were allowed toequilibrate at 400 ppm to induce stomatal opening and the netCO2 assimilation per leaf area was determined at Ci steps of400 300 200 100 50 400 400 500 600 800 1200 14001600 ppm with 3ndash4min equilibration time at each step Wedetermined maximum rate of carboxylation and maximum rateof electron transport per leaf area (Vcmax and Jmax) from plots ofCi vs A corrected to 25C (Farquhar and von Caemmerer 1980)
Cuticular conductance (ie minimumepidermal conductancegmin sensu Kerstiens 1996) was determined for 3ndash4 maturephylloclades and 10 cm lengths of stems from each of threeindividuals of each species Phylloclade and stem sampleswere hydrated and then the cut ends were sealed with waxSamples were dried for at least 30min on a laboratory bench atPAR of lt10mmol photons mndash2 sndash1 to induce stomatal closurethen samples were weighed for at least eight intervals of 30minduring which the slope of water loss versus time was highlylinear (R2 gt 0995) and therefore taken to represent transpirationafter stomata had closed fully The gmin was calculated as thetranspiration rate divided by the mole fraction vapour pressuredeficit (VPD determined from a weather station HOBO MicroStation with Smart Sensors Onset Bourne MA USA)
Pressurendashvolume curve parametersPressurendashvolume curve parameters were determined for matureshoots using the bench-drying method (Koide et al 2000 Sacket al 2003a) Shoots were ~10ndash15 cm long with about fourphylloclades per shoot for R microglossum and 15ndash20phylloclades for R aculeatus Shoots were progressively driedon a laboratory bench and measured at intervals by equilibratingfor 10min in a Whirlpak bag (Whirl-Pak Nasco Fort AtkinsonWIUSA) beforeweighing andmeasuring for leafwater potential(Yleaf) with a pressure chamber (Model 1000 Plant MoistureStress Instruments Albany OR USA) Subsequently dry masswas determined after more than 48 h in an oven at 70C Wedetermined the leaf dry matter content (LDMC) saturated watercontent (SWC) turgor loss point (Ytlp) relative water content atturgor loss point (RWCtlp) and osmotic potential at full turgor
(po) relative capacitance at full turgor (Cft DRWCDYleaf) andrelative capacitance at turgor loss point (Ctlp DRWCDYleaf) Wedetermined the modulus of elasticity (e) as the linear slope of theline fitted for pressure potential versus relative water contentabove and including turgor loss point (Sack et al 2013b)
Shoot hydraulic conductance
We measured the hydraulic conductance of mature shoots(Kshoot) ~10ndash15 cm long bearing about four phylloclades pershoot for R microglossum and 15ndash20 phylloclades forR aculeatus using the evaporative flux method (Sack et al2002) Samples were harvested and the ends were re-cut underdistilled degassed water with a fresh razor blade then hydratedovernight Shoots were connected to tubing containing distilleddegassed water running to a graduated cylinder on a balanceinterfaced to a computer logging data every minute to calculatethe flow rate of water from the balance into the sample Sampleswere held in place above a fan on wood frames strung withfishing line Lights were arranged above a plexiglass containerof water that acted as a heat trap producing PAR ofgt1200mmolmndash2 sndash1 at shoot level When steady-statetranspiration was achieved samples were covered with aplastic bag and removed from the tubing and the leaf waterpotential was determined with a pressure chamber (Model 1000Plant Moisture Stress Instruments) Kshoot was calculated as thesteady-state transpirational flow rate (E mmolmndash2 sndash1) dividedby the water potential driving force (DYleaF= ndashYleaf MPa)further normalised by total phylloclade area Kshoot valueswere standardised to 25C to correct for the temperaturedependence of the viscosity of water (Sack et al 2003a)
Foliar nitrogen concentration and carbon isotope ratio
To analyse total tissue nitrogen concentration and carbon isotopecomposition (d13C) for three replicates from each of threeindividuals for each species phylloclade samples were oven-dried at gt70C for more than 48 h and ground by mortar andpestle The nitrogen concentration and d13C were determined atthe UCDavis Stable Isotope Facility using an elemental analyser(PDZEuropaANCA-GSL SerconLtd Cheshire UK) interfacedto an isotope ratiomass spectrometer (PDZEuropa20ndash20 SerconLtd) at the UC Davis Stable Isotope Facility Final d13C contentvalues are expressed relative to international standardVienna PeeDee belemnite (V-PDB) for carbon
d13CethpermilTHORN frac14 etheth13C=12C of sampleTHORN=eth13C=12C of standardTHORN 1THORN 1000
eth4THORN
d13C of plant tissues can provide a measure of intrinsic WUE(Ags) at the time that carbon was assimilated giving long-termwater-use efficiency
Comparative data compilation and trait comparison
To consider Ruscus trait values in a global context we compileddata from the literature for (1) temperate and tropical broadleafevergreen species (2) Mediterranean species and (3) woodyangiosperms in general When available we considered data forplants grown in the shade and for species that are shade tolerant
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology I
however eco-physiological trait data is generally collected forsun leaves or plants making a comparison solely for shade grownplants not possible Formore commonly studied traits (egAmax)comparative data was taken from studies with large databasesand multiple traits (eg GLOPNET Wright et al 2004) Wedetermined minimum mean and maximum values for traitsfrom comparative studies or the mean and standard error ifraw species values were not available Studies that wereincluded for comparative data are referenced in the captionsfor Table 1ndash5 in association with the specific traits wecompared Differences between Ruscus trait values andcomparative data from the literature were determined for bothR aculeatus and R microglossum Hypotheses were deemedsupported for a Ruscus species if the trait value was higher orlower than the mean comparator value in the way predicted
Results
Both Ruscus species studied had leaf morphology consistentwith adaptation to shade and drought relative to comparator
species (Tables 1ndash5)We constructed a radar graph to encapsulatethe key traits that would confer adaptation to shade drought andthe combination for R aculeatus which had the more extremeadaptation of the two Ruscus species considered (Fig 3) Thisfigure summarises the major results of our study with values forcomparator species appearing as the inner circle and R aculeatustrait values displayed as the bold outer line
Gross morphology of phylloclades
Both R aculeatus and R microglossum had LMA valueslower than comparator species and R microglossum hadphylloclades larger than the mean leaf area for comparatorspecies Both Ruscus species had lower bulk tissue density thancomparator species consistent with the water storage in theRuscus phylloclades Notably despite its water storage tissuethe LDMC and SWC values of R aculeatus were typical ofthose for comparator species whereas R microglossum had alower LDMC and a higher SWC than for comparator speciessets (Table 1)
Fig 3 Radar graph illustrating percent difference between selected traits of Ruscus aculeatuswithcomparative data (see Tables 1ndash5 for symbols and sources of comparative data) Values forR aculeatus outside the circle indicate shade andor drought tolerance Traits are arrangedaccording to whether they would contribute shade tolerance drought tolerance or both The innerdisc represents the mean for comparative species for given traits and the values for R aculeatus isscaled relative to the magnitude of that value () with a value outside the disc representing greatershade andor drought tolerance (+) and (ndash) indicate if the axis is scaled such that a value outside thecircle represents percent higher or lower than comparative values respectively For traits expressed asnegative values (+) and (ndash) indicate more negative or less negative respectively
J Functional Plant Biology A Pivovaroff et al
Anatomy of phylloclades
The Ruscus species possessed numerous anatomical traitsconsistent with benefits for both shade and drought tolerance(Table 2) The phylloclade cross-sections were symmetrical(Fig 2) and thus the cross-sectional anatomy of the adaxialand abaxial halves were not different for all traits (paired t-testP gt 005) and values were averaged within species The twospecies were similar in their substantial leaf thickness and inthe thickness of cuticle epidermis cell walls and water storagecompartment (Table 2) The fractions of the lamina occupied bythe epidermis mesophyll and water storage tissues were 1552ndash56 and 29ndash32 respectively
Both species had parallel longitudinal veins of three sizes(midrib intermediate and small veins) Consistent with droughtadaptation the two species had low Dm R aculeatus having thelower value andRmicroglossumhadahigher IVD (Table 2)Thetwo species had on average the same maximum conduitdiameters in their midribs intermediate veins and small veinsand the maximum conduit diameter decreased ~35 from themidrib to the small veins The average theoretical hydraulicconductivity of xylem conduits did not differ between thespecies and decreased by up to 86 from the midrib to theminor vein R aculeatus had on average walls that were 85thicker in the fibrous bundle sheath (Table 2)
Gas-exchange measurements
Consistent with adaptation to simultaneous shade and droughtthe Ruscus species had very low values for Rarea Rmass and gs(Table 3) relative to comparator temperate broadleaf evergreenspecies (Fig 3) The Aarea Amass LCP Vcmax and Jmax were alsovery low relative to comparator species (Fig 3 Table 3)consistent with shade adaptation The two Ruscus species hadvery high values for Ags (Table 3) consistent with excellentWUE Consistent with strong drought tolerance via retention ofstored water both species had very low values for leaf and stemgmin especially relative to comparator vascular plant species(Fig 3 Table 3)
Hydraulic conductance pressure volume curveparameters and leaf water storage
Consistent with expectations for combined drought and shadetolerance both Ruscus species had low Kshoot relative tocomparator tropical and temperate woody angiosperms (Fig 3Table 4) The pressurendashvolume curve parameters of Ruscuswereconsistent with achieving drought tolerance through tissue waterstorage Both species had less negative po and ytlp than meanvalues for comparative evergreen woody species (Fig 3Table 4) Notably both species had lower e values thancomparative evergreen woody species and higher RWCtlp andCft values (Fig 3 Table 4) consistent with drought tolerance
Nitrogen concentration and carbon isotope composition
Both species had high values for phyllyclade Narea and Nmass
relative to comparative temperate broadleaf evergreen speciesconsistent with drought adaptation (Fig 3) The d13C valueswerevery negative and typical of values often observed for understoryplants (da Silveira et al 1989) consistent with shade tolerance
Indeed the d13C values were notably strongly negative given thehigh WUE found for these species
Testing hypotheses for shade and drought tolerancewith trait survey data
Overall we quantified 57 traits for the twoRuscus species and for21 traits we had a priori hypotheses for a benefit for shadetolerance and for 24 traits we had a priori hypotheses for abenefit for drought tolerance Using comparative data we foundthat 16 of 21 hypotheses were supported for shade tolerance and22 of 24 were supported for drought tolerance Of the ninehypotheses for traits that would contribute to both shade anddrought tolerance simultaneously (ie expectations were both forhigher or lower values than comparative species) eight weresupported by trait data All these proportions were significantlyhigher than the 50 support that would have been expected toarise only from chance (P = 0001ndash0058 proportion tests)Notably in the seven cases when shade tolerance traits wouldconflict with drought tolerance traits four indicated a benefit fordrought tolerance rather than shade tolerance for both species(Tables 1ndash5)
Discussion
Both R aculeatus (Fig 3) and R microglossum showed traitvalues consistent with combined shade and drought toleranceNumerous traits were consistent with a combined shade anddrought tolerance through improving carbon balance enablinga conservative resource use ie via slow respiration and long-lived parts Other traits would contribute specifically to droughttolerance via reduced demand for water during activephotosynthesis and the ability to survive strong drought afterstomatal closure Notably many such traits were related to waterstorage providingnew insights into effective formsof succulencein shaded habitats This suite of traits would contributeimportantly to the ability of Ruscus to occupy shaded sitesprone to strong drought across a wide geographical range
Traits contributing to simultaneous drought and shadetolerance
Ruscus species showed specialisation associated withconservative resource use consistent with tolerance of shadeand drought These specialised traits included thick lamina andcomponent tissues that contribute to long tissue life-spans (shootslastgt5 years Sack et al 2003bWright et al 2004) AdditionallyRuscus species had very low gas-exchange rates including lowgs and lowKshoot whichwould correspond to a low investment invascular tissue (Tyree andZimmermann1983 Sack et al 2003b)and low Rarea Rmass Aarea and Amass all representing an ability tomaintain photosynthesis and growth with low requirements forlight and water
Traits contributing to drought tolerance
According to Jones et al (1992) drought tolerance can beachieved through avoidance of plant water deficits toleranceof plant water deficits or efficiency mechanisms Ruscus speciesshowed traits associated with drought tolerance either byproviding the ability to maintain photosynthesis and growth indrying soil andor the ability to survive chronic drought as
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology K
previously shown experimentally for R aculeatus (Sack 2004)Traits that would contribute to the ability to maintain gasexchange in drying soil include small leaf size high WUE andlow IVD High WUE achieved in part with high Narea meansthese Ruscus species can attain positive carbon balance evenwith extremely low gs Low IVD and water storage capacitanceallow the phylloclades to maintain water supply to the mesophylland tolerate transiently high evaporation rates for example dueto sunflecks without desiccating the leaf (Sack et al 2003a)Traits contributing to the ability of the phylloclades to surviveextended drought included those enabling a low evaporationrate per leaf area once stomata have shut such as low gmin inleaf and stem and those related to specialised water storagetissue linked with low leaf density low e and po values thatwere low in magnitude
Ruscus water storage
The water storage tissue of Ruscus that occupied a third of theleaf thickness although contributing most directly to droughttolerance is also consistent with shade tolerance given itscontribution to reduced tissue costs for the phylloclade as awhole The water storage tissue had thin cell walls reflected inthe low bulk e and low solute concentration contributing tobulk po values that were low in magnitude
This water storage tissue would also contribute to both typesof drought tolerance ndash the ability to maintain photosynthesis indrying soil and to survive after stomata have shut duringextended drought The lsquosucculencersquo of Ruscus phylloclades isdistinctive relative to more typical succulent-leafed andsucculent-stemmed species which tend to have high leaf watercontent and capacitance values (Vendramini et al 2002 Ogburnand Edwards 2012) In contrast in Ruscus species the SWCwas low relative to typical leaf succulent species and forR aculeatus Cft fell within the range of typical evergreenleaves We note that the strong tissue differentiation in Ruscus(ie separation of mesophyll cell and water storage in spaceand their distinctions in anatomy) would contribute to higheffectiveness of water storage even if the bulk tissue overallhad low SWC and capacitance Indeed across species theretends to be no relationship between the magnitude of SWC orcapacitance and the degree of within-leaf tissue differentiation(Ogburn and Edwards 2012) Notably such differentiationwould contribute special advantages for supply of waterwhether stomata are open or closed as the large thin-walledwater storage cells with low solute concentration can yield theirwater to supply the evaporative load while the photosynthetictissues can maintain their volume according to their thickerwalls and stronger solute concentration
Although the capacitance and SWC values were low forRuscus species these values would be more substantial ifconsidered relative to water demand Across several speciesCft has been found to correlate with Kshoot and with gs (Sacket al 2003a Blackman et al 2010) indicating that leaves tendto be built with capacitance to match their maximum flux ratesand thus to buffer the leaf water potential against surges intranspiration Thus because Ruscus has low gs when stomataare open and low Kshoot the capacitance would be expectedto supply transpiration transiently during sunflecks or high
VPD Likewise when stomata close the capacitance suppliesongoing water loss via cuticular conductance Given theextremely low gmin of Ruscus even its moderate Ctlp canenable survival for weeks (Sack et al 2003b Sack 2004)Further at turgor loss the water content would equalSWCRWCtlp and the relatively high RWCtlp wouldcontribute to the high water content once turgor is lost Thusthe lsquosucculencersquo of Ruscus is moderate in absolute terms butcombined with its other very strong mechanisms to reducetranspiration when stomata are open or closed ie low gs andgmin even this moderate capacitance would provide strongfunctionality
Water-use efficiency and carbon isotope composition
It was noteworthy that despite extremely high WUE valuesphylloclade d13C values of the Ruscus species were verynegative This presents a strong anomaly worthy of furtherinvestigation as species with high WUE typically have higherd13C (less negative ie closer to zero) The d13C can beinfluenced by a host of processes including source CO2 storedplant carbon and time-integrated CO2 concentration at the siteof carboxylation (Farquhar et al 1989) For Ruscus althoughd13C values were typical of an understory plant they did notappear to be drivenby internalCO2 concentration because the leafisotopic values were depleted in 13C whereas the high WUEdetermined by gas exchange would likely promote enrichedisotopic values It is more likely that d13C in this species wasdetermined by source CO2 or stored carbon or recycling ofrespired CO2 (da Silveira et al 1989) Our study individualsof Ruscus were cultivated in a shaded understory similar totheir natural habitat Previous studies have shown that there canbe higher concentrations of respired CO2 in the forest understorythan in the canopy resulting in more negative carbon isotoperatios in understory plant tissue than canopy plant tissue (daSilveira et al 1989) This is a function of decomposing leavesand litter cover slow air mixing as well as plant environmentalresponses
A second possible explanation for the d13C values ofRuscus is related to its growth form and phenology asRuscus has extensive rhizomes (Sack et al 2003b) whichact as a carbon store for the plant Using this recycled carbonduring the growth season when the plant may not be able tomeet its carbon requirement by photosynthesis alone becauseof low maximum rates under light limitation may alsocontribute to more negative d13C values (Vizzini 2003)Notably Ruscus stems are hollow and thus can also storerelatively large amounts of CO2 for use during a growth periodwhen carbon is otherwise limiting especially given very lowgs Some of this stored carbon might be photosyntheticallyfixed by the stem (Nilsen and Sharifi 1997) In each of thesecases respired stored or recycled CO2 would supply carbonthat was previously fixed by Rubisco with more negative d13Cvalues (ndash27o) than air (ndash8o)
There was also a dissonance between the d13C value and CiCa The fully mature phylloclades which were selected andused for gas-exchange and d13C measurements were producedduring late winter and early spring with mild temperatures(average at midday 190ndash220C) and low atmospheric VPD
L Functional Plant Biology A Pivovaroff et al
(average at midday 06ndash15 kPa) However the gas-exchangemeasurements were taken during summer with hightemperature (average at midday 300ndash355C) and high VPD(average at midday 25ndash35 kPa) We harvested thephylloclades that we used for gas exchange to determine thecarbon isotope ratio Although the low value of instantaneousCiCa may be caused by high temperature and high atmosphericVPD the very low value of d13C may represent the time whenthe phyllocladersquos carbon was assimilated (during mildtemperature and low VPD)
Implications for drought and shade tolerance Ruscusas a model
We found strong support for a large number of hypotheses fortrait-based shade and drought tolerance providing a strong traitbasis for combined tolerance Although the detailed functionaltrait survey conducted here is relatively novel in its breadth(see also Pasquet-Kok et al 2010) this is a logical extensionof the traditional approach for understanding the basis forplant adaptation to environment ie testing expectations forindividual traits established by previous studies of thefunctional significance of these traits in other species Weacknowledge there is some degree of uncertainty ininterpreting a large number of traits simultaneously based onstudies of other species First the interpretation of the value oftraits based on other species may not be in all cases equally validfor Ruscus Some trait variation may relate to other functionsFurther studies for example using mutants would be necessaryas conclusive evidence of the value of specific traits in a givenspecies However one advantage of testing numerousexpectations for each hypothesis is that the key finding will berobust to the removal of some traits from the analysis if thoseare later found to be inappropriate Ideally when a model forestimating plant performance from leaf traits becomes availableone could determine how the specific quantitative combinationsof traits presented here scales up to plant shade and droughttolerance
The shade and drought tolerance of Ruscus consistent withthe suite of traits examined here is one case demonstratinghow plants can avoid a general trade-off between shade anddrought tolerance Further Ruscus is noteworthy as one of onlya few stem photosynthetic plants that occupy a shaded habitatHistorically it is likely that shade tolerance preceded droughttolerance given this speciesrsquo ancestors were species of moisttropical forests (Kim et al 2010) and thus Ruscus or itsancestor apparently evolved drought tolerance whileexpanding its range into drier habitat or during past climatechange Considering its unique adaptations and trait valuesRuscus can serve as an excellent model for the basis ofcombined shade and drought tolerance
Acknowledgements
We thank the Mildred E Mathias Botanical Garden for access to garden andplants Andy Truong for assistance with measurements Howard GriffithsLouis Santiago and Cheryl Swift for helpful discussions and comments onthe manuscript This work was supported by National Science FoundationGrant IOB-0546784
References
Bartlett MK Scoffoni C Sack L (2012) The determinants of leaf turgor losspoint and prediction of drought tolerance of species and biomes a globalmeta-analysis Ecology Letters 15 393ndash405 doi101111j1461-0248201201751x
Blackman CJ Brodribb TJ Jordan GJ (2010) Leaf hydraulic vulnerability isrelated to conduit dimensions and drought resistance across a diverserange of woody angiosperms New Phytologist 188 1113ndash1123doi101111j1469-8137201003439x
Brodribb TJ Feild TS Jordan GJ (2007) Leaf maximum photosynthetic rateand venation are linked by hydraulicsPlant Physiology 144 1890ndash1898doi101104pp107101352
Caspersen JP (2001) Interspecific variation in sapling mortality in relationto growth and soil moisture Oikos 92 160ndash168 doi101034j1600-07062001920119x
Cochard H Nardini A Coll L (2004) Hydraulic architecture of leaf bladeswhere is the main resistance Plant Cell amp Environment 27 1257ndash1267doi101111j1365-3040200401233x
Cooney-Sovetts C Sattler R (1987) Phylloclade development in theAsparagaceae an example of homoeosis Botanical Journal of theLinnean Society 94 327ndash371 doi101111j1095-83391986tb01053x
Cowling R Rundel P Lamont BB ArroyoMK ArianoutsouM (1996) Plantdiversity in Mediterranean-climate regions Trends in Ecology ampEvolution 11 362ndash366 doi1010160169-5347(96)10044-6
da Silveira L Sternberg L Mulkey SS Wright SJ (1989) Ecologicalinterpretation of leaf carbon isotope ratios influence of respired carbondioxide Ecology 70 1317ndash1324 doi1023071938191
de Lillis M Fontanella A (1992) Comparative phenology and growth indifferent species of the Mediterranean maquis of central Italy PlantEcology 99-100 83ndash96 doi101007BF00118213
Dracup JA (1991)DroughtmonitoringStochasticHydrologyandHydraulics5 261ndash266 doi101007BF01543134
Engelbrecht BMJ Kursar TA (2003) Comparative drought-resistance ofseedlings of 28 species of co-occurring tropical woody plantsOecologia 136 383ndash393 doi101007s00442-003-1290-8
Farquhar GD von Caemmerer S (1980) A biochemical model ofphotosynthetic CO2 assimilation in leaves of C3 species Planta 14978ndash90 doi101007BF00386231
FarquharGD Ehleringer JR HubickK (1989) Carbon isotope discriminationand photosynthesis Annual Review of Plant Physiology and PlantMolecular Biology 40 503ndash537 doi101146annurevpp40060189002443
Givnish TJ (1988) Adaptation to sun and shade a whole-plant perspectiveAustralian Journal of Plant Physiology 15 63ndash92 doi101071PP9880063
Gulias J Flexas J Mus M Cifre J Lefi E Medrano H (2003) Relationshipbetweenmaximum leaf photosynthesis nitrogen content and specific leafarea inBalearic endemic and non-endemicMediterranean speciesAnnalsof Botany 92 215ndash222 doi101093aobmcg123
Haberlandt G (1914) lsquoPhysiological plant anatomyrsquo (MacMillan London)Hallik L Niinemets U Wright J (2009) Are species shade and drought
tolerance reflected in leaf-level structural and functional differentiationin Northern Hemisphere temperate woody flora New Phytologist 184257ndash274 doi101111j1469-8137200902918x
Jones HG (1992) lsquoPlants and microclimate a quantitative approach toenvironmental plant physiologyrsquo (Cambridge University PressCambridge)
Kerstiens G (1996) Cuticular water permeability and its physiologicalsignificance Journal of Experimental Botany 47 1813ndash1832doi101093jxb47121813
Kim JH Kim DK Forest F Fay MF Chase MW (2010) Molecularphylogenetics of Ruscaceae sensu lato and related families(Asparagales) based on plastid and nuclear DNA sequences Annals ofBotany 106 775ndash790 doi101093aobmcq167
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology M
Koide RT Robichaux RH Morse SR Smith CM (2000) Plant water statushydraulic resistance and capacitance In lsquoPlant physiological ecologyfield methods and instrumentationrsquo (Eds R Pearcy JR Ehleringer HMooney P Rundel) pp 161ndash183 (Kluwer Dordrecht The Netherlands)
Koumlrner C Farquhar GD Wong SC (1991) Carbon isotope discrimination byplants follows latitudinal and altitudinal trends Oecologia 88 30ndash40doi101007BF00328400
Lewis AM Boose ER (1995) Estimating volume flow rates through xylemconduits American Journal of Botany 82 1112ndash1116 doi1023072446063
Martiacutenez-Palleacute E Aronne G (1999) Flower development and reproductivecontinuity inMediterraneanRuscusaculeatusL (Liliaceae)Protoplasma208 58ndash64 doi101007BF01279075
Martiacutenez-Tilleriacutea K Loayza AP Sandquist DR Squeo FA (2012) Noevidence of a trade-off between drought and shade tolerance inseedlings of six coastal desert shrub species in north-central ChileJournal of Vegetation Science 23 1051ndash1061 doi101111j1654-1103201201427x
Matalas NC (1963) Probability distribution of low flows USGS ProfessionalPapers 434-A US Government Printing Office Washington DC
Niinemets U (1999) Research review components of leaf dry mass per area ndashthickness and density ndash alter leaf photosynthetic capacity in reversedirections in woody plants New Phytologist 144 35ndash47 doi101046j1469-8137199900466x
Niinemets U Valladares F (2006) Tolerance to shade drought andwaterlogging of temperate northern hemisphere trees and shrubsEcological Monographs 76 521ndash547 doi1018900012-9615(2006)076[0521TTSDAW]20CO2
Nilsen ET Sharifi MR (1997) Carbon isotope composition of legumes withphotosynthetic stems from Mediterranean and desert habitats AmericanJournal of Botany 84 1707ndash1713 doi1023072446469
Ogburn RM Edwards EJ (2012) Quantifying succulence a rapidphysiologically meaningful metric of plant water storage Plant Cell ampEnvironment 35 1533ndash1542 doi101111j1365-3040201202503x
Pasquet-Kok J Creese C Sack L (2010) Turning over a new lsquoleafrsquo multiplefunctional significances of leaves versus phyllodes in Hawaiian Acaciakoa Plant Cell amp Environment 33 2084ndash2100 doi101111j1365-3040201002207x
SackL (2004)Responses of temperatewoody seedlings to shade and droughtdo trade-offs limit potential niche differentiation Oikos 107 110ndash127doi101111j0030-1299200413184x
SackLFroleK (2006)Leaf structural diversity is related to hydraulic capacityin tropical rain forest trees Ecology 87 483ndash491 doi10189005-0710
Sack L Holbrook NM (2006) Leaf hydraulics Annual Review of PlantBiology 57 361ndash381 doi101146annurevarplant56032604144141
Sack L Melcher PJ Zwieniecki MA Holbrook NM (2002) The hydraulicconductance of the angiosperm leaf lamina a comparison of threemeasurement methods Journal of Experimental Botany 532177ndash2184 doi101093jxberf069
Sack L Cowan P Jaikumar N Holbrook NM (2003a) The ldquohydrologyrdquo ofleaves co-ordination of structure and function in temperate woodyspecies Plant Cell amp Environment 26 1343ndash1356 doi101046j0016-8025200301058x
Sack L Grubb P Maronon T (2003b) The functional morphology of juvenileplants tolerant of strong summer drought in shaded forest understoriesin southern Spain Plant Ecology 168 139ndash163 doi101023A1024423820136
SackLScoffoniCMcKownADFroleKRawlsMHavran JCTranHTranT (2012) Developmentally based scaling of leaf venation architectureexplains global ecological patterns Nature Communications 3 837doi101038ncomms1835
Sack L Chatelet D Scoffoni C PrometheusWiki contributors (2013a)ldquoEstimating the mesophyll surface area per leaf area from leaf celland tissue dimensions measured from transverse cross-sectionsrdquoPrometheusWiki Available at httpwwwpublishcsiroauprometheuswikitiki-pagehistoryphppage=Estimating the mesophyllsurface area per leaf area from leaf cell and tissue dimensionsmeasured from transverse cross-sectionsamppreview=16 [accessed June18 2013]
SackL Pasquet-Kok JPrometheusWiki contributors (2013b) ldquoLeaf pressure-volume curve parametersrdquo PrometheusWiki Available at httpprometheuswikipublishcsiroautiki-indexphppage=Leaf+pressure-volume+curve+parameters
Scoffoni C Pou A Aasamaa K Sack L (2008) The rapid light response ofleaf hydraulic conductance new evidence from two experimentalmethods Plant Cell amp Environment 31 1803ndash1812 doi101111j1365-3040200801884x
Smith T Huston M (1989) A theory of the spatial and temporal dynamics ofplant communities Vegetatio 83 49ndash69 doi101007BF00031680
Sterck F Markesteijn L Schieving F Poorter L (2011) Functional traitsdetermine trade-offs and niches in a tropical forest communityProceedings of the National Academy of Sciences of the United Statesof America 108 20 627ndash20 632 doi101073pnas1106950108
Thomas GS (1992) lsquoOrnamental shrubs climbers and bamboosrsquo (JohnMurray Publishers Great Britain)
TyreeMT ZimmermannMH (1983) lsquoXylem structure and the ascent of saprsquo(Springer-Verlag Berlin)
USDA ARS National Genetic Resources Program (2009) GermplasmResources Information Network (GRIN) online database NationalGermplasm Resources Laboratory Beltsville Maryland Availableathttpwwwars-gringovcgi-binnpgshtmltaxonpl410577[Accessed 20 July 2009]
Vendramini F Diacuteaz S Gurvich DE Wilson PJ Thompson K Hodgson JG(2002) Leaf traits as indicators of resource-use strategy in floras withsucculent species New Phytologist 154 147ndash157 doi101046j1469-8137200200357x
Vile D (2005) Specific leaf area and dry matter content estimate thicknessin laminar leaves Annals of Botany 96 1129ndash1136 doi101093aobmci264
Vizzini S (2003) d13C and d15N variability in Posidonia oceanica associatedwith seasonality and plant fraction Aquatic Botany 76 195ndash202doi101016S0304-3770(03)00052-4
Walters M Reich PB (1999) Research review low-light carbon balance andshade tolerance in the seedlings of woody plants dowinter deciduous andbroad-leaved evergreen species differ New Phytologist 143 143ndash154doi101046j1469-8137199900425x
Witkowski E Lamont BB (1991) Leaf specific mass confounds leaf densityand thickness Oecologia 88 486ndash493
Wright IJWestobyM (2003)Nutrient concentration resorption and lifespanleaf traits of Australian sclerophyll species Functional Ecology 1710ndash19 doi101046j1365-2435200300694x
Wright IJ Reich PB Westoby M Ackerly DD Baruch Z Bongers FCavender-Bares J Chapin T Cornelissen JHC Diemer M Flexas JGarnier E Groom PK Gulias J Hikosaka K Lamont BB Lee T LeeWLusk C Midgley JJ Navas M-L Niinemets U Oleksyn J Osada NPoorter H Poot P Prior L Pyankov VI Roumet C Thomas SC TjoelkerMG Veneklaas EJ Villar R (2004) The worldwide leaf economicsspectrum Nature 428 821ndash827 doi101038nature02403
Wullschleger SD (1993) Biochemical limitations to carbon assimilation inC3 plants ndash a retrospective analysis of the ACi curves from 109 speciesJournal of Experimental Botany 44 907ndash920 doi101093jxb445907
N Functional Plant Biology A Pivovaroff et al
wwwpublishcsiroaujournalsfpb
by a red-blue light source (6400ndash02B no SI-710 Li-CorBiosciences ) Gas-exchange measurements were made on atleast 1ndash2 phylloclades per individual
For light-response curves phylloclades were acclimatedfor at least 5min at 1500mmolmndash2 sndash1 PAR at temperatures of2527C with RH maintained at ~50 and CO2 concentrationof 400mmolmolndash1 Then phylloclades were measured for netCO2 assimilation per leaf area at PAR steps of 1600 1400 12001000 800 600 500 400 300 200 100 50 and 0mmolmndash2 sndash1with 180ndash240 s stabilisation at each irradiance step Wedetermined light-saturated photosynthetic rate per area andmass (Aarea and Amass) dark respiration rate per area and mass(Rarea and Rmass) ie the negative A at zero PAR maximumstomatal conductance per area (gs) light compensation point(LCP) as the x-intercept intrinsic water use efficiency (WUEAareags) and the ratio of intercellular to ambient CO2
concentration (CiCa) at 100mmolmndash2 sndash1 PAR We thenharvested the phylloclades measured for gas exchange todetermine the nitrogen concentration and carbon isotope ratio(see below)
For CO2-response curves phylloclades were allowed toequilibrate at 400 ppm to induce stomatal opening and the netCO2 assimilation per leaf area was determined at Ci steps of400 300 200 100 50 400 400 500 600 800 1200 14001600 ppm with 3ndash4min equilibration time at each step Wedetermined maximum rate of carboxylation and maximum rateof electron transport per leaf area (Vcmax and Jmax) from plots ofCi vs A corrected to 25C (Farquhar and von Caemmerer 1980)
Cuticular conductance (ie minimumepidermal conductancegmin sensu Kerstiens 1996) was determined for 3ndash4 maturephylloclades and 10 cm lengths of stems from each of threeindividuals of each species Phylloclade and stem sampleswere hydrated and then the cut ends were sealed with waxSamples were dried for at least 30min on a laboratory bench atPAR of lt10mmol photons mndash2 sndash1 to induce stomatal closurethen samples were weighed for at least eight intervals of 30minduring which the slope of water loss versus time was highlylinear (R2 gt 0995) and therefore taken to represent transpirationafter stomata had closed fully The gmin was calculated as thetranspiration rate divided by the mole fraction vapour pressuredeficit (VPD determined from a weather station HOBO MicroStation with Smart Sensors Onset Bourne MA USA)
Pressurendashvolume curve parametersPressurendashvolume curve parameters were determined for matureshoots using the bench-drying method (Koide et al 2000 Sacket al 2003a) Shoots were ~10ndash15 cm long with about fourphylloclades per shoot for R microglossum and 15ndash20phylloclades for R aculeatus Shoots were progressively driedon a laboratory bench and measured at intervals by equilibratingfor 10min in a Whirlpak bag (Whirl-Pak Nasco Fort AtkinsonWIUSA) beforeweighing andmeasuring for leafwater potential(Yleaf) with a pressure chamber (Model 1000 Plant MoistureStress Instruments Albany OR USA) Subsequently dry masswas determined after more than 48 h in an oven at 70C Wedetermined the leaf dry matter content (LDMC) saturated watercontent (SWC) turgor loss point (Ytlp) relative water content atturgor loss point (RWCtlp) and osmotic potential at full turgor
(po) relative capacitance at full turgor (Cft DRWCDYleaf) andrelative capacitance at turgor loss point (Ctlp DRWCDYleaf) Wedetermined the modulus of elasticity (e) as the linear slope of theline fitted for pressure potential versus relative water contentabove and including turgor loss point (Sack et al 2013b)
Shoot hydraulic conductance
We measured the hydraulic conductance of mature shoots(Kshoot) ~10ndash15 cm long bearing about four phylloclades pershoot for R microglossum and 15ndash20 phylloclades forR aculeatus using the evaporative flux method (Sack et al2002) Samples were harvested and the ends were re-cut underdistilled degassed water with a fresh razor blade then hydratedovernight Shoots were connected to tubing containing distilleddegassed water running to a graduated cylinder on a balanceinterfaced to a computer logging data every minute to calculatethe flow rate of water from the balance into the sample Sampleswere held in place above a fan on wood frames strung withfishing line Lights were arranged above a plexiglass containerof water that acted as a heat trap producing PAR ofgt1200mmolmndash2 sndash1 at shoot level When steady-statetranspiration was achieved samples were covered with aplastic bag and removed from the tubing and the leaf waterpotential was determined with a pressure chamber (Model 1000Plant Moisture Stress Instruments) Kshoot was calculated as thesteady-state transpirational flow rate (E mmolmndash2 sndash1) dividedby the water potential driving force (DYleaF= ndashYleaf MPa)further normalised by total phylloclade area Kshoot valueswere standardised to 25C to correct for the temperaturedependence of the viscosity of water (Sack et al 2003a)
Foliar nitrogen concentration and carbon isotope ratio
To analyse total tissue nitrogen concentration and carbon isotopecomposition (d13C) for three replicates from each of threeindividuals for each species phylloclade samples were oven-dried at gt70C for more than 48 h and ground by mortar andpestle The nitrogen concentration and d13C were determined atthe UCDavis Stable Isotope Facility using an elemental analyser(PDZEuropaANCA-GSL SerconLtd Cheshire UK) interfacedto an isotope ratiomass spectrometer (PDZEuropa20ndash20 SerconLtd) at the UC Davis Stable Isotope Facility Final d13C contentvalues are expressed relative to international standardVienna PeeDee belemnite (V-PDB) for carbon
d13CethpermilTHORN frac14 etheth13C=12C of sampleTHORN=eth13C=12C of standardTHORN 1THORN 1000
eth4THORN
d13C of plant tissues can provide a measure of intrinsic WUE(Ags) at the time that carbon was assimilated giving long-termwater-use efficiency
Comparative data compilation and trait comparison
To consider Ruscus trait values in a global context we compileddata from the literature for (1) temperate and tropical broadleafevergreen species (2) Mediterranean species and (3) woodyangiosperms in general When available we considered data forplants grown in the shade and for species that are shade tolerant
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology I
however eco-physiological trait data is generally collected forsun leaves or plants making a comparison solely for shade grownplants not possible Formore commonly studied traits (egAmax)comparative data was taken from studies with large databasesand multiple traits (eg GLOPNET Wright et al 2004) Wedetermined minimum mean and maximum values for traitsfrom comparative studies or the mean and standard error ifraw species values were not available Studies that wereincluded for comparative data are referenced in the captionsfor Table 1ndash5 in association with the specific traits wecompared Differences between Ruscus trait values andcomparative data from the literature were determined for bothR aculeatus and R microglossum Hypotheses were deemedsupported for a Ruscus species if the trait value was higher orlower than the mean comparator value in the way predicted
Results
Both Ruscus species studied had leaf morphology consistentwith adaptation to shade and drought relative to comparator
species (Tables 1ndash5)We constructed a radar graph to encapsulatethe key traits that would confer adaptation to shade drought andthe combination for R aculeatus which had the more extremeadaptation of the two Ruscus species considered (Fig 3) Thisfigure summarises the major results of our study with values forcomparator species appearing as the inner circle and R aculeatustrait values displayed as the bold outer line
Gross morphology of phylloclades
Both R aculeatus and R microglossum had LMA valueslower than comparator species and R microglossum hadphylloclades larger than the mean leaf area for comparatorspecies Both Ruscus species had lower bulk tissue density thancomparator species consistent with the water storage in theRuscus phylloclades Notably despite its water storage tissuethe LDMC and SWC values of R aculeatus were typical ofthose for comparator species whereas R microglossum had alower LDMC and a higher SWC than for comparator speciessets (Table 1)
Fig 3 Radar graph illustrating percent difference between selected traits of Ruscus aculeatuswithcomparative data (see Tables 1ndash5 for symbols and sources of comparative data) Values forR aculeatus outside the circle indicate shade andor drought tolerance Traits are arrangedaccording to whether they would contribute shade tolerance drought tolerance or both The innerdisc represents the mean for comparative species for given traits and the values for R aculeatus isscaled relative to the magnitude of that value () with a value outside the disc representing greatershade andor drought tolerance (+) and (ndash) indicate if the axis is scaled such that a value outside thecircle represents percent higher or lower than comparative values respectively For traits expressed asnegative values (+) and (ndash) indicate more negative or less negative respectively
J Functional Plant Biology A Pivovaroff et al
Anatomy of phylloclades
The Ruscus species possessed numerous anatomical traitsconsistent with benefits for both shade and drought tolerance(Table 2) The phylloclade cross-sections were symmetrical(Fig 2) and thus the cross-sectional anatomy of the adaxialand abaxial halves were not different for all traits (paired t-testP gt 005) and values were averaged within species The twospecies were similar in their substantial leaf thickness and inthe thickness of cuticle epidermis cell walls and water storagecompartment (Table 2) The fractions of the lamina occupied bythe epidermis mesophyll and water storage tissues were 1552ndash56 and 29ndash32 respectively
Both species had parallel longitudinal veins of three sizes(midrib intermediate and small veins) Consistent with droughtadaptation the two species had low Dm R aculeatus having thelower value andRmicroglossumhadahigher IVD (Table 2)Thetwo species had on average the same maximum conduitdiameters in their midribs intermediate veins and small veinsand the maximum conduit diameter decreased ~35 from themidrib to the small veins The average theoretical hydraulicconductivity of xylem conduits did not differ between thespecies and decreased by up to 86 from the midrib to theminor vein R aculeatus had on average walls that were 85thicker in the fibrous bundle sheath (Table 2)
Gas-exchange measurements
Consistent with adaptation to simultaneous shade and droughtthe Ruscus species had very low values for Rarea Rmass and gs(Table 3) relative to comparator temperate broadleaf evergreenspecies (Fig 3) The Aarea Amass LCP Vcmax and Jmax were alsovery low relative to comparator species (Fig 3 Table 3)consistent with shade adaptation The two Ruscus species hadvery high values for Ags (Table 3) consistent with excellentWUE Consistent with strong drought tolerance via retention ofstored water both species had very low values for leaf and stemgmin especially relative to comparator vascular plant species(Fig 3 Table 3)
Hydraulic conductance pressure volume curveparameters and leaf water storage
Consistent with expectations for combined drought and shadetolerance both Ruscus species had low Kshoot relative tocomparator tropical and temperate woody angiosperms (Fig 3Table 4) The pressurendashvolume curve parameters of Ruscuswereconsistent with achieving drought tolerance through tissue waterstorage Both species had less negative po and ytlp than meanvalues for comparative evergreen woody species (Fig 3Table 4) Notably both species had lower e values thancomparative evergreen woody species and higher RWCtlp andCft values (Fig 3 Table 4) consistent with drought tolerance
Nitrogen concentration and carbon isotope composition
Both species had high values for phyllyclade Narea and Nmass
relative to comparative temperate broadleaf evergreen speciesconsistent with drought adaptation (Fig 3) The d13C valueswerevery negative and typical of values often observed for understoryplants (da Silveira et al 1989) consistent with shade tolerance
Indeed the d13C values were notably strongly negative given thehigh WUE found for these species
Testing hypotheses for shade and drought tolerancewith trait survey data
Overall we quantified 57 traits for the twoRuscus species and for21 traits we had a priori hypotheses for a benefit for shadetolerance and for 24 traits we had a priori hypotheses for abenefit for drought tolerance Using comparative data we foundthat 16 of 21 hypotheses were supported for shade tolerance and22 of 24 were supported for drought tolerance Of the ninehypotheses for traits that would contribute to both shade anddrought tolerance simultaneously (ie expectations were both forhigher or lower values than comparative species) eight weresupported by trait data All these proportions were significantlyhigher than the 50 support that would have been expected toarise only from chance (P = 0001ndash0058 proportion tests)Notably in the seven cases when shade tolerance traits wouldconflict with drought tolerance traits four indicated a benefit fordrought tolerance rather than shade tolerance for both species(Tables 1ndash5)
Discussion
Both R aculeatus (Fig 3) and R microglossum showed traitvalues consistent with combined shade and drought toleranceNumerous traits were consistent with a combined shade anddrought tolerance through improving carbon balance enablinga conservative resource use ie via slow respiration and long-lived parts Other traits would contribute specifically to droughttolerance via reduced demand for water during activephotosynthesis and the ability to survive strong drought afterstomatal closure Notably many such traits were related to waterstorage providingnew insights into effective formsof succulencein shaded habitats This suite of traits would contributeimportantly to the ability of Ruscus to occupy shaded sitesprone to strong drought across a wide geographical range
Traits contributing to simultaneous drought and shadetolerance
Ruscus species showed specialisation associated withconservative resource use consistent with tolerance of shadeand drought These specialised traits included thick lamina andcomponent tissues that contribute to long tissue life-spans (shootslastgt5 years Sack et al 2003bWright et al 2004) AdditionallyRuscus species had very low gas-exchange rates including lowgs and lowKshoot whichwould correspond to a low investment invascular tissue (Tyree andZimmermann1983 Sack et al 2003b)and low Rarea Rmass Aarea and Amass all representing an ability tomaintain photosynthesis and growth with low requirements forlight and water
Traits contributing to drought tolerance
According to Jones et al (1992) drought tolerance can beachieved through avoidance of plant water deficits toleranceof plant water deficits or efficiency mechanisms Ruscus speciesshowed traits associated with drought tolerance either byproviding the ability to maintain photosynthesis and growth indrying soil andor the ability to survive chronic drought as
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology K
previously shown experimentally for R aculeatus (Sack 2004)Traits that would contribute to the ability to maintain gasexchange in drying soil include small leaf size high WUE andlow IVD High WUE achieved in part with high Narea meansthese Ruscus species can attain positive carbon balance evenwith extremely low gs Low IVD and water storage capacitanceallow the phylloclades to maintain water supply to the mesophylland tolerate transiently high evaporation rates for example dueto sunflecks without desiccating the leaf (Sack et al 2003a)Traits contributing to the ability of the phylloclades to surviveextended drought included those enabling a low evaporationrate per leaf area once stomata have shut such as low gmin inleaf and stem and those related to specialised water storagetissue linked with low leaf density low e and po values thatwere low in magnitude
Ruscus water storage
The water storage tissue of Ruscus that occupied a third of theleaf thickness although contributing most directly to droughttolerance is also consistent with shade tolerance given itscontribution to reduced tissue costs for the phylloclade as awhole The water storage tissue had thin cell walls reflected inthe low bulk e and low solute concentration contributing tobulk po values that were low in magnitude
This water storage tissue would also contribute to both typesof drought tolerance ndash the ability to maintain photosynthesis indrying soil and to survive after stomata have shut duringextended drought The lsquosucculencersquo of Ruscus phylloclades isdistinctive relative to more typical succulent-leafed andsucculent-stemmed species which tend to have high leaf watercontent and capacitance values (Vendramini et al 2002 Ogburnand Edwards 2012) In contrast in Ruscus species the SWCwas low relative to typical leaf succulent species and forR aculeatus Cft fell within the range of typical evergreenleaves We note that the strong tissue differentiation in Ruscus(ie separation of mesophyll cell and water storage in spaceand their distinctions in anatomy) would contribute to higheffectiveness of water storage even if the bulk tissue overallhad low SWC and capacitance Indeed across species theretends to be no relationship between the magnitude of SWC orcapacitance and the degree of within-leaf tissue differentiation(Ogburn and Edwards 2012) Notably such differentiationwould contribute special advantages for supply of waterwhether stomata are open or closed as the large thin-walledwater storage cells with low solute concentration can yield theirwater to supply the evaporative load while the photosynthetictissues can maintain their volume according to their thickerwalls and stronger solute concentration
Although the capacitance and SWC values were low forRuscus species these values would be more substantial ifconsidered relative to water demand Across several speciesCft has been found to correlate with Kshoot and with gs (Sacket al 2003a Blackman et al 2010) indicating that leaves tendto be built with capacitance to match their maximum flux ratesand thus to buffer the leaf water potential against surges intranspiration Thus because Ruscus has low gs when stomataare open and low Kshoot the capacitance would be expectedto supply transpiration transiently during sunflecks or high
VPD Likewise when stomata close the capacitance suppliesongoing water loss via cuticular conductance Given theextremely low gmin of Ruscus even its moderate Ctlp canenable survival for weeks (Sack et al 2003b Sack 2004)Further at turgor loss the water content would equalSWCRWCtlp and the relatively high RWCtlp wouldcontribute to the high water content once turgor is lost Thusthe lsquosucculencersquo of Ruscus is moderate in absolute terms butcombined with its other very strong mechanisms to reducetranspiration when stomata are open or closed ie low gs andgmin even this moderate capacitance would provide strongfunctionality
Water-use efficiency and carbon isotope composition
It was noteworthy that despite extremely high WUE valuesphylloclade d13C values of the Ruscus species were verynegative This presents a strong anomaly worthy of furtherinvestigation as species with high WUE typically have higherd13C (less negative ie closer to zero) The d13C can beinfluenced by a host of processes including source CO2 storedplant carbon and time-integrated CO2 concentration at the siteof carboxylation (Farquhar et al 1989) For Ruscus althoughd13C values were typical of an understory plant they did notappear to be drivenby internalCO2 concentration because the leafisotopic values were depleted in 13C whereas the high WUEdetermined by gas exchange would likely promote enrichedisotopic values It is more likely that d13C in this species wasdetermined by source CO2 or stored carbon or recycling ofrespired CO2 (da Silveira et al 1989) Our study individualsof Ruscus were cultivated in a shaded understory similar totheir natural habitat Previous studies have shown that there canbe higher concentrations of respired CO2 in the forest understorythan in the canopy resulting in more negative carbon isotoperatios in understory plant tissue than canopy plant tissue (daSilveira et al 1989) This is a function of decomposing leavesand litter cover slow air mixing as well as plant environmentalresponses
A second possible explanation for the d13C values ofRuscus is related to its growth form and phenology asRuscus has extensive rhizomes (Sack et al 2003b) whichact as a carbon store for the plant Using this recycled carbonduring the growth season when the plant may not be able tomeet its carbon requirement by photosynthesis alone becauseof low maximum rates under light limitation may alsocontribute to more negative d13C values (Vizzini 2003)Notably Ruscus stems are hollow and thus can also storerelatively large amounts of CO2 for use during a growth periodwhen carbon is otherwise limiting especially given very lowgs Some of this stored carbon might be photosyntheticallyfixed by the stem (Nilsen and Sharifi 1997) In each of thesecases respired stored or recycled CO2 would supply carbonthat was previously fixed by Rubisco with more negative d13Cvalues (ndash27o) than air (ndash8o)
There was also a dissonance between the d13C value and CiCa The fully mature phylloclades which were selected andused for gas-exchange and d13C measurements were producedduring late winter and early spring with mild temperatures(average at midday 190ndash220C) and low atmospheric VPD
L Functional Plant Biology A Pivovaroff et al
(average at midday 06ndash15 kPa) However the gas-exchangemeasurements were taken during summer with hightemperature (average at midday 300ndash355C) and high VPD(average at midday 25ndash35 kPa) We harvested thephylloclades that we used for gas exchange to determine thecarbon isotope ratio Although the low value of instantaneousCiCa may be caused by high temperature and high atmosphericVPD the very low value of d13C may represent the time whenthe phyllocladersquos carbon was assimilated (during mildtemperature and low VPD)
Implications for drought and shade tolerance Ruscusas a model
We found strong support for a large number of hypotheses fortrait-based shade and drought tolerance providing a strong traitbasis for combined tolerance Although the detailed functionaltrait survey conducted here is relatively novel in its breadth(see also Pasquet-Kok et al 2010) this is a logical extensionof the traditional approach for understanding the basis forplant adaptation to environment ie testing expectations forindividual traits established by previous studies of thefunctional significance of these traits in other species Weacknowledge there is some degree of uncertainty ininterpreting a large number of traits simultaneously based onstudies of other species First the interpretation of the value oftraits based on other species may not be in all cases equally validfor Ruscus Some trait variation may relate to other functionsFurther studies for example using mutants would be necessaryas conclusive evidence of the value of specific traits in a givenspecies However one advantage of testing numerousexpectations for each hypothesis is that the key finding will berobust to the removal of some traits from the analysis if thoseare later found to be inappropriate Ideally when a model forestimating plant performance from leaf traits becomes availableone could determine how the specific quantitative combinationsof traits presented here scales up to plant shade and droughttolerance
The shade and drought tolerance of Ruscus consistent withthe suite of traits examined here is one case demonstratinghow plants can avoid a general trade-off between shade anddrought tolerance Further Ruscus is noteworthy as one of onlya few stem photosynthetic plants that occupy a shaded habitatHistorically it is likely that shade tolerance preceded droughttolerance given this speciesrsquo ancestors were species of moisttropical forests (Kim et al 2010) and thus Ruscus or itsancestor apparently evolved drought tolerance whileexpanding its range into drier habitat or during past climatechange Considering its unique adaptations and trait valuesRuscus can serve as an excellent model for the basis ofcombined shade and drought tolerance
Acknowledgements
We thank the Mildred E Mathias Botanical Garden for access to garden andplants Andy Truong for assistance with measurements Howard GriffithsLouis Santiago and Cheryl Swift for helpful discussions and comments onthe manuscript This work was supported by National Science FoundationGrant IOB-0546784
References
Bartlett MK Scoffoni C Sack L (2012) The determinants of leaf turgor losspoint and prediction of drought tolerance of species and biomes a globalmeta-analysis Ecology Letters 15 393ndash405 doi101111j1461-0248201201751x
Blackman CJ Brodribb TJ Jordan GJ (2010) Leaf hydraulic vulnerability isrelated to conduit dimensions and drought resistance across a diverserange of woody angiosperms New Phytologist 188 1113ndash1123doi101111j1469-8137201003439x
Brodribb TJ Feild TS Jordan GJ (2007) Leaf maximum photosynthetic rateand venation are linked by hydraulicsPlant Physiology 144 1890ndash1898doi101104pp107101352
Caspersen JP (2001) Interspecific variation in sapling mortality in relationto growth and soil moisture Oikos 92 160ndash168 doi101034j1600-07062001920119x
Cochard H Nardini A Coll L (2004) Hydraulic architecture of leaf bladeswhere is the main resistance Plant Cell amp Environment 27 1257ndash1267doi101111j1365-3040200401233x
Cooney-Sovetts C Sattler R (1987) Phylloclade development in theAsparagaceae an example of homoeosis Botanical Journal of theLinnean Society 94 327ndash371 doi101111j1095-83391986tb01053x
Cowling R Rundel P Lamont BB ArroyoMK ArianoutsouM (1996) Plantdiversity in Mediterranean-climate regions Trends in Ecology ampEvolution 11 362ndash366 doi1010160169-5347(96)10044-6
da Silveira L Sternberg L Mulkey SS Wright SJ (1989) Ecologicalinterpretation of leaf carbon isotope ratios influence of respired carbondioxide Ecology 70 1317ndash1324 doi1023071938191
de Lillis M Fontanella A (1992) Comparative phenology and growth indifferent species of the Mediterranean maquis of central Italy PlantEcology 99-100 83ndash96 doi101007BF00118213
Dracup JA (1991)DroughtmonitoringStochasticHydrologyandHydraulics5 261ndash266 doi101007BF01543134
Engelbrecht BMJ Kursar TA (2003) Comparative drought-resistance ofseedlings of 28 species of co-occurring tropical woody plantsOecologia 136 383ndash393 doi101007s00442-003-1290-8
Farquhar GD von Caemmerer S (1980) A biochemical model ofphotosynthetic CO2 assimilation in leaves of C3 species Planta 14978ndash90 doi101007BF00386231
FarquharGD Ehleringer JR HubickK (1989) Carbon isotope discriminationand photosynthesis Annual Review of Plant Physiology and PlantMolecular Biology 40 503ndash537 doi101146annurevpp40060189002443
Givnish TJ (1988) Adaptation to sun and shade a whole-plant perspectiveAustralian Journal of Plant Physiology 15 63ndash92 doi101071PP9880063
Gulias J Flexas J Mus M Cifre J Lefi E Medrano H (2003) Relationshipbetweenmaximum leaf photosynthesis nitrogen content and specific leafarea inBalearic endemic and non-endemicMediterranean speciesAnnalsof Botany 92 215ndash222 doi101093aobmcg123
Haberlandt G (1914) lsquoPhysiological plant anatomyrsquo (MacMillan London)Hallik L Niinemets U Wright J (2009) Are species shade and drought
tolerance reflected in leaf-level structural and functional differentiationin Northern Hemisphere temperate woody flora New Phytologist 184257ndash274 doi101111j1469-8137200902918x
Jones HG (1992) lsquoPlants and microclimate a quantitative approach toenvironmental plant physiologyrsquo (Cambridge University PressCambridge)
Kerstiens G (1996) Cuticular water permeability and its physiologicalsignificance Journal of Experimental Botany 47 1813ndash1832doi101093jxb47121813
Kim JH Kim DK Forest F Fay MF Chase MW (2010) Molecularphylogenetics of Ruscaceae sensu lato and related families(Asparagales) based on plastid and nuclear DNA sequences Annals ofBotany 106 775ndash790 doi101093aobmcq167
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology M
Koide RT Robichaux RH Morse SR Smith CM (2000) Plant water statushydraulic resistance and capacitance In lsquoPlant physiological ecologyfield methods and instrumentationrsquo (Eds R Pearcy JR Ehleringer HMooney P Rundel) pp 161ndash183 (Kluwer Dordrecht The Netherlands)
Koumlrner C Farquhar GD Wong SC (1991) Carbon isotope discrimination byplants follows latitudinal and altitudinal trends Oecologia 88 30ndash40doi101007BF00328400
Lewis AM Boose ER (1995) Estimating volume flow rates through xylemconduits American Journal of Botany 82 1112ndash1116 doi1023072446063
Martiacutenez-Palleacute E Aronne G (1999) Flower development and reproductivecontinuity inMediterraneanRuscusaculeatusL (Liliaceae)Protoplasma208 58ndash64 doi101007BF01279075
Martiacutenez-Tilleriacutea K Loayza AP Sandquist DR Squeo FA (2012) Noevidence of a trade-off between drought and shade tolerance inseedlings of six coastal desert shrub species in north-central ChileJournal of Vegetation Science 23 1051ndash1061 doi101111j1654-1103201201427x
Matalas NC (1963) Probability distribution of low flows USGS ProfessionalPapers 434-A US Government Printing Office Washington DC
Niinemets U (1999) Research review components of leaf dry mass per area ndashthickness and density ndash alter leaf photosynthetic capacity in reversedirections in woody plants New Phytologist 144 35ndash47 doi101046j1469-8137199900466x
Niinemets U Valladares F (2006) Tolerance to shade drought andwaterlogging of temperate northern hemisphere trees and shrubsEcological Monographs 76 521ndash547 doi1018900012-9615(2006)076[0521TTSDAW]20CO2
Nilsen ET Sharifi MR (1997) Carbon isotope composition of legumes withphotosynthetic stems from Mediterranean and desert habitats AmericanJournal of Botany 84 1707ndash1713 doi1023072446469
Ogburn RM Edwards EJ (2012) Quantifying succulence a rapidphysiologically meaningful metric of plant water storage Plant Cell ampEnvironment 35 1533ndash1542 doi101111j1365-3040201202503x
Pasquet-Kok J Creese C Sack L (2010) Turning over a new lsquoleafrsquo multiplefunctional significances of leaves versus phyllodes in Hawaiian Acaciakoa Plant Cell amp Environment 33 2084ndash2100 doi101111j1365-3040201002207x
SackL (2004)Responses of temperatewoody seedlings to shade and droughtdo trade-offs limit potential niche differentiation Oikos 107 110ndash127doi101111j0030-1299200413184x
SackLFroleK (2006)Leaf structural diversity is related to hydraulic capacityin tropical rain forest trees Ecology 87 483ndash491 doi10189005-0710
Sack L Holbrook NM (2006) Leaf hydraulics Annual Review of PlantBiology 57 361ndash381 doi101146annurevarplant56032604144141
Sack L Melcher PJ Zwieniecki MA Holbrook NM (2002) The hydraulicconductance of the angiosperm leaf lamina a comparison of threemeasurement methods Journal of Experimental Botany 532177ndash2184 doi101093jxberf069
Sack L Cowan P Jaikumar N Holbrook NM (2003a) The ldquohydrologyrdquo ofleaves co-ordination of structure and function in temperate woodyspecies Plant Cell amp Environment 26 1343ndash1356 doi101046j0016-8025200301058x
Sack L Grubb P Maronon T (2003b) The functional morphology of juvenileplants tolerant of strong summer drought in shaded forest understoriesin southern Spain Plant Ecology 168 139ndash163 doi101023A1024423820136
SackLScoffoniCMcKownADFroleKRawlsMHavran JCTranHTranT (2012) Developmentally based scaling of leaf venation architectureexplains global ecological patterns Nature Communications 3 837doi101038ncomms1835
Sack L Chatelet D Scoffoni C PrometheusWiki contributors (2013a)ldquoEstimating the mesophyll surface area per leaf area from leaf celland tissue dimensions measured from transverse cross-sectionsrdquoPrometheusWiki Available at httpwwwpublishcsiroauprometheuswikitiki-pagehistoryphppage=Estimating the mesophyllsurface area per leaf area from leaf cell and tissue dimensionsmeasured from transverse cross-sectionsamppreview=16 [accessed June18 2013]
SackL Pasquet-Kok JPrometheusWiki contributors (2013b) ldquoLeaf pressure-volume curve parametersrdquo PrometheusWiki Available at httpprometheuswikipublishcsiroautiki-indexphppage=Leaf+pressure-volume+curve+parameters
Scoffoni C Pou A Aasamaa K Sack L (2008) The rapid light response ofleaf hydraulic conductance new evidence from two experimentalmethods Plant Cell amp Environment 31 1803ndash1812 doi101111j1365-3040200801884x
Smith T Huston M (1989) A theory of the spatial and temporal dynamics ofplant communities Vegetatio 83 49ndash69 doi101007BF00031680
Sterck F Markesteijn L Schieving F Poorter L (2011) Functional traitsdetermine trade-offs and niches in a tropical forest communityProceedings of the National Academy of Sciences of the United Statesof America 108 20 627ndash20 632 doi101073pnas1106950108
Thomas GS (1992) lsquoOrnamental shrubs climbers and bamboosrsquo (JohnMurray Publishers Great Britain)
TyreeMT ZimmermannMH (1983) lsquoXylem structure and the ascent of saprsquo(Springer-Verlag Berlin)
USDA ARS National Genetic Resources Program (2009) GermplasmResources Information Network (GRIN) online database NationalGermplasm Resources Laboratory Beltsville Maryland Availableathttpwwwars-gringovcgi-binnpgshtmltaxonpl410577[Accessed 20 July 2009]
Vendramini F Diacuteaz S Gurvich DE Wilson PJ Thompson K Hodgson JG(2002) Leaf traits as indicators of resource-use strategy in floras withsucculent species New Phytologist 154 147ndash157 doi101046j1469-8137200200357x
Vile D (2005) Specific leaf area and dry matter content estimate thicknessin laminar leaves Annals of Botany 96 1129ndash1136 doi101093aobmci264
Vizzini S (2003) d13C and d15N variability in Posidonia oceanica associatedwith seasonality and plant fraction Aquatic Botany 76 195ndash202doi101016S0304-3770(03)00052-4
Walters M Reich PB (1999) Research review low-light carbon balance andshade tolerance in the seedlings of woody plants dowinter deciduous andbroad-leaved evergreen species differ New Phytologist 143 143ndash154doi101046j1469-8137199900425x
Witkowski E Lamont BB (1991) Leaf specific mass confounds leaf densityand thickness Oecologia 88 486ndash493
Wright IJWestobyM (2003)Nutrient concentration resorption and lifespanleaf traits of Australian sclerophyll species Functional Ecology 1710ndash19 doi101046j1365-2435200300694x
Wright IJ Reich PB Westoby M Ackerly DD Baruch Z Bongers FCavender-Bares J Chapin T Cornelissen JHC Diemer M Flexas JGarnier E Groom PK Gulias J Hikosaka K Lamont BB Lee T LeeWLusk C Midgley JJ Navas M-L Niinemets U Oleksyn J Osada NPoorter H Poot P Prior L Pyankov VI Roumet C Thomas SC TjoelkerMG Veneklaas EJ Villar R (2004) The worldwide leaf economicsspectrum Nature 428 821ndash827 doi101038nature02403
Wullschleger SD (1993) Biochemical limitations to carbon assimilation inC3 plants ndash a retrospective analysis of the ACi curves from 109 speciesJournal of Experimental Botany 44 907ndash920 doi101093jxb445907
N Functional Plant Biology A Pivovaroff et al
wwwpublishcsiroaujournalsfpb
however eco-physiological trait data is generally collected forsun leaves or plants making a comparison solely for shade grownplants not possible Formore commonly studied traits (egAmax)comparative data was taken from studies with large databasesand multiple traits (eg GLOPNET Wright et al 2004) Wedetermined minimum mean and maximum values for traitsfrom comparative studies or the mean and standard error ifraw species values were not available Studies that wereincluded for comparative data are referenced in the captionsfor Table 1ndash5 in association with the specific traits wecompared Differences between Ruscus trait values andcomparative data from the literature were determined for bothR aculeatus and R microglossum Hypotheses were deemedsupported for a Ruscus species if the trait value was higher orlower than the mean comparator value in the way predicted
Results
Both Ruscus species studied had leaf morphology consistentwith adaptation to shade and drought relative to comparator
species (Tables 1ndash5)We constructed a radar graph to encapsulatethe key traits that would confer adaptation to shade drought andthe combination for R aculeatus which had the more extremeadaptation of the two Ruscus species considered (Fig 3) Thisfigure summarises the major results of our study with values forcomparator species appearing as the inner circle and R aculeatustrait values displayed as the bold outer line
Gross morphology of phylloclades
Both R aculeatus and R microglossum had LMA valueslower than comparator species and R microglossum hadphylloclades larger than the mean leaf area for comparatorspecies Both Ruscus species had lower bulk tissue density thancomparator species consistent with the water storage in theRuscus phylloclades Notably despite its water storage tissuethe LDMC and SWC values of R aculeatus were typical ofthose for comparator species whereas R microglossum had alower LDMC and a higher SWC than for comparator speciessets (Table 1)
Fig 3 Radar graph illustrating percent difference between selected traits of Ruscus aculeatuswithcomparative data (see Tables 1ndash5 for symbols and sources of comparative data) Values forR aculeatus outside the circle indicate shade andor drought tolerance Traits are arrangedaccording to whether they would contribute shade tolerance drought tolerance or both The innerdisc represents the mean for comparative species for given traits and the values for R aculeatus isscaled relative to the magnitude of that value () with a value outside the disc representing greatershade andor drought tolerance (+) and (ndash) indicate if the axis is scaled such that a value outside thecircle represents percent higher or lower than comparative values respectively For traits expressed asnegative values (+) and (ndash) indicate more negative or less negative respectively
J Functional Plant Biology A Pivovaroff et al
Anatomy of phylloclades
The Ruscus species possessed numerous anatomical traitsconsistent with benefits for both shade and drought tolerance(Table 2) The phylloclade cross-sections were symmetrical(Fig 2) and thus the cross-sectional anatomy of the adaxialand abaxial halves were not different for all traits (paired t-testP gt 005) and values were averaged within species The twospecies were similar in their substantial leaf thickness and inthe thickness of cuticle epidermis cell walls and water storagecompartment (Table 2) The fractions of the lamina occupied bythe epidermis mesophyll and water storage tissues were 1552ndash56 and 29ndash32 respectively
Both species had parallel longitudinal veins of three sizes(midrib intermediate and small veins) Consistent with droughtadaptation the two species had low Dm R aculeatus having thelower value andRmicroglossumhadahigher IVD (Table 2)Thetwo species had on average the same maximum conduitdiameters in their midribs intermediate veins and small veinsand the maximum conduit diameter decreased ~35 from themidrib to the small veins The average theoretical hydraulicconductivity of xylem conduits did not differ between thespecies and decreased by up to 86 from the midrib to theminor vein R aculeatus had on average walls that were 85thicker in the fibrous bundle sheath (Table 2)
Gas-exchange measurements
Consistent with adaptation to simultaneous shade and droughtthe Ruscus species had very low values for Rarea Rmass and gs(Table 3) relative to comparator temperate broadleaf evergreenspecies (Fig 3) The Aarea Amass LCP Vcmax and Jmax were alsovery low relative to comparator species (Fig 3 Table 3)consistent with shade adaptation The two Ruscus species hadvery high values for Ags (Table 3) consistent with excellentWUE Consistent with strong drought tolerance via retention ofstored water both species had very low values for leaf and stemgmin especially relative to comparator vascular plant species(Fig 3 Table 3)
Hydraulic conductance pressure volume curveparameters and leaf water storage
Consistent with expectations for combined drought and shadetolerance both Ruscus species had low Kshoot relative tocomparator tropical and temperate woody angiosperms (Fig 3Table 4) The pressurendashvolume curve parameters of Ruscuswereconsistent with achieving drought tolerance through tissue waterstorage Both species had less negative po and ytlp than meanvalues for comparative evergreen woody species (Fig 3Table 4) Notably both species had lower e values thancomparative evergreen woody species and higher RWCtlp andCft values (Fig 3 Table 4) consistent with drought tolerance
Nitrogen concentration and carbon isotope composition
Both species had high values for phyllyclade Narea and Nmass
relative to comparative temperate broadleaf evergreen speciesconsistent with drought adaptation (Fig 3) The d13C valueswerevery negative and typical of values often observed for understoryplants (da Silveira et al 1989) consistent with shade tolerance
Indeed the d13C values were notably strongly negative given thehigh WUE found for these species
Testing hypotheses for shade and drought tolerancewith trait survey data
Overall we quantified 57 traits for the twoRuscus species and for21 traits we had a priori hypotheses for a benefit for shadetolerance and for 24 traits we had a priori hypotheses for abenefit for drought tolerance Using comparative data we foundthat 16 of 21 hypotheses were supported for shade tolerance and22 of 24 were supported for drought tolerance Of the ninehypotheses for traits that would contribute to both shade anddrought tolerance simultaneously (ie expectations were both forhigher or lower values than comparative species) eight weresupported by trait data All these proportions were significantlyhigher than the 50 support that would have been expected toarise only from chance (P = 0001ndash0058 proportion tests)Notably in the seven cases when shade tolerance traits wouldconflict with drought tolerance traits four indicated a benefit fordrought tolerance rather than shade tolerance for both species(Tables 1ndash5)
Discussion
Both R aculeatus (Fig 3) and R microglossum showed traitvalues consistent with combined shade and drought toleranceNumerous traits were consistent with a combined shade anddrought tolerance through improving carbon balance enablinga conservative resource use ie via slow respiration and long-lived parts Other traits would contribute specifically to droughttolerance via reduced demand for water during activephotosynthesis and the ability to survive strong drought afterstomatal closure Notably many such traits were related to waterstorage providingnew insights into effective formsof succulencein shaded habitats This suite of traits would contributeimportantly to the ability of Ruscus to occupy shaded sitesprone to strong drought across a wide geographical range
Traits contributing to simultaneous drought and shadetolerance
Ruscus species showed specialisation associated withconservative resource use consistent with tolerance of shadeand drought These specialised traits included thick lamina andcomponent tissues that contribute to long tissue life-spans (shootslastgt5 years Sack et al 2003bWright et al 2004) AdditionallyRuscus species had very low gas-exchange rates including lowgs and lowKshoot whichwould correspond to a low investment invascular tissue (Tyree andZimmermann1983 Sack et al 2003b)and low Rarea Rmass Aarea and Amass all representing an ability tomaintain photosynthesis and growth with low requirements forlight and water
Traits contributing to drought tolerance
According to Jones et al (1992) drought tolerance can beachieved through avoidance of plant water deficits toleranceof plant water deficits or efficiency mechanisms Ruscus speciesshowed traits associated with drought tolerance either byproviding the ability to maintain photosynthesis and growth indrying soil andor the ability to survive chronic drought as
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology K
previously shown experimentally for R aculeatus (Sack 2004)Traits that would contribute to the ability to maintain gasexchange in drying soil include small leaf size high WUE andlow IVD High WUE achieved in part with high Narea meansthese Ruscus species can attain positive carbon balance evenwith extremely low gs Low IVD and water storage capacitanceallow the phylloclades to maintain water supply to the mesophylland tolerate transiently high evaporation rates for example dueto sunflecks without desiccating the leaf (Sack et al 2003a)Traits contributing to the ability of the phylloclades to surviveextended drought included those enabling a low evaporationrate per leaf area once stomata have shut such as low gmin inleaf and stem and those related to specialised water storagetissue linked with low leaf density low e and po values thatwere low in magnitude
Ruscus water storage
The water storage tissue of Ruscus that occupied a third of theleaf thickness although contributing most directly to droughttolerance is also consistent with shade tolerance given itscontribution to reduced tissue costs for the phylloclade as awhole The water storage tissue had thin cell walls reflected inthe low bulk e and low solute concentration contributing tobulk po values that were low in magnitude
This water storage tissue would also contribute to both typesof drought tolerance ndash the ability to maintain photosynthesis indrying soil and to survive after stomata have shut duringextended drought The lsquosucculencersquo of Ruscus phylloclades isdistinctive relative to more typical succulent-leafed andsucculent-stemmed species which tend to have high leaf watercontent and capacitance values (Vendramini et al 2002 Ogburnand Edwards 2012) In contrast in Ruscus species the SWCwas low relative to typical leaf succulent species and forR aculeatus Cft fell within the range of typical evergreenleaves We note that the strong tissue differentiation in Ruscus(ie separation of mesophyll cell and water storage in spaceand their distinctions in anatomy) would contribute to higheffectiveness of water storage even if the bulk tissue overallhad low SWC and capacitance Indeed across species theretends to be no relationship between the magnitude of SWC orcapacitance and the degree of within-leaf tissue differentiation(Ogburn and Edwards 2012) Notably such differentiationwould contribute special advantages for supply of waterwhether stomata are open or closed as the large thin-walledwater storage cells with low solute concentration can yield theirwater to supply the evaporative load while the photosynthetictissues can maintain their volume according to their thickerwalls and stronger solute concentration
Although the capacitance and SWC values were low forRuscus species these values would be more substantial ifconsidered relative to water demand Across several speciesCft has been found to correlate with Kshoot and with gs (Sacket al 2003a Blackman et al 2010) indicating that leaves tendto be built with capacitance to match their maximum flux ratesand thus to buffer the leaf water potential against surges intranspiration Thus because Ruscus has low gs when stomataare open and low Kshoot the capacitance would be expectedto supply transpiration transiently during sunflecks or high
VPD Likewise when stomata close the capacitance suppliesongoing water loss via cuticular conductance Given theextremely low gmin of Ruscus even its moderate Ctlp canenable survival for weeks (Sack et al 2003b Sack 2004)Further at turgor loss the water content would equalSWCRWCtlp and the relatively high RWCtlp wouldcontribute to the high water content once turgor is lost Thusthe lsquosucculencersquo of Ruscus is moderate in absolute terms butcombined with its other very strong mechanisms to reducetranspiration when stomata are open or closed ie low gs andgmin even this moderate capacitance would provide strongfunctionality
Water-use efficiency and carbon isotope composition
It was noteworthy that despite extremely high WUE valuesphylloclade d13C values of the Ruscus species were verynegative This presents a strong anomaly worthy of furtherinvestigation as species with high WUE typically have higherd13C (less negative ie closer to zero) The d13C can beinfluenced by a host of processes including source CO2 storedplant carbon and time-integrated CO2 concentration at the siteof carboxylation (Farquhar et al 1989) For Ruscus althoughd13C values were typical of an understory plant they did notappear to be drivenby internalCO2 concentration because the leafisotopic values were depleted in 13C whereas the high WUEdetermined by gas exchange would likely promote enrichedisotopic values It is more likely that d13C in this species wasdetermined by source CO2 or stored carbon or recycling ofrespired CO2 (da Silveira et al 1989) Our study individualsof Ruscus were cultivated in a shaded understory similar totheir natural habitat Previous studies have shown that there canbe higher concentrations of respired CO2 in the forest understorythan in the canopy resulting in more negative carbon isotoperatios in understory plant tissue than canopy plant tissue (daSilveira et al 1989) This is a function of decomposing leavesand litter cover slow air mixing as well as plant environmentalresponses
A second possible explanation for the d13C values ofRuscus is related to its growth form and phenology asRuscus has extensive rhizomes (Sack et al 2003b) whichact as a carbon store for the plant Using this recycled carbonduring the growth season when the plant may not be able tomeet its carbon requirement by photosynthesis alone becauseof low maximum rates under light limitation may alsocontribute to more negative d13C values (Vizzini 2003)Notably Ruscus stems are hollow and thus can also storerelatively large amounts of CO2 for use during a growth periodwhen carbon is otherwise limiting especially given very lowgs Some of this stored carbon might be photosyntheticallyfixed by the stem (Nilsen and Sharifi 1997) In each of thesecases respired stored or recycled CO2 would supply carbonthat was previously fixed by Rubisco with more negative d13Cvalues (ndash27o) than air (ndash8o)
There was also a dissonance between the d13C value and CiCa The fully mature phylloclades which were selected andused for gas-exchange and d13C measurements were producedduring late winter and early spring with mild temperatures(average at midday 190ndash220C) and low atmospheric VPD
L Functional Plant Biology A Pivovaroff et al
(average at midday 06ndash15 kPa) However the gas-exchangemeasurements were taken during summer with hightemperature (average at midday 300ndash355C) and high VPD(average at midday 25ndash35 kPa) We harvested thephylloclades that we used for gas exchange to determine thecarbon isotope ratio Although the low value of instantaneousCiCa may be caused by high temperature and high atmosphericVPD the very low value of d13C may represent the time whenthe phyllocladersquos carbon was assimilated (during mildtemperature and low VPD)
Implications for drought and shade tolerance Ruscusas a model
We found strong support for a large number of hypotheses fortrait-based shade and drought tolerance providing a strong traitbasis for combined tolerance Although the detailed functionaltrait survey conducted here is relatively novel in its breadth(see also Pasquet-Kok et al 2010) this is a logical extensionof the traditional approach for understanding the basis forplant adaptation to environment ie testing expectations forindividual traits established by previous studies of thefunctional significance of these traits in other species Weacknowledge there is some degree of uncertainty ininterpreting a large number of traits simultaneously based onstudies of other species First the interpretation of the value oftraits based on other species may not be in all cases equally validfor Ruscus Some trait variation may relate to other functionsFurther studies for example using mutants would be necessaryas conclusive evidence of the value of specific traits in a givenspecies However one advantage of testing numerousexpectations for each hypothesis is that the key finding will berobust to the removal of some traits from the analysis if thoseare later found to be inappropriate Ideally when a model forestimating plant performance from leaf traits becomes availableone could determine how the specific quantitative combinationsof traits presented here scales up to plant shade and droughttolerance
The shade and drought tolerance of Ruscus consistent withthe suite of traits examined here is one case demonstratinghow plants can avoid a general trade-off between shade anddrought tolerance Further Ruscus is noteworthy as one of onlya few stem photosynthetic plants that occupy a shaded habitatHistorically it is likely that shade tolerance preceded droughttolerance given this speciesrsquo ancestors were species of moisttropical forests (Kim et al 2010) and thus Ruscus or itsancestor apparently evolved drought tolerance whileexpanding its range into drier habitat or during past climatechange Considering its unique adaptations and trait valuesRuscus can serve as an excellent model for the basis ofcombined shade and drought tolerance
Acknowledgements
We thank the Mildred E Mathias Botanical Garden for access to garden andplants Andy Truong for assistance with measurements Howard GriffithsLouis Santiago and Cheryl Swift for helpful discussions and comments onthe manuscript This work was supported by National Science FoundationGrant IOB-0546784
References
Bartlett MK Scoffoni C Sack L (2012) The determinants of leaf turgor losspoint and prediction of drought tolerance of species and biomes a globalmeta-analysis Ecology Letters 15 393ndash405 doi101111j1461-0248201201751x
Blackman CJ Brodribb TJ Jordan GJ (2010) Leaf hydraulic vulnerability isrelated to conduit dimensions and drought resistance across a diverserange of woody angiosperms New Phytologist 188 1113ndash1123doi101111j1469-8137201003439x
Brodribb TJ Feild TS Jordan GJ (2007) Leaf maximum photosynthetic rateand venation are linked by hydraulicsPlant Physiology 144 1890ndash1898doi101104pp107101352
Caspersen JP (2001) Interspecific variation in sapling mortality in relationto growth and soil moisture Oikos 92 160ndash168 doi101034j1600-07062001920119x
Cochard H Nardini A Coll L (2004) Hydraulic architecture of leaf bladeswhere is the main resistance Plant Cell amp Environment 27 1257ndash1267doi101111j1365-3040200401233x
Cooney-Sovetts C Sattler R (1987) Phylloclade development in theAsparagaceae an example of homoeosis Botanical Journal of theLinnean Society 94 327ndash371 doi101111j1095-83391986tb01053x
Cowling R Rundel P Lamont BB ArroyoMK ArianoutsouM (1996) Plantdiversity in Mediterranean-climate regions Trends in Ecology ampEvolution 11 362ndash366 doi1010160169-5347(96)10044-6
da Silveira L Sternberg L Mulkey SS Wright SJ (1989) Ecologicalinterpretation of leaf carbon isotope ratios influence of respired carbondioxide Ecology 70 1317ndash1324 doi1023071938191
de Lillis M Fontanella A (1992) Comparative phenology and growth indifferent species of the Mediterranean maquis of central Italy PlantEcology 99-100 83ndash96 doi101007BF00118213
Dracup JA (1991)DroughtmonitoringStochasticHydrologyandHydraulics5 261ndash266 doi101007BF01543134
Engelbrecht BMJ Kursar TA (2003) Comparative drought-resistance ofseedlings of 28 species of co-occurring tropical woody plantsOecologia 136 383ndash393 doi101007s00442-003-1290-8
Farquhar GD von Caemmerer S (1980) A biochemical model ofphotosynthetic CO2 assimilation in leaves of C3 species Planta 14978ndash90 doi101007BF00386231
FarquharGD Ehleringer JR HubickK (1989) Carbon isotope discriminationand photosynthesis Annual Review of Plant Physiology and PlantMolecular Biology 40 503ndash537 doi101146annurevpp40060189002443
Givnish TJ (1988) Adaptation to sun and shade a whole-plant perspectiveAustralian Journal of Plant Physiology 15 63ndash92 doi101071PP9880063
Gulias J Flexas J Mus M Cifre J Lefi E Medrano H (2003) Relationshipbetweenmaximum leaf photosynthesis nitrogen content and specific leafarea inBalearic endemic and non-endemicMediterranean speciesAnnalsof Botany 92 215ndash222 doi101093aobmcg123
Haberlandt G (1914) lsquoPhysiological plant anatomyrsquo (MacMillan London)Hallik L Niinemets U Wright J (2009) Are species shade and drought
tolerance reflected in leaf-level structural and functional differentiationin Northern Hemisphere temperate woody flora New Phytologist 184257ndash274 doi101111j1469-8137200902918x
Jones HG (1992) lsquoPlants and microclimate a quantitative approach toenvironmental plant physiologyrsquo (Cambridge University PressCambridge)
Kerstiens G (1996) Cuticular water permeability and its physiologicalsignificance Journal of Experimental Botany 47 1813ndash1832doi101093jxb47121813
Kim JH Kim DK Forest F Fay MF Chase MW (2010) Molecularphylogenetics of Ruscaceae sensu lato and related families(Asparagales) based on plastid and nuclear DNA sequences Annals ofBotany 106 775ndash790 doi101093aobmcq167
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology M
Koide RT Robichaux RH Morse SR Smith CM (2000) Plant water statushydraulic resistance and capacitance In lsquoPlant physiological ecologyfield methods and instrumentationrsquo (Eds R Pearcy JR Ehleringer HMooney P Rundel) pp 161ndash183 (Kluwer Dordrecht The Netherlands)
Koumlrner C Farquhar GD Wong SC (1991) Carbon isotope discrimination byplants follows latitudinal and altitudinal trends Oecologia 88 30ndash40doi101007BF00328400
Lewis AM Boose ER (1995) Estimating volume flow rates through xylemconduits American Journal of Botany 82 1112ndash1116 doi1023072446063
Martiacutenez-Palleacute E Aronne G (1999) Flower development and reproductivecontinuity inMediterraneanRuscusaculeatusL (Liliaceae)Protoplasma208 58ndash64 doi101007BF01279075
Martiacutenez-Tilleriacutea K Loayza AP Sandquist DR Squeo FA (2012) Noevidence of a trade-off between drought and shade tolerance inseedlings of six coastal desert shrub species in north-central ChileJournal of Vegetation Science 23 1051ndash1061 doi101111j1654-1103201201427x
Matalas NC (1963) Probability distribution of low flows USGS ProfessionalPapers 434-A US Government Printing Office Washington DC
Niinemets U (1999) Research review components of leaf dry mass per area ndashthickness and density ndash alter leaf photosynthetic capacity in reversedirections in woody plants New Phytologist 144 35ndash47 doi101046j1469-8137199900466x
Niinemets U Valladares F (2006) Tolerance to shade drought andwaterlogging of temperate northern hemisphere trees and shrubsEcological Monographs 76 521ndash547 doi1018900012-9615(2006)076[0521TTSDAW]20CO2
Nilsen ET Sharifi MR (1997) Carbon isotope composition of legumes withphotosynthetic stems from Mediterranean and desert habitats AmericanJournal of Botany 84 1707ndash1713 doi1023072446469
Ogburn RM Edwards EJ (2012) Quantifying succulence a rapidphysiologically meaningful metric of plant water storage Plant Cell ampEnvironment 35 1533ndash1542 doi101111j1365-3040201202503x
Pasquet-Kok J Creese C Sack L (2010) Turning over a new lsquoleafrsquo multiplefunctional significances of leaves versus phyllodes in Hawaiian Acaciakoa Plant Cell amp Environment 33 2084ndash2100 doi101111j1365-3040201002207x
SackL (2004)Responses of temperatewoody seedlings to shade and droughtdo trade-offs limit potential niche differentiation Oikos 107 110ndash127doi101111j0030-1299200413184x
SackLFroleK (2006)Leaf structural diversity is related to hydraulic capacityin tropical rain forest trees Ecology 87 483ndash491 doi10189005-0710
Sack L Holbrook NM (2006) Leaf hydraulics Annual Review of PlantBiology 57 361ndash381 doi101146annurevarplant56032604144141
Sack L Melcher PJ Zwieniecki MA Holbrook NM (2002) The hydraulicconductance of the angiosperm leaf lamina a comparison of threemeasurement methods Journal of Experimental Botany 532177ndash2184 doi101093jxberf069
Sack L Cowan P Jaikumar N Holbrook NM (2003a) The ldquohydrologyrdquo ofleaves co-ordination of structure and function in temperate woodyspecies Plant Cell amp Environment 26 1343ndash1356 doi101046j0016-8025200301058x
Sack L Grubb P Maronon T (2003b) The functional morphology of juvenileplants tolerant of strong summer drought in shaded forest understoriesin southern Spain Plant Ecology 168 139ndash163 doi101023A1024423820136
SackLScoffoniCMcKownADFroleKRawlsMHavran JCTranHTranT (2012) Developmentally based scaling of leaf venation architectureexplains global ecological patterns Nature Communications 3 837doi101038ncomms1835
Sack L Chatelet D Scoffoni C PrometheusWiki contributors (2013a)ldquoEstimating the mesophyll surface area per leaf area from leaf celland tissue dimensions measured from transverse cross-sectionsrdquoPrometheusWiki Available at httpwwwpublishcsiroauprometheuswikitiki-pagehistoryphppage=Estimating the mesophyllsurface area per leaf area from leaf cell and tissue dimensionsmeasured from transverse cross-sectionsamppreview=16 [accessed June18 2013]
SackL Pasquet-Kok JPrometheusWiki contributors (2013b) ldquoLeaf pressure-volume curve parametersrdquo PrometheusWiki Available at httpprometheuswikipublishcsiroautiki-indexphppage=Leaf+pressure-volume+curve+parameters
Scoffoni C Pou A Aasamaa K Sack L (2008) The rapid light response ofleaf hydraulic conductance new evidence from two experimentalmethods Plant Cell amp Environment 31 1803ndash1812 doi101111j1365-3040200801884x
Smith T Huston M (1989) A theory of the spatial and temporal dynamics ofplant communities Vegetatio 83 49ndash69 doi101007BF00031680
Sterck F Markesteijn L Schieving F Poorter L (2011) Functional traitsdetermine trade-offs and niches in a tropical forest communityProceedings of the National Academy of Sciences of the United Statesof America 108 20 627ndash20 632 doi101073pnas1106950108
Thomas GS (1992) lsquoOrnamental shrubs climbers and bamboosrsquo (JohnMurray Publishers Great Britain)
TyreeMT ZimmermannMH (1983) lsquoXylem structure and the ascent of saprsquo(Springer-Verlag Berlin)
USDA ARS National Genetic Resources Program (2009) GermplasmResources Information Network (GRIN) online database NationalGermplasm Resources Laboratory Beltsville Maryland Availableathttpwwwars-gringovcgi-binnpgshtmltaxonpl410577[Accessed 20 July 2009]
Vendramini F Diacuteaz S Gurvich DE Wilson PJ Thompson K Hodgson JG(2002) Leaf traits as indicators of resource-use strategy in floras withsucculent species New Phytologist 154 147ndash157 doi101046j1469-8137200200357x
Vile D (2005) Specific leaf area and dry matter content estimate thicknessin laminar leaves Annals of Botany 96 1129ndash1136 doi101093aobmci264
Vizzini S (2003) d13C and d15N variability in Posidonia oceanica associatedwith seasonality and plant fraction Aquatic Botany 76 195ndash202doi101016S0304-3770(03)00052-4
Walters M Reich PB (1999) Research review low-light carbon balance andshade tolerance in the seedlings of woody plants dowinter deciduous andbroad-leaved evergreen species differ New Phytologist 143 143ndash154doi101046j1469-8137199900425x
Witkowski E Lamont BB (1991) Leaf specific mass confounds leaf densityand thickness Oecologia 88 486ndash493
Wright IJWestobyM (2003)Nutrient concentration resorption and lifespanleaf traits of Australian sclerophyll species Functional Ecology 1710ndash19 doi101046j1365-2435200300694x
Wright IJ Reich PB Westoby M Ackerly DD Baruch Z Bongers FCavender-Bares J Chapin T Cornelissen JHC Diemer M Flexas JGarnier E Groom PK Gulias J Hikosaka K Lamont BB Lee T LeeWLusk C Midgley JJ Navas M-L Niinemets U Oleksyn J Osada NPoorter H Poot P Prior L Pyankov VI Roumet C Thomas SC TjoelkerMG Veneklaas EJ Villar R (2004) The worldwide leaf economicsspectrum Nature 428 821ndash827 doi101038nature02403
Wullschleger SD (1993) Biochemical limitations to carbon assimilation inC3 plants ndash a retrospective analysis of the ACi curves from 109 speciesJournal of Experimental Botany 44 907ndash920 doi101093jxb445907
N Functional Plant Biology A Pivovaroff et al
wwwpublishcsiroaujournalsfpb
Anatomy of phylloclades
The Ruscus species possessed numerous anatomical traitsconsistent with benefits for both shade and drought tolerance(Table 2) The phylloclade cross-sections were symmetrical(Fig 2) and thus the cross-sectional anatomy of the adaxialand abaxial halves were not different for all traits (paired t-testP gt 005) and values were averaged within species The twospecies were similar in their substantial leaf thickness and inthe thickness of cuticle epidermis cell walls and water storagecompartment (Table 2) The fractions of the lamina occupied bythe epidermis mesophyll and water storage tissues were 1552ndash56 and 29ndash32 respectively
Both species had parallel longitudinal veins of three sizes(midrib intermediate and small veins) Consistent with droughtadaptation the two species had low Dm R aculeatus having thelower value andRmicroglossumhadahigher IVD (Table 2)Thetwo species had on average the same maximum conduitdiameters in their midribs intermediate veins and small veinsand the maximum conduit diameter decreased ~35 from themidrib to the small veins The average theoretical hydraulicconductivity of xylem conduits did not differ between thespecies and decreased by up to 86 from the midrib to theminor vein R aculeatus had on average walls that were 85thicker in the fibrous bundle sheath (Table 2)
Gas-exchange measurements
Consistent with adaptation to simultaneous shade and droughtthe Ruscus species had very low values for Rarea Rmass and gs(Table 3) relative to comparator temperate broadleaf evergreenspecies (Fig 3) The Aarea Amass LCP Vcmax and Jmax were alsovery low relative to comparator species (Fig 3 Table 3)consistent with shade adaptation The two Ruscus species hadvery high values for Ags (Table 3) consistent with excellentWUE Consistent with strong drought tolerance via retention ofstored water both species had very low values for leaf and stemgmin especially relative to comparator vascular plant species(Fig 3 Table 3)
Hydraulic conductance pressure volume curveparameters and leaf water storage
Consistent with expectations for combined drought and shadetolerance both Ruscus species had low Kshoot relative tocomparator tropical and temperate woody angiosperms (Fig 3Table 4) The pressurendashvolume curve parameters of Ruscuswereconsistent with achieving drought tolerance through tissue waterstorage Both species had less negative po and ytlp than meanvalues for comparative evergreen woody species (Fig 3Table 4) Notably both species had lower e values thancomparative evergreen woody species and higher RWCtlp andCft values (Fig 3 Table 4) consistent with drought tolerance
Nitrogen concentration and carbon isotope composition
Both species had high values for phyllyclade Narea and Nmass
relative to comparative temperate broadleaf evergreen speciesconsistent with drought adaptation (Fig 3) The d13C valueswerevery negative and typical of values often observed for understoryplants (da Silveira et al 1989) consistent with shade tolerance
Indeed the d13C values were notably strongly negative given thehigh WUE found for these species
Testing hypotheses for shade and drought tolerancewith trait survey data
Overall we quantified 57 traits for the twoRuscus species and for21 traits we had a priori hypotheses for a benefit for shadetolerance and for 24 traits we had a priori hypotheses for abenefit for drought tolerance Using comparative data we foundthat 16 of 21 hypotheses were supported for shade tolerance and22 of 24 were supported for drought tolerance Of the ninehypotheses for traits that would contribute to both shade anddrought tolerance simultaneously (ie expectations were both forhigher or lower values than comparative species) eight weresupported by trait data All these proportions were significantlyhigher than the 50 support that would have been expected toarise only from chance (P = 0001ndash0058 proportion tests)Notably in the seven cases when shade tolerance traits wouldconflict with drought tolerance traits four indicated a benefit fordrought tolerance rather than shade tolerance for both species(Tables 1ndash5)
Discussion
Both R aculeatus (Fig 3) and R microglossum showed traitvalues consistent with combined shade and drought toleranceNumerous traits were consistent with a combined shade anddrought tolerance through improving carbon balance enablinga conservative resource use ie via slow respiration and long-lived parts Other traits would contribute specifically to droughttolerance via reduced demand for water during activephotosynthesis and the ability to survive strong drought afterstomatal closure Notably many such traits were related to waterstorage providingnew insights into effective formsof succulencein shaded habitats This suite of traits would contributeimportantly to the ability of Ruscus to occupy shaded sitesprone to strong drought across a wide geographical range
Traits contributing to simultaneous drought and shadetolerance
Ruscus species showed specialisation associated withconservative resource use consistent with tolerance of shadeand drought These specialised traits included thick lamina andcomponent tissues that contribute to long tissue life-spans (shootslastgt5 years Sack et al 2003bWright et al 2004) AdditionallyRuscus species had very low gas-exchange rates including lowgs and lowKshoot whichwould correspond to a low investment invascular tissue (Tyree andZimmermann1983 Sack et al 2003b)and low Rarea Rmass Aarea and Amass all representing an ability tomaintain photosynthesis and growth with low requirements forlight and water
Traits contributing to drought tolerance
According to Jones et al (1992) drought tolerance can beachieved through avoidance of plant water deficits toleranceof plant water deficits or efficiency mechanisms Ruscus speciesshowed traits associated with drought tolerance either byproviding the ability to maintain photosynthesis and growth indrying soil andor the ability to survive chronic drought as
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology K
previously shown experimentally for R aculeatus (Sack 2004)Traits that would contribute to the ability to maintain gasexchange in drying soil include small leaf size high WUE andlow IVD High WUE achieved in part with high Narea meansthese Ruscus species can attain positive carbon balance evenwith extremely low gs Low IVD and water storage capacitanceallow the phylloclades to maintain water supply to the mesophylland tolerate transiently high evaporation rates for example dueto sunflecks without desiccating the leaf (Sack et al 2003a)Traits contributing to the ability of the phylloclades to surviveextended drought included those enabling a low evaporationrate per leaf area once stomata have shut such as low gmin inleaf and stem and those related to specialised water storagetissue linked with low leaf density low e and po values thatwere low in magnitude
Ruscus water storage
The water storage tissue of Ruscus that occupied a third of theleaf thickness although contributing most directly to droughttolerance is also consistent with shade tolerance given itscontribution to reduced tissue costs for the phylloclade as awhole The water storage tissue had thin cell walls reflected inthe low bulk e and low solute concentration contributing tobulk po values that were low in magnitude
This water storage tissue would also contribute to both typesof drought tolerance ndash the ability to maintain photosynthesis indrying soil and to survive after stomata have shut duringextended drought The lsquosucculencersquo of Ruscus phylloclades isdistinctive relative to more typical succulent-leafed andsucculent-stemmed species which tend to have high leaf watercontent and capacitance values (Vendramini et al 2002 Ogburnand Edwards 2012) In contrast in Ruscus species the SWCwas low relative to typical leaf succulent species and forR aculeatus Cft fell within the range of typical evergreenleaves We note that the strong tissue differentiation in Ruscus(ie separation of mesophyll cell and water storage in spaceand their distinctions in anatomy) would contribute to higheffectiveness of water storage even if the bulk tissue overallhad low SWC and capacitance Indeed across species theretends to be no relationship between the magnitude of SWC orcapacitance and the degree of within-leaf tissue differentiation(Ogburn and Edwards 2012) Notably such differentiationwould contribute special advantages for supply of waterwhether stomata are open or closed as the large thin-walledwater storage cells with low solute concentration can yield theirwater to supply the evaporative load while the photosynthetictissues can maintain their volume according to their thickerwalls and stronger solute concentration
Although the capacitance and SWC values were low forRuscus species these values would be more substantial ifconsidered relative to water demand Across several speciesCft has been found to correlate with Kshoot and with gs (Sacket al 2003a Blackman et al 2010) indicating that leaves tendto be built with capacitance to match their maximum flux ratesand thus to buffer the leaf water potential against surges intranspiration Thus because Ruscus has low gs when stomataare open and low Kshoot the capacitance would be expectedto supply transpiration transiently during sunflecks or high
VPD Likewise when stomata close the capacitance suppliesongoing water loss via cuticular conductance Given theextremely low gmin of Ruscus even its moderate Ctlp canenable survival for weeks (Sack et al 2003b Sack 2004)Further at turgor loss the water content would equalSWCRWCtlp and the relatively high RWCtlp wouldcontribute to the high water content once turgor is lost Thusthe lsquosucculencersquo of Ruscus is moderate in absolute terms butcombined with its other very strong mechanisms to reducetranspiration when stomata are open or closed ie low gs andgmin even this moderate capacitance would provide strongfunctionality
Water-use efficiency and carbon isotope composition
It was noteworthy that despite extremely high WUE valuesphylloclade d13C values of the Ruscus species were verynegative This presents a strong anomaly worthy of furtherinvestigation as species with high WUE typically have higherd13C (less negative ie closer to zero) The d13C can beinfluenced by a host of processes including source CO2 storedplant carbon and time-integrated CO2 concentration at the siteof carboxylation (Farquhar et al 1989) For Ruscus althoughd13C values were typical of an understory plant they did notappear to be drivenby internalCO2 concentration because the leafisotopic values were depleted in 13C whereas the high WUEdetermined by gas exchange would likely promote enrichedisotopic values It is more likely that d13C in this species wasdetermined by source CO2 or stored carbon or recycling ofrespired CO2 (da Silveira et al 1989) Our study individualsof Ruscus were cultivated in a shaded understory similar totheir natural habitat Previous studies have shown that there canbe higher concentrations of respired CO2 in the forest understorythan in the canopy resulting in more negative carbon isotoperatios in understory plant tissue than canopy plant tissue (daSilveira et al 1989) This is a function of decomposing leavesand litter cover slow air mixing as well as plant environmentalresponses
A second possible explanation for the d13C values ofRuscus is related to its growth form and phenology asRuscus has extensive rhizomes (Sack et al 2003b) whichact as a carbon store for the plant Using this recycled carbonduring the growth season when the plant may not be able tomeet its carbon requirement by photosynthesis alone becauseof low maximum rates under light limitation may alsocontribute to more negative d13C values (Vizzini 2003)Notably Ruscus stems are hollow and thus can also storerelatively large amounts of CO2 for use during a growth periodwhen carbon is otherwise limiting especially given very lowgs Some of this stored carbon might be photosyntheticallyfixed by the stem (Nilsen and Sharifi 1997) In each of thesecases respired stored or recycled CO2 would supply carbonthat was previously fixed by Rubisco with more negative d13Cvalues (ndash27o) than air (ndash8o)
There was also a dissonance between the d13C value and CiCa The fully mature phylloclades which were selected andused for gas-exchange and d13C measurements were producedduring late winter and early spring with mild temperatures(average at midday 190ndash220C) and low atmospheric VPD
L Functional Plant Biology A Pivovaroff et al
(average at midday 06ndash15 kPa) However the gas-exchangemeasurements were taken during summer with hightemperature (average at midday 300ndash355C) and high VPD(average at midday 25ndash35 kPa) We harvested thephylloclades that we used for gas exchange to determine thecarbon isotope ratio Although the low value of instantaneousCiCa may be caused by high temperature and high atmosphericVPD the very low value of d13C may represent the time whenthe phyllocladersquos carbon was assimilated (during mildtemperature and low VPD)
Implications for drought and shade tolerance Ruscusas a model
We found strong support for a large number of hypotheses fortrait-based shade and drought tolerance providing a strong traitbasis for combined tolerance Although the detailed functionaltrait survey conducted here is relatively novel in its breadth(see also Pasquet-Kok et al 2010) this is a logical extensionof the traditional approach for understanding the basis forplant adaptation to environment ie testing expectations forindividual traits established by previous studies of thefunctional significance of these traits in other species Weacknowledge there is some degree of uncertainty ininterpreting a large number of traits simultaneously based onstudies of other species First the interpretation of the value oftraits based on other species may not be in all cases equally validfor Ruscus Some trait variation may relate to other functionsFurther studies for example using mutants would be necessaryas conclusive evidence of the value of specific traits in a givenspecies However one advantage of testing numerousexpectations for each hypothesis is that the key finding will berobust to the removal of some traits from the analysis if thoseare later found to be inappropriate Ideally when a model forestimating plant performance from leaf traits becomes availableone could determine how the specific quantitative combinationsof traits presented here scales up to plant shade and droughttolerance
The shade and drought tolerance of Ruscus consistent withthe suite of traits examined here is one case demonstratinghow plants can avoid a general trade-off between shade anddrought tolerance Further Ruscus is noteworthy as one of onlya few stem photosynthetic plants that occupy a shaded habitatHistorically it is likely that shade tolerance preceded droughttolerance given this speciesrsquo ancestors were species of moisttropical forests (Kim et al 2010) and thus Ruscus or itsancestor apparently evolved drought tolerance whileexpanding its range into drier habitat or during past climatechange Considering its unique adaptations and trait valuesRuscus can serve as an excellent model for the basis ofcombined shade and drought tolerance
Acknowledgements
We thank the Mildred E Mathias Botanical Garden for access to garden andplants Andy Truong for assistance with measurements Howard GriffithsLouis Santiago and Cheryl Swift for helpful discussions and comments onthe manuscript This work was supported by National Science FoundationGrant IOB-0546784
References
Bartlett MK Scoffoni C Sack L (2012) The determinants of leaf turgor losspoint and prediction of drought tolerance of species and biomes a globalmeta-analysis Ecology Letters 15 393ndash405 doi101111j1461-0248201201751x
Blackman CJ Brodribb TJ Jordan GJ (2010) Leaf hydraulic vulnerability isrelated to conduit dimensions and drought resistance across a diverserange of woody angiosperms New Phytologist 188 1113ndash1123doi101111j1469-8137201003439x
Brodribb TJ Feild TS Jordan GJ (2007) Leaf maximum photosynthetic rateand venation are linked by hydraulicsPlant Physiology 144 1890ndash1898doi101104pp107101352
Caspersen JP (2001) Interspecific variation in sapling mortality in relationto growth and soil moisture Oikos 92 160ndash168 doi101034j1600-07062001920119x
Cochard H Nardini A Coll L (2004) Hydraulic architecture of leaf bladeswhere is the main resistance Plant Cell amp Environment 27 1257ndash1267doi101111j1365-3040200401233x
Cooney-Sovetts C Sattler R (1987) Phylloclade development in theAsparagaceae an example of homoeosis Botanical Journal of theLinnean Society 94 327ndash371 doi101111j1095-83391986tb01053x
Cowling R Rundel P Lamont BB ArroyoMK ArianoutsouM (1996) Plantdiversity in Mediterranean-climate regions Trends in Ecology ampEvolution 11 362ndash366 doi1010160169-5347(96)10044-6
da Silveira L Sternberg L Mulkey SS Wright SJ (1989) Ecologicalinterpretation of leaf carbon isotope ratios influence of respired carbondioxide Ecology 70 1317ndash1324 doi1023071938191
de Lillis M Fontanella A (1992) Comparative phenology and growth indifferent species of the Mediterranean maquis of central Italy PlantEcology 99-100 83ndash96 doi101007BF00118213
Dracup JA (1991)DroughtmonitoringStochasticHydrologyandHydraulics5 261ndash266 doi101007BF01543134
Engelbrecht BMJ Kursar TA (2003) Comparative drought-resistance ofseedlings of 28 species of co-occurring tropical woody plantsOecologia 136 383ndash393 doi101007s00442-003-1290-8
Farquhar GD von Caemmerer S (1980) A biochemical model ofphotosynthetic CO2 assimilation in leaves of C3 species Planta 14978ndash90 doi101007BF00386231
FarquharGD Ehleringer JR HubickK (1989) Carbon isotope discriminationand photosynthesis Annual Review of Plant Physiology and PlantMolecular Biology 40 503ndash537 doi101146annurevpp40060189002443
Givnish TJ (1988) Adaptation to sun and shade a whole-plant perspectiveAustralian Journal of Plant Physiology 15 63ndash92 doi101071PP9880063
Gulias J Flexas J Mus M Cifre J Lefi E Medrano H (2003) Relationshipbetweenmaximum leaf photosynthesis nitrogen content and specific leafarea inBalearic endemic and non-endemicMediterranean speciesAnnalsof Botany 92 215ndash222 doi101093aobmcg123
Haberlandt G (1914) lsquoPhysiological plant anatomyrsquo (MacMillan London)Hallik L Niinemets U Wright J (2009) Are species shade and drought
tolerance reflected in leaf-level structural and functional differentiationin Northern Hemisphere temperate woody flora New Phytologist 184257ndash274 doi101111j1469-8137200902918x
Jones HG (1992) lsquoPlants and microclimate a quantitative approach toenvironmental plant physiologyrsquo (Cambridge University PressCambridge)
Kerstiens G (1996) Cuticular water permeability and its physiologicalsignificance Journal of Experimental Botany 47 1813ndash1832doi101093jxb47121813
Kim JH Kim DK Forest F Fay MF Chase MW (2010) Molecularphylogenetics of Ruscaceae sensu lato and related families(Asparagales) based on plastid and nuclear DNA sequences Annals ofBotany 106 775ndash790 doi101093aobmcq167
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology M
Koide RT Robichaux RH Morse SR Smith CM (2000) Plant water statushydraulic resistance and capacitance In lsquoPlant physiological ecologyfield methods and instrumentationrsquo (Eds R Pearcy JR Ehleringer HMooney P Rundel) pp 161ndash183 (Kluwer Dordrecht The Netherlands)
Koumlrner C Farquhar GD Wong SC (1991) Carbon isotope discrimination byplants follows latitudinal and altitudinal trends Oecologia 88 30ndash40doi101007BF00328400
Lewis AM Boose ER (1995) Estimating volume flow rates through xylemconduits American Journal of Botany 82 1112ndash1116 doi1023072446063
Martiacutenez-Palleacute E Aronne G (1999) Flower development and reproductivecontinuity inMediterraneanRuscusaculeatusL (Liliaceae)Protoplasma208 58ndash64 doi101007BF01279075
Martiacutenez-Tilleriacutea K Loayza AP Sandquist DR Squeo FA (2012) Noevidence of a trade-off between drought and shade tolerance inseedlings of six coastal desert shrub species in north-central ChileJournal of Vegetation Science 23 1051ndash1061 doi101111j1654-1103201201427x
Matalas NC (1963) Probability distribution of low flows USGS ProfessionalPapers 434-A US Government Printing Office Washington DC
Niinemets U (1999) Research review components of leaf dry mass per area ndashthickness and density ndash alter leaf photosynthetic capacity in reversedirections in woody plants New Phytologist 144 35ndash47 doi101046j1469-8137199900466x
Niinemets U Valladares F (2006) Tolerance to shade drought andwaterlogging of temperate northern hemisphere trees and shrubsEcological Monographs 76 521ndash547 doi1018900012-9615(2006)076[0521TTSDAW]20CO2
Nilsen ET Sharifi MR (1997) Carbon isotope composition of legumes withphotosynthetic stems from Mediterranean and desert habitats AmericanJournal of Botany 84 1707ndash1713 doi1023072446469
Ogburn RM Edwards EJ (2012) Quantifying succulence a rapidphysiologically meaningful metric of plant water storage Plant Cell ampEnvironment 35 1533ndash1542 doi101111j1365-3040201202503x
Pasquet-Kok J Creese C Sack L (2010) Turning over a new lsquoleafrsquo multiplefunctional significances of leaves versus phyllodes in Hawaiian Acaciakoa Plant Cell amp Environment 33 2084ndash2100 doi101111j1365-3040201002207x
SackL (2004)Responses of temperatewoody seedlings to shade and droughtdo trade-offs limit potential niche differentiation Oikos 107 110ndash127doi101111j0030-1299200413184x
SackLFroleK (2006)Leaf structural diversity is related to hydraulic capacityin tropical rain forest trees Ecology 87 483ndash491 doi10189005-0710
Sack L Holbrook NM (2006) Leaf hydraulics Annual Review of PlantBiology 57 361ndash381 doi101146annurevarplant56032604144141
Sack L Melcher PJ Zwieniecki MA Holbrook NM (2002) The hydraulicconductance of the angiosperm leaf lamina a comparison of threemeasurement methods Journal of Experimental Botany 532177ndash2184 doi101093jxberf069
Sack L Cowan P Jaikumar N Holbrook NM (2003a) The ldquohydrologyrdquo ofleaves co-ordination of structure and function in temperate woodyspecies Plant Cell amp Environment 26 1343ndash1356 doi101046j0016-8025200301058x
Sack L Grubb P Maronon T (2003b) The functional morphology of juvenileplants tolerant of strong summer drought in shaded forest understoriesin southern Spain Plant Ecology 168 139ndash163 doi101023A1024423820136
SackLScoffoniCMcKownADFroleKRawlsMHavran JCTranHTranT (2012) Developmentally based scaling of leaf venation architectureexplains global ecological patterns Nature Communications 3 837doi101038ncomms1835
Sack L Chatelet D Scoffoni C PrometheusWiki contributors (2013a)ldquoEstimating the mesophyll surface area per leaf area from leaf celland tissue dimensions measured from transverse cross-sectionsrdquoPrometheusWiki Available at httpwwwpublishcsiroauprometheuswikitiki-pagehistoryphppage=Estimating the mesophyllsurface area per leaf area from leaf cell and tissue dimensionsmeasured from transverse cross-sectionsamppreview=16 [accessed June18 2013]
SackL Pasquet-Kok JPrometheusWiki contributors (2013b) ldquoLeaf pressure-volume curve parametersrdquo PrometheusWiki Available at httpprometheuswikipublishcsiroautiki-indexphppage=Leaf+pressure-volume+curve+parameters
Scoffoni C Pou A Aasamaa K Sack L (2008) The rapid light response ofleaf hydraulic conductance new evidence from two experimentalmethods Plant Cell amp Environment 31 1803ndash1812 doi101111j1365-3040200801884x
Smith T Huston M (1989) A theory of the spatial and temporal dynamics ofplant communities Vegetatio 83 49ndash69 doi101007BF00031680
Sterck F Markesteijn L Schieving F Poorter L (2011) Functional traitsdetermine trade-offs and niches in a tropical forest communityProceedings of the National Academy of Sciences of the United Statesof America 108 20 627ndash20 632 doi101073pnas1106950108
Thomas GS (1992) lsquoOrnamental shrubs climbers and bamboosrsquo (JohnMurray Publishers Great Britain)
TyreeMT ZimmermannMH (1983) lsquoXylem structure and the ascent of saprsquo(Springer-Verlag Berlin)
USDA ARS National Genetic Resources Program (2009) GermplasmResources Information Network (GRIN) online database NationalGermplasm Resources Laboratory Beltsville Maryland Availableathttpwwwars-gringovcgi-binnpgshtmltaxonpl410577[Accessed 20 July 2009]
Vendramini F Diacuteaz S Gurvich DE Wilson PJ Thompson K Hodgson JG(2002) Leaf traits as indicators of resource-use strategy in floras withsucculent species New Phytologist 154 147ndash157 doi101046j1469-8137200200357x
Vile D (2005) Specific leaf area and dry matter content estimate thicknessin laminar leaves Annals of Botany 96 1129ndash1136 doi101093aobmci264
Vizzini S (2003) d13C and d15N variability in Posidonia oceanica associatedwith seasonality and plant fraction Aquatic Botany 76 195ndash202doi101016S0304-3770(03)00052-4
Walters M Reich PB (1999) Research review low-light carbon balance andshade tolerance in the seedlings of woody plants dowinter deciduous andbroad-leaved evergreen species differ New Phytologist 143 143ndash154doi101046j1469-8137199900425x
Witkowski E Lamont BB (1991) Leaf specific mass confounds leaf densityand thickness Oecologia 88 486ndash493
Wright IJWestobyM (2003)Nutrient concentration resorption and lifespanleaf traits of Australian sclerophyll species Functional Ecology 1710ndash19 doi101046j1365-2435200300694x
Wright IJ Reich PB Westoby M Ackerly DD Baruch Z Bongers FCavender-Bares J Chapin T Cornelissen JHC Diemer M Flexas JGarnier E Groom PK Gulias J Hikosaka K Lamont BB Lee T LeeWLusk C Midgley JJ Navas M-L Niinemets U Oleksyn J Osada NPoorter H Poot P Prior L Pyankov VI Roumet C Thomas SC TjoelkerMG Veneklaas EJ Villar R (2004) The worldwide leaf economicsspectrum Nature 428 821ndash827 doi101038nature02403
Wullschleger SD (1993) Biochemical limitations to carbon assimilation inC3 plants ndash a retrospective analysis of the ACi curves from 109 speciesJournal of Experimental Botany 44 907ndash920 doi101093jxb445907
N Functional Plant Biology A Pivovaroff et al
wwwpublishcsiroaujournalsfpb
previously shown experimentally for R aculeatus (Sack 2004)Traits that would contribute to the ability to maintain gasexchange in drying soil include small leaf size high WUE andlow IVD High WUE achieved in part with high Narea meansthese Ruscus species can attain positive carbon balance evenwith extremely low gs Low IVD and water storage capacitanceallow the phylloclades to maintain water supply to the mesophylland tolerate transiently high evaporation rates for example dueto sunflecks without desiccating the leaf (Sack et al 2003a)Traits contributing to the ability of the phylloclades to surviveextended drought included those enabling a low evaporationrate per leaf area once stomata have shut such as low gmin inleaf and stem and those related to specialised water storagetissue linked with low leaf density low e and po values thatwere low in magnitude
Ruscus water storage
The water storage tissue of Ruscus that occupied a third of theleaf thickness although contributing most directly to droughttolerance is also consistent with shade tolerance given itscontribution to reduced tissue costs for the phylloclade as awhole The water storage tissue had thin cell walls reflected inthe low bulk e and low solute concentration contributing tobulk po values that were low in magnitude
This water storage tissue would also contribute to both typesof drought tolerance ndash the ability to maintain photosynthesis indrying soil and to survive after stomata have shut duringextended drought The lsquosucculencersquo of Ruscus phylloclades isdistinctive relative to more typical succulent-leafed andsucculent-stemmed species which tend to have high leaf watercontent and capacitance values (Vendramini et al 2002 Ogburnand Edwards 2012) In contrast in Ruscus species the SWCwas low relative to typical leaf succulent species and forR aculeatus Cft fell within the range of typical evergreenleaves We note that the strong tissue differentiation in Ruscus(ie separation of mesophyll cell and water storage in spaceand their distinctions in anatomy) would contribute to higheffectiveness of water storage even if the bulk tissue overallhad low SWC and capacitance Indeed across species theretends to be no relationship between the magnitude of SWC orcapacitance and the degree of within-leaf tissue differentiation(Ogburn and Edwards 2012) Notably such differentiationwould contribute special advantages for supply of waterwhether stomata are open or closed as the large thin-walledwater storage cells with low solute concentration can yield theirwater to supply the evaporative load while the photosynthetictissues can maintain their volume according to their thickerwalls and stronger solute concentration
Although the capacitance and SWC values were low forRuscus species these values would be more substantial ifconsidered relative to water demand Across several speciesCft has been found to correlate with Kshoot and with gs (Sacket al 2003a Blackman et al 2010) indicating that leaves tendto be built with capacitance to match their maximum flux ratesand thus to buffer the leaf water potential against surges intranspiration Thus because Ruscus has low gs when stomataare open and low Kshoot the capacitance would be expectedto supply transpiration transiently during sunflecks or high
VPD Likewise when stomata close the capacitance suppliesongoing water loss via cuticular conductance Given theextremely low gmin of Ruscus even its moderate Ctlp canenable survival for weeks (Sack et al 2003b Sack 2004)Further at turgor loss the water content would equalSWCRWCtlp and the relatively high RWCtlp wouldcontribute to the high water content once turgor is lost Thusthe lsquosucculencersquo of Ruscus is moderate in absolute terms butcombined with its other very strong mechanisms to reducetranspiration when stomata are open or closed ie low gs andgmin even this moderate capacitance would provide strongfunctionality
Water-use efficiency and carbon isotope composition
It was noteworthy that despite extremely high WUE valuesphylloclade d13C values of the Ruscus species were verynegative This presents a strong anomaly worthy of furtherinvestigation as species with high WUE typically have higherd13C (less negative ie closer to zero) The d13C can beinfluenced by a host of processes including source CO2 storedplant carbon and time-integrated CO2 concentration at the siteof carboxylation (Farquhar et al 1989) For Ruscus althoughd13C values were typical of an understory plant they did notappear to be drivenby internalCO2 concentration because the leafisotopic values were depleted in 13C whereas the high WUEdetermined by gas exchange would likely promote enrichedisotopic values It is more likely that d13C in this species wasdetermined by source CO2 or stored carbon or recycling ofrespired CO2 (da Silveira et al 1989) Our study individualsof Ruscus were cultivated in a shaded understory similar totheir natural habitat Previous studies have shown that there canbe higher concentrations of respired CO2 in the forest understorythan in the canopy resulting in more negative carbon isotoperatios in understory plant tissue than canopy plant tissue (daSilveira et al 1989) This is a function of decomposing leavesand litter cover slow air mixing as well as plant environmentalresponses
A second possible explanation for the d13C values ofRuscus is related to its growth form and phenology asRuscus has extensive rhizomes (Sack et al 2003b) whichact as a carbon store for the plant Using this recycled carbonduring the growth season when the plant may not be able tomeet its carbon requirement by photosynthesis alone becauseof low maximum rates under light limitation may alsocontribute to more negative d13C values (Vizzini 2003)Notably Ruscus stems are hollow and thus can also storerelatively large amounts of CO2 for use during a growth periodwhen carbon is otherwise limiting especially given very lowgs Some of this stored carbon might be photosyntheticallyfixed by the stem (Nilsen and Sharifi 1997) In each of thesecases respired stored or recycled CO2 would supply carbonthat was previously fixed by Rubisco with more negative d13Cvalues (ndash27o) than air (ndash8o)
There was also a dissonance between the d13C value and CiCa The fully mature phylloclades which were selected andused for gas-exchange and d13C measurements were producedduring late winter and early spring with mild temperatures(average at midday 190ndash220C) and low atmospheric VPD
L Functional Plant Biology A Pivovaroff et al
(average at midday 06ndash15 kPa) However the gas-exchangemeasurements were taken during summer with hightemperature (average at midday 300ndash355C) and high VPD(average at midday 25ndash35 kPa) We harvested thephylloclades that we used for gas exchange to determine thecarbon isotope ratio Although the low value of instantaneousCiCa may be caused by high temperature and high atmosphericVPD the very low value of d13C may represent the time whenthe phyllocladersquos carbon was assimilated (during mildtemperature and low VPD)
Implications for drought and shade tolerance Ruscusas a model
We found strong support for a large number of hypotheses fortrait-based shade and drought tolerance providing a strong traitbasis for combined tolerance Although the detailed functionaltrait survey conducted here is relatively novel in its breadth(see also Pasquet-Kok et al 2010) this is a logical extensionof the traditional approach for understanding the basis forplant adaptation to environment ie testing expectations forindividual traits established by previous studies of thefunctional significance of these traits in other species Weacknowledge there is some degree of uncertainty ininterpreting a large number of traits simultaneously based onstudies of other species First the interpretation of the value oftraits based on other species may not be in all cases equally validfor Ruscus Some trait variation may relate to other functionsFurther studies for example using mutants would be necessaryas conclusive evidence of the value of specific traits in a givenspecies However one advantage of testing numerousexpectations for each hypothesis is that the key finding will berobust to the removal of some traits from the analysis if thoseare later found to be inappropriate Ideally when a model forestimating plant performance from leaf traits becomes availableone could determine how the specific quantitative combinationsof traits presented here scales up to plant shade and droughttolerance
The shade and drought tolerance of Ruscus consistent withthe suite of traits examined here is one case demonstratinghow plants can avoid a general trade-off between shade anddrought tolerance Further Ruscus is noteworthy as one of onlya few stem photosynthetic plants that occupy a shaded habitatHistorically it is likely that shade tolerance preceded droughttolerance given this speciesrsquo ancestors were species of moisttropical forests (Kim et al 2010) and thus Ruscus or itsancestor apparently evolved drought tolerance whileexpanding its range into drier habitat or during past climatechange Considering its unique adaptations and trait valuesRuscus can serve as an excellent model for the basis ofcombined shade and drought tolerance
Acknowledgements
We thank the Mildred E Mathias Botanical Garden for access to garden andplants Andy Truong for assistance with measurements Howard GriffithsLouis Santiago and Cheryl Swift for helpful discussions and comments onthe manuscript This work was supported by National Science FoundationGrant IOB-0546784
References
Bartlett MK Scoffoni C Sack L (2012) The determinants of leaf turgor losspoint and prediction of drought tolerance of species and biomes a globalmeta-analysis Ecology Letters 15 393ndash405 doi101111j1461-0248201201751x
Blackman CJ Brodribb TJ Jordan GJ (2010) Leaf hydraulic vulnerability isrelated to conduit dimensions and drought resistance across a diverserange of woody angiosperms New Phytologist 188 1113ndash1123doi101111j1469-8137201003439x
Brodribb TJ Feild TS Jordan GJ (2007) Leaf maximum photosynthetic rateand venation are linked by hydraulicsPlant Physiology 144 1890ndash1898doi101104pp107101352
Caspersen JP (2001) Interspecific variation in sapling mortality in relationto growth and soil moisture Oikos 92 160ndash168 doi101034j1600-07062001920119x
Cochard H Nardini A Coll L (2004) Hydraulic architecture of leaf bladeswhere is the main resistance Plant Cell amp Environment 27 1257ndash1267doi101111j1365-3040200401233x
Cooney-Sovetts C Sattler R (1987) Phylloclade development in theAsparagaceae an example of homoeosis Botanical Journal of theLinnean Society 94 327ndash371 doi101111j1095-83391986tb01053x
Cowling R Rundel P Lamont BB ArroyoMK ArianoutsouM (1996) Plantdiversity in Mediterranean-climate regions Trends in Ecology ampEvolution 11 362ndash366 doi1010160169-5347(96)10044-6
da Silveira L Sternberg L Mulkey SS Wright SJ (1989) Ecologicalinterpretation of leaf carbon isotope ratios influence of respired carbondioxide Ecology 70 1317ndash1324 doi1023071938191
de Lillis M Fontanella A (1992) Comparative phenology and growth indifferent species of the Mediterranean maquis of central Italy PlantEcology 99-100 83ndash96 doi101007BF00118213
Dracup JA (1991)DroughtmonitoringStochasticHydrologyandHydraulics5 261ndash266 doi101007BF01543134
Engelbrecht BMJ Kursar TA (2003) Comparative drought-resistance ofseedlings of 28 species of co-occurring tropical woody plantsOecologia 136 383ndash393 doi101007s00442-003-1290-8
Farquhar GD von Caemmerer S (1980) A biochemical model ofphotosynthetic CO2 assimilation in leaves of C3 species Planta 14978ndash90 doi101007BF00386231
FarquharGD Ehleringer JR HubickK (1989) Carbon isotope discriminationand photosynthesis Annual Review of Plant Physiology and PlantMolecular Biology 40 503ndash537 doi101146annurevpp40060189002443
Givnish TJ (1988) Adaptation to sun and shade a whole-plant perspectiveAustralian Journal of Plant Physiology 15 63ndash92 doi101071PP9880063
Gulias J Flexas J Mus M Cifre J Lefi E Medrano H (2003) Relationshipbetweenmaximum leaf photosynthesis nitrogen content and specific leafarea inBalearic endemic and non-endemicMediterranean speciesAnnalsof Botany 92 215ndash222 doi101093aobmcg123
Haberlandt G (1914) lsquoPhysiological plant anatomyrsquo (MacMillan London)Hallik L Niinemets U Wright J (2009) Are species shade and drought
tolerance reflected in leaf-level structural and functional differentiationin Northern Hemisphere temperate woody flora New Phytologist 184257ndash274 doi101111j1469-8137200902918x
Jones HG (1992) lsquoPlants and microclimate a quantitative approach toenvironmental plant physiologyrsquo (Cambridge University PressCambridge)
Kerstiens G (1996) Cuticular water permeability and its physiologicalsignificance Journal of Experimental Botany 47 1813ndash1832doi101093jxb47121813
Kim JH Kim DK Forest F Fay MF Chase MW (2010) Molecularphylogenetics of Ruscaceae sensu lato and related families(Asparagales) based on plastid and nuclear DNA sequences Annals ofBotany 106 775ndash790 doi101093aobmcq167
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology M
Koide RT Robichaux RH Morse SR Smith CM (2000) Plant water statushydraulic resistance and capacitance In lsquoPlant physiological ecologyfield methods and instrumentationrsquo (Eds R Pearcy JR Ehleringer HMooney P Rundel) pp 161ndash183 (Kluwer Dordrecht The Netherlands)
Koumlrner C Farquhar GD Wong SC (1991) Carbon isotope discrimination byplants follows latitudinal and altitudinal trends Oecologia 88 30ndash40doi101007BF00328400
Lewis AM Boose ER (1995) Estimating volume flow rates through xylemconduits American Journal of Botany 82 1112ndash1116 doi1023072446063
Martiacutenez-Palleacute E Aronne G (1999) Flower development and reproductivecontinuity inMediterraneanRuscusaculeatusL (Liliaceae)Protoplasma208 58ndash64 doi101007BF01279075
Martiacutenez-Tilleriacutea K Loayza AP Sandquist DR Squeo FA (2012) Noevidence of a trade-off between drought and shade tolerance inseedlings of six coastal desert shrub species in north-central ChileJournal of Vegetation Science 23 1051ndash1061 doi101111j1654-1103201201427x
Matalas NC (1963) Probability distribution of low flows USGS ProfessionalPapers 434-A US Government Printing Office Washington DC
Niinemets U (1999) Research review components of leaf dry mass per area ndashthickness and density ndash alter leaf photosynthetic capacity in reversedirections in woody plants New Phytologist 144 35ndash47 doi101046j1469-8137199900466x
Niinemets U Valladares F (2006) Tolerance to shade drought andwaterlogging of temperate northern hemisphere trees and shrubsEcological Monographs 76 521ndash547 doi1018900012-9615(2006)076[0521TTSDAW]20CO2
Nilsen ET Sharifi MR (1997) Carbon isotope composition of legumes withphotosynthetic stems from Mediterranean and desert habitats AmericanJournal of Botany 84 1707ndash1713 doi1023072446469
Ogburn RM Edwards EJ (2012) Quantifying succulence a rapidphysiologically meaningful metric of plant water storage Plant Cell ampEnvironment 35 1533ndash1542 doi101111j1365-3040201202503x
Pasquet-Kok J Creese C Sack L (2010) Turning over a new lsquoleafrsquo multiplefunctional significances of leaves versus phyllodes in Hawaiian Acaciakoa Plant Cell amp Environment 33 2084ndash2100 doi101111j1365-3040201002207x
SackL (2004)Responses of temperatewoody seedlings to shade and droughtdo trade-offs limit potential niche differentiation Oikos 107 110ndash127doi101111j0030-1299200413184x
SackLFroleK (2006)Leaf structural diversity is related to hydraulic capacityin tropical rain forest trees Ecology 87 483ndash491 doi10189005-0710
Sack L Holbrook NM (2006) Leaf hydraulics Annual Review of PlantBiology 57 361ndash381 doi101146annurevarplant56032604144141
Sack L Melcher PJ Zwieniecki MA Holbrook NM (2002) The hydraulicconductance of the angiosperm leaf lamina a comparison of threemeasurement methods Journal of Experimental Botany 532177ndash2184 doi101093jxberf069
Sack L Cowan P Jaikumar N Holbrook NM (2003a) The ldquohydrologyrdquo ofleaves co-ordination of structure and function in temperate woodyspecies Plant Cell amp Environment 26 1343ndash1356 doi101046j0016-8025200301058x
Sack L Grubb P Maronon T (2003b) The functional morphology of juvenileplants tolerant of strong summer drought in shaded forest understoriesin southern Spain Plant Ecology 168 139ndash163 doi101023A1024423820136
SackLScoffoniCMcKownADFroleKRawlsMHavran JCTranHTranT (2012) Developmentally based scaling of leaf venation architectureexplains global ecological patterns Nature Communications 3 837doi101038ncomms1835
Sack L Chatelet D Scoffoni C PrometheusWiki contributors (2013a)ldquoEstimating the mesophyll surface area per leaf area from leaf celland tissue dimensions measured from transverse cross-sectionsrdquoPrometheusWiki Available at httpwwwpublishcsiroauprometheuswikitiki-pagehistoryphppage=Estimating the mesophyllsurface area per leaf area from leaf cell and tissue dimensionsmeasured from transverse cross-sectionsamppreview=16 [accessed June18 2013]
SackL Pasquet-Kok JPrometheusWiki contributors (2013b) ldquoLeaf pressure-volume curve parametersrdquo PrometheusWiki Available at httpprometheuswikipublishcsiroautiki-indexphppage=Leaf+pressure-volume+curve+parameters
Scoffoni C Pou A Aasamaa K Sack L (2008) The rapid light response ofleaf hydraulic conductance new evidence from two experimentalmethods Plant Cell amp Environment 31 1803ndash1812 doi101111j1365-3040200801884x
Smith T Huston M (1989) A theory of the spatial and temporal dynamics ofplant communities Vegetatio 83 49ndash69 doi101007BF00031680
Sterck F Markesteijn L Schieving F Poorter L (2011) Functional traitsdetermine trade-offs and niches in a tropical forest communityProceedings of the National Academy of Sciences of the United Statesof America 108 20 627ndash20 632 doi101073pnas1106950108
Thomas GS (1992) lsquoOrnamental shrubs climbers and bamboosrsquo (JohnMurray Publishers Great Britain)
TyreeMT ZimmermannMH (1983) lsquoXylem structure and the ascent of saprsquo(Springer-Verlag Berlin)
USDA ARS National Genetic Resources Program (2009) GermplasmResources Information Network (GRIN) online database NationalGermplasm Resources Laboratory Beltsville Maryland Availableathttpwwwars-gringovcgi-binnpgshtmltaxonpl410577[Accessed 20 July 2009]
Vendramini F Diacuteaz S Gurvich DE Wilson PJ Thompson K Hodgson JG(2002) Leaf traits as indicators of resource-use strategy in floras withsucculent species New Phytologist 154 147ndash157 doi101046j1469-8137200200357x
Vile D (2005) Specific leaf area and dry matter content estimate thicknessin laminar leaves Annals of Botany 96 1129ndash1136 doi101093aobmci264
Vizzini S (2003) d13C and d15N variability in Posidonia oceanica associatedwith seasonality and plant fraction Aquatic Botany 76 195ndash202doi101016S0304-3770(03)00052-4
Walters M Reich PB (1999) Research review low-light carbon balance andshade tolerance in the seedlings of woody plants dowinter deciduous andbroad-leaved evergreen species differ New Phytologist 143 143ndash154doi101046j1469-8137199900425x
Witkowski E Lamont BB (1991) Leaf specific mass confounds leaf densityand thickness Oecologia 88 486ndash493
Wright IJWestobyM (2003)Nutrient concentration resorption and lifespanleaf traits of Australian sclerophyll species Functional Ecology 1710ndash19 doi101046j1365-2435200300694x
Wright IJ Reich PB Westoby M Ackerly DD Baruch Z Bongers FCavender-Bares J Chapin T Cornelissen JHC Diemer M Flexas JGarnier E Groom PK Gulias J Hikosaka K Lamont BB Lee T LeeWLusk C Midgley JJ Navas M-L Niinemets U Oleksyn J Osada NPoorter H Poot P Prior L Pyankov VI Roumet C Thomas SC TjoelkerMG Veneklaas EJ Villar R (2004) The worldwide leaf economicsspectrum Nature 428 821ndash827 doi101038nature02403
Wullschleger SD (1993) Biochemical limitations to carbon assimilation inC3 plants ndash a retrospective analysis of the ACi curves from 109 speciesJournal of Experimental Botany 44 907ndash920 doi101093jxb445907
N Functional Plant Biology A Pivovaroff et al
wwwpublishcsiroaujournalsfpb
(average at midday 06ndash15 kPa) However the gas-exchangemeasurements were taken during summer with hightemperature (average at midday 300ndash355C) and high VPD(average at midday 25ndash35 kPa) We harvested thephylloclades that we used for gas exchange to determine thecarbon isotope ratio Although the low value of instantaneousCiCa may be caused by high temperature and high atmosphericVPD the very low value of d13C may represent the time whenthe phyllocladersquos carbon was assimilated (during mildtemperature and low VPD)
Implications for drought and shade tolerance Ruscusas a model
We found strong support for a large number of hypotheses fortrait-based shade and drought tolerance providing a strong traitbasis for combined tolerance Although the detailed functionaltrait survey conducted here is relatively novel in its breadth(see also Pasquet-Kok et al 2010) this is a logical extensionof the traditional approach for understanding the basis forplant adaptation to environment ie testing expectations forindividual traits established by previous studies of thefunctional significance of these traits in other species Weacknowledge there is some degree of uncertainty ininterpreting a large number of traits simultaneously based onstudies of other species First the interpretation of the value oftraits based on other species may not be in all cases equally validfor Ruscus Some trait variation may relate to other functionsFurther studies for example using mutants would be necessaryas conclusive evidence of the value of specific traits in a givenspecies However one advantage of testing numerousexpectations for each hypothesis is that the key finding will berobust to the removal of some traits from the analysis if thoseare later found to be inappropriate Ideally when a model forestimating plant performance from leaf traits becomes availableone could determine how the specific quantitative combinationsof traits presented here scales up to plant shade and droughttolerance
The shade and drought tolerance of Ruscus consistent withthe suite of traits examined here is one case demonstratinghow plants can avoid a general trade-off between shade anddrought tolerance Further Ruscus is noteworthy as one of onlya few stem photosynthetic plants that occupy a shaded habitatHistorically it is likely that shade tolerance preceded droughttolerance given this speciesrsquo ancestors were species of moisttropical forests (Kim et al 2010) and thus Ruscus or itsancestor apparently evolved drought tolerance whileexpanding its range into drier habitat or during past climatechange Considering its unique adaptations and trait valuesRuscus can serve as an excellent model for the basis ofcombined shade and drought tolerance
Acknowledgements
We thank the Mildred E Mathias Botanical Garden for access to garden andplants Andy Truong for assistance with measurements Howard GriffithsLouis Santiago and Cheryl Swift for helpful discussions and comments onthe manuscript This work was supported by National Science FoundationGrant IOB-0546784
References
Bartlett MK Scoffoni C Sack L (2012) The determinants of leaf turgor losspoint and prediction of drought tolerance of species and biomes a globalmeta-analysis Ecology Letters 15 393ndash405 doi101111j1461-0248201201751x
Blackman CJ Brodribb TJ Jordan GJ (2010) Leaf hydraulic vulnerability isrelated to conduit dimensions and drought resistance across a diverserange of woody angiosperms New Phytologist 188 1113ndash1123doi101111j1469-8137201003439x
Brodribb TJ Feild TS Jordan GJ (2007) Leaf maximum photosynthetic rateand venation are linked by hydraulicsPlant Physiology 144 1890ndash1898doi101104pp107101352
Caspersen JP (2001) Interspecific variation in sapling mortality in relationto growth and soil moisture Oikos 92 160ndash168 doi101034j1600-07062001920119x
Cochard H Nardini A Coll L (2004) Hydraulic architecture of leaf bladeswhere is the main resistance Plant Cell amp Environment 27 1257ndash1267doi101111j1365-3040200401233x
Cooney-Sovetts C Sattler R (1987) Phylloclade development in theAsparagaceae an example of homoeosis Botanical Journal of theLinnean Society 94 327ndash371 doi101111j1095-83391986tb01053x
Cowling R Rundel P Lamont BB ArroyoMK ArianoutsouM (1996) Plantdiversity in Mediterranean-climate regions Trends in Ecology ampEvolution 11 362ndash366 doi1010160169-5347(96)10044-6
da Silveira L Sternberg L Mulkey SS Wright SJ (1989) Ecologicalinterpretation of leaf carbon isotope ratios influence of respired carbondioxide Ecology 70 1317ndash1324 doi1023071938191
de Lillis M Fontanella A (1992) Comparative phenology and growth indifferent species of the Mediterranean maquis of central Italy PlantEcology 99-100 83ndash96 doi101007BF00118213
Dracup JA (1991)DroughtmonitoringStochasticHydrologyandHydraulics5 261ndash266 doi101007BF01543134
Engelbrecht BMJ Kursar TA (2003) Comparative drought-resistance ofseedlings of 28 species of co-occurring tropical woody plantsOecologia 136 383ndash393 doi101007s00442-003-1290-8
Farquhar GD von Caemmerer S (1980) A biochemical model ofphotosynthetic CO2 assimilation in leaves of C3 species Planta 14978ndash90 doi101007BF00386231
FarquharGD Ehleringer JR HubickK (1989) Carbon isotope discriminationand photosynthesis Annual Review of Plant Physiology and PlantMolecular Biology 40 503ndash537 doi101146annurevpp40060189002443
Givnish TJ (1988) Adaptation to sun and shade a whole-plant perspectiveAustralian Journal of Plant Physiology 15 63ndash92 doi101071PP9880063
Gulias J Flexas J Mus M Cifre J Lefi E Medrano H (2003) Relationshipbetweenmaximum leaf photosynthesis nitrogen content and specific leafarea inBalearic endemic and non-endemicMediterranean speciesAnnalsof Botany 92 215ndash222 doi101093aobmcg123
Haberlandt G (1914) lsquoPhysiological plant anatomyrsquo (MacMillan London)Hallik L Niinemets U Wright J (2009) Are species shade and drought
tolerance reflected in leaf-level structural and functional differentiationin Northern Hemisphere temperate woody flora New Phytologist 184257ndash274 doi101111j1469-8137200902918x
Jones HG (1992) lsquoPlants and microclimate a quantitative approach toenvironmental plant physiologyrsquo (Cambridge University PressCambridge)
Kerstiens G (1996) Cuticular water permeability and its physiologicalsignificance Journal of Experimental Botany 47 1813ndash1832doi101093jxb47121813
Kim JH Kim DK Forest F Fay MF Chase MW (2010) Molecularphylogenetics of Ruscaceae sensu lato and related families(Asparagales) based on plastid and nuclear DNA sequences Annals ofBotany 106 775ndash790 doi101093aobmcq167
Trait-based shade and drought tolerance in Ruscus Functional Plant Biology M
Koide RT Robichaux RH Morse SR Smith CM (2000) Plant water statushydraulic resistance and capacitance In lsquoPlant physiological ecologyfield methods and instrumentationrsquo (Eds R Pearcy JR Ehleringer HMooney P Rundel) pp 161ndash183 (Kluwer Dordrecht The Netherlands)
Koumlrner C Farquhar GD Wong SC (1991) Carbon isotope discrimination byplants follows latitudinal and altitudinal trends Oecologia 88 30ndash40doi101007BF00328400
Lewis AM Boose ER (1995) Estimating volume flow rates through xylemconduits American Journal of Botany 82 1112ndash1116 doi1023072446063
Martiacutenez-Palleacute E Aronne G (1999) Flower development and reproductivecontinuity inMediterraneanRuscusaculeatusL (Liliaceae)Protoplasma208 58ndash64 doi101007BF01279075
Martiacutenez-Tilleriacutea K Loayza AP Sandquist DR Squeo FA (2012) Noevidence of a trade-off between drought and shade tolerance inseedlings of six coastal desert shrub species in north-central ChileJournal of Vegetation Science 23 1051ndash1061 doi101111j1654-1103201201427x
Matalas NC (1963) Probability distribution of low flows USGS ProfessionalPapers 434-A US Government Printing Office Washington DC
Niinemets U (1999) Research review components of leaf dry mass per area ndashthickness and density ndash alter leaf photosynthetic capacity in reversedirections in woody plants New Phytologist 144 35ndash47 doi101046j1469-8137199900466x
Niinemets U Valladares F (2006) Tolerance to shade drought andwaterlogging of temperate northern hemisphere trees and shrubsEcological Monographs 76 521ndash547 doi1018900012-9615(2006)076[0521TTSDAW]20CO2
Nilsen ET Sharifi MR (1997) Carbon isotope composition of legumes withphotosynthetic stems from Mediterranean and desert habitats AmericanJournal of Botany 84 1707ndash1713 doi1023072446469
Ogburn RM Edwards EJ (2012) Quantifying succulence a rapidphysiologically meaningful metric of plant water storage Plant Cell ampEnvironment 35 1533ndash1542 doi101111j1365-3040201202503x
Pasquet-Kok J Creese C Sack L (2010) Turning over a new lsquoleafrsquo multiplefunctional significances of leaves versus phyllodes in Hawaiian Acaciakoa Plant Cell amp Environment 33 2084ndash2100 doi101111j1365-3040201002207x
SackL (2004)Responses of temperatewoody seedlings to shade and droughtdo trade-offs limit potential niche differentiation Oikos 107 110ndash127doi101111j0030-1299200413184x
SackLFroleK (2006)Leaf structural diversity is related to hydraulic capacityin tropical rain forest trees Ecology 87 483ndash491 doi10189005-0710
Sack L Holbrook NM (2006) Leaf hydraulics Annual Review of PlantBiology 57 361ndash381 doi101146annurevarplant56032604144141
Sack L Melcher PJ Zwieniecki MA Holbrook NM (2002) The hydraulicconductance of the angiosperm leaf lamina a comparison of threemeasurement methods Journal of Experimental Botany 532177ndash2184 doi101093jxberf069
Sack L Cowan P Jaikumar N Holbrook NM (2003a) The ldquohydrologyrdquo ofleaves co-ordination of structure and function in temperate woodyspecies Plant Cell amp Environment 26 1343ndash1356 doi101046j0016-8025200301058x
Sack L Grubb P Maronon T (2003b) The functional morphology of juvenileplants tolerant of strong summer drought in shaded forest understoriesin southern Spain Plant Ecology 168 139ndash163 doi101023A1024423820136
SackLScoffoniCMcKownADFroleKRawlsMHavran JCTranHTranT (2012) Developmentally based scaling of leaf venation architectureexplains global ecological patterns Nature Communications 3 837doi101038ncomms1835
Sack L Chatelet D Scoffoni C PrometheusWiki contributors (2013a)ldquoEstimating the mesophyll surface area per leaf area from leaf celland tissue dimensions measured from transverse cross-sectionsrdquoPrometheusWiki Available at httpwwwpublishcsiroauprometheuswikitiki-pagehistoryphppage=Estimating the mesophyllsurface area per leaf area from leaf cell and tissue dimensionsmeasured from transverse cross-sectionsamppreview=16 [accessed June18 2013]
SackL Pasquet-Kok JPrometheusWiki contributors (2013b) ldquoLeaf pressure-volume curve parametersrdquo PrometheusWiki Available at httpprometheuswikipublishcsiroautiki-indexphppage=Leaf+pressure-volume+curve+parameters
Scoffoni C Pou A Aasamaa K Sack L (2008) The rapid light response ofleaf hydraulic conductance new evidence from two experimentalmethods Plant Cell amp Environment 31 1803ndash1812 doi101111j1365-3040200801884x
Smith T Huston M (1989) A theory of the spatial and temporal dynamics ofplant communities Vegetatio 83 49ndash69 doi101007BF00031680
Sterck F Markesteijn L Schieving F Poorter L (2011) Functional traitsdetermine trade-offs and niches in a tropical forest communityProceedings of the National Academy of Sciences of the United Statesof America 108 20 627ndash20 632 doi101073pnas1106950108
Thomas GS (1992) lsquoOrnamental shrubs climbers and bamboosrsquo (JohnMurray Publishers Great Britain)
TyreeMT ZimmermannMH (1983) lsquoXylem structure and the ascent of saprsquo(Springer-Verlag Berlin)
USDA ARS National Genetic Resources Program (2009) GermplasmResources Information Network (GRIN) online database NationalGermplasm Resources Laboratory Beltsville Maryland Availableathttpwwwars-gringovcgi-binnpgshtmltaxonpl410577[Accessed 20 July 2009]
Vendramini F Diacuteaz S Gurvich DE Wilson PJ Thompson K Hodgson JG(2002) Leaf traits as indicators of resource-use strategy in floras withsucculent species New Phytologist 154 147ndash157 doi101046j1469-8137200200357x
Vile D (2005) Specific leaf area and dry matter content estimate thicknessin laminar leaves Annals of Botany 96 1129ndash1136 doi101093aobmci264
Vizzini S (2003) d13C and d15N variability in Posidonia oceanica associatedwith seasonality and plant fraction Aquatic Botany 76 195ndash202doi101016S0304-3770(03)00052-4
Walters M Reich PB (1999) Research review low-light carbon balance andshade tolerance in the seedlings of woody plants dowinter deciduous andbroad-leaved evergreen species differ New Phytologist 143 143ndash154doi101046j1469-8137199900425x
Witkowski E Lamont BB (1991) Leaf specific mass confounds leaf densityand thickness Oecologia 88 486ndash493
Wright IJWestobyM (2003)Nutrient concentration resorption and lifespanleaf traits of Australian sclerophyll species Functional Ecology 1710ndash19 doi101046j1365-2435200300694x
Wright IJ Reich PB Westoby M Ackerly DD Baruch Z Bongers FCavender-Bares J Chapin T Cornelissen JHC Diemer M Flexas JGarnier E Groom PK Gulias J Hikosaka K Lamont BB Lee T LeeWLusk C Midgley JJ Navas M-L Niinemets U Oleksyn J Osada NPoorter H Poot P Prior L Pyankov VI Roumet C Thomas SC TjoelkerMG Veneklaas EJ Villar R (2004) The worldwide leaf economicsspectrum Nature 428 821ndash827 doi101038nature02403
Wullschleger SD (1993) Biochemical limitations to carbon assimilation inC3 plants ndash a retrospective analysis of the ACi curves from 109 speciesJournal of Experimental Botany 44 907ndash920 doi101093jxb445907
N Functional Plant Biology A Pivovaroff et al
wwwpublishcsiroaujournalsfpb
Koide RT Robichaux RH Morse SR Smith CM (2000) Plant water statushydraulic resistance and capacitance In lsquoPlant physiological ecologyfield methods and instrumentationrsquo (Eds R Pearcy JR Ehleringer HMooney P Rundel) pp 161ndash183 (Kluwer Dordrecht The Netherlands)
Koumlrner C Farquhar GD Wong SC (1991) Carbon isotope discrimination byplants follows latitudinal and altitudinal trends Oecologia 88 30ndash40doi101007BF00328400
Lewis AM Boose ER (1995) Estimating volume flow rates through xylemconduits American Journal of Botany 82 1112ndash1116 doi1023072446063
Martiacutenez-Palleacute E Aronne G (1999) Flower development and reproductivecontinuity inMediterraneanRuscusaculeatusL (Liliaceae)Protoplasma208 58ndash64 doi101007BF01279075
Martiacutenez-Tilleriacutea K Loayza AP Sandquist DR Squeo FA (2012) Noevidence of a trade-off between drought and shade tolerance inseedlings of six coastal desert shrub species in north-central ChileJournal of Vegetation Science 23 1051ndash1061 doi101111j1654-1103201201427x
Matalas NC (1963) Probability distribution of low flows USGS ProfessionalPapers 434-A US Government Printing Office Washington DC
Niinemets U (1999) Research review components of leaf dry mass per area ndashthickness and density ndash alter leaf photosynthetic capacity in reversedirections in woody plants New Phytologist 144 35ndash47 doi101046j1469-8137199900466x
Niinemets U Valladares F (2006) Tolerance to shade drought andwaterlogging of temperate northern hemisphere trees and shrubsEcological Monographs 76 521ndash547 doi1018900012-9615(2006)076[0521TTSDAW]20CO2
Nilsen ET Sharifi MR (1997) Carbon isotope composition of legumes withphotosynthetic stems from Mediterranean and desert habitats AmericanJournal of Botany 84 1707ndash1713 doi1023072446469
Ogburn RM Edwards EJ (2012) Quantifying succulence a rapidphysiologically meaningful metric of plant water storage Plant Cell ampEnvironment 35 1533ndash1542 doi101111j1365-3040201202503x
Pasquet-Kok J Creese C Sack L (2010) Turning over a new lsquoleafrsquo multiplefunctional significances of leaves versus phyllodes in Hawaiian Acaciakoa Plant Cell amp Environment 33 2084ndash2100 doi101111j1365-3040201002207x
SackL (2004)Responses of temperatewoody seedlings to shade and droughtdo trade-offs limit potential niche differentiation Oikos 107 110ndash127doi101111j0030-1299200413184x
SackLFroleK (2006)Leaf structural diversity is related to hydraulic capacityin tropical rain forest trees Ecology 87 483ndash491 doi10189005-0710
Sack L Holbrook NM (2006) Leaf hydraulics Annual Review of PlantBiology 57 361ndash381 doi101146annurevarplant56032604144141
Sack L Melcher PJ Zwieniecki MA Holbrook NM (2002) The hydraulicconductance of the angiosperm leaf lamina a comparison of threemeasurement methods Journal of Experimental Botany 532177ndash2184 doi101093jxberf069
Sack L Cowan P Jaikumar N Holbrook NM (2003a) The ldquohydrologyrdquo ofleaves co-ordination of structure and function in temperate woodyspecies Plant Cell amp Environment 26 1343ndash1356 doi101046j0016-8025200301058x
Sack L Grubb P Maronon T (2003b) The functional morphology of juvenileplants tolerant of strong summer drought in shaded forest understoriesin southern Spain Plant Ecology 168 139ndash163 doi101023A1024423820136
SackLScoffoniCMcKownADFroleKRawlsMHavran JCTranHTranT (2012) Developmentally based scaling of leaf venation architectureexplains global ecological patterns Nature Communications 3 837doi101038ncomms1835
Sack L Chatelet D Scoffoni C PrometheusWiki contributors (2013a)ldquoEstimating the mesophyll surface area per leaf area from leaf celland tissue dimensions measured from transverse cross-sectionsrdquoPrometheusWiki Available at httpwwwpublishcsiroauprometheuswikitiki-pagehistoryphppage=Estimating the mesophyllsurface area per leaf area from leaf cell and tissue dimensionsmeasured from transverse cross-sectionsamppreview=16 [accessed June18 2013]
SackL Pasquet-Kok JPrometheusWiki contributors (2013b) ldquoLeaf pressure-volume curve parametersrdquo PrometheusWiki Available at httpprometheuswikipublishcsiroautiki-indexphppage=Leaf+pressure-volume+curve+parameters
Scoffoni C Pou A Aasamaa K Sack L (2008) The rapid light response ofleaf hydraulic conductance new evidence from two experimentalmethods Plant Cell amp Environment 31 1803ndash1812 doi101111j1365-3040200801884x
Smith T Huston M (1989) A theory of the spatial and temporal dynamics ofplant communities Vegetatio 83 49ndash69 doi101007BF00031680
Sterck F Markesteijn L Schieving F Poorter L (2011) Functional traitsdetermine trade-offs and niches in a tropical forest communityProceedings of the National Academy of Sciences of the United Statesof America 108 20 627ndash20 632 doi101073pnas1106950108
Thomas GS (1992) lsquoOrnamental shrubs climbers and bamboosrsquo (JohnMurray Publishers Great Britain)
TyreeMT ZimmermannMH (1983) lsquoXylem structure and the ascent of saprsquo(Springer-Verlag Berlin)
USDA ARS National Genetic Resources Program (2009) GermplasmResources Information Network (GRIN) online database NationalGermplasm Resources Laboratory Beltsville Maryland Availableathttpwwwars-gringovcgi-binnpgshtmltaxonpl410577[Accessed 20 July 2009]
Vendramini F Diacuteaz S Gurvich DE Wilson PJ Thompson K Hodgson JG(2002) Leaf traits as indicators of resource-use strategy in floras withsucculent species New Phytologist 154 147ndash157 doi101046j1469-8137200200357x
Vile D (2005) Specific leaf area and dry matter content estimate thicknessin laminar leaves Annals of Botany 96 1129ndash1136 doi101093aobmci264
Vizzini S (2003) d13C and d15N variability in Posidonia oceanica associatedwith seasonality and plant fraction Aquatic Botany 76 195ndash202doi101016S0304-3770(03)00052-4
Walters M Reich PB (1999) Research review low-light carbon balance andshade tolerance in the seedlings of woody plants dowinter deciduous andbroad-leaved evergreen species differ New Phytologist 143 143ndash154doi101046j1469-8137199900425x
Witkowski E Lamont BB (1991) Leaf specific mass confounds leaf densityand thickness Oecologia 88 486ndash493
Wright IJWestobyM (2003)Nutrient concentration resorption and lifespanleaf traits of Australian sclerophyll species Functional Ecology 1710ndash19 doi101046j1365-2435200300694x
Wright IJ Reich PB Westoby M Ackerly DD Baruch Z Bongers FCavender-Bares J Chapin T Cornelissen JHC Diemer M Flexas JGarnier E Groom PK Gulias J Hikosaka K Lamont BB Lee T LeeWLusk C Midgley JJ Navas M-L Niinemets U Oleksyn J Osada NPoorter H Poot P Prior L Pyankov VI Roumet C Thomas SC TjoelkerMG Veneklaas EJ Villar R (2004) The worldwide leaf economicsspectrum Nature 428 821ndash827 doi101038nature02403
Wullschleger SD (1993) Biochemical limitations to carbon assimilation inC3 plants ndash a retrospective analysis of the ACi curves from 109 speciesJournal of Experimental Botany 44 907ndash920 doi101093jxb445907
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wwwpublishcsiroaujournalsfpb
