UvA-DARE (Digital Academic Repository) A novel approach to ...Submitted 7 March 2018 Accepted 1 June...

UvA-DARE is a service provided by the library of the University of Amsterdam (httpsdareuvanl)

UvA-DARE (Digital Academic Repository)

A novel approach to study the morphology and chemistry of pollen in aphylogenetic context applied to the halophytic taxon Nitraria L (Nitrariaceae)

Woutersen A Jardine PE Bogotaacute-Angel RG Zhang H-X Silvestro D Antonelli AGogna E Erkens RHJ Gosling WD Dupont-Nivet G Hoorn CDOI107717peerj5055Publication date2018Document VersionFinal published versionPublished inPeerJLicenseCC BY

Link to publication

Citation for published version (APA)Woutersen A Jardine P E Bogotaacute-Angel R G Zhang H-X Silvestro D Antonelli AGogna E Erkens R H J Gosling W D Dupont-Nivet G amp Hoorn C (2018) A novelapproach to study the morphology and chemistry of pollen in a phylogenetic context appliedto the halophytic taxon Nitraria L (Nitrariaceae) PeerJ 6 [e5055]httpsdoiorg107717peerj5055

General rightsIt is not permitted to download or to forwarddistribute the text or part of it without the consent of the author(s)andor copyright holder(s) other than for strictly personal individual use unless the work is under an opencontent license (like Creative Commons)

DisclaimerComplaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests pleaselet the Library know stating your reasons In case of a legitimate complaint the Library will make the materialinaccessible andor remove it from the website Please Ask the Library httpsubauvanlencontact or a letterto Library of the University of Amsterdam Secretariat Singel 425 1012 WP Amsterdam The Netherlands Youwill be contacted as soon as possible

Download date24 Aug 2021

Submitted 7 March 2018Accepted 1 June 2018Published 19 July 2018

Corresponding authorsAmber WoutersenamberwoutersengmailcomCarina Hoorn mchoornuvanl

Academic editorAlastair Potts

Additional Information andDeclarations can be found onpage 25

DOI 107717peerj5055

Copyright2018 Woutersen et al

Distributed underCreative Commons CC-BY 40

OPEN ACCESS

A novel approach to study the morphologyand chemistry of pollen in a phylogeneticcontext applied to the halophytic taxonNitraria L(Nitrariaceae)Amber Woutersen1 Phillip E Jardine23 Raul Giovanni Bogotaacute-Angel14Hong-Xiang Zhang5 Daniele Silvestro6 Alexandre Antonelli6789 Elena Gogna10Roy HJ Erkens10 William D Gosling1 Guillaume Dupont-Nivet211 andCarina Hoorn1

1University of Amsterdam Institute for Biodiversity and Ecosystem Dynamics (IBED) AmsterdamThe Netherlands

2University of Potsdam Institute of Earth and Environmental Science Potsdam Germany3University of Muumlnster Institute of Geology and Palaeontology Muumlnster Germany4Universidad Distrital Francisco Joseacute de Caldas Facultad del Medio Ambiente y Recursos Naturales BogotaacuteColombia

5Key Laboratory of Biogeography and Bioresource in Arid Land XinjiangInstitute of Ecology and Geography China Academy of Sciences Urumqi China

6Gothenburg Global Biodiversity Centre Goumlteborg Sweden7University of Gothenburg Department of Biological and Environmental Sciences Goumlteborg Sweden8Gothenburg Botanical Garden Goumlteborg Sweden9Harvard University Department of Organismic and Evolutionary Biology Cambridge MAUnited States of America

10Maastricht University Maastricht Science Programme Maastricht The Netherlands11Universiteacute de Rennes Geosciences Rennes UMR-CNRS Rennes FranceThese authors contributed equally to this work

ABSTRACTNitraria is a halophytic taxon (ie adapted to saline environments) that belongs tothe plant family Nitrariaceae and is distributed from the Mediterranean across Asiainto the south-eastern tip of Australia This taxon is thought to have originated in Asiaduring the Paleogene (66ndash23 Ma) alongside the proto-Paratethys epicontinental seaThe evolutionary history of Nitraria might hold important clues on the links betweenclimatic and biotic evolution but limited taxonomic documentation of this taxon hasthus far hindered this line of research Here we investigate if the pollen morphologyand the chemical composition of the pollen wall are informative of the evolutionaryhistory of Nitraria and could explain if origination along the proto-Paratethys anddispersal to the Tibetan Plateau was simultaneous or a secondary process To answerthese questions we applied a novel approach consisting of a combination of FourierTransform Infrared spectroscopy (FTIR) to determine the chemical composition ofthe pollen wall and pollen morphological analyses using Light Microscopy (LM)and Scanning Electron Microscopy (SEM) We analysed our data using ordinations(principal components analysis and non-metricmultidimensional scaling) and directlymapped it on the Nitrariaceae phylogeny to produce a phylomorphospace and aphylochemospace Our LM SEM and FTIR analyses show clear morphological andchemical differences between the sister groups Peganum andNitraria Differences in the

How to cite this article Woutersen et al (2018) A novel approach to study the morphology and chemistry of pollen in a phylogeneticcontext applied to the halophytic taxon Nitraria L(Nitrariaceae) PeerJ 6e5055 DOI 107717peerj5055

morphological and chemical characteristics of highland species (Nitraria schoberi Nsphaerocarpa N sibirica and N tangutorum) and lowland species (Nitraria billardiereiand N retusa) are very subtle with phylogenetic history appearing to be a moreimportant control on Nitraria pollen than local environmental conditions Ourapproach shows a compelling consistency between the chemical and morphologicalcharacteristics of the eight studiedNitrariaceae species and these traits are in agreementwith the phylogenetic tree Taken together this demonstrates how novel methods forstudying fossil pollen can facilitate the evolutionary investigation of living and extincttaxa and the environments they represent

Subjects Biogeography Evolutionary Studies Molecular Biology PaleontologyKeywords FTIR LM SEM Paratethys Tibet Sporopollenin Mediterranean Steppe-desertAustralia Palynology

INTRODUCTIONThe steppe biome occurs all around the world covering large areas in Africa the MiddleEast Australia and Eurasia (see lsquoDesert and Xeric Shrublandsrsquo and lsquoMediterranean ForestsWoodlands and Scrubsrsquo distribution in Olson et al 2001 Fig 1) In particular the originsand development of the Eurasian steppe are of great interest since they have been influencedbymdashand therefore may be a tracer ofmdashpast Asian monsoon intensification and inlanddrying of central Asia since the Paleogene The Asian steppes are thought to have formed inresponse to the India-Asia collision Tibetan Plateau uplift the retreat of the epicontinentalproto-Paratethys sea (which is called the Paratethys sea from 339ndash281 Ma Popov et al2004) and global cooling (eg Han et al 2016 Bosboom et al 2014 Bougeois et al 2018)Although these processes have shaped present-day population structures and distributionslittle is known about the evolution of the Tibetan steppe environment

Palynological records suggest that the taxonomic composition in the Tibetan regionduring the Paleogene is distinctly different from Present and in the Paleogene the Tibetansteppe is characterized by a predominance of shrubs such as Ephedraceae and Nitrariaceae(Wang 1990 Hoorn et al 2012 Han et al 2016) How the steppe-desert evolved throughtime is insufficiently known and a better understanding of the ancestral steppe taxa maytherefore help us to clarify the overall evolution of the Tibetan steppe environment

Nitrariaceae is a family of flowering plants within the order Sapindales that are commonin arid climates (Noble amp Whalley 1978 APG Angiosperm Phylogeny Group Sheahan2010) The family includes the genera Peganum Tetradiclis and Nitraria Peganum occursin arid to semiarid parts of Africa theMiddle East and central Asia and has been introducedin theUnited StatesMexico andAustralia (Abbott Lepak amp Daniel 2007Zhao et al 2011)Peganum currently comprises four accepted species (Peganum harmala P nigellastrumP mexicanum and P multisectum The Plant List 2013) with Peganum harmala being wellknown because of its medicinal properties (Moloudizargari et al 2013Niroumand Farzaeiamp Amin 2015) Tetradiclis tenella is the only accepted species within the genus Tetradiclis(The Plant List 2013) and is mainly distributed in the Middle East (GBIF 2018) Nitraria

Woutersen et al (2018) PeerJ DOI 107717peerj5055 231

N billardierei

N retusa

N schoberiN sibiricaN sphaerocarpaN tangutorum

N roborowskii

Genus Nitraria

Legend

N pamirica

Figure 1 Map showing the present distribution of the genusNitraria The climate map was modifiedafter Peel Finlayson amp McMahon (2007) Coordinates of the occurrences obtained from GBIF and Tropi-cos can be found in Appendix S1

Full-size DOI 107717peerj5055fig-1

comprises seven accepted species namely Nitraria pamirica N retusa N roborowskiiN schoberi N sibirica N sphaerocarpa and N tangutorum (The Plant List 2013) Thedistributional range of this taxon is Central Asia southern Europe and west to northAfrica However the genus is also present in Papua New Guinea and Australia (Fig 1)where it is reported as N schoberi and N billardierei with the latter remaining unresolved(ie neither lsquoAcceptedrsquo or lsquoSynonymrsquo The Plant List 2013)

All extant species of Nitraria are halophytes (ie adapted to saline environments)and tend to grow in coastal regions of the Mediterranean the Middle-East andSouthern Australia In the Mediterranean and Middle-East region Nitraria retusa co-occurs with species like Artemisia herba-alba Cousinia stenocephala Phlomis bruguieriand Capparis ovata (httpswwwworldwildlifeorgecoregionspa0805) and in SouthernAustralia Nitraria schoberi and N billardierei co-occur among others with Arthrocnemumhalocnemoides Atriplex paludosa Disphyma blackii and Suaeda australis (State Herbariumof South Australia 1921-2001) A diversion from this coastal pattern is the occurrence ofNitraria in the steppe-desert vegetation of the Tibetan Plateau (Noble amp Whalley 1978Zhang et al 2015) This steppe is characterized by Poaceae Asteraceae Chenopodiaceae(Haloxylon ammodendron Salsola collina Kalidium foliatum and Ceratoides lateens) andminor amounts of Ephedraceae (Ephedra przewalski) and Nitrariaceae among others(Zhou Sun amp Chen 1990 in Cai et al 2012)

Based on molecular data Nitraria is believed to have originated along the coast of theproto-Paratethys Sea and in the Tibetan region during the early Paleogene or perhaps evenin the Cretaceous (Temirbayeva amp Zhang 2015 Zhang et al 2015) From here the taxon


is thought to have dispersed westward to western central Asia and Africa (Temirbayeva ampZhang 2015) Subsequent diversification of modernNitraria is thought to have occurred inthe Miocene (896 Ma) with a dispersal to Australia only taking place during the Pliocene(261 Ma) (Zhang et al 2015) Nevertheless the palynological record suggests thatNitrariapeaked in abundance and taxonomic diversity during the late Cretaceous-Paleogene(Wang 1990 Hoorn et al 2012 Miao et al 2016 Han et al 2016) The latter is in goodagreement with the current consensus on the paleogeographic regional evolution with theIndia-Asia collision occurring around 50ndash60 Ma and contributing to the retreat of theproto-Paratethys sea until 35 Ma and the growth of the Tibetan Plateau until today (egMolnar Boos amp Battisti 2010 Favre et al 2015 Najman et al 2017 Bosboom et al 2017)

The Paleogene pollen floras of todayrsquos Tibetan Plateau area were once dominated byEphedraceae and Nitrariaceae but at the end of the Eocene (34 Ma) the two familieswere gradually replaced by Chenopodiaceae Poaceae and Asteraceae (Song Wang amp Mao2008 in Han et al 2016 Dupont-Nivet Hoorn amp Konert 2008 Hoorn et al 2012) Thiscompositional change coincides with the shift from greenhouse to icehouse conditionsduring the Cenozoic which in turn resulted in the aridification of central Asia (DeContoamp Pollard 2003 Dupont-Nivet et al 2007 Abels et al 2011) Moreover the retreat of theproto-Paratethys Sea and the impact of the uplift of the NE Tibetan Plateau might havealso played a role in the Eocene-Oligocene vegetation shift (Wang et al 2012 Bosboom etal 2014)

Nitraria has proven to be an important taxon in tracing the evolution of the Tibetansteppe environment (Wang 1990) and pollen of extant Nitraria were previouslydocumented by Agababian amp Tumanian (in Russian 1972 original source not found)Xi amp Sun (in Chinese 1987) Xi amp Zhang (in Chinese 1991) Nurbay amp Pan (in Chinese2003) Perveen amp Qaiser (2006) and Hoorn et al (2012) Yet there are extensive knowledgegaps in the identification and documentation of extant and fossil Nitraria pollen and onlyrecently a phylogeny of the genus was published by Zhang et al (2015)

Molecular and palynological data together form a powerful toolset to explore theevolutionary history (Chung Elisens amp Skvarla 2010 Xie amp Li 2012 Kriebel Khabbazianamp Sytsma 2017) by constructing a phylomorphospace In this approach a projection of thephylogenetic tree is plotted into a morphospace which can lead to a better understandingof the history and direction of morphological diversification within taxa (Sidlauskas 2008Stone 2003)

The aim of this study is to assess whether the morphological chemical and geneticfeatures of extant Nitraria pollen permit us to differentiate between species andcould provide keys for identification of fossil pollen Furthermore we investigated ifmorphological and chemical evolution are concomitant with molecular evolution Toresolve the relation between the morphology and distribution of the taxon we documenthere the ecology and distribution of extant Nitraria and present a systematic descriptionof their pollen morphology accompanied by LM and SEM photography In addition wepresent data on the organic chemistry of the pollen wall which was analysed by meansof Fourier Transformed Infrared (FTIR) microspectroscopy We integrated both themorphological and the chemical data sets with the molecular phylogeny of Zhang et al


(2015) resulting in a phylomorphospace and lsquolsquophylochemospacersquorsquo This direct combinationof data types has to our knowledge never been used before and allows us to integratetaxonomic and palaeoclimatic information inferred from the chemistry of the pollen wall

MATERIALS AND METHODSDistribution of modern NitrariaOccurrence data and altitudinal range of Nitraria were collected from GBIF (httpsdoiorg1015468dlyvwv2n) and Tropicos (httpwwwtropicosorg 5012611534600191 50126114 50126119) lsquoObservation-onlyrsquo and lsquoPreserved specimenrsquo recordswere used in order to reduce false occurrences Data were obtained for the speciesNitraria billardierei N retusa N roborowskii N schoberi N sibirica N sphaerocarpa andN tangutorum No data were available for N pamirica Coordinates of the data points aregiven in Appendix S1 Additional occurrences of N schoberi were obtained from Zhang etal (2015) and the occurrence of N pamirica was obtained from Pan Shen amp Chen (1999)Species distributions of Nitraria pamirica N roborowskii N sibirica N sphaerocarpa andN tangutorum published in Fang Wang amp Tang (2011) were also taken into accountOccurrence data were supplemented with altitudinal data of the specimens we obtainedourselves for pollen analysis (Appendix S2)

Processing methodPlant material was collected from various herbaria and collections (see Appendix S2)but for N pamirica no samples could be obtained Pollen was collected from anthers andstandard acetolysis (Erdman 1986) was applied to remove the cytoplasmic content and toclean the exine residues were oven-dried while placed in glycerine Pollen was mounted inglycerine jelly on slides and sealed with paraffin

Palynological description of NitrariaPollen description in general followed the format proposed for the Northwest EuropeanPollen Flora which is based on the terminology of Punt et al (2007) MoreoverHesse et al(2009) was also taken into account This was complemented with extra information whennecessary The description included information on polarity polar symmetry equatorialand polar shape number position and type of apertures exine thickness structure andsculpture All measurements are given in micrometers (microm) average and maximum andminimum ranges are also presented

Light microscopy (LM) and Scanning Electron Microscopy (SEM)For LM analysis the pollen was described using a Leitz microscope under 1000times mag-nification In total 20 grains per species were measured in polar and equatorial viewsMicrophotographs were taken with a Normarski Differential Interference Contrast (DIC)microscope (Bercovici Hadley amp Villanueva-Amadoz 2009) While taking the photos thevarying z-axis was recorded and images were later combined through manual z-stackingin Adobe Rcopy Photoshop Rcopy This stacking technique combines different layers to provide asense of depth to the images with a result comparable to 3D photography The position


of the pollen grains that were photographed in LM are recorded with England Finder (EF)and positions indicated in the figure caption

SEM images of Nitraria were obtained at the Servei de Microscopia (UniversitatAutogravenoma de Barcelona Spain) After standard acetolysis the pollen were depositedin stubs with a carbon adhesive disc coated with a mixture composed of Au-Pt and thespecimens were scanned with a Zeiss EVO and kV ranging from 5 to 15 We also usedField Emission Scanning Electron Microscopy (FESEM) on a Zeiss Merlin to obtainmicrophotography of Nitraria schoberi

In addition SEM is applied to selected specimens of Nitraria at the Microscopy Depart-ment of the Faculty of Health Medicine and Life Sciences Maastricht University (Maas-tricht the Netherlands) using a Philips XL30 Scanning ElectronMicroscope (Philips Eind-hoven TheNetherlands) In the figure caption we indicated which photographs weremadeat Maastricht University (UM) and which at Universitat Autogravenoma de Barcelona (UAB)

Morphological analysisWe compiled a character matrix comprising 24 morphological characters describing20 individual specimens from each of the eight Nitraria species included in this study(Supplemental Information 1) We used Gowerrsquos coefficient (Gower 1971) to calculatedissimilarity between pairs of individuals because the morphological characters are a mixof continuous and discrete variables We then ordinated the Gower distance matrix usingnon-metric multidimensional scaling (NMDS) with the number of axes set to two NMDSseeks to find the optimal arrangement of points in ordination space such that the rankorder of dissimilarities is preserved (Kruskal 1964) To directly relate pollen morphologyto the molecular phylogeny of Zhang et al (2015 Fig 2) we plotted a phylomorphospace(Sidlauskas 2008Hopkins amp Smith 2015) We did this by first calculating the species meanscore for each NMDS axis We then mapped the phylogeny onto the ordination using thelsquophylomorphospacersquo function from the R package phytools (Revell 2012) which plots aprojection of the tree into a two-dimensional morphospace (Sidlauskas 2008 Hopkins ampSmith 2015) using estimated maximum likelihood ancestral character states (here NMDSaxis scores) for each internal node on the phylogeny (Revell 2012Hopkins amp Smith 2015)

Chemical palynologyWe used FTIR microspectroscopy to characterise the chemistry of the extant Nitrariaand Peganum pollen FTIR is a vibrational spectroscopic technique that is increasinglybeing used by palynologists as a taxonomic (Zimmermann 2010 Steemans et al2010 Zimmermann amp Kohler 2014 Bonhomme Prasad amp Gaucherel 2013 BağcıoğluZimmermann amp Kohler 2015 Zimmermann et al 2015 Julier et al 2016 Zimmermann etal 2016) and palaeoclimatic (Lomax et al 2008 Fraser et al 2014 Lomax amp Fraser 2015Jardine et al 2016 Jardine et al 2017) tool By directly analysing the chemical signatureof palynomorphs it is possible to identify and classify plant taxa with high accuracy(Zimmermann et al 2016) including distinguishing between closely related species that aremorphologically highly similar (Zimmermann 2010 Julier et al 2016) FTIR spectroscopyprovides a non-destructive and time-efficient way of pollen analysis that can operate at


Nitraria billardierei

Nitraria retusa

Nitraria schoberi

Nitraria sibirica

Nitraria sphaerocarpa

Nitraria tangutorum

Peganum harmala

Peganum nigellastrum

80 60 40 20 0

Ma

Figure 2 Molecular phylogeny ofNitraria and Peganummodified after Zhang et al (2015)Full-size DOI 107717peerj5055fig-2

small sample sizes including down to individual pollen grains (Zimmermann et al 2015)and can add valuable information to the knowledge obtained by classical morphologicalanalysis (Zimmermann amp Kohler 2014)Chemical data were generated using a Thermo Scientific Nicolet iN10 MX Dual detector

IR microscope at the University of Amsterdam Individual pollen grains were mountedonto ZnSe windows and analysed using 256 scans and a resolution of 4 We used thesame acetolysed pollen residues as for the morphological analysis because acetolysis doesnot impact upon the pollen wall chemistry (Jardine et al 2017 Jardine et al 2015) Abackground spectrum was taken prior to each measurement and automatically subtractedfrom the pollen spectrum The system was purged using a dry nitrogen feed to removeatmospheric CO2 and H2O variations

Chemical spectra were generated from individual pollen grains with a target of 20 grainsper species For both N sibirica and N sphaerocarpa a lack of material meant that this wasnot possible and these species are represented by nine and three spectra respectively Sinceno high-quality spectra were obtained for P harmala this species is not present in thechemical dataset


The spectra were processed in a number of ways First baseline drift was corrected bysubtracting a 2nd order polynomial baseline from each spectrum The spectra were thenz-score standardised (ie so that each had amean of 0 and a variance of 1) by subtracting themean spectrum value and dividing by the standard deviation this removes any differencesin absolute absorbance caused by differences in material thickness (Jardine et al 2015) Wealso treated the spectra using Savitzky-Golay (SG) smoothing and differentiation (Savitzkyamp Golay 1964) since these steps have been shown to improve signal detectability inmultivariate chemical analyses (Julier et al 2016) To find the best combination of spectralprocessing parameters we treated the spectra in four different ways no processing SGsmoothing SG smoothing plus first derivative and SG smoothing plus second derivativewith the SG smoothing window size varying between 5 and 43 We then used k-nearestneighbour (k-nn) classification coupled with leave one out cross validation (Varmuza ampFilzmoser 2009 Julier et al 2016) and calculated the classification success rate (ie thepercentage of spectra that were correctly classified to species level) for each treatmentcombination with the rationale that the parameter combination that produces the bestclassification success rate will also produce the best separation of taxa in multivariateanalyses

We ordinated the processed spectra using principal components analysis (PCA)which has been widely used with chemometric and environmental data (Varmuza ampFilzmoser 2009) As with other ordination techniques PCA finds axes of variation sothat complex multivariate data can be viewed on a small number of axes Unlike NMDSPCA uses absolute values rather than ranked distances ordinating the data such that theEuclidean distance between objects (in the present case chemical spectra) is preserved(Varmuza amp Filzmoser 2009) The continuous nature of chemical spectral data makesPCA an appropriate ordination technique and unlike the morphometric data a separatedistance matrix does not need to be computed and then ordinated We also produced aphylochemospace plot by running a PCA on the mean spectrum for each species and thenmapping on the phylogeny following the same procedure as for the phylomorphospace

All data analyses were carried out using the programme R v342 (R Core Team 2017)with RStudio v10143 (RStudio Team 2016) with the packages ape v50 (Paradis Claudeamp Strimmer 2004) baseline v12-1 (Liland amp Mevik 2015) class v73-14 (Venables ampRipley 2002) FD v10-12 (Laliberteacute Legendre amp Shipley 2014) prospectr v013 (Stevensamp Ramirez-Lopez 2013) and vegan v24-4 (Oksanen et al 2017) Datasets and R code areprovided in the Supplemental Information 3

List of studied species materialNitraria billardierei DC New South Wales South-Western Plains Australia SourcesTrinity College Dublin-Herbarium (Ireland) Royal Botanic Gardens amp Domain Trust andState Herbarium of South Australia Adelaide Australia (see Appendix S2) Slide IBED R8510Nitraria retusa (Forssk) Asch Northern shore of the Dead Sea Israel SourceUniversitatisHebraicae Hiersolymitanae (Flora Palaestinae Exciccata) (see Appendix S2) Slide IBED R8507


Nitraria roborowski Kom Slides borrowed from F Schultz in 2011 and reported in Hoornet al (2012) Description from microphotographyNitraria schoberi L South of Teheran Qom province Iran SourceMiddle East collectionMorteza Djamali (see Appendix S2) Slide Hugo de Vries 6934Nitraria sibirica Pall Toudaohu Alxa Inner Mongolia China Source Institute of Botany(PE herbarium) Chinese Academy of Sciences Beijing China (see Appendix S2) SlideIBED R 8503Nitraria sphaerocarpa Maxim Yinjisha Kashgar Xinjiang China Source Institute ofBotany (PE herbarium) Chinese Academy of Sciences Beijing China (see Appendix S2)Slide IBED R 8504Nitraria tangutorumBobrov Sanshenggong Alxa InnerMongolia China Source Instituteof Botany (PE herbarium) Chinese Academy of Sciences Beijing China (see AppendixS2) Slide IBED R 8505Peganum harmala var multisectum Maxim Guozhigou Huocheng Xinjiang ChinaSource Institute of Botany (PE herbarium) Chinese Academy of Sciences Beijing China(see Appendix S2) Slide IBED R 8506Peganum nigellastrum Bunge Toudaohu Alxa Inner Mongolia China Source Instituteof Botany (PE herbarium) Chinese Academy of Sciences Beijing China (see AppendixS2) Slide IBED R 8503

RESULTSModern distribution of NitrariaOur data compilation of extant records shows that Nitraria is mostly distributed in centralAsia with N pamirica N roborowskii N sphaerocarpa N sibirica and N tangutorumoccurring only at the Tibetan Plateau and northwest China (Fig 1) According to ouroccurrence data and distributional data from Fang Wang amp Tang (2011) the speciesN roborowskii N sphaerocarpa N sibirica and N tangutorum are all well representedacross the Tibetan Plateau whereas N pamirica only occurs in the most northwesternpart of China (Fang Wang amp Tang 2011) and in southwest China (Pan Shen amp Chen1999) Furthermore N schoberi occurs on the Tibetan Plateau as well as in Iran PapuaNew Guinea and south Australia Outside the Tibetan Plateau N billardierei solely occursin south Australia and N retusa is restricted to the northern part of Africa Notably Nbillardierei and N retusa occur in coastal (lowland) areas N sibirica and N tangutorumboth occur in lowland and highland environments and N roborowskii N schoberi and Nsphaerocarpa are restricted to mountain areas (Table 1)

Systematic palynologyBelow we report on the pollen morphological characterisation of all the taxa surveyedproviding measurements and listing all specimens analysed

Family Nitrariaceae Bercht amp J PresAccording to this study and the general pollen characterization of Tetradiclis (Sheahan2011) pollen of Nitrariaceae is monad isopolar and radially symmetric Pollen shape


Table 1 Altitudinal range ofNitraria and Peganum Range obtained from GBIF and Tropicos and sup-plemented with altitudes of our obtained specimens

Species Altitudinal range (m)

Nitraria billardierei DC 1 ndash 155Nitraria retusa (Forssk) Asch 0 ndash 115Nitraria roborowskii Kom 910 ndash 4402Nitraria schoberi L 820 ndash 3670Nitraria sibirica Pall 6 ndash 4368Nitraria sphaerocarpa Maxim 912 ndash 1867Nitraria tangutorum Bobrov 497 ndash 4560

varies from circular to triangular or quandrangular convex in polar view and ellipticprolate subprolate to oblate spheroidal or spheroidal in equatorial view The aperture istricolporate or hexacolpate (Tetradiclis) with a lalongate endoaperture The exine is tectateor semitectate with a finely striate (Tetradiclis) striate striate perforate or reticulate surface

Genus Nitraria LPollen is monad isopolar radially symmetric The pollen shape is circular pseudo-hexagonal hexagonal or triangular convex in polar view and elliptic prolate to subprolatein equatorial view The aperture is tricolporate and colpi are long and narrow usuallyconstricted at the equator and have costae colpi and a fastigium The endoapertures areformed by lalongate pores which are elliptical to rhomboidal in shape The exine is tectatenexine thicker than sexine surface striate to striate-perforate

Species Nitraria billardierei DCLM description Pollen class Tricolporate Monad isopolar radially symmetric prolate(Fig 3a AndashE) Aperture Tricolporate Ectoaperturemdashcolpus long (sim56 of polar axis)straight narrow equatorially constricted with acute ends apocolpia asymmetric Margin(defined as an area of exine around an ectocolpus that is differentiated from the remainder ofthe sexine either in ornamentation or by difference in thickness Punt et al 2007) observedin polar view costae colpi and fastigium conspicuous in equatorial view Endoaperturemdashporus lalongate rhomboidal to elliptic in shape Exine Tectate exine slightly thicker inpolar areas in relation with the equatorial region nexine thicker than sexine Columellaehardly visible tectum thin Sculpture Surface striate hardly observed in LM Outline Polarview circular to pseudo-hexagonal Equatorial view elliptical Measurements length 5068(458ndash54) microm width 3038 (28ndash365) microm (see summary in Appendix S3)

SEM descriptionMonads are elliptic radially symmetric (Fig 4A) The colpus is almostas long as the polar axis slightly open pore eventually conspicuous (Figs 4Bndash4C) Howeverin 4D is it clearly visible that the colpi do not quite meet at the pole Exine ornamentation isstriate with some perforations (Fig 4B2) Striae are tightly (densely) packed and relativelyshort Striae in colpus area are running parallel to the polar axis (Fig 4A2) whereas in thearea of the mesocolpus and apocolpia they are running slightly counter clock direction toslightly perpendicular (Figs 4A1 4B1 4C)


A1 B1 B2A2 A3 C1 C2

D1 D2 E1 E2

F1 F2

G H1 H2

I1 I2 I3

J1 J2

D3 E3

F3 F4

J3

L1 L2K1 K2 K3

10 mm

Figure 3 a LMmicrographs of recentNitraria and Peganum pollen Specimen courtesy and EnglandFinder reference are given for each specimen (A1-E3)N billardierei (A1-3) equatorial view (TrinityCollege Dublin (TCD) Ireland Q45-1) (B1-2) semipolar view (TCD Ireland V38-3) (C1-2) polar viewsmall morphotype (Royal Botanic Gardens amp Domain Trust and State Herbarium of South Australia 43-3) (D1-3) equatorial view at mesocolpus area (Royal Botanic Gardens amp Domain Trust and State Herbar-ium of South Australia L-M 29-24) (E1-3) equatorial view at colporus area (Royal Botanic Gardens ampDomain Trust and State Herbarium of South Australia M29-2) (F1-H2)N retusa (F1-4) equatorial viewat mesocolpus and colporus area (Martin Luther Universitat Germany N42-2) (G) equatorial view smallmorphotype (Martin Luther Universitat Germany H46-1) (H1-2) polar view (Martin Luther Universi-tat Germany N42) (I1-J3)N roborowskii (J1-3) equatorial view small morphotype reproduced fromHoorn et al (2012) (J1-2) equatorial view big morphotype reproduced from Hoorn et al (2012) (K1-L2)N schoberi (Middle East Pollen Reference Collection (MEPRC) France K1-3) equatorial view at col-porus area (MEPRC France EF coordinates not recorded) (L1-2) polar view (MEPRC France V19-2)(continued on next page )



A1 B1 B2A2 A3

C1 C2 C3 D1 D2 E1 E2

F1 F2 G1 G2 H1 H2

I1 I2 I3 J1 J2 K1 K2J3

L1 L2 L3 M1 M2

10 mm

Figure 3 ( continued)b LMmicrographs of recentNitraria pollen (A1-B2)N sibirica (A1-3) equatorial view (ChineseAcademy of Sciences China P45-4) (B1-2) polar view (Chinese Academy of Sciences China M43-2)(C1-E2)N sphaerocarpa (C1-3) equatorial view at mesocolpuscolporus area (Chinese Academy ofSciences China K43-2) (D1-2) equatorial view at mesocolpus area (Chinese Academy of Sciences ChinaM44-1) (E1-2) polar view (Chinese Academy of Sciences China L42-3) (F1-H2)N tangutorum (F1-2)equatorial view at mesocolpus area (Chinese Academy of Sciences China N48-2) (G1-2) equatorialview at colporus area small morphotype (Chinese Academy of Sciences China Q37-2) (H1-2) polarview (Chinese Academy of Sciences China H-G 44-13) (I1-K2) P harmala (I1-3) equatorial view atcolporus-mesocolporus area of a tricolporate grain (Chinese Academy of Sciences China L53-1) (J1-3)equatorial view at colporusmesocolpus area of a tetracolporate grain (Chinese Academy of SciencesChina E-F 47-24) (K1-2) polar view of a tetracolporate grain (Chinese Academy of Sciences ChinaG42-2) (L1-M2) P nigellastrum (L1-3) equatorial view at colporus area (Chinese Academy of SciencesChina Q43-4) (M1-2) polar view (Chinese Academy of Sciences China Q43-4)


A1 A2 B1 B2

C D E1 E2

F G H1 H2

I J K1 K2

2 mm 2 mm 2 mm 2 mm

2 mm

2 mm

2 mm 2 mm

2 mm 2 mm 2 mm 2 mm

2 mm2 mm 2 mm 2 mm

Figure 4 SEMmicrographs of extantNitraria pollen Location of photograph indicated (A1-D)Nbillardierei (A1) Equatorial view note parallel orientation of striae near the colpus (MU) (A2) Detailof lsquolsquotightrsquorsquo striae (MU) (B1) Equatorial view note transversal striae orientation in mesocolpus area (MU)(B2) note presence of perforations in exine surface (MU) (C) Equatorial view on the mesocolpus area(UAB) (D) Polar view (UAB) (E1-G)Nretusa (continued on next page )



Figure 4 ( continued)(E1) Equatorial view (UAB) (E2) detail of perforation on the exine surface (UAB) (F) Equatorial view onthe mesocolpus area (MU) (G) Equatorial view showing colpus and porus (MU) (H1-J)Nschoberi (H1)Equatorial view clearly showing the colpus note parallel orientation of the striae near the colpus (UABFESEM) (H2) Detail of exine surface note transversal orientation of the striae in the mesocolpus area(UAB FESEM) (I) Equatorial view showing colpus and open porus (UAB FESEM) (J) Equatorial viewshowing rotation in striae orientation in the mesoculpus area three main lsquolsquospiralsrsquorsquo can be distinguished(UAB FESEM) (K)Nsibirica (K1) Equatorial view (MU) (K2) Detail of the exine surface note a loosepacked striae pattern and some perforations (MU)

Species Nitraria retusa (Forssk) AschLM description Pollen class Tricolporate Monad isopolar radially symmetric prolate tosubprolate (Fig 3a FndashH) Aperture Tricolporate Ectoaperturemdashcolpus long (sim78 ofpolar axis) straight narrow usually constricted at equator with ends acute apocolpiaasymmetric Margin observed in polar view costae colpi and fastigium conspicuous inequatorial view Endoaperturemdashporus lalongate elliptic to rhomboidal in shape ExineTectate exine slightly thicker in polar areas in relation with the equatorial region nexinethicker than sexine Columellae hardly visible tectum thin Sculpture Surface striate withsome perforations hardly observed in LMOutline Polar view triangular convex to circularEquatorial view ellipticalMeasurements length 3896 (36ndash42) microm width 2835 (268ndash30)microm (see summary in Appendix S3)

SEM descriptionMonads are elliptic in equatorial view radially symmetric (Figs 4Endash4G)Colpus almost as long as the polar axis slightly intruded (margin of colpi pointing inwards)and open pore conspicuous (Fig 4G) Exine ornamentation is striate and perforate (Figs4E1ndash4E2) Striae relatively loose packed in the mesocolpia and short running parallel tothe polar axis near the colpus while running slightly counter clock in the mesocolpus areaSometimes striae follow perpendicular orientation in the area close to one of the poles inthe mesocolpus area (Fig 4F)

Species Nitraria roborowski KomLM description Pollen class Tricolporate Monad isopolar radially symmetric subprolateto prolate (Fig 3a I-J) Aperture Tricolporate Ectoaperturemdashcolpus long (sim56 ofpolar axis) straight narrow with ends acute Costae colpi conspicuous in equatorialview Endoaperturemdashporus lalongate elliptic shape Exine Tectate exine slightly thickColumellae hardly visible tectum thin Sculpture Surface striate with some perforationsOutline Polar view triangular convex Equatorial view elliptic

Species Nitraria schoberi LLM description Pollen class Tricolporate Monad isopolar radially symmetric subprolateto prolate (Fig 3a K-L) Aperture Tricolporate Ectoaperturemdashcolpus long (sim45 ofpolar axis) straight narrow commonly constricted at equator with ends acute apocolpiaasymmetric Margin observed in polar view costae colpi and fastigium conspicuous inequatorial view Endoaperturemdashporus lalongate elliptic to rhomboidal in shape ExineTectate exine slightly thicker in polar areas in relation with the equatorial region nexinethicker than sexine Columellae hardly visible tectum thin Sculpture Surface striate


with some perforations hardly observed in LM Outline Polar view triangular convex tohexagonal Equatorial view ellipticMeasurements length 4549 (42ndash59) microm width 3471(30ndash39) microm (see summary in Appendix S3)

SEM description Monads are elliptic radially symmetric (Figs 4Hndash 4J) Colpus almostas long as the polar axis open pore conspicuous (Fig 4I) Exine ornamentation is striateno perforations observed (Figs 4Hndash 4J) Striae relatively tight and short running parallelto the polar axis near the colpus (Fig 4I) while running counter clock sometimes formingtwo to three lsquolsquospiralrsquorsquo clusters in the equatorial mesocolpus area (Fig 4J) Striae followperpendicular orientation in the area close to one of the poles in the mesocolpus area

Species Nitraria sibirica PallLM description Pollen class Monad isopolar radially symmetric prolate to subprolate(Figs 3Andash3B) Aperture Tricolporate Ectoaperturemdashcolpus long (sim78 of polar axis)straight narrow constricted at equator or not with ends acute apocolpia asymmetricMargin prominent observed in polar view costae colpi and fastigium conspicuous inequatorial view Endoaperturemdashporus lalongate ellipsoidal in shape Exine Tectateexine slightly thicker in polar areas in relation with the equatorial region nexine thickerthan sexine Columellae hardly visible tectum thin Sculpture Surface finely striate withperforations hardly observed in LM Outline Polar view pseudohexagonal Equatorialview elliptic Measurements length 3984 (36ndash418) microm width 2753 (24ndash298) microm (seesummary in Appendix S3)

SEM description Monads are elliptic radially symmetric (Figs 4K) Colpus almost aslong as the polar axis intruded pore conspicuous (Fig 4K1) Exine ornamentation isstriate no perforations observed Striae relatively loosely packed and short running fairlyparallel to the polar axis near the colpus and in the mesocolpus area (Fig 4K2)

Species Nitraria sphaerocarpa MaximLM description Pollen class Tricolporate Monad isopolar radially symmetric prolate tosubprolate (Fig 3b C-E) Aperture Tricolporate Ectoaperturemdashcolpus long (sim56 ofpolar axis) straight narrow constricted at equator or not with ends acute apocolpiaasymmetric Margin observed in polar view costae colpi and fastigium conspicuous inequatorial view Endoaperturemdashporus lalongate ellipsoidal in shape Exine Tectateexine slightly thicker in polar areas in relation with the equatorial region nexine thickerthan sexine Columellae hardly visible tectum thin Sculpture Surface finely striate withperforations hardly observed in LM Outline Polar view triangular convex to pseudo-hexagonal Equatorial view elliptic Measurements length 3666 (345ndash40) microm width2569 (24ndash28) microm (see summary in Appendix S3)

SEM description Monads are elliptic radially symmetric (Figs 5Andash5B) Colpus almost aslong as the polar axis slightly intruded pore not observed (Fig 5B1) Exine ornamentationis striate perforations observed near the colpus Striae relatively tight and short runningparallel to the polar axis near the colpus while running slightly counter clock in themesocolpus area (Figs 5B2 5B1) Striae follow perpendicular orientation in the area closeto one of the poles in the mesocolpus area (Fig 5A)


A B1 B2

C D E1 E2

F G H1 H2

J1 J2

I

2 mm 2 mm 2 mm

2 mm 2 mm 2 mm 2 mm

2 mm

2 mm2 mm2 mm

2 mm 2 mm 2 mm

Figure 5 SEMmicrographs of extantNitraria and Peganum pollen Location of photograph indicated(A-B2)Nsphaerocarpa (A1) Detail of the polar area on an equatorial view (MU) (B1) Equatorial viewon mesocolpus area note parallel orientation of the striae near the colpus and more oblique orientation inthe mesoculpus area (UAB) (continued on next page )



Figure 5 ( continued)(B2) Detail of the exine surface striae looks lsquolsquotightrsquorsquo (UAB) (C-E2)Ntangutorum (C) Equatorial viewshowing colpus and open porus note parallel orientation of the striae near the colpus and transversal ori-entation on the polar area (UAB) (D) Polar view (UAB) (E1) Equatorial view showing the mesoculpusarea (UAB) (E2) detail of the exine surface striae looks lsquolsquotightrsquorsquo (UAB) (F-H2) Pharmala (F) Equato-rial view (MU) (G) Polar view of a tetracolporate pollen (MU) (H1) Equatorial view (MU) (H2) Detailof the exine surface note the heterobrochate microreticulate pattern muri thin in relation to the brochidiameter (MU) (I-J2) Pnigellastrum (I) Polar view of a tricolporate pollen (MU) (J1) Equatorial viewshowing a mesoculpus area (MU) (J2) Detail of the exine surface note the heterobrochate microreticulatepattern muri thick in relation to the brochi diameter (MU)

Species Nitraria tangutorum BobrovLM description Pollen class Tricolporate Monad isopolar radially symmetric prolate(Fig 3b F-H) Aperture Tricolporate Ectoaperturemdashcolpus long (sim78 of polar axis)straight narrow occasionally constricted at equator with ends acute apocolpia asymmetricMargin observed in polar view costae colpi and fastigium conspicuous in equatorial viewEndoaperturemdashporus lalongate rhomboidal in shape Exine Tectate exine slightly thickerin polar areas in relation with the equatorial region nexine thicker than sexine Columellaehardly visible tectum thin Sculpture Surface slightly striate with some perforations hardlyobserved in LM Outline Polar view triangular convex to circular Equatorial view ellipticMeasurements length 4217 (39ndash455) microm width 2724 (24ndash29) microm (see summary inAppendix S3)

SEM description Monads are elliptic radially symmetric (Fig 5Cndash5E) Colpus almostas long as the polar axis slightly intruded and open pore conspicuous (Fig 5C) Exineornamentation is striate no perforations observed (Fig 5E1) Striae relatively tight andshort running parallel to the polar axis near the colpus (Fig 5E2) while running slightlycounter clock in the mesocolpus area eventually forming a spiral lsquolsquoclusterrsquorsquo in one area closeto a pole or running almost parallel Striae follow perpendicular orientation in the areaclose to one of the poles in the mesocolpus area (Fig 5E2)

Genus Peganum LPollen is monad isopolar radially symmetric Pollen shape circular to triangular convex orquadrangular in polar view and elliptic prolate spheroidal to oblate spheroidal or suboblatein equatorial view Tricolporate eventually tetracolporate colpi long open with operculumEndoaperturemdashporus lalongate quadrangular in shape Exine semitectate sexine slightlythicker than nexine surface microreticulate heterobrochate

Species Peganum harmala var multisectum MaximLM description Pollen class Tricolporate Monad isopolar radially symmetric (Fig 3bIndashK) Aperture Tricolporate Ectoaperturemdashcolpus long (sim45 of polar axis) straightopen with ends acute apocolpia asymmetric Operculum present margin observedin polar view Endoaperturemdashporus lalongate quadrangular with outlines not clearlydifferentiated Exine Semitectate microreticulate heterobrochate sexine slightly thicker ornot than nexine Columellae not visible tectum thin Sculpture Surface psilate OutlinePolar view circular to triangular convex eventually quadrangular Equatorial view elliptic


oblate spheroidal to suboblate Measurements length 244 (22ndash265) microm width 2702(24ndash29) microm (see summary in Appendix S3)

SEM description Monads are elliptic radially symmetric (Figs 5Fndash5H) Colpus almostas long as the polar axis intruded pore not observed (Fig 5F) Exine ornamentation ismicroreticulate heterobrochate (Fig 5H) Reticulum with lumens decreasing near thecolpus and polar areas and relatively narrow muri (Fig 5H2)

Species Peganum nigellastrum BungeLM description Pollen class Tricolporate Monad isopolar radially symmetric (Fig 3bL-M) Aperture Tricolporate Ectoaperturemdashcolpus long (sim45 of polar axis) straightopen with ends acute apocolpia asymmetric Operculum present margin observedin polar view Endoaperturemdashporus lalongate quadrangular with outlines not clearlydifferentiated Exine Semitectate microreticulate heterobrochate sexine slightly thicker ornot than nexine Columellae not visible tectum thin Sculpture Surface psilate OutlinePolar view circular to triangular convex eventually quadrangular Equatorial view ellipticprolate spheroidal to oblate spheroidal or even spheroidalMeasurementslength 203 (185ndash215) microm width 2025 (185ndash215) microm (see summary in Appendix S3)

SEM description Monads are elliptic radially symmetric (Figs 5Indash5J) Colpus almost aslong as the polar axis slightly intruded pore not observed (Fig 5J1) Exine ornamentationis microreticulate to perforate (Fig 5J1) muri heterobrochate Reticulum with broad muriand lumens irregular in shape and decreasing near the colpus and polar areas (Fig 5J2)

Summary of Nitraria and Peganum pollen morphologyThepollenmorphology betweenNitraria andPeganum shows that the tricolporate characterof the grains is a shared character However Peganum differs from Nitraria because of itssmaller size and its semitectate microreticulate exine Instead Nitraria is tectate to tectate-perforate with a striate exine A clear distinction between P harmala var multisectum andP nigellastrum are the irregular borders of the muri in the reticulum of the latter Thischaracter was only observed with SEM

Measured morphometric characters of Nitraria show considerable overlap among the6 studied taxa (see boxplots Appendix S4) Our LM observations indicate that the striatepattern varies from faint (N billardierei) to striate-perforated or hardly recognizable (Nschoberi N retusa N tangutorum N sphaerocarpa and N sibirica) Nevertheless SEManalysis confirms a perforate exine inN retusa and to some degree also inN sphaerocarpain particular near the colpus A lsquolsquoloosersquorsquo striate pattern is found in N retusa and to somedegree also in N sibirica (seemingly related to a perforate exine) contrary to the lsquolsquotightrsquorsquostriate pattern in N schoberi and N tangutorum

Multivariate analysis of the pollen morphological dataOrdination of the morphological character data using NMDS shows a clear within-taxongrouping of the specimens both at the genus and species level (Fig 6A) NMDS axis 1primarily separates out the two genera with some additional separation of Nitraria speciesat the lower end of the axis Further species-level separation occurs on NMDS axis 2


minus02 minus01 00 01 02 03

NMDS 1

minus03 minus02 minus01 00 01 02 03

NMDS 1

N billardiereiN retusaN schoberiN sibiricaN sphaerocarpaN tangutorumP harmalaP nigellastrum

minus015

minus010

minus005

000

005

010

015

020

NM

DS

2

minus015

minus010

minus005

000

005

010

015

020

NM

DS

2

N billardierei

N retusa

N schoberi N sibirica

N sphaerocarpa

N tangutorum

P harmala

P nigellastrum

A

B

Figure 6 Morphological characteristics ofNitraria and Peganum (A) Non-metric multidimensionalscaling (NMDS) ordination of Nitraria and Peganum species morphologies The ordination is based onpairwise Gower dissimilarities (Gower 1971) of coded morphological characters for 20 specimens perspecies (B) Phylomorphospace based on the mean NMDS axis scores for each species using the phy-logeny of Zhang et al (2015)



especially between N sphaerocarpa and N retusa at the upper end of the axis and the otherNitraria species at the lower end

The phylomorphospace (Fig 6B) where the phylogeny is mapped directly onto theordination space demonstrates a phylogenetic signal in the morphological data This ismanifested not only with the separation ofNitraria and Peganum but also between the earlydiverging Nitraria species N sphaerocarpa and N retusa and those that appeared duringthe late Miocene to Pliocene Nitraria diversification (Zhang et al 2015)

Chemical palynologyIn commonwith previous studies of sporopollenin chemistry (eg Bağcıoğlu Zimmermannamp Kohler 2015 Fraser et al 2012 Jardine et al 2015 Jardine et al 2017 Julier et al 2016)plots of the mean FTIR spectrum for each species show absorbance bands associatedwith hydroxyl (sim3400 cmminus1) and carboxyl (1710 cmminus1) groups aliphatic compounds(2925 and 2850 cmminus1) and aromatic compounds (1600 1510 1440 1370 1170 and1030 cmminus1) (Fig 7) Principal differences among the spectra relate to the relative heightsof these peaks especially among the carboxyl and aromatic peaks in the region below1800 cmminus1

The highest classification success rate for the FTIR spectra was 96 (Table 2)which means that sim108 of the 112 spectra were correctly classified to species levelThe combination of processing parameters that produced this classification success ratewas SG smoothing plus first or second derivative (see Methods) Both ways of processingthe spectra led to highly similar PCA ordinations and here we present those for the SGsmoothing plus second derivative spectra with the smoothing window size set to 37

The first two axes of the PCA of the processed spectra account for 67 of the variation inthe dataset (Fig 8A)Nitraria and Peganum are separated on the first axis with theNitrariaspectra being distributed across the second axis The Nitraria spectra show within-speciesgroupings although there is considerable overlap among several of the species in themiddle of axis 2 The PCA of the species mean spectra with the phylogeny mapped onshows that while a phylogenetic signal is present in the pollen chemistry it is less clear thanwith the morphological data and three PCA axes are needed to reveal the full structure(Figs 8Bndash8C) Together these three axes account for 94 of the variation in the chemicaldata

DISCUSSIONMorphology and chemistry as diagnostic featuresThe consistency of within-species groupings for both the morphological and chemical datasuggest that the multivariate approach used here is useful for assigning fossil specimensto modern taxa especially following the diversification of extant Nitraria species in thelate Miocene (Zhang et al 2015) The 96 classification success rate demonstrated by thechemical data suggests that there is potential to use pollen chemistry either on its ownor in combination with pollen morphology as a tool for classifying fossil Nitraria pollenHowever we acknowledge that this rate was obtained from a relatively small dataset which


4000 3500 3000 2500 2000 1500 1000

Abs

orba

nce

Wavenumber (cmminus1)

P nigellastrum

N tangutorum

N sphaerocarpa

N sibirica

N schoberi

N retusa

N billardierei

3390 2925 117013702850 17101600

15101440

1030

Figure 7 Within-species mean Fourier Transform infrared (FTIR) spectra forNitraria and Peganumspecies Positions of peaks mentioned in the text are shown as vertical dashed lines with wavenumbersgiven above


means that this is likely an overestimate of true classification success if more individualsrepresenting greater environmental variation were sampled

Nitraria and Peganum pollen morphology show a clear phylogenetic structure (Fig 6)This indicates that fossil specimens can be scored for similarmorphological traits to identifyextinct lineages and assigned to major lineages within the Nitraria phylogeny This in turnmay improve the estimation of divergence times between extant species In particularboth N sphaerocarpa and N retusa have pollen that has a shorter polar axis than the laterdiverging taxa as well as a perforated exine However the latter character is only reliably


Table 2 Chemical classification success rates from different processing of the spectra

Treatment Highest classificationsuccess

w for highestclassification success

Unprocessed 839 ndashSG smoothing 848 21 to 43SG + first derivative 955 9 to 27SG + second derivative 964 37 and 39

NotesSG Savitzky-Golay smoothing w window size for smoothing

determined with SEM analysis Even so this finding suggests that with careful observationthe split between N retusa and the more recently appearing taxa may be discernible fromthe fossil record Themorphospace inferred for extant taxa also provides the basis for futureresearch to assess morphological disparity (Lupia 1999) using fossil Nitraria specimens

Our morphological and chemical data suggest that there are no obvious differencesbetween the modern lowland species (N billardierei and N retusa) and the other Nitrariataxa that all occur in highland environments Similarly no individual morphologicalcharacters appear to distinguish highland and lowland species Phylogeny appears tobe a more important control on Nitraria pollen form than the local environmentalconditions A possible environmental or biogeographic signal is recorded on axis 2 of thephylochemospace (Fig 8B) with the species having positive axis scores (N sphaerocarpaN sibirica P nigellastrum and to a lesser extent N tangutorum) represented by specimensfrom northern China In particular N sphaerocarpa and N sibirica have highly similarchemical spectra (Fig 7) The links between environmental conditions most obviouslyUV-B exposure (Jardine et al 2016) and phylogeny in controlling sporopollenin chemistryrequire further research but our results suggest that both play an important role

Methodological advancesThe chemical analysis aspect of this study is novel for two reasons First most studies usingextant pollen and spore chemistry for taxonomic or classification purposes have used freshor herbarium specimens (egZimmermann 2010Zimmermann amp Kohler 2014BağcıoğluZimmermann amp Kohler 2015 Zimmermann et al 2015 Julier et al 2016 Zimmermann etal 2016) In these cases the chemical signal includes proteins lipids and carbohydratesthat are not present in the fossil record where only the sporopollenin is preserved (Jardineet al 2015 Julier et al 2016) There is therefore a limited understanding of how closelyrelated taxa will be chemically-distinguishable in fossil samples Since our study has usedisolated sporopollenin following acetolysis it provides new information on the taxonomicand phylogenetic signal that can be applied to fossil pollen grains

Second to our knowledge this is the first time that the phylomorphospace approachhas been applied to chemical data of pollen and the phylogenetic structure presentin sporopollenin chemistry directly assessed Applying a similar approach to largerdatasets comprising a greater range of taxa will therefore enable a better understandingof sporopollenin evolution (Fraser et al 2012) and the phylogenetic underpinnings of


minus004 minus003 minus002 minus001 000 001 002

minus002

minus001

000

001

002

PCA 1 (47)

PCA2(20

)

N billardiereiN retusaN schoberiN sibiricaN sphaerocarpaN tangutorumP nigellastrum

minus003 minus002 minus001 000 001 002

minus0015

minus0010

minus0005

0000

0005

0010

PCA 1 (56)

PCA2(27

)

N billardierei

N retusa

N schoberi

N sibirica

N sphaero-carpa

N tangutorum

P nigellastrum

A

B


minus0010

minus0005

0000

0005

0010

PCA 1 (56)

PCA3(11

)

N billardierei

N retusa

N schoberi

N sibirica

N sphaerocarpa

N tangutorum

P nigellastrum

C

Figure 8 Chemical characteristics ofNitraria and Peganum (A) Principal components analysis (PCA)ordination of Nitraria and Peganum FTIR spectra (BndashC) Phylochemospace based on the mean FTIR spec-trum for each species using the phylogeny of Zhang et al (2015) Values in parentheses give the percent-age of variance in the data explained by each PCA axis


the palaeoclimatic and taxonomic indicators that are currently being developed based onpollen and spore chemistry (Zimmermann et al 2015 Zimmermann et al 2016)

We found a clear differentiation between species based on pollen morphology andchemical composition in particular between the earliest diverging species N sphaerocarpaand the more recently derived species N schoberi and N billardierei (Figs 6 and 8) Thetwo taxa show distinct dispersal patterns N sphaerocarpa originated in central Asia andis today only found in this region whereas N schoberi today is also found in Iran PapuaNew-Guinea and Australia and N billardierei is restricted to Australia The reflection ofthe biogeographic history of species in the morphological and chemical characteristics ofthe pollen suggests that either morphological and chemical evolution resulted in or wasdriven by processes related to taxon dispersal However further research is required toclarify this relationship


While the phylogenetic analysis of Zhang et al (2015) shows that most of the extantNitraria species originated in the late Miocene or Pliocene (ca 9ndash5 Ma) the palynologicalrecord suggests that diversity was much higher in the Paleocene and Eocene (66ndash34Ma) (Wang 1990 Hoorn et al 2012 Han et al 2016) This apparent contradiction callsfor future research to focus on understanding this earlier phase in Nitraria evolutionincluding its first appearance and diversification in the proto-ParatethysTibetan Plateauregion and its subsequent decline in the late Eocene at 34 Ma and how this fits in withthe late Miocene (ca 9 Ma) diversification and dispersal phase (Zhang et al 2015) A firststep has already been made by Hoorn et al (2012) who noticed the similarity betweenfossil Nitraria specimens and the earliest diverging Nitraria species N sphaerocarpa Theinformation presented here on the extant species ofNitraria provides a valuable comparisondataset for further interrogation of the fossil record and offers the potential to further tiefossil specimens to the extant taxon molecular phylogeny (eg Barreda et al 2015)

CONCLUSIONNitraria is an important halophytic taxon of wide current distribution in Asia Africa andAustralia This taxon has an intriguing past with predominance in the Paleogene andsubsequent dwindling at the end of this period However the history of this taxonmdashand ofthe Asian steppe biomemdashis poorly resolved and to address this we looked into novel waysof applying palynology

In this study we have tested a new method that consists of combining pollenmorphological and chemical data sets by producing the phylomorphospace andphylochemospace in which the palynological data are directly plotted on the Nitrariaceaephylogeny We conclude that together they form a powerful tool for the identification ofmodern Nitraria species and hold great prospects for exploring the fossil record in general

Our data indicate that differences between highland and lowland species are subtle andwe conclude that phylogenetic history has a more important control on morphology andchemistry of the pollen than local environmental conditions Future research could focuson the early Paleogene history of this genus and why it went through a major bottleneckat the Eocene-Oligocene Transition Such study has the potential to provide insights notonly on the evolution of the genus but of the steppe-desert biome as a whole

ACKNOWLEDGEMENTSThis paper is dedicated to the memory of professor Ming-Li Zhang molecularphylogeneticist botanist and prominent arid land researcher We thank all the followingpeople and their institutions for facilitating plant material for this study First of allwe are grateful to the late Ming-Li Zhang (PE Herbarium Institute of Botany ChineseAcademy of Sciences Beijing China) whorsquos work was an inspiration and whom prior tohis untimely death was involved in this study We also thank Uwe Braun (Martin-LutherUniversitat Halle Saale Germany) John Parnell (Herbarium Trinity College DublinDublin Ireland) Helen Vonow and Robyn Barker (State Herbarium of South AustraliaAdelaide Botanic Garden Adelaide Australia) and Morteza Djamali (Middle East Pollen


Reference Collection Aix en Provence France) for providing us with the essential plantsamples that form the basis of this study Furthermore we are indebted to Jan van Arkelfor providing us with high quality microphotography Annemarie Philip for processingthe plant material Huasheng Huang for his help with literature research (all at IBEDUniversity of Amsterdam) We also acknowledge Alejandro Saacutenchez-Chardi and EmmaRossinyol-Casals from the Servei de Microscopia (Universitat Autogravenoma de BarcelonaSpain) for their help during SEM imaging

ADDITIONAL INFORMATION AND DECLARATIONS

FundingGuillaume Dupont-Nivet received ERC consolidator grant MAGIC 649081 which alsoprovided funding for Phillip E Jardine Daniele Silvestro received funding from theSwedish Research Council (2015-04748) Alexandre Antonelli is supported by the Knutand Alice Wallenberg Foundation the Swedish Research Council (B0569601) the SwedishFoundation for Strategic Research the Faculty of Sciences at the University of Gothenburgand the David Rockefeller Center for Latin American Studies at Harvard University Thefunders had no role in study design data collection and analysis decision to publish orpreparation of the manuscript

Grant DisclosuresThe following grant information was disclosed by the authorsERC consolidator MAGIC 649081Swedish Research Council 2015-04748Knut and Alice Wallenberg Foundation the Swedish Research Council B0569601The Swedish Foundation for Strategic ResearchThe Faculty of Sciences at the University of GothenburgThe David Rockefeller Center for Latin American Studies at Harvard University

Competing InterestsThe authors declare there are no competing interests

Author Contributionsbull AmberWoutersen conceived and designed the experiments performed the experimentsanalyzed the data contributed reagentsmaterialsanalysis tools prepared figures andortables authored or reviewed drafts of the paper approved the final draftbull Phillip E Jardine conceived and designed the experiments performed the experimentsanalyzed the data prepared figures andor tables authored or reviewed drafts of thepaper approved the final draftbull Raul Giovanni Bogotaacute-Angel and Elena Gogna performed the experiments preparedfigures andor tables authored or reviewed drafts of the paper approved the final draftbull Hong-Xiang Zhang contributed reagentsmaterialsanalysis tools authored or revieweddrafts of the paper approved the final draft


bull Daniele Silvestro performed the experiments authored or reviewed drafts of the paperapproved the final draftbull Alexandre Antonelli Roy HJ Erkens William D Gosling and Guillaume Dupont-Nivetauthored or reviewed drafts of the paper approved the final draftbull Carina Hoorn conceived and designed the experiments performed the experimentscontributed reagentsmaterialsanalysis tools prepared figures andor tables authoredor reviewed drafts of the paper approved the final draft

Data AvailabilityThe following information was supplied regarding data availability

The raw data are provided in the Supplemental Files

Supplemental InformationSupplemental information for this article can be found online at httpdxdoiorg107717peerj5055supplemental-information

REFERENCESAbbott LB Lepak D Daniel DL 2007 Vegetative and reproductive phenology of African

rue (Peganum harmala) in the northern Chihuahuan Desert The SouthwesternNaturalist 52209ndash218 DOI 1018940038-4909(2007)52[209VARPOA]20CO2

Abels HA Dupont-Nivet G Xiao G Bosboom R KrijgsmanW 2011 Step-wisechange of Asian interior climate preceding the EocenendashOligocene Transi-tion (EOT) Palaeogeography Palaeoclimatology Palaeoecology 299399ndash412DOI 101016jpalaeo201011028

Agababyan VS Tumanyan KT 1972 Palynomorphology of the genus Nitraria Bio-logicheskiy Zhurnal 25(4)36ndash41

Angiosperm Phylogeny Group (APG) 2009 An update of the angiosperm phylogenygroup classification for the orders and families of flowering plants APG III BotanicalJournal of the Linnean Society 161105ndash121 DOI 101111j1095-8339200900996x

BağcıoğluM Zimmermann B Kohler A 2015 A multiscale vibrational spectroscopicapproach for identification and biochemical characterization of pollen PLOS ONE101ndash19 DOI 101371journalpone0137899

Barreda VD Palazzesi L Telleria MC Olivero EB Raine JI Forest F 2015 Earlyevolution of the angiosperm clade Asteraceae in the Cretaceous of AntarcticaProceedings of the National Academy of Sciences of the United States of America1210989ndash10994 DOI 101073pnas1423653112

Bercovici A Hadley A Villanueva-Amadoz U 2009 Improving depth of field resolutionfor palynological photomicrography Palaeontologia Electronica 12(2)1ndash12

Bonhomme V Prasad S Gaucherel C 2013 Intraspecific variability of pollen mor-phology as revealed by elliptic Fourier analysis Plant Systematics and Evolution299811ndash816 DOI 101007s00606-013-0762-5

Bosboom RE Abels HA Hoorn C Van den Berg BC Guo Z Dupont-Nivet G 2014Aridification in continental Asia after the Middle Eocene Climatic Optimum


(MECO) Earth and Planetary Science Letters 38934ndash42DOI 101016jepsl201312014

Bosboom R Mandic O Dupont-Nivet G Proust JN Ormukov C Aminov J 2017Late Eocene palaeogeography of the proto-Paratethys Sea in Central Asia (NWChina southern Kyrgyzstan and SW Tajikistan) Geological Society London SpecialPublications 427565ndash588 DOI 101144SP42711

Bougeois L Dupont-Nivet G De Rafeacutelis M Tindall JC Proust JN Reichart GJDe Nooijer LJ Guo Z Ormukov C 2018 Asian monsoons and aridificationresponse to Paleogene sea retreat and Neogene westerly shielding indicated byseasonality in Paratethys oysters Earth and Planetary Science Letters 48599ndash110DOI 101016jepsl201712036

Cai M Fang XWu F Miao Y Appel E 2012 PliocenendashPleistocene stepwise dryingof Central Asia evidence from paleomagnetism and sporopollen record of thedeep borehole SG-3 in the western Qaidam Basin NE Tibetan Plateau Global andPlanetary Change 9472ndash81 DOI 101016jgloplacha201207002

Chung KS ElisensWJ Skvarla JJ 2010 Pollen morphology and its phylogeneticsignificance in tribe Sanguisorbeae (Rosaceae) Plant Systematics and Evolution285139ndash148 DOI 101007s00606-009-0262-9

DeConto RM Pollard D 2003 Rapid Cenozoic glaciation of Antarctica induced bydeclining atmospheric CO2 Nature 421245ndash249 DOI 101038nature01290

Dupont-Nivet G Hoorn C Konert M 2008 Tibetan uplift prior to the Eocene-Oligocene climate transition evidence from pollen analysis of the Xining BasinGeology 36987ndash990 DOI 101130G25063A1

Dupont-Nivet G KrijgsmanW Langereis CG Abels HA Dai S Fang X 2007 Tibetanplateau aridification linked to global cooling at the EocenendashOligocene transitionNature 445635ndash638 DOI 101038nature05516

Erdman G 1986 Pollen and plant taxonomy angiosperms New York Hafner Publ Co553

Fang J Wang Z Tang Z (eds) 2011 Atlas of woody plants in China distribution andclimate Vol 1 Berlin Springer Science amp Business Media

Favre A Paumlckert M Pauls SU Jaumlhnig SC Uhl D Michalak I Muellner-Riehl AN 2015The role of the uplift of the Qinghai-Tibetan Plateau for the evolution of Tibetanbiotas Biological Reviews 90236ndash253 DOI 101111brv12107

FraserWT Lomax BH Jardine PE GoslingWD SephtonMA 2014 Pollen and sporesas a passive monitor of ultraviolet radiation Frontiers in Ecology and Evolution 21ndash3DOI 103389fevo201400012

FraserWT Scott AC Forbes AES Glasspool IJ Plotnick RE Kenig F Lomax BH 2012Evolutionary stasis of sporopollenin biochemistry revealed by unaltered Pennsylva-nian spores New Phytologist 196397ndash401 DOI 101111j1469-8137201204301x

GBIF 2018 GBIF Occurrence Download Available at https doiorg1015468dlj0ehrr(accessed on 25 February 2018)

Gower JC 1971 A general coefficient of similarity and some of its properties Biometrics27857ndash874 DOI 1023072528823


Han F Rydin C Bolinder K Dupont-Nivet G Abels HA Koutsodendris A Zhang KHoorn C 2016 Steppe development on the Northern Tibetan Plateau inferred fromPaleogene ephedroid pollen Grana 5571ndash100 DOI 1010800017313420151120343

Hesse M Halbritter H Zetter RWeber M Buchner R Frosch-Radivo A Ulrich S2009 Pollen terminology an illustrated handbook Wien Springer 266

Hoorn C Straathof J Abels HA Xu Y Utescher T Dupont-Nivet G 2012 A lateEocene palynological record of climate change and Tibetan Plateau uplift (XiningBasin China) Palaeogeography Palaeoclimatology Palaeoecology 34416ndash38DOI 101016jpalaeo201205011

Hopkins MJ Smith AB 2015 Dynamic evolutionary change in post-Paleozoic echinoidsand the importance of scale when interpreting changes in rates of evolutionProceedings of the National Academy of Sciences of the United States of America1123758ndash3763 DOI 101073pnas1418153112

Jardine PE Abernethy FAJ Lomax BH GoslingWD FraserWT 2017 Sheddinglight on sporopollenin chemistry with reference to UV reconstructions Review ofPalaeobotany and Palynology 2381ndash6 DOI 101016jrevpalbo201611014

Jardine PE FraserWT Lomax BH GoslingWD 2015 The impact of oxidationon spore and pollen chemistry Journal of Micropalaeontology 34139ndash149DOI 101144jmpaleo2014-022

Jardine PE FraserWT Lomax BH SephtonMA Shanahan TMMiller CS GoslingWD 2016 Pollen and spores as biological recorders of past ultraviolet irradianceScientific Reports 6(39269)1ndash8 DOI 101038srep39269

Julier ACM Jardine PE Coe AL GoslingWD Lomax BH FraserWT 2016 Chemo-taxonomy as a tool for interpreting the cryptic diversity of Poaceae pollen Review ofPalaeobotany and Palynology 235140ndash147 DOI 101016jrevpalbo201608004

Kriebel R KhabbazianM Sytsma KJ 2017 A continuous morphological approach tostudy the evolution of pollen in a phylogenetic context an example with the orderMyrtales PLOS ONE 12e0187228 DOI 101371journalpone0187228

Kruskal JB 1964 Nonmetric multidimensional scaling a numerical method Psychome-trika 29115ndash129 DOI 101007BF02289694

Laliberteacute E Legendre P Shipley B 2014 FD measuring functional diversity frommultiple traits and other tools for functional ecology R package version 10-12Available at httpsCRANR-projectorgpackage=FD

Liland KHMevik BH 2015 baseline baseline correction of spectra R package version12-1 Available at httpsCRANR-projectorgpackage=baseline

Lomax BH FraserWT 2015 Palaeoproxies botanical monitors and recorders ofatmospheric change Palaeontology 58759ndash768 DOI 101111pala12180

Lomax BH FraserWT SephtonMA Callaghan TV Self S Harfoot M Pyle JAWell-man CH Beerling DJ et al 2008 Plant spore walls as a record of long-term changesin ultraviolet-B radiation Nature Geoscience 1592ndash596 DOI 101038ngeo278

Lupia R 1999 Discordant morphological disparity and taxonomic diversity duringthe Cretaceous angiosperm radiation North American pollen record Paleobiology251ndash28


Miao YWu F Chang H Fang X Deng T Sun J Jin C 2016 A Late-Eocene palynologi-cal record from the Hoh Xil Basin northern Tibetan Plateau and its implications forstratigraphic age paleoclimate and paleoelevation Gondwana Research 31241ndash252DOI 101016jgr201501007

Molnar P BoosWR Battisti DS 2010 Orographic controls on climate and paleoclimateof Asia thermal and mechanical roles for the Tibetan Plateau Annual Review ofEarth and Planetary Sciences 3877ndash102 DOI 101146annurev-earth-040809-152456

Moloudizargari M Mikaili P Aghajanshakeri S Asghari MH Shayegh J 2013Pharmacological and therapeutic effects of Peganum harmala and its main alkaloidsPharmacognosy Reviews 7199ndash212 DOI 1041030973-7847120524

Najman Y Jenks D Godin L Boudagher-Fadel M Millar I Garzanti E HorstwoodM Bracciali L 2017 The Tethyan Himalayan detrital record shows that IndiandashAsiaterminal collision occurred by 54 Ma in the Western Himalaya Earth and PlanetaryScience Letters 459301ndash310 DOI 101016jepsl201611036

NiroumandMC Farzaei MH Amin G 2015Medicinal properties of Peganum harmalaL in traditional Iranian medicine and modern phytotherapy a review Journal ofTraditional Chinese Medicine 35104ndash109 DOI 101016S0254-6272(15)30016-9

Noble JCWhalley RDB 1978 The biology and autecology of Nitraria L in AustraliaI Distribution morphology and potential utilization Austral Ecology 3141ndash163DOI 101111j1442-99931978tb01166x

Nurbay A Pan X 2003 Pollen morphology and taxonomy of Nitraria and its alliedgenera in west China Arid Zone Research 20(1)16ndash19

Oksanen J Blanchet FG Friendly M Kindt R Legendre P McGlinn D Minchin PROrsquoHara RB Simpson GL Solymos P Stevens MHH Szoecs E Wagner H 2017vegan community ecology package R package version 24-4 Available at httpsCRANR-projectorgpackage=vegan

Olson DM Dinerstein EWikramanayake ED Burgess ND Powell GV UnderwoodEC Drsquoamico JA Itoua I Strand HE Morrison JC Loucks CJ Allnutt TF RickettsCH Kura Y Lamoreux JF Wettengel WW Hedao P Kassem KR 2001 Terrestrialecoregions of the world a new map of life on earth a new global map of terrestrialecoregions provides an innovative tool for conserving biodiversity BioScience51933ndash938 DOI 1016410006-3568(2001)051[0933TEOTWA]20CO2

Pan X Shen G Chen P 1999 A preliminary research on taxonomy and systematics ofgenus Nitraria Acta Botanica Yunnanica 21287ndash295

Paradis E Claude J Strimmer K 2004 APE analyses of phylogenetics and evolution inR language Bioinformatics 20289ndash290 DOI 101093bioinformaticsbgt412

Peel MC Finlayson BL McMahon TA 2007 Updated world map of the Koumlppen-Geigerclimate classification Hydrology and Earth System Sciences Discussions 4439ndash473DOI 105194hessd-4-439-2007

Perveen A Qaiser M 2006 Pollen flora of Pakistan ndashXlix Zygophyllaceae PakistanJournal of Botany 38(2)225ndash232


Popov SV Roumlgl F Rozanov AY Steininger FF Shcherba IG Kovac M 2004Lithological-Paleogeographic maps of Paratethys-10 maps Late Eocene to PlioceneStuttgart Schweizerbart science publishers

PuntW Hoen PP Blackmore S Nilsson S Le Thomas A 2007 Glossary of pollenand spore terminology Review of Palaeobotany and Palynology 1431ndash81DOI 101016jrevpalbo200606008

R Core Team 2017 R a language and environment for statistical computing Vienna RFoundation for Statistical Computing Available at httpswwwR-projectorg

Revell LJ 2012 phytools an R package for phylogenetic comparative biology (and otherthings)Methods in Ecology and Evolution 3217ndash223DOI 101111j2041-210X201100169x

RStudio Team 2016 RStudio integrated development for R Boston RStudio IncAvailable at httpwwwrstudiocom

Savitzky A Golay MJE 1964 Smoothing and differentiation of data by simplified leastsquares procedure Analytical Chemistry 361627ndash1639 DOI 101021ac60214a047

SheahanMC 2010 Nitrariaceae In Kubitzki K ed Flowering plants Eudicots Thefamilies and genera of vascular plants Berlin Springer

SheahanMC 2011 Tetradiclidaceae In Kubitski K ed The families and genera ofvascular plants volume X flowering plants Eudicots Sapindales Cucurbitales MyrtaceaeBerlin Springer 424ndash429

Sidlauskas B 2008 Continuous and arrested morphological diversification insister clades of characiform fishes a phylomorphospace approach Evolution623135ndash3156 DOI 101111j1558-5646200800519x

Song ZCWangWMMao FY 2008 Palynological implications for relationship betweenaridification and monsoon climate in the Tertiary of NW China Acta PalaeontologicaSinica 47265ndash272

State Herbarium of South Australia 1921ndash2001Handbooks to the flora of SouthAustralia Adelaide The Flora and Fauna of South Australia Handbooks Committee

Steemans P Lepot K Marshall CP Le Herisse A Javaux EJ 2010 FTIR characterisationof the chemical composition of Silurian miospores (cryptospore and trilete spores)from Gotland Sweden Review of Palaeobotany and Palynology 162577ndash590DOI 101016jrevpalbo2010

Stevens A Ramirez-Lopez L 2013 An introduction to the prospectr package R packageVignette R package version 013 Available at httpsCRANR-projectorgpackage=prospectr

Stone JR 2003Mapping cladograms into morphospaces Acta Zoologica 8463ndash68DOI 101046j1463-6395200300131x

Temirbayeva K ZhangML 2015Molecular phylogenetic and biogeographical analysisof Nitraria based on nuclear and chloroplast DNA sequences Plant Systematics andEvolution 3011897ndash1906 DOI 101007s00606-015-1202-5

The Plant List 2013 Version 11 Available at httpwwwtheplantlistorg (accessed on 29January 2018)


Varmuza K Filzmoser P 2009 Introduction to multivariate statistical analysis inchemometrics Boca Raton CRC Press

VenablesWN Ripley BD 2002Modern applied statistics with S 4th edition New YorkSpringer

Wang P 1990 The ice-age China Sea-research results and problems In Proceedingsof the First International Conference on Asian Marine Geology China Ocean PressBeijing 181ndash197

Wang J Fang X Appel E Song C 2012 PliocenendashPleistocene climate change at theNE Tibetan plateau deduced from lithofacies variation in the drill core SG-1Western Qaidam Basin China Journal of Sedimentary Research 82933ndash952DOI 102110jsr201276

Xi Y SunM 1987 Pollen morphology of Nitraria and its geological distributionBotanical Research 2235ndash243 (In Chinese with English summary)

Xi Y Zhang J 1991 The comparative studies of pollen morphology between Nitraria andMeliaceae Botanical Research 547ndash58 (In Chinese with English summary)

Xie L Li LQ 2012 Variation of pollen morphology and its implications in the phylogenyof Clematis (Ranunculaceae) Plant Systematics and Evolution 2981437ndash1453DOI 101007s00606-012-0648-y

ZhangML Temirbayeva K Sanderson SC Chen X 2015 Young dispersal of xerophilNitraria lineages in intercontinental disjunctions of the Old World Scientific Reports513840 DOI 101038srep13840

Zhao TWang ZT Branford-White CJ Xu HWang CH 2011 Classification anddifferentiation of the genus Peganum indigenous to China based on chloroplast trnL-F and psbA-trnH sequences and seed coat morphology Plant Biology 13940ndash947DOI 101111j1438-8677201100455x

Zhou LH Sun SZ Chen GC 1990 Vegetation of Qinghai Province (1 1000000) BeijingChina Science and Technology Press 23ndash24

Zimmermann B 2010 Characterization of pollen by vibrational spectroscopy AppliedSpectroscopy 641364ndash1373 DOI 101366000370210793561664

Zimmermann B BağcıoğluM Sandt C Kohler A 2015 Vibrational microspectroscopyenables chemical characterization of single pollen grains as well as comparativeanalysis of plant species based on pollen ultrastructure Planta 2421237ndash1250DOI 101007s00425-015-2380-7

Zimmermann B Kohler A 2014 Infrared spectroscopy of pollen identifies plantspecies and genus as well as environmental conditions PLOS ONE 9e95417DOI 101371journalpone0095417

Zimmermann B Tafintseva V BağcıoğluM Hoslashegh Berdahl M Kohler A 2016Analysis of allergenic pollen by FTIR microspectroscopy Analytical Chemistry88803ndash811 DOI 101021acsanalchem5b03208


Submitted 7 March 2018Accepted 1 June 2018Published 19 July 2018

Corresponding authorsAmber WoutersenamberwoutersengmailcomCarina Hoorn mchoornuvanl

Academic editorAlastair Potts

Additional Information andDeclarations can be found onpage 25

DOI 107717peerj5055

Copyright2018 Woutersen et al

Distributed underCreative Commons CC-BY 40

OPEN ACCESS

A novel approach to study the morphologyand chemistry of pollen in a phylogeneticcontext applied to the halophytic taxonNitraria L(Nitrariaceae)Amber Woutersen1 Phillip E Jardine23 Raul Giovanni Bogotaacute-Angel14Hong-Xiang Zhang5 Daniele Silvestro6 Alexandre Antonelli6789 Elena Gogna10Roy HJ Erkens10 William D Gosling1 Guillaume Dupont-Nivet211 andCarina Hoorn1

1University of Amsterdam Institute for Biodiversity and Ecosystem Dynamics (IBED) AmsterdamThe Netherlands

2University of Potsdam Institute of Earth and Environmental Science Potsdam Germany3University of Muumlnster Institute of Geology and Palaeontology Muumlnster Germany4Universidad Distrital Francisco Joseacute de Caldas Facultad del Medio Ambiente y Recursos Naturales BogotaacuteColombia

5Key Laboratory of Biogeography and Bioresource in Arid Land XinjiangInstitute of Ecology and Geography China Academy of Sciences Urumqi China

6Gothenburg Global Biodiversity Centre Goumlteborg Sweden7University of Gothenburg Department of Biological and Environmental Sciences Goumlteborg Sweden8Gothenburg Botanical Garden Goumlteborg Sweden9Harvard University Department of Organismic and Evolutionary Biology Cambridge MAUnited States of America

10Maastricht University Maastricht Science Programme Maastricht The Netherlands11Universiteacute de Rennes Geosciences Rennes UMR-CNRS Rennes FranceThese authors contributed equally to this work

ABSTRACTNitraria is a halophytic taxon (ie adapted to saline environments) that belongs tothe plant family Nitrariaceae and is distributed from the Mediterranean across Asiainto the south-eastern tip of Australia This taxon is thought to have originated in Asiaduring the Paleogene (66ndash23 Ma) alongside the proto-Paratethys epicontinental seaThe evolutionary history of Nitraria might hold important clues on the links betweenclimatic and biotic evolution but limited taxonomic documentation of this taxon hasthus far hindered this line of research Here we investigate if the pollen morphologyand the chemical composition of the pollen wall are informative of the evolutionaryhistory of Nitraria and could explain if origination along the proto-Paratethys anddispersal to the Tibetan Plateau was simultaneous or a secondary process To answerthese questions we applied a novel approach consisting of a combination of FourierTransform Infrared spectroscopy (FTIR) to determine the chemical composition ofthe pollen wall and pollen morphological analyses using Light Microscopy (LM)and Scanning Electron Microscopy (SEM) We analysed our data using ordinations(principal components analysis and non-metricmultidimensional scaling) and directlymapped it on the Nitrariaceae phylogeny to produce a phylomorphospace and aphylochemospace Our LM SEM and FTIR analyses show clear morphological andchemical differences between the sister groups Peganum andNitraria Differences in the

How to cite this article Woutersen et al (2018) A novel approach to study the morphology and chemistry of pollen in a phylogeneticcontext applied to the halophytic taxon Nitraria L(Nitrariaceae) PeerJ 6e5055 DOI 107717peerj5055







N billardierei

N retusa


N roborowskii

Genus Nitraria

Legend

N pamirica


























Nitraria retusa

Nitraria schoberi

Nitraria sibirica


Nitraria tangutorum

Peganum harmala


80 60 40 20 0

Ma
























A1 B1 B2A2 A3 C1 C2

D1 D2 E1 E2

F1 F2

G H1 H2

I1 I2 I3

J1 J2

D3 E3

F3 F4

J3

L1 L2K1 K2 K3

10 mm




A1 B1 B2A2 A3

C1 C2 C3 D1 D2 E1 E2

F1 F2 G1 G2 H1 H2

I1 I2 I3 J1 J2 K1 K2J3

L1 L2 L3 M1 M2

10 mm



A1 A2 B1 B2

C D E1 E2

F G H1 H2

I J K1 K2

2 mm 2 mm 2 mm 2 mm

2 mm

2 mm

2 mm 2 mm

2 mm 2 mm 2 mm 2 mm

2 mm2 mm 2 mm 2 mm

















A B1 B2

C D E1 E2

F G H1 H2

J1 J2

I

2 mm 2 mm 2 mm

2 mm 2 mm 2 mm 2 mm

2 mm

2 mm2 mm2 mm

2 mm 2 mm 2 mm


















minus02 minus01 00 01 02 03

NMDS 1


NMDS 1


minus015

minus010

minus005

000

005

010

015

020

NM

DS

2

minus015

minus010

minus005

000

005

010

015

020

NM

DS

2

N billardierei

N retusa


N sphaerocarpa

N tangutorum

P harmala

P nigellastrum

A

B











4000 3500 3000 2500 2000 1500 1000

Abs

orba

nce


P nigellastrum

N tangutorum

N sphaerocarpa

N sibirica

N schoberi

N retusa

N billardierei

3390 2925 117013702850 17101600

15101440

1030

















minus002

minus001

000

001

002

PCA 1 (47)

PCA2(20

)



minus0015

minus0010

minus0005

0000

0005

0010

PCA 1 (56)

PCA2(27

)

N billardierei

N retusa

N schoberi

N sibirica

N sphaero-carpa

N tangutorum

P nigellastrum

A

B


minus0010

minus0005

0000

0005

0010

PCA 1 (56)

PCA3(11

)

N billardierei

N retusa

N schoberi

N sibirica

N sphaerocarpa

N tangutorum

P nigellastrum

C






















































































































N billardierei

N retusa


N roborowskii

Genus Nitraria

Legend

N pamirica


























Nitraria retusa

Nitraria schoberi

Nitraria sibirica


Nitraria tangutorum

Peganum harmala


80 60 40 20 0

Ma
























A1 B1 B2A2 A3 C1 C2

D1 D2 E1 E2

F1 F2

G H1 H2

I1 I2 I3

J1 J2

D3 E3

F3 F4

J3

L1 L2K1 K2 K3

10 mm




A1 B1 B2A2 A3

C1 C2 C3 D1 D2 E1 E2

F1 F2 G1 G2 H1 H2

I1 I2 I3 J1 J2 K1 K2J3

L1 L2 L3 M1 M2

10 mm



A1 A2 B1 B2

C D E1 E2

F G H1 H2

I J K1 K2

2 mm 2 mm 2 mm 2 mm

2 mm

2 mm

2 mm 2 mm

2 mm 2 mm 2 mm 2 mm

2 mm2 mm 2 mm 2 mm

















A B1 B2

C D E1 E2

F G H1 H2

J1 J2

I

2 mm 2 mm 2 mm

2 mm 2 mm 2 mm 2 mm

2 mm

2 mm2 mm2 mm

2 mm 2 mm 2 mm


















minus02 minus01 00 01 02 03

NMDS 1


NMDS 1


minus015

minus010

minus005

000

005

010

015

020

NM

DS

2

minus015

minus010

minus005

000

005

010

015

020

NM

DS

2

N billardierei

N retusa


N sphaerocarpa

N tangutorum

P harmala

P nigellastrum

A

B











4000 3500 3000 2500 2000 1500 1000

Abs

orba

nce


P nigellastrum

N tangutorum

N sphaerocarpa

N sibirica

N schoberi

N retusa

N billardierei

3390 2925 117013702850 17101600

15101440

1030

















minus002

minus001

000

001

002

PCA 1 (47)

PCA2(20

)



minus0015

minus0010

minus0005

0000

0005

0010

PCA 1 (56)

PCA2(27

)

N billardierei

N retusa

N schoberi

N sibirica

N sphaero-carpa

N tangutorum

P nigellastrum

A

B


minus0010

minus0005

0000

0005

0010

PCA 1 (56)

PCA3(11

)

N billardierei

N retusa

N schoberi

N sibirica

N sphaerocarpa

N tangutorum

P nigellastrum

C



































































































































Nitraria retusa

Nitraria schoberi

Nitraria sibirica


Nitraria tangutorum

Peganum harmala


80 60 40 20 0

Ma
























A1 B1 B2A2 A3 C1 C2

D1 D2 E1 E2

F1 F2

G H1 H2

I1 I2 I3

J1 J2

D3 E3

F3 F4

J3

L1 L2K1 K2 K3

10 mm




A1 B1 B2A2 A3

C1 C2 C3 D1 D2 E1 E2

F1 F2 G1 G2 H1 H2

I1 I2 I3 J1 J2 K1 K2J3

L1 L2 L3 M1 M2

10 mm



A1 A2 B1 B2

C D E1 E2

F G H1 H2

I J K1 K2

2 mm 2 mm 2 mm 2 mm

2 mm

2 mm

2 mm 2 mm

2 mm 2 mm 2 mm 2 mm

2 mm2 mm 2 mm 2 mm

















A B1 B2

C D E1 E2

F G H1 H2

J1 J2

I

2 mm 2 mm 2 mm

2 mm 2 mm 2 mm 2 mm

2 mm

2 mm2 mm2 mm

2 mm 2 mm 2 mm


















minus02 minus01 00 01 02 03

NMDS 1


NMDS 1


minus015

minus010

minus005

000

005

010

015

020

NM

DS

2

minus015

minus010

minus005

000

005

010

015

020

NM

DS

2

N billardierei

N retusa


N sphaerocarpa

N tangutorum

P harmala

P nigellastrum

A

B











4000 3500 3000 2500 2000 1500 1000

Abs

orba

nce


P nigellastrum

N tangutorum

N sphaerocarpa

N sibirica

N schoberi

N retusa

N billardierei

3390 2925 117013702850 17101600

15101440

1030

















minus002

minus001

000

001

002

PCA 1 (47)

PCA2(20

)



minus0015

minus0010

minus0005

0000

0005

0010

PCA 1 (56)

PCA2(27

)

N billardierei

N retusa

N schoberi

N sibirica

N sphaero-carpa

N tangutorum

P nigellastrum

A

B


minus0010

minus0005

0000

0005

0010

PCA 1 (56)

PCA3(11

)

N billardierei

N retusa

N schoberi

N sibirica

N sphaerocarpa

N tangutorum

P nigellastrum

C


































































































































A1 B1 B2A2 A3 C1 C2

D1 D2 E1 E2

F1 F2

G H1 H2

I1 I2 I3

J1 J2

D3 E3

F3 F4

J3

L1 L2K1 K2 K3

10 mm




A1 B1 B2A2 A3

C1 C2 C3 D1 D2 E1 E2

F1 F2 G1 G2 H1 H2

I1 I2 I3 J1 J2 K1 K2J3

L1 L2 L3 M1 M2

10 mm



A1 A2 B1 B2

C D E1 E2

F G H1 H2

I J K1 K2

2 mm 2 mm 2 mm 2 mm

2 mm

2 mm

2 mm 2 mm

2 mm 2 mm 2 mm 2 mm

2 mm2 mm 2 mm 2 mm

















A B1 B2

C D E1 E2

F G H1 H2

J1 J2

I

2 mm 2 mm 2 mm

2 mm 2 mm 2 mm 2 mm

2 mm

2 mm2 mm2 mm

2 mm 2 mm 2 mm


















minus02 minus01 00 01 02 03

NMDS 1


NMDS 1


minus015

minus010

minus005

000

005

010

015

020

NM

DS

2

minus015

minus010

minus005

000

005

010

015

020

NM

DS

2

N billardierei

N retusa


N sphaerocarpa

N tangutorum

P harmala

P nigellastrum

A

B











4000 3500 3000 2500 2000 1500 1000

Abs

orba

nce


P nigellastrum

N tangutorum

N sphaerocarpa

N sibirica

N schoberi

N retusa

N billardierei

3390 2925 117013702850 17101600

15101440

1030

















minus002

minus001

000

001

002

PCA 1 (47)

PCA2(20

)



minus0015

minus0010

minus0005

0000

0005

0010

PCA 1 (56)

PCA2(27

)

N billardierei

N retusa

N schoberi

N sibirica

N sphaero-carpa

N tangutorum

P nigellastrum

A

B


minus0010

minus0005

0000

0005

0010

PCA 1 (56)

PCA3(11

)

N billardierei

N retusa

N schoberi

N sibirica

N sphaerocarpa

N tangutorum

P nigellastrum

C
















































































































A1 B1 B2A2 A3

C1 C2 C3 D1 D2 E1 E2

F1 F2 G1 G2 H1 H2

I1 I2 I3 J1 J2 K1 K2J3

L1 L2 L3 M1 M2

10 mm



A1 A2 B1 B2

C D E1 E2

F G H1 H2

I J K1 K2

2 mm 2 mm 2 mm 2 mm

2 mm

2 mm

2 mm 2 mm

2 mm 2 mm 2 mm 2 mm

2 mm2 mm 2 mm 2 mm

















A B1 B2

C D E1 E2

F G H1 H2

J1 J2

I

2 mm 2 mm 2 mm

2 mm 2 mm 2 mm 2 mm

2 mm

2 mm2 mm2 mm

2 mm 2 mm 2 mm


















minus02 minus01 00 01 02 03

NMDS 1


NMDS 1


minus015

minus010

minus005

000

005

010

015

020

NM

DS

2

minus015

minus010

minus005

000

005

010

015

020

NM

DS

2

N billardierei

N retusa


N sphaerocarpa

N tangutorum

P harmala

P nigellastrum

A

B











4000 3500 3000 2500 2000 1500 1000

Abs

orba

nce


P nigellastrum

N tangutorum

N sphaerocarpa

N sibirica

N schoberi

N retusa

N billardierei

3390 2925 117013702850 17101600

15101440

1030

















minus002

minus001

000

001

002

PCA 1 (47)

PCA2(20

)



minus0015

minus0010

minus0005

0000

0005

0010

PCA 1 (56)

PCA2(27

)

N billardierei

N retusa

N schoberi

N sibirica

N sphaero-carpa

N tangutorum

P nigellastrum

A

B


minus0010

minus0005

0000

0005

0010

PCA 1 (56)

PCA3(11

)

N billardierei

N retusa

N schoberi

N sibirica

N sphaerocarpa

N tangutorum

P nigellastrum

C
















































































































A1 A2 B1 B2

C D E1 E2

F G H1 H2

I J K1 K2

2 mm 2 mm 2 mm 2 mm

2 mm

2 mm

2 mm 2 mm

2 mm 2 mm 2 mm 2 mm

2 mm2 mm 2 mm 2 mm

















A B1 B2

C D E1 E2

F G H1 H2

J1 J2

I

2 mm 2 mm 2 mm

2 mm 2 mm 2 mm 2 mm

2 mm

2 mm2 mm2 mm

2 mm 2 mm 2 mm


















minus02 minus01 00 01 02 03

NMDS 1


NMDS 1


minus015

minus010

minus005

000

005

010

015

020

NM

DS

2

minus015

minus010

minus005

000

005

010

015

020

NM

DS

2

N billardierei

N retusa


N sphaerocarpa

N tangutorum

P harmala

P nigellastrum

A

B











4000 3500 3000 2500 2000 1500 1000

Abs

orba

nce


P nigellastrum

N tangutorum

N sphaerocarpa

N sibirica

N schoberi

N retusa

N billardierei

3390 2925 117013702850 17101600

15101440

1030

















minus002

minus001

000

001

002

PCA 1 (47)

PCA2(20

)



minus0015

minus0010

minus0005

0000

0005

0010

PCA 1 (56)

PCA2(27

)

N billardierei

N retusa

N schoberi

N sibirica

N sphaero-carpa

N tangutorum

P nigellastrum

A

B


minus0010

minus0005

0000

0005

0010

PCA 1 (56)

PCA3(11

)

N billardierei

N retusa

N schoberi

N sibirica

N sphaerocarpa

N tangutorum

P nigellastrum

C





























































































































A B1 B2

C D E1 E2

F G H1 H2

J1 J2

I

2 mm 2 mm 2 mm

2 mm 2 mm 2 mm 2 mm

2 mm

2 mm2 mm2 mm

2 mm 2 mm 2 mm


















minus02 minus01 00 01 02 03

NMDS 1


NMDS 1


minus015

minus010

minus005

000

005

010

015

020

NM

DS

2

minus015

minus010

minus005

000

005

010

015

020

NM

DS

2

N billardierei

N retusa


N sphaerocarpa

N tangutorum

P harmala

P nigellastrum

A

B











4000 3500 3000 2500 2000 1500 1000

Abs

orba

nce


P nigellastrum

N tangutorum

N sphaerocarpa

N sibirica

N schoberi

N retusa

N billardierei

3390 2925 117013702850 17101600

15101440

1030

















minus002

minus001

000

001

002

PCA 1 (47)

PCA2(20

)



minus0015

minus0010

minus0005

0000

0005

0010

PCA 1 (56)

PCA2(27

)

N billardierei

N retusa

N schoberi

N sibirica

N sphaero-carpa

N tangutorum

P nigellastrum

A

B


minus0010

minus0005

0000

0005

0010

PCA 1 (56)

PCA3(11

)

N billardierei

N retusa

N schoberi

N sibirica

N sphaerocarpa

N tangutorum

P nigellastrum

C






























































































































minus02 minus01 00 01 02 03

NMDS 1


NMDS 1


minus015

minus010

minus005

000

005

010

015

020

NM

DS

2

minus015

minus010

minus005

000

005

010

015

020

NM

DS

2

N billardierei

N retusa


N sphaerocarpa

N tangutorum

P harmala

P nigellastrum

A

B











4000 3500 3000 2500 2000 1500 1000

Abs

orba

nce


P nigellastrum

N tangutorum

N sphaerocarpa

N sibirica

N schoberi

N retusa

N billardierei

3390 2925 117013702850 17101600

15101440

1030

















minus002

minus001

000

001

002

PCA 1 (47)

PCA2(20

)



minus0015

minus0010

minus0005

0000

0005

0010

PCA 1 (56)

PCA2(27

)

N billardierei

N retusa

N schoberi

N sibirica

N sphaero-carpa

N tangutorum

P nigellastrum

A

B


minus0010

minus0005

0000

0005

0010

PCA 1 (56)

PCA3(11

)

N billardierei

N retusa

N schoberi

N sibirica

N sphaerocarpa

N tangutorum

P nigellastrum

C























































































































4000 3500 3000 2500 2000 1500 1000

Abs

orba

nce


P nigellastrum

N tangutorum

N sphaerocarpa

N sibirica

N schoberi

N retusa

N billardierei

3390 2925 117013702850 17101600

15101440

1030

















minus002

minus001

000

001

002

PCA 1 (47)

PCA2(20

)



minus0015

minus0010

minus0005

0000

0005

0010

PCA 1 (56)

PCA2(27

)

N billardierei

N retusa

N schoberi

N sibirica

N sphaero-carpa

N tangutorum

P nigellastrum

A

B


minus0010

minus0005

0000

0005

0010

PCA 1 (56)

PCA3(11

)

N billardierei

N retusa

N schoberi

N sibirica

N sphaerocarpa

N tangutorum

P nigellastrum

C



























































































































minus002

minus001

000

001

002

PCA 1 (47)

PCA2(20

)



minus0015

minus0010

minus0005

0000

0005

0010

PCA 1 (56)

PCA2(27

)

N billardierei

N retusa

N schoberi

N sibirica

N sphaero-carpa

N tangutorum

P nigellastrum

A

B


minus0010

minus0005

0000

0005

0010

PCA 1 (56)

PCA3(11

)

N billardierei

N retusa

N schoberi

N sibirica

N sphaerocarpa

N tangutorum

P nigellastrum

C
















































































































UvA-DARE (Digital Academic Repository) A novel approach to ...Submitted 7 March 2018 Accepted 1 June...

Documents

Transcript of UvA-DARE (Digital Academic Repository) A novel approach to ...Submitted 7 March 2018 Accepted 1 June...