Annals of Botany 82 (Supplement A): 99–105, 1998Article No. bo980751

Genome Size is Negatively Correlated with Altitude in Natural Populations of

Dactylis glomerata

GAIL REEVES*, DENNIS FRANCIS†, M. STUART DAVIES, HILARY J. ROGERSand TREVOR R. HODKINSON‡§

School of Biosciences, Cardiff Uni�ersity, PO Box 915, Cardiff CF1 3TL, UK

and ‡The Jodrell Laboratory, Royal Botanic Gardens, Kew, Surrey TW9 3DS, UK

Received: 29 October 1997 Returned for revision: 17 June 1998 Accepted: 2 July 1998

Previously, we found a significant negative correlation between DNA C-value and altitude among eight naturalpopulations of Dactylis glomerata L. (Creber et al., New Phytologist 128 : 555–561, 1994). We have examined theextent to which similar negative relationships exist in other altitudinal transects, one in southern France and the otherin Italy. Using Feulgen microdensitometry, C-values were negatively correlated with altitude both for the French andItalian populations. A combined plot of DNA C-values against altitude for all of the transects (representing C-valuesfor 17 natural populations), exhibited a highly significant negative relationship; there was a 1±3-fold variation in DNAC-value from the largest genome-lowest altitude to the smallest genome-highest altitude natural population. Such aconsistent marked altitudinal cline suggests strong nucleotypic selection acting upon these populations with increasingaltitude. Preliminary examination of amplified fragment length polymorphisms between populations selected from theupper and lower limits of the French and Italian transects has shown that these populations are genetically distinct.The extent to which this genetic separation is related to altitude or genome size, or both, is discussed.

# 1998 Annals of Botany Company

Key words : Altitude, AFLPs, Dactylis glomerata, DNA C-value.

INTRODUCTION

The DNA C-value for a species is the amount of nuclearDNA in the unreplicated haploid genome of a gamete(Swift, 1950). Angiosperms exhibit at least a 2500-foldvariation in DNA C-value from 0±05 pg in Cardamine amarato 124±7 pg in Fritillaria assyriaca (Bennett, 1987; Bennettand Smith, 1991). However, this may not represent theabsolute extent of C-value variation since it encompasses amere 1% of the world’s angiosperm flora (Bennett andLeitch, 1995).

DNA C-value is positively related to a number of cellularcharacters including mitotic cell cycle time, rate of nuclearDNA synthesis and cell and nuclear size through to wholeplant characters, such as minimum generation time andgeographic distribution (Bennett, 1972, 1976, 1987; Kidd,Francis and Bennett, 1987). Important ecological charac-teristics of plant species in natural habitats also vary withgenome size, e.g. timing of spring growth (Grime, Shacklockand Band, 1985), cell size and rate of leaf expansion in earlyseason growth (Grime et al., 1985) and frost resistance(Macgillivray and Grime, 1995). Importantly, some of theseso-called nucleotypic correlations, e.g. the relationshipbetween genome size and cell cycle duration, are sustainablewithin a ploidy level but break down in comparisons of C-

Present address : *NERC Centre for Population Biology, ImperialCollege at Silwood Park, Ascot, Berks, SL5 7PY, UK, §Departmentof Botany, University of Dublin, Trinity College, Dublin 2, Eire.

† For correspondence. E-mail francisd!cardiff.ac.uk

values between species of different ploidy levels (Kaltsikes,1972, Bennett, 1972; Kidd, Francis and Bennett, 1992).

The majority of published DNA C-values of angiosperms,which cover about 3000 species to date (Bennett and Leitch,1997), indicate interspecific variation which is neithercorrelated with chromosome number nor ploidy level ;higher C values exist in some diploids compared with sometetraploids. The majority of published C-value data is basedon single samples and hence might favour the idea of DNAC-value constancy for a species. Papers first describingintraspecific variation appeared in the mid 1960s. Forexample, a 16% variation in DNA C-values, induced byenvironmental treatments, was reported for Linum usitati-ssimum (Evans, Durrant and Rees, 1966). Since then,intraspecific variation has been reported for a range ofunrelated species and taxa e.g. 20% variation in Aegilopssquarrosa (Furuta, Nishikawa and Makino, 1975), 29 to67% in two Microseris species (Price, Chambers andBachmann, 1981a, b) and 37% in different cultivars of Zeamays (Laurie and Bennett, 1985; Rayburn et al., 1985).Moreover, Bennett (1985) listed 24 angiosperm species inwhich intraspecific variation ranged between 4 and astaggering 288%, leading to the conclusion that the conceptof the �ery constant genome be refined to the fairly constantgenome (Bennett and Leitch, 1995). However, the occurrenceand extent of intraspecific variation in genome size continuesto be debated because, despite the many reports of suchvariation in the literature, others have failed to substantiateit. For example, a 1±29-fold variation in genome size inPisum sati�um (Cavallini and Natali, 1990; Cavallini et al.,

0305-7364}98}0A0099­07 $30.00}0 # 1998 Annals of Botany Company

100 Ree�es et al.—Genome Size is Negati�ely Correlated with Altitude in D. glomerata Populations

1993), was not confirmed by other workers (Greilhuber andEbert, 1994).

Some of the reported intraspecific variation in DNA C-values shows significant relationships with geographiclocation. For example, cultivars of Zea mays in NorthAmerica exhibited a decline in C-value with increasinglatitude (Rayburn et al., 1985). Similarly, a negativecorrelation between genome size and latitude of origin wasfound in European populations of Festuca arundinacea(Ceccarelli, Falisfocco and Cionini, 1992). However, thesecorrelations are not found consistently because in naturalpopulations of Picea glauca (Teoh and Rees, 1976) andDactylis glomerata (Creber et al., 1994) there were noconsistent relationships between C-value and latitude oforigin. Similarly, conflicting accounts exist of possiblerelationships between altitude and genome size. In maize,heterochromatic knob number decreases with altitude butfluorometric measurements of DNA amount revealedincreasing genome size in accession numbers from higherelevations (Rayburn and Auger, 1990, Rosato et al., 1998).Importantly, selection of cultivars which mature morerapidly in progressively shorter growing seasons since thebeginning of maize cultivation probably confounds theserelationships. In our view, studies of natural populations,which are free of conscious or unconscious artificialselection, provide a better source of material to examinepossible relationships between intraspecific DNA C-valuevariation and geographic distribution. As mentioned earlier,ten natural populations of D. glomerata from various sitesin mainland Europe did not show a meaningful relationshipbetween C-value variation and latitude (Creber et al., 1994).However, in the same study, eight populations from analtitudinal transect in northern Spain [350–1120 m abovesea level (asl)] showed a significant negative relationshipwith altitude. In other words, the higher the origin of thepopulation, the lower the DNA C-value. To examinewhether this pattern of change was more widespread in thisspecies, natural populations were analysed from twocompletely separate altitudinal transects, one in southernFrance and the other in Sicily ; we present these data here.

Over the past decade, the extent of genetic variability inplant populations has been studied by methods which detectDNA polymorphisms. These genetic fingerprints can bedetected by the presence or absence of fragments inelectrophoretic banding patterns generated by restrictionenzyme digestion or DNA amplification procedures, orboth. Using amplified fragment length polymorphisms(AFLPs) (Vos et al., 1995), we have determined the extent towhich intraspecific DNA C-value variation and altitude oforigin was matched by genetic differences between thesealtitudinally contrasting populations of D. glomerata.

MATERIALS AND METHODS

Plant material

Seeds of natural populations of Dactylis glomerata werecollected from altitudinal transects in France (500 to2500 m asl) and Italy (80 to 1500 m asl) by the GeneticResources Unit, Institute of Grassland and Environmental

F. 1. Map showing the origin of the altitudinal transects described inTable 1.

T 1. Details of the sites of origin and DNA C-�alues (pg)of the populations of Dactylis glomerata collected from three

altitudinal transects in France, Italy and Spain

IGER accessionnumber

Latitude(°N)

Longitude(°E)

Altitude(m asl)

Mean C-value (pg)(³s.e.)

French TransectBC 5734 44±28 3±21 450 5±35³0±10BC 5442 44±34 2±40 800 5±03³0±08BC 5449 44±15 2±50 975 5±10³0±10BC 5445 44±41 2±50 1040 4±70³0±01BC 5446 44±36 2±59 1320 4±56³0±11BC 5471 44±41 6±20 2500 4±37³0±20

Italian TransectBC 7216 37±43 12±56 80 4±99³0±07BC 7226 37±06 14±46 520 4±74³0±07BC 7227 37±08 14±55 700 5±00³0±07BC 7245 37±52 14±23 1050 4±51³0±06BC 7246 37±52 14±01 1430 4±49³0±05

Spanish TransectBC 6930 42±52 8±03W 350 5±39³0±04BC 7041 42±15 8±13W 400 5±46³0±04BC 6924 42±32 8±06W 550 5±60³0±04BC 7003 42±28 8±16W 650 5±27³0±03BC 6922 42±32 8±06W 740 5±32³0±05BC 6925 42±28 8±16W 920 5±46³0±03BC 6949 42±44 7±01W 1000 4±98³0±03BC 6974 42±01 7±59W 1120 4±88³0±02

Research, Aberystwyth, UK (Fig. 1, Table 1). A preliminarycytological analysis confirmed that all of these populationswere tetraploid (2n¯ 4x¯ 28).

On experimental day 0, seed from the five French and fiveItalian populations together with a natural population fromsouthern Portugal of known DNA C-value (2n¯ 2x¯ 14,C value¯ 3±3 pg, 38°53« N, 7°10« W), were sown on fibreglass matting supported on a polystyrene raft. Subsequently,the material was grown in exactly the same way as describedpreviously (Creber et al., 1994). On experimental day 5, the

Ree�es et al.—Genome Size is Negati�ely Correlated with Altitude in D. glomerata Populations 101

seedlings were fixed immediately in 3:1 (v}v) ethanol :glacialacetic acid at 4 °C for a minimum of 24 h.

C-�alue determination by Feulgen microdensitometry

DNA C-value determinations were undertaken as de-scribed previously (Creber et al., 1994). All of the procedureswere carried out at exactly the same time, using the samereagents throughout. The fixed root tips were washed indistilled water and then hydrolysed in 5 HCl for exactly62 min at 25 °C. Previously, we found that deviationby³5 min from this optimum hydrolysis time for D.glomerata reduced the staining intensity by about 5%(Creber et al., 1994). Immediately after hydrolysis, rootswere washed in ice-cold distilled water (2¬5 min) and thenimmersed in Feulgen stain at 25 °C for exactly 2 h. Theroots were then transferred to 45% (v}v) acetic acid. Theprimary root meristems were placed in droplets of 45%acetic acid, dissected with needles and squash preparationsmade. The preparations were then made permanent asdescribed previously (Powell, Davies and Francis, 1986).The density of each of ten prophases, selected at random,was measured immediately for each of three meristems pernatural population using a Vickers M85A integratingmicrodensitometer at a wavelength of 560 nm. Densityreadings in arbitrary units were converted to DNA amountby reference to the amount of DNA in the Portuguesepopulation [referred to by Creber et al. (1994) by itsaccession number, 6265], the standard used throughoutthese measurements.

Analysis of amplified fragment length polymorphisms(AFLPs)

AFLPs were analysed between 12 individuals in theFrench transect, five from the population at 2500 m asl, andseven from the population at 800 m asl. Similarly, AFLPswere examined between 16 individuals from the Italiantransect, seven from the population at 1430 m asl and ninefrom the population at 80 m asl. In addition, one individualof D. glomerata from a grassland scrub near the laboratoryat Cardiff, was analysed alongside the French individuals,and two local genotypes were included in the analysis of theItalian genotypes. For each genotype, genomic DNA wasextracted from approx. 0±5 g of fresh leaf tissue using the2¬CTABmethod ofDoyle andDoyle (1987). Subsequently,all DNA extracts were purified by caesium chloride-ethidiumbromide equilibrium gradients (1±55 g ml−"). Purified DNAwas resuspended in 1¬TE buffer and stored at ®20 °C.

All protocols relating to the preparation and detection ofAFLPs were performed as described by PE AppliedBiosystems Inc. (ABI) (1996). Briefly, purified DNA wasdigested with the restriction enzymes: EcoR1 (Promega)and Mse1 (New England BioLabs) and double-strandedadaptor pairs (ABI) ligated to the restriction fragments withT4 ligase (Promega) in a single reaction. These taggedrestriction fragments were used as template DNAs forpreselective PCR directed by preselective primers 5«GCA-TCTGACGCATGGTTAAG-3« (EcoR1) and 5«-TACTCA-GGACTCATT®3« (Mse1) (selective base at the 3« ends of

the primers is underlined). The preselective amplifiedproducts were then further amplified by PCR using thefollowing selective primers, EcoR1: 5«-CGTAGACGTC-GTACCAATTCCA-3« with attached fluorescent dye FAM(blue) ; Mse1: 5«ATGAGTCCTTGAGTAACG-3«.

2±5 µl formamide dye [98% (v}v) formamide, 10 m

EDTA, 0±1% dextran blue, GS-500 ROX-labelled sizestandard] were added to 0±75 µl FAM-labelled PCR pro-ducts. Samples were then denatured at 90 °C for 3 min.Subsequently, entire samples were loaded onto a 5% (w}v)denaturing polyacrylamide gel using 1¬TBE runningbuffer. All samples were run on an ABI PRISM 377 DNAsequencer according to manufacturer’s protocols (ABI).

AFLP data analysis

The AFLP data were analysed by GeneScan}Genotypersoftware (ABI). The software identified all DNA bands,compared the mobility of each fragment to the internal GS-500 ROX-labelled size standard, and then sized thefragments, in base pairs, accordingly. The area and intensityof each DNA band was quantified and represented as peaks.Any fragment with an amplitude at or below 50 arbitraryfluorescent units was excluded from the analysis due to theimpossibility of discerning background signal from a weaklyamplified band.

Phylograms were generated from a matrix of pairwisedistances using the UPGMA (unweighted pair groupmethod using arithmetic averages) or Neighbour-Joininganalysis (Saitou and Nei, 1987).

RESULTS

For the French transect, the C-values were negativelycorrelated with altitude [Fig. 2B, y¯ 5±43®0±00045(³0±0001)x (r¯®0±871; P¯ 0±024)] ; there was 22±7%variation in DNA C-values. Similarly, C-values were nega-tively correlated with altitude for the Italian transect [Fig.2C, y¯ 5±04®0±00040 (³0±00015)x (r¯®0±824; P¯0±08)]. Previously, we showed that populations taken from atransect in the Galician mountains of northern Spain alsoexhibited a negative relationship between genome size andaltitude [Fig. 2A, y¯ 5±76®0±0007 (³0±0002)x (r¯®0±74,P¯ 0±037, Creber et al., 1994)]. A combined plot of DNAC-values against altitude from all three transects (Fig. 3)representing 17 measurements, exhibited a highly significantnegative relationship between altitude and C-value (y¯5±44®0±00049 (³0±00013)x (r¯®0±69, P¯ 0±001). Such aconsistent marked altitudinal cline in three geographicallyseparate transects in mainland Europe suggests that strongnucleotypic selection is acting upon these populations withincreasing altitude (Table 1).

AFLP data analysis

UPGMA is a clustering technique from which a tree(dendogram) is generated that portrays linking of pairs oftaxa (genotypes) exhibiting least difference in AFLPs (leastdifferent pairs of genotypes) followed by successively greaterdifferences in AFLPs (successively more distant pairs of

102 Ree�es et al.—Genome Size is Negati�ely Correlated with Altitude in D. glomerata Populations

2500

5.4

4.20

Altitude (m asl)

DN

A C

-val

ue

(pg)

4.8

5.2

5

4.6

4.4

500 1000 1500 2000

C

Italy

2500

5.4

4.20

DN

A C

-val

ue

(pg)

4.8

5.2

5

4.6

4.4

500 1000 1500 2000

B

France

2500

5.8

4.20

DN

A C

-val

ue

(pg)

4.8

5.2

5

4.6

4.4

500 1000 1500 2000

A

Spain

5.4

5.6

F. 2. The relationship between mean DNA C-value (pg) (³s.e., n¯30) and altitude of origin [metres above sea level m (asl)] for naturalpopulations of D. glomerata. A, Eight populations in Galicia, Spain(Creber et al., 1994) ; B, six populations in southern France; C, five

populations in Sicily, Italy. See text for regressions.

genotypes or groups of genotypes). An unrooted phylogramusing UPGMA on the individuals from the Italian popu-lations (Fig. 4A) showed a distinct clustering of the lowaltitude genotypes which was clearly separated from aclustering of the high-altitude genotypes. The phylogram isconsistent with a clear genetic separation of the smaller-genome, high-altitude population from the larger genome,low-altitude population. The two genotypes from the Cardiffarea, depicted in the phylogram as outgroups 1 and 2,

Altitude (m asl)

2500

5.8

4.20

DN

A C

-val

ue

(pg)

4.8

5.2

5

4.6

4.4

500 1000 1500 2000

5.4

5.6

F. 3. A combined plot of C-value against altitude for all populationsshown in Fig. 2. See text for regressions. +, Spanish; _, French; E,

Italian.

respectively, were grouped by this technique into a separatecluster from both the high and low altitude Italianpopulations.

The AFLP patterns of these genotypes were also analysedby Neighbour-Joining (Fig. 4B). Unlike clustering tech-niques, this method allows for unequal rates of molecularchange among branches. However, it does seek to fit thedata to an additive tree. In contrast to cluster analysis,Neighbour-Joining monitors nodes on a tree as opposed totaxa or clusters of taxa. The Neighbour-Joining algorithmhas been described as a special case of a star decompositionmethod whereby a tree is constructed by linking leastdistant pairs of nodes (Swofford et al., 1996). Applying thismethod to the genotypes from the Italian transect resultedin the separation of most of the low altitude genotypes fromthe high altitude genotypes, but genotype 12 from highaltitude shared a common node with genotype 1 from lowaltitude, which is consistent with a limited genetic overlapbetween sites. Overall, this analysis resulted in a remarkablysimilar separation pattern to that generated by UPGMA.

An unrooted Neighbour-Joining phylogram of the Frenchgenotypes (Fig. 5) shows a common node for four of the2500 m asl genotypes (1, 2, 4 and 6) and another commonnode for six of the 800 m asl genotypes (8–13). However,genotype 5 from 2500 m asl shared a common node withgenotype 7 from 800 m asl. The Cardiff genotype formed aclear outgroup. The UPGMA phylogram showed exactlythe same pattern (data not shown). Hence, both techniquesshowed a clear genetic separation of all of the larger-genome, low-altitude genotypes from four out of the fivesmaller-genome, high-altitude genotypes.

DISCUSSION

Clear negative correlations were established between DNAC-value and altitude for both the French and Italiantransects. Thus the genome size data for the French andItalian altitudinal transects matched our previous obser-vations on populations from an altitudinal transect in

Ree�es et al.—Genome Size is Negati�ely Correlated with Altitude in D. glomerata Populations 103

1430 m no. 8

1430 m no. 12

1430 m no. 6

1430 m no. 10

1430 m no. 7

1430 m no. 11

1430 m no. 9

80 m no. 9

80 m no. 8

80 m no. 4

80 m no. 780 m no. 5

80 m no. 3

80 m no. 10

80 m no. 6

80 m no. 1

Outgroup 1

Outgroup 2

A. UPMGA unrooted phylogram

Italy 80 m: 9 individualsItaly 1430 m: 7 individuals

B. Neighbour-joining unrooted phylogram

80 m no. 8

1430 m no. 9

1430 m no. 7

1430 m no. 10

1430 m no. 11

80 m no. 5

80 m no. 4

1430 m no. 6

80 m no. 380 m no. 1

80 m no. 6

80 m no. 10

80 m no. 780 m no. 9

1430 m no. 8

1430 m no. 12

Outgroup 1

Outgroup 2

N-J genetic distance of 0.1

F. 4. Italian transect AFLP data analysed by unrooted phylogramsfor seven genotypes from 1430 m asl ( ) and nine genotypes from80 m asl ( ), Outgroups 1 and 2 are two genotypes from Cardiff. A,

UPGMA; B, Neighbour-Joining.

Galicia, northern Spain (Creber et al., 1994). Moreover, thecombined plot of all three transects is consistent with theconclusion that the higher the altitude the lower the genomesize. Similar results have been reported for Argentineanraces of maize (Rosato et al., 1997) where populationslacking B chromosomes and encompassing a 36% variationin genome size also exhibited a negative correlation betweengenome size and altitude. The intraspecific variation inDNA C-value may be explicable in terms of differences inthe level of heterochromatin between populations. In otherwords, whilst genic content would be expected to berelatively constant for the species, variations in non-coding

France 2500 m: 5 individualsFrance 800 m: 7 individuals

800 m 13

800 m 11

N-J genetic distance of 0.1

800 m 10

800 m 8

800 m 9

800 m 12

2500 m 5

800 m 72500 m 2

2500 m 4

2500 m 6

2500 m 1

Outgroup

F. 5. French transect AFLP data analysed by an unrooted Neighbour-Joining phylogram of five genotypes from 2500 m asl ( ) and sevengenotypes ( ) from 800 m asl. Outgroup 1 is one Cardiff genotype.

repetitive DNA might account for the differences that wedetect. ‘DNA makes RNA makes protein’ describes asimplistic relationship which is at the heart of studies thataim to provide a greater understanding of the control ofgene expression in living organisms. However, it ignoresfeatures of the topology of DNA which are known to affectboth gene expression and the timing of various eventsthroughout the life of a cell. DNA topology will depend, atleast partly, on the conformation of chromatin whereregions of active gene expression (euchromatin) can bedistinguished cytologically from non-transcribed regions ofthe genome.

Notwithstanding the current debate about intraspecificvariation in genome size (Bennett and Leitch, 1997), howcould variation in DNA C-value in these transects be linkedto important cellular determinants of growth such as celldivision? Nucleotypic correlations within a ploidy levelindicate that compared with large genome size species,species with smaller genomes exhibit cells with rapid rates ofnuclear DNA replication and cell division. For example, at20 °C, the duration of the cell cycle was 11 h in Oryza sati�a(DNA C-value¯ 0±6 pg) compared with 30 h in Tulipakaufmanniana (C-value¯ 22±6 pg; Kidd et al., 1987). Notethat the 33-fold increase in C-values between these specieswas accompanied by a three-fold increase in the duration ofthe cell cycle. The intraspecific variation in C-values reportedhere only represents a 1±3-fold increase from the lowest to thehighest natural population. This relatively small increase inC-value may not impact greatly on cell cycle duration.

104 Ree�es et al.—Genome Size is Negati�ely Correlated with Altitude in D. glomerata Populations

However, we concluded elsewhere that in a meristem, theproportion of rapidly cycling cells is related positively togrowth rates in D. glomerata exposed to elevated CO

#

(Kinsman et al., 1997) and in newly formed leaves inLolium temulentum (Moses, Ougham and Francis, 1997).Hence, we are now examining the possibility that theharsher climates and shorter growing seasons at highaltitude favour genotypes of D. glomerata with smallergenomes whose meristems exhibit a higher proportion ofrapidly cycling cells compared with lower altitude-largergenome size genotypes.

Closer scrutiny of the combined plot (Fig. 3) reveals thatgenotypes from the lower-altitude sites on the Italiantransect had smaller genomes compared with the lowerlatitude populations from the French and Spanish transects.It is notable that the Sicilian transect was located at a loweraltitude than the other two; the relatively smaller genomesize of the low-altitude populations from this more southerlysite is consistent with a trend of species from southernlatitudes having smaller genomes than those from northernlatitudes (Bennett, 1987). This general cline of increasinggenome size with increasing latitude is reversed in verynorthern latitudes and it has been argued that this mayresult from selection against individuals with high C-valuein these habitats with harsh climates and short growingseasons (Grime and Mowforth, 1982; Bennett, 1987). Dothe same forces operate at the higher altitudes of ourtransects? In the absence of primary cell cycle measurementsin the natural populations from our altitudinal transectsand, given the complexities of prevailing climate andfluctuations in climatic conditions which are likely atdifferent times of the year (e.g. Grace, 1987), furtherpredictions about the relationship between genome size andrate and pattern of cell division, and their relationship toplant growth and development, would be highly speculative.However, we believe that a study of these natural popu-lations of D. glomerata, which originate from contrastingclimates and which also vary in genome size, are idealexperimental tools with which to investigate the impact ofDNA C-value with environmental factors on the adaptationof plants to contrasting environments.

The AFLPs detected in this study are consistent with agenetic separation of high and low altitude genotypes inboth the French and Italian transects. Application of otherDNA fingerprinting techniques e.g. random amplifiedpolymorphic DNA (RAPDs) and single sequence repeats(SSR), carried out on the French 800 m asl and 2500 m aslpopulations also revealed near identical genetic separationpatterns (G. Reeves unpubl. res.). We have demonstratedthat AFLP technology can be used to resolve differentgenotypes and we can now examine the extent to whichthese DNA polymorphisms are related to tolerance ofclimatic factors or C-value, or both, at the genotypic level.

Our results describing a maximum 1±30-fold variation inDNA C-value in D. glomerata from three different alti-tudinal sites adds to the debate on intraspecific variation ingenome size. Clearly more measurements of natural popu-lations from a wider range of species are required to fullyresolve the influence of the environment on genome sizewithin plant species.

ACKNOWLEDGEMENTS

We thank Drs I. Thomas and K. H. Chorlton of the GeneticResources Unit, Institute of Grassland and EnvironmentalResearch, Aberystwyth for supplying seed of all populationsused in this study. We also thank Drs Mark Chase andMichael Fay, Jodrell Laboratory, Royal Botanic Gardens,Kew for their help and advice with the AFLP technique andthe associated phylograms.

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