Genome size evolution and polyploidy in the Daphniapulex complex (Cladocera: Daphniidae)

ROLAND VERGILINO1, CLAUDE BELZILE2 and FRANCE DUFRESNE1*

1Département de Biologie, Université du Québec à Rimouski, Québec G5L 3A1, Canada2Institut des sciences de la mer de Rimouski (ISMER), Université du Québec à Rimouski, Rimouski,Québec G5L 3A1, Canada

Received 16 June 2008; accepted for publication 19 September 2008

Genome size was estimated in 49 clones of the Daphnia pulex complex from temperate and subarctic locationsusing flow cytometry and microsatellite DNA analyses. Significant genome size differences were found in diploidspecies belonging to the two genetically distinct groups (the pulicaria and the tenebrosa groups), with clones fromthe tenebrosa group having genome sizes 22% larger than those in the pulicaria group. Combined flow cytometryand microsatellite DNA analyses revealed that nearly all polyploid clones in the D. pulex complex are triploid andnot tetraploid, as was previously suggested. Sequencing analyses of the ND5 gene to position clones in theirrespective clades within the D. pulex complex have uncovered three triploid clones of Daphnia middendorffianawith a D. pulex maternal parent. This result was unexpected because Daphnia pulicaria has always been identifiedas the maternal parent of these hybrid polyploid clones. Triploid clones likely owe their origins to interactionsbetween sexual and asexual populations. Further interactions in the tenebrosa group have generated tetraploidclones but these events have been rare. © 2009 The Linnean Society of London, Biological Journal of the LinneanSociety, 2009, 97, 68–79.

ADDITIONAL KEYWORDS: C-value – flow cytometry – microsatellite – triploidy.

INTRODUCTION

The overall nuclear DNA quantity in cells may evolvein different ways. Genome size (the C-value) alter-ations can be gradual and involve small-scale inser-tions and deletions of nucleotides or large-scalealterations such as gene duplications or transposoninsertions (Petrov, 2001; Bennetzen, 2002). Additionsof chromosomes (aneuploidy) or complete genomes(polyploidy) can abruptly increase the DNA quantitycontained in the nucleus. Nuclear DNA contentincreases (whether they arise by polyploidization ornot) are frequently accompanied by phenotypic modi-fications (Levin, 1983; Vinogradov, 1995, 1997; Otto &Whitton, 2000; Gregory, 2002, 2005). Polyploids oftenhave a wider geographical range than their diploidrelatives and are often more prevalent at high lati-tudes (Levin, 1983; Otto & Whitton, 2000; Brochmann

et al., 2004). It has been suggested that elevatedploidy could be positively selected in marginal habi-tats (Dufresne & Hebert, 1995; Little & Hebert, 1997;Stenberg et al., 2003).

Members of the Daphnia pulex complex providegood models to assess the evolutionary consequencesof changes in nuclear DNA content because they varyboth in genome size and in ploidy levels. Previousstudies have revealed a four-fold variation in nuclearDNA content in the D. pulex complex, with the largestvalue arising from polyploidy (Beaton, 1995). Al-though all diploid individuals of this complex possess24 chromosomes (Beaton & Hebert, 1994; Beaton,1995), their C-values vary from 0.37 ± 0.010 pg to0.58 ± 0.013 pg. (Beaton & Hebert, 1989; Beaton,1995). Diploid clones of Daphnia tenebrosa have thelargest genome size in the genus Daphnia (Beaton,1995; Dufresne & Hebert, 1995). The D. pulexcomplex shows a geographical polyploidy pattern(Hobaek, Weider & Wolf, 1993; Ward et al.,*Corresponding author. E-mail: france_dufresne@uqar.qc.ca

Biological Journal of the Linnean Society, 2009, 97, 68–79. With 2 figures

© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 97, 68–7968

1994; Dufresne & Hebert, 1995, 1997; Weider et al.,1999a, b), with polyploid populations found at highlatitudes (53 °N and higher) and altitudes (BolivianAndes) and diploid clones being prevalent in temper-ate regions (Beaton & Hebert, 1988; Dufresne &Hebert, 1995; Aguilera et al., 2007). An apparentexception to this pattern is the discovery of two poly-ploid clones, closely related to the North Americanarctic clones, in Patagonia (within a latitudinal rangeof 46–54 °S; Adamowicz et al., 2002). Members ofpolyploid populations of the D. pulex complex areobligate apomicts (i.e. parthenogens that developfrom unfertilized egg by a mitosis-like cell division)and have multiple hybrid origins (Dufresne & Hebert,1995, 1997). Two species of the complex, Daphniamiddendorffiana and D. tenebrosa, are composedentirely or partially of polyploid populations (Duf-resne & Hebert, 1994, 1995, 1997) and have acircumarctic distribution (Weider et al., 1999a, b).Polyploid clones of this complex are thought to betetraploids due to their unbalanced phenotypes atallozyme loci (Dufresne & Hebert, 1995, 1997) andtheir larger genome sizes (1.7-fold greater than thediploid values) as estimated by Feulgen densitometry(Beaton, 1995; Dufresne & Hebert, 1995). Combinedallozymes and mitochondrial gene sequences haveshown that tetraploid clones of D. middendorffianaand D. tenebrosa result from several events of hybrid-izations (Dufresne & Hebert, 1997).

Genome size estimates of D. pulex, obtainedthrough Feulgen densitometry, often vary dependingon the study. For example, E. M. Rasch, D. Stanton &P. D. N. Hebert (unpublished data; cited in Rasch,1985) obtained C-values in the range 0.23–0.25 pg forD. pulex, whereas Beaton & Hebert (1989) andBeaton (1995) found values of 0.37 ± 0.010 pg and0.38 ± 0.003 pg, respectively. The recent Daphniagenome sequencing project suggests that the C-valueof D. pulex is near 0.20 pg (Cristescu et al., 2006),which is much smaller than the values reported forthe diploid D. pulex clones that served as referencesfor the estimation of ploidy levels (Beaton & Hebert,1988).

The present study aimed to revisit genome size andploidy level estimates in members of the D. pulexcomplex using alternative techniques. Microsatelliteloci are more variable than allozyme loci and moreamenable to assess allele numbers and hence ploidylevel. The majority of studies on Daphnia genomesizes have been carried out with Feulgen staining(Beaton & Hebert, 1988; Beaton, 1995; Dufresne &Hebert, 1997; Adamowicz et al., 2002). Flow cytom-etry is an accurate and rapid technique that allowsthe evaluation of genome size of thousands of cells inthe same run (Galbraith et al., 1983; Bennett et al.,2003). The present study provides new insights into

genome size and ploidy levels of 49 clones of fivespecies in the D. pulex complex using both flow cytom-etry and microsatellite markers.

MATERIAL AND METHODSDAPHNIA COLLECTIONS

Forty-nine Daphnia clones were reared in the labora-tory for several generations prior to the experiments(Table 1). Each clone was kept in a 500 mL cultureflask at a constant temperature and was fed two timesper week with the green alga Selenastrum capricornu-tum. Clones K9, K154, K52, K207, K228, K230, K232,K86, K92, K95, and K31 were sampled from rock poolsand ponds during the summers of 2006 and 2007 atKuujjuarapik, Québec, Canada. Clones A17-200, A35-201, B141-201, B141-202, A, B, C, D, E, F, G, and Hwere sampled in ponds located near Churchill, Mani-toba, Canada, during the summers of 2005, 2006, and2007. Clone Disp1-21 was provided by Dr D. J. Innes,Memorial University. Daphnia pulicaria clones C2,Puli 9, Puli 13, Puli 15, Puli 21, and Puli 22 wereprovided by Dr M. Cristescu, University of Windsor,and Dr C. E. Càceres, University of Illinois.

CLONE IDENTIFICATION

Total DNA was extracted from individual Daphniausing 50 mL of 10% CHELEX 100 solution (Bio-Rad)or using 50 mL of the QuickExtract DNA extractionsolution (Epicentre Biotechnologies). A fragment of amitochondrial gene NADH dehydrogenase subunit 5(ND5) was amplified and sequenced to confirm theidentity of the maternal lineage. DpuND5b (5′-GGGGTGTATCTATTAATTCG-3′) as reverse primerand DpuND5a (5′-ATAAAACTCCAATCAACCTTG-3′)or ND5ColbF (5′-AACTTAGTATCACCAGCAGG-3′) asforward primers were used to amplify approximately850-bp and 700-bp fragments, respectively, in thepulicaria group. Clones C2, Puli 9, Puli 13, Puli 15,Puli 21, and Puli 22 were identified through lactatedehydrogenase allozyme analysis (e.g. homozygosityof the fast ‘F’ allele, was considered to be of D.pulicaria). The primers ND5tene-F (5′-TTTCAACGAAAACCCCTGAC-3′) and ND5tene-R (5′-AGGCCCTGCTCTTTCTTTGT-3′) were needed to amplify a600-bp fragment in the tenebrosa group. Amplifica-tions were carried out using the conditions: one cycleof denaturation at 94 °C for 3 min, and 40 cycles ofdenaturation at 94 °C for 1 min, annealing at 50 °Cfor 1 min, and extension at 72 °C for 2 min, with afinal extension at 72 °C for 15 min. ND5 partialsequences were corrected manually and aligned usingthe CLUSTALW module of BIOEDIT, version 7.0.5(T. Hall, unpublished data; http://www.mbio.ncsu.edu/BioEdit/bioedit.html).

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Table 1. Mitochondrial lineages (ND5 sequences), geographic origins, and habitats of Daphnia clones used in the presentstudy

StocksIdentification (formethods, see text) Reproduction Localization Habitats

CHQ6 pulex Cyclical parthenogen Wisconsin, USA PondEB1 pulex Cyclical parthenogen Minnesota, USA PondPOV1-102 pulex Cyclical parthenogen Michigan, USA PondDISP13 pulex Cyclical parthenogen Ontario, Canada PondPET pulex Obligate parthenogen Quebec, Canada PondLIS3 pulex Obligate parthenogen Ontario, Canada PondWG1 pulex Obligate parthenogen Michigan, USA PondCU4-1 pulex Obligate parthenogen Quebec, Canada PondA17-200 pulex Obligate parthenogen Churchill, Manitoba, Canada Rock bluffMetis-3 pulex Obligate parthenogen Metis, Quebec, Canada PondDisp1-21 pulex Obligate parthenogen Ontario, Canada PondK86 pulex Obligate parthenogen Kuujjuarappik, Quebec, Canada Rock bluffK92 pulex Obligate parthenogen Kuujjuarappik Quebec, Canada Rock bluffK52 pulex Obligate parthenogen Kuujjuarappik Quebec, Canada Rock bluffK9 pulex Obligate parthenogen Kuujjuarappik Quebec, Canada Rock bluffK154 pulex Obligate parthenogen Kuujjuarappik Quebec, Canada Rock bluffK230 Eastern pulicaria Obligate parthenogen Kuujjuarappik Quebec, Canada Rock bluffC2 pulicaria Cyclical parthenogen Michigan, USA LakePuli9 pulicaria Cyclical parthenogen Michigan, USA LakePuli13 pulicaria Cyclical parthenogen Michigan, USA LakePuli15 pulicaria Cyclical parthenogen Michigan, USA LakePuli21 pulicaria Cyclical parthenogen Michigan, USA LakePuli22 pulicaria Cyclical parthenogen Michigan, USA LakeW4-111-b1 Western pulicaria Cyclical parthenogen Indiana, USA LakeW4-95 Western pulicaria Cyclical parthenogen Indiana, USA LakeK207 Western pulicaria Obligate parthenogen Kuujjuarappik Quebec, Canada Rock bluffA35-201 middendorffiana (clade I) Obligate parthenogen Churchill, Manitoba, Canada Rock bluffB141-201 middendorffiana (clade I) Obligate parthenogen Churchill, Manitoba, Canada Rock bluffMIDA20 middendorffiana (clade IIA) Obligate parthenogen Churchill, Manitoba, Canada Rock bluffB141-202 middendorffiana (clade IIA) Obligate parthenogen Churchill, Manitoba, Canada Rock bluffK31 middendorffiana (clade IIB) Obligate parthenogen Kuujjuarappik Quebec, Canada Rock bluffK95 middendorffiana (clade III) Obligate parthenogen Kuujjuarappik Quebec, Canada Rock bluffK228 middendorffiana (clade III) Obligate parthenogen Kuujjuarappik Quebec, Canada Rock bluffK232 middendorffiana (clade III) Obligate parthenogen Kuujjuarappik Quebec, Canada Rock bluffCC3 arenata Cyclical parthenogen Oregon, USA Coastal pondOP1 arenata Cyclical parthenogen Oregon, USA Coastal pondClone A tenebrosa Obligate parthenogen Churchill, Manitoba, Canada PondClone B tenebrosa Obligate parthenogen Churchill, Manitoba, Canada PondClone C tenebrosa Obligate parthenogen Churchill, Manitoba, Canada PondClone D tenebrosa Obligate parthenogen Churchill, Manitoba, Canada PondClone E tenebrosa Obligate parthenogen Churchill, Manitoba, Canada PondClone F tenebrosa Obligate parthenogen Churchill, Manitoba, Canada Thermokast

pondClone G tenebrosa Obligate parthenogen Churchill, Manitoba, Canada PondClone H tenebrosa Obligate parthenogen Churchill, Manitoba, Canada PondClone 13 tenebrosa Obligate parthenogen Churchill, Manitoba, Canada Rock bluffGerber 1a European pulicaria Cyclical parthenogen Central Pyrenees, Catalonia, Spain Mountain lakeGerber 3 European pulicaria Cyclical parthenogen Central Pyrenees, Catalonia, Spain Mountain lakeChmelnice European pulicaria Cyclical parthenogen Chmelnice, Czech republic Lowland lakeMalá Kuš 5-1 European pulicaria Cyclical parthenogen Malá Kuš, Czech republic Lowland lake

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PHYLOGENETIC ANALYSIS

Phylogenetic analyses were carried out to assignclones to their respective lineages and to estimateevolutionary relationships between diploid and poly-ploid clones in the D. pulex complex.

Twenty-nine ND5 sequences (from CU4-1, K9,K154, K52, K207, K228, K230, K232, K86, K92, K95,K31, A17-200, A35-201, B141-201, B141-202,MIDA20, Disp1-21, W4-95, W4-111-b1, Clones 13, A,B, C, D, E, F, H, and Chmelnice clones; accessionnumbers FJ591096 to FJ591124) out of the 49 clonesanalysed for genome size were added to the Markováet al. (2007) data set, yielding a total of 93 distincthaplotypes.

The HKY + G model of DNA substitution witha gamma substitution parameter (a) of 0.3739,transition/tranversion ratios of 5.2071, and basefrequencies of A = 0.1965, C = 0.2025, G = 0.2173, andT = 0.3837, were identified by the hierarchical like-lihood ratio test performed using MODELTEST,version 3.07 (Posada & Crandall, 1998) and PAUP*4.0b10 (Swofford, 2000).

To classify polyploid clones into the phylogeny ofthe complex D. pulex containing the different groupsdefined by Dufresne & Hebert (1995, 1997), Col-bourne et al. (1998) and Weider et al. (1999a), weperformed phylogenetic analyses with PAUP* 4.0b10(Swofford, 2000) and PHYML (Guindon & Gascuel,2003). Gene-tree reconstruction was accomplishedusing Neighbour-joining (NJ) (Saitou & Nei, 1987),with the respective optimal distance model identifiedwith MODELTEST; and maximum parsimony (MP)using heuristic searches with the default option inPAUP*. Maximum likelihood (ML) analysis wascarried out with PHYML, version 2.4.4 (Guindon &Gascuel, 2003), again using the best fit DNA substi-tution model chosen by the MODELTEST program.Confidence in the partitioning within the ML tree wasestimated with 1000 nonparametric bootstrap repli-cates (Felsenstein, 1985).

Bayesian inference was performed with MrBayes,version 3.1.1 (Ronquist & Huelsenbeck, 2003; Huel-senbeck & Ronquist, 2005) assuming the gamma-HKY model of sequence evolution (two substitutiontypes with gamma-distributed rates across sites) andusing the same parameters as those used in theBayesian phylogenetic analysis of Marková et al.(2007).

MULTILOCUS GENOTYPES

Each individual was genotyped at nine microsatelliteloci: Dp183 (Wfms0000187), Dp502 (Wfms0000519),Dp512 (Wfms0000529), Dp513 (Wfms0000530),Dp514 (Wfms0000531), Dp514alt (Wfms0000532),Dp519 (Wfms0000537), Dp523 (Wfms0000541), and

Dp525alt (Wfms0000544) as described in Colbourneet al. (2004) and Cristescu et al. (2006). Polymerasechain reactions (PCR) were performed in 12-mL reac-tions with 10 ng of DNA template, 1¥ PCR bufferwith 25 nmol of MgCl2, 0.5 units of Taq polymerase,2.5 nmol of each dNTP, 2 pmol of the forward primerend-labeled with fluorescent dyes HEX, FAM or TET(Alpha DNA), and 2 pmol of the reverse primer. Eachprimer pair was used separately and PCR reactionsfor each locus were not pooled. Thermal cycle pro-grams included an initial denaturation step of 3 minat 95 °C followed by ten cycles of denaturation at94 °C for 35 s, an initial annealing temperature of55 °C for 35 s for Dp183, Dp502, Dp512, Dp514,Dp514alt, Dp519, Dp523, Dp525alt, and 64 °C forDp513 (the annealing temperature was decreased by1 °C every cycle during each of the six followingcycles), extension at 72 °C for 45 s, followed by 30cycles of denaturation at 94 °C for 35 s, an annealingtemperature at 48 °C or 59 °C for 35 s, and extensionat 72 °C for 45 s, with a final extension at 72 °C for10 min. PCR products were denatured at 95 °C for3 min and alleles were separated on a denatur-ing polyacrylamide gel containing 6% acrylamide/bis-acrylamide (19 : 1) at 50 W and 40 °C for 2–3 h.Fluorescent dye-labeled alleles were detected on aFMBIO III fluorescent scanner (Hitachi SoftwareEngineering America Ltd) and analysed using thesoftware ImageAnalysis, version 3.0.0.21 (Miraibio).Ploidy level for each clone was estimated by countingthe number of alleles at each locus. The assumption ofploidy level n is rejected when level n + 1 is met at agiven locus. With this method, there is a risk oferroneously rejecting level n + 1, but this riskdecreases with the number of loci used and thenumber of alleles per locus. The large number ofpolymorphic loci used in this study (i.e. nine) shouldprovide an accurate estimate of ploidy level.

FLOW CYTOMETRY

The genome size of each of our 49 clones was deter-mined using flow cytometry (Galbraith et al., 1983). Astock solution of chicken erythrocytes was preparedfor use as an internal standard. Freshly-drawnchicken blood was transferred to a sterile tube con-taining a 20-mL heparin solution (100 U mL-1). In aKontes Dounce tissue grinder, 25 mL of blood wasadded to 1 mL of modified Galbraith buffer (Galbraithet al., 1983; Bennett et al., 2003) and was groundgently for 20 strokes with an ‘A’ pestle. This solutionwas filtered through a 40-mm mesh and stored at 4 °Cfor less than 1 week.

Five females of each Daphnia clone were centri-fuged in distilled water at 600 g and washed twicewith distilled water. They were subsequently ground

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gently in a Kontes Dounce tissue grinder for 20strokes with an ‘A’ pestle in 1 mL of modified Gal-braith buffer (Bennett et al., 2003; Rees et al., 2007).After filtering twice through a 40-mm mesh, 2 mL ofthe chicken erythrocyte cell suspension were added toeach sample. Cell suspensions were stained with pro-pidium iodide (50 p.p.m.; Invitrogen) at 4 °C in thedark for about 10 h. The measurements wererepeated two to five times on different individualsover a period of less than one month for the majorityof clones.

The nuclear DNA content of each clone wasassessed using an Epics Altra flow cytometer(Beckman-Coulter) with an argon laser emitting14 mW of light at 488 nm. DNA-PI fluorescence emis-sion was measured at 600–640 nm. Instrument align-ment and stability were monitored by adding 5 mL ofa solution of red-fluorescing beads (Linear FlowCarmine; Molecular Probes). Due to endopolyploidy(somatic cell lines with different ploidy level), only thefirst peak, which has an amplitude (e.g. number ofnucleus measured) always greater than twice the‘endopolyploid peaks’, of the cytograms was taken intoaccount to estimate genome sizes. Nuclear DNAcontent was calculated from the mean fluorescenceintensity (FL, arbitrary units) on the gated data ofthe first peak as:

Total genome size sample FL chicken FL pg= ( ) × ( )2 5 2.

where 2.5 pg corresponds to the diploid genome size ofchicken erythrocytes (Rasch, Barr & Rasch, 1971).The monoploid genome size of each clone (i.e. the1Cx-value corresponding to the 2C-value dividedby the ploidy level; Greilhuber et al., 2005) wascalculated.

Differences between 2C-values obtained for eachdiploid species were analysed with a nonparametricKruskal–Wallis test due to unequal sample sizesusing SYSTAT, version 11 (SYSTAT Software Inc.).The repeated measurements from each clone belong-ing to the same species were averaged. The GT2-method of comparison of means (Hochberg, 1974) wasperformed to determine which pairwise comparisonswere significantly different. The GT2-method givesonly a threshold level of significance (e.g. P < 0.1,P < 0.05 or P < 0.01). Means were considered differentwhen P < 0.05.

RESULTSCLONE IDENTIFICATION

The Bayesian phylogeny inferred from the ND5sequences (Fig. 1) is consistent with the phylogeniesof Colbourne et al. (1998) and Marková et al. (2007).Phylogenetic reconstructions obtained with the ML,NJ, and MP approaches showed similar topologies to

the Bayesian tree (results not shown). They consis-tently recovered all nine evolutionary lineages withinthe D. pulex complex, which fell into two major groups(pulicaria and tenebrosa) as described by Colbourneet al. (1998) and Weider et al. (1999a). The tenebrosagroup consists of European D. pulicaria and D. tene-brosa. The D. tenebrosa lineage included both poly-ploid (A, B, C, G, and H) and diploid (E, D, and F)clones, whereas European D. pulicaria included onlydiploid clones. The pulicaria group consisted of threeD. pulicaria mitochondrial subgroups (Eastern D.pulicaria, Western D. pulicaria, and Polar D. puli-caria, all of which are found in lakes), the D. mid-dendorffiana lineage, the Panarctic D. pulex lineage,and the D. melanica lineage (Fig. 1). A fifth species,Daphnia arenata, belongs to the pulicaria group(Colbourne, Hebert & Taylor, 1997) but their ND5sequences were not included in our phylogeneticanalysis. Polyploid clones of D. middendorffiana havemultiple origins, as described by Dufresne & Hebert(1997). Clones (A35-201 and B141-201) belongedto the D. middendorffiana lineage (Fig. 1). ClonesmidA20 and B141-202 had mitochondrial (mt)DNAhaplotypes that were closely allied to Polar D. puli-caria, whereas clone K31 clustered with the westernlineage of D. pulicaria. Clones (K95, K228, and K232)were closely related to eastern populations of D. puli-caria. Finally, three polyploid clones (Disp1-21, K9,and K154) had mtDNA haplotypes that clustered withPanarctic D. pulex haplotypes (Fig. 1).

POLYPLOID DIVERSITY AND PLOIDY LEVEL

All microsatellite loci used in the present studywere polymorphic in the 49 clones of the D. pulexcomplex, with 4 to 24 alleles per locus and anaverage of 11.56 alleles per locus (see Supportinginformation, Table S1). A maximum of three allelesper locus was observed within clones. Seventeenclones of the 49 tested in this study were polyploids(Table 2). Due to incongruence between ploidy levelsestimated from the first nine microsatellite lociand that from flow cytometric measurements, sixadditional loci were screened; Dp612 (DQ249459),Dp936 (DQ249392), Dp1005 (DQ249458), DP1155(DQ249427), Dp1232 (DQ249380), and Dp1351(DQ249397), as described in Cristescu et al. (2006).These additional loci failed to reveal more thanthree alleles per locus, indicating that these seven-teen polyploid clones are at least triploid. In addi-tion to the forty-nine clones tested in the presentstudy, we obtained multilocus genotypes at micro-satellite loci for another set of 34 polyploid clones ofthe pulex complex and none of them had more thanthree alleles per locus (data not shown).

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GENOME SIZES AND PLOIDY LEVEL

Nuclear DNA content and monoploid genome sizeestimates of the 49 clones are presented in Table 2. Theaverage monoploid genome size (which is equivalent tothe C-value) from different D. pulex clones found in thepresent study (0.23 ± 0.002 pg) is the closest estimateto the value of 0.2035 pg estimated through the D.pulex genome sequencing [(199 ¥ 106)/(0.978 ¥ 109),where 199 ¥ 106 is the number of base pairs in theD. pulex haploid genome and 0.978 ¥ 109 is the numberof base pairs contained in 1 pg; Doležel et al., 2003].The low standard errors (±0.001 pg to ±0.044 pg) forrepeated measurements conducted over 1 month ondifferent individuals from the same clone (Table 2),also indicate the consistency of flow cytometric genomesize estimates. Significant genome size differenceswere found among diploid species (P < 0.001, Kruskal–Wallis test statistic = 39.66; Fig. 2). Average nuclearDNA content for the five diploid species and signifi-cance of test for differences between 2C-values(GT2-method) are presented in Table 3.

Daphnia middendorffiana clones had nuclear DNAcontent in the range 0.64–0.38 pg (Table 2). Due tothe limited number of D. middendorffiana clones, nostatistical test was applied to compare genome sizesof D. middendorffiana clones clustering in differentlineages. Triploid clones (A35-201 and B141-201)from D. middendorffiana s.s., had a 2C-value of0.71 ± 0.006 pg (mean ± SE). Daphnia middendio-rffiana in the polar pulicaria lineage (MIDA20and B141-202) had a mean overall 2C-value of0.69 ± 0.012 pg. Clone K31 from the western puli-caria lineage had a 2C-value of 0.76 ± 0.031 pg.Daphnia middendorffiana with eastern pulicaria

Figure 1. Bayesian reconstruction of the phylogeneticrelationships among individuals of the Daphnia pulexcomplex. Polyploid and diploid Daphnia tenebrosa haplo-types determined in the present study were added to thedatasets of Colbourne et al. (1998) and Marková et al.(2007).The tree is rooted through the European D. pulexgroup Bayesian posterior probabilities are indicated formajor groups. Polyploids tested in the present study areshown in bold.

Figure 2. Nuclear DNA content of diploid clones of theDaphnia pulex complex. Mean ± SE 2C-values are shown.Significant differences in C-value (P < 0.05) are indicatedby different letters.

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Table 2. Genetic and cytogenetic characteristics of Daphnia clones

StocksIdentification (formethods, see text)

Ploidy level(inferred frommicrosatellitedata) N

Nuclear DNAcontent 2C-value

Monoploid genomesize 1Cx-value

Mean ± SE (pg) Mean ± SE (pg)

CHQ6 pulex 2x 2 0.44 ± 0.003 0.22 ± 0.002EB1 pulex 2x 1 0.44 0.22POV1-102 pulex 2x 4 0.47 ± 0.012 0.23 ± 0.006DISP13 pulex 2x 3 0.44 ± 0.015 0.22 ± 0.007PET pulex 2x 1 0.48 0.24LIS3 pulex 2x 1 0.41 0.20WG1 pulex 2x 1 0.46 0.23CU4-1 pulex 2x 7 0.44 ± 0.009 0.22 ± 0.004A17-200 pulex 2x 1 0.48 0.24Metis-3 pulex 2x 1 0.48 0.24Disp1-21 pulex 3x 3 0.62 ± 0.033 0.21 ± 0.011K52 pulex 2x 2 0.45 ± 0.029 0.22 ± 0.014K86 pulex 2x 7 0.46 ± 0.007 0.23 ± 0.004K92 pulex 2x 1 0.47 0.23K9 pulex 3x 2 0.61 ± 0.002 0.20 ± 0.001K154 pulex 3x 3 0.70 ± 0.044 0.23 ± 0.014K230 Eastern pulicaria 2x 2 0.44 ± 0.018 0.22 ± 0.009C2 pulicaria 2x 1 0.45 0.23Puli9 pulicaria 2x 1 0.44 0.22Puli13 pulicaria 2x 1 0.50 0.25Puli15 pulicaria 2x 1 0.49 0.24Puli21 pulicaria 2x 1 0.46 0.23Puli22 pulicaria 2x 1 0.46 0.23W4-111-b1 Western pulicaria 2x 3 0.49 ± 0.007 0.25 ± 0.004W4-95 Western pulicaria 2x 3 0.49 ± 0.024 0.25 ± 0.012K207 Western pulicaria 2x 2 0.47 ± 0.012 0.23 ± 0.006A35-201 middendorffiana (Clade I) 3x 1 0.72 0.24B141-201 middendorffiana (Clade I) 3x 1 0.70 0.23MIDA20 middendorffiana (Clade IIA) 3x 5 0.70 ± 0.013 0.23 ± 0.005B141-202 middendorffiana (Clade IIA) 3x 1 0.69 0.23K31 middendorffiana (Clade IIB) 3x 2 0.76 ± 0.031 0.25 ± 0.010K95 middendorffiana (Clade III) 3x 1 0.67 0.22K228 middendorffiana (Clade III) 3x 1 0.65 0.22K232 middendorffiana (Clade III) 3x 1 0.64 0.21CC3 arenata 2x 1 0.69 0.24OP1 arenata 2x 5 0.48 ± 0.011 0.24 ± 0.005Clone A tenebrosa 3x 2 0.89 ± 0.031 0.30 ± 0.010Clone B tenebrosa 3x 2 0.71 ± 0.012 0.24 ± 0.004Clone C tenebrosa 3x 1 1.07 0.36Clone D tenebrosa 2x 2 0.62 ± 0.021 0.31 ± 0.010Clone E tenebrosa 2x 4 0.54 ± 0.018 0.27 ± 0.009Clone F tenebrosa 2x 2 0.57 ± 0.024 0.28 ± 0.012Clone G tenebrosa 3x 1 1.00 0.33Clone H tenebrosa 3x 1 1.03 0.34Clone 13 tenebrosa 3x 4 0.66 ± 0.026 0.22 ± 0.009Gerber 1a European pulicaria 2x 1 0.46 0.23Gerber 3 European pulicaria 2x 1 0.46 0.23Chmelnice European pulicaria 2x 5 0.57 ± 0.022 0.28 ± 0.011Malá Kuš 5-1 European pulicaria 2x 1 0.52 0.26

N, number of repeated measurements.

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mitochondria (K95, K228, and K232) had the smallest2C-values (0.65 ± 0.007 pg) of all the D. middendorf-fiana clones. The D. middendorffiana clones with D.pulex mtDNA had 2C-values in the range 0.61 ± 0.002to 0.70 ± 0.044 pg (Table 2). Nuclear DNA contentsshowed large variation among the five D. tenebrosapolyploid clones with values in the range 0.71 ± 0.012to 1.07 pg (Table 2). The average 2C-value of clone 13(0.66 ± 0.026 pg), known as an introgressed D. pulexwith D. tenebrosa mitochondria (Dufresne & Hebert,1994), is concordant with 1.5 times the average2C-value of D. pulex diploids (Tables 2, 3).

DISCUSSIONGENOME SIZE IN DIPLOID MEMBERS OF THE

D. PULEX COMPLEX

The results obtained in the present study have shownthat diploid clones belonging to the two major groupsin the D. pulex complex (pulicaria and tenebrosa)have significantly different nuclear DNA content. Asshown in Figure 1, European populations of D. puli-caria have mtDNA haplotypes closely related to thoseof D. tenebrosa and a nuclear DNA content (0.54 pg;Table 3) similar in size to the one of diploid clones ofD. tenebrosa (0.57 pg). Diploid species from the puli-caria group all have smaller 2C-values: D. pulex(0.45 pg), D. arenata (0.48 pg), and D. pulicaria(0.47 pg). Clones in the tenebrosa group have thelargest genomes (not arising as a result of polyploidy)in the whole genus Daphnia (Beaton, 1995), whichindicates a genome size increase of 22% in this par-ticular group. Larger genome sizes in the tenebrosagroup may be due to a greater amount of repetitiveDNA or to transposons (Petrov, 2001). Futureresearch will aim to understand the mechanismsunderlying genome size differences in Daphnia.

EVOLUTION OF POLYPLOIDY IN THE

D. PULICARIA GROUP

Nuclear DNA content values for polyploid clones ofthe pulicaria group were approximately 1.5¥ larger

than those of diploid members of this group, sug-gesting a triploid status for these clones and nottetraploid status as was previously inferred (Beaton& Hebert, 1988; Dufresne & Hebert, 1994, 1997;Beaton, 1995; but see also Ward et al., 1994). Previ-ous measurements of nuclear DNA contents based onFeulgen staining suggested that polyploids had 1.7times more DNA in their cells than diploid clones(Beaton, 1995; Dufresne & Hebert, 1995). Tetraploidstatus was inferred by assuming that these cloneshad lost DNA through genome rearrangementssubsequent to polyploidy. However, results from thepresent study indicate that previous measures ofDNA content values, estimated by Feulgen staining,have been overestimated (for D. pulex, C-values were0.38 pg: Beaton, 1995; 0.37 pg: Beaton & Hebert,1989; 0.23–0.25 pg: Rasch, 1985). We report agenome size (1Cx-value in Table 2) of 0.23 ± 0.002 pgfor D. pulex clone. Comparisons of genome sizemeasurements obtained by both flow cytometry andFeulgen imaging in 42 species of moss have shownto be very similar (Voglmayr, 2000). Overestimationof DNA content based on Feulgen image analyseswas found in marine crustaceans (Rees et al., 2007).It is possible that the smaller number of nucleiavailable in certain animal groups (e.g. exopoditecells of the thoracic limbs, epidermal cells and otherareas underlying the rostrum, the central core areaof the limbs and the heart in Daphnia) may repre-sent a substantial bias for genome size measuresobtained with Feulgen analyses. It is noteworthythat only five individuals were needed for genomesize measurements, which are much less than the20–160 used by Korpelainen, Ketola & Hietala(1997). Propidium iodide, rather than ethidiumbromide, as well as the type of instrument used, mayexplain these differences. Our genome size values,combined with the microsatellite results revealing nomore than three alleles per locus in this group, indi-cate that these clones are triploids and not tetrap-loids. Polyploid clones belonging to the D. pulexcomplex have recently been found in Bolivia and inArgentina. Argentinean Daphnia have been assigned

Table 3. Average 2C-values of Daphnia diploid species

2C-value mean ± SE (pg) Pulex Pulicaria Arenata TenebrosaEuropeanPulicaria

Daphnia pulex 0.45 ± 0.003 –Daphnia pulicaria 0.47 ± 0.007 NS –Daphnia arenata 0.48 ± 0.010 NS NS –Daphnia tenebrosa 0.57 ± 0.017 ** ** ** –European Daphnia pulicaria 0.54 ± 0.021 ** ** * NS –

*0.01 < P < 0.05; **P < 0.01; not significant (NS), P > 0.05.

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a tetraploid status based on Feulgen staining(Adamowicz et al., 2002), whereas the BolivianDaphnia have also been inferred a tetraploid statusbut based on allele dosage at microsatellite loci(Aguilera et al., 2007). The analysis of the twoArgentinean Daphnia revealed a maximum of threealleles at nine microsatellite loci (see Supportinginformation, Table S2). This coupled with the factthat they are very closely related to D. middendor-ffiana clones strongly suggests that they are alsotriploids and not tetraploids. We attempted to obtainploidy level estimates based on microsatellite alleledosages using two methods: the microsatellite DNAallele counting-peak ratios (Esselink, Nybom &Vosman, 2004) and the method used by Aguileraet al. (2007). The former method consists of calculat-ing ratios between the amplitude of the differentpeaks representing different alleles at one locus forone individual. The obtained ratios are then com-pared with the expected ratios under the differentploidy level hypotheses (triploidy, tetraploidy, penta-ploidy, and so on) with different allele configurations(e.g. one locus with three alleles, x, y and z, in atetraploid may present different phenotypes, xxyz,xyyz and xyzz). In the second method, relative peaksizes were assessed in each individual PCR amplifi-cation. To account for differences in amplificationintensity between PCRs, allele peak sizes are stan-dardized to the allele with the smallest peak size foreach PCR and locus, and relative peak sizes of allalleles per locus are averaged for each genotype. Wetested both methods using two different clones (A35-201 and MIDA20). Results for the clone A35-201indicated one dosage for allele 147, two dosages forallele 145, and one dosage for allele 143 at theDp519 locus, congruent with a tetraploid status. Forthe clone MIDA20 at the Dp512 locus, the resultsrevealed a phenotype with two dosages of the 128allele, one of the 135 allele, and two of the 137alleles, thus indicating a pentaploid status. However,the nuclear DNA contents of A35-201 and MIDA20were 0.72 pg and 0.70 pg, respectively, thus clearlyindicating a triploid status. Therefore, the resultsobtained based solely on microsatellite allele dosagewould have proved erroneous in our case. Oneimportant problem with these methods is the hybridorigins of many of these clones. We observed severalcases of unequal amplification efficiency at microsat-ellite loci between the pulicaria and the tenebrosagroups. Thus, clones that are hybrids between thesetwo groups may not possess the same allele dosageand hence may be falsely classified as tetraploids.Therefore, we believe that ploidy level assessmentsobtained through microsatellite dosage alleles mustbe corroborated with DNA quantification analysesand/or chromosome analyses when feasible.

GENOME SIZE EVOLUTION OF D. TENEBROSA

POLYPLOIDS

Daphnia tenebrosa clones never showed more thanthree alleles per locus, as found in D. middendorffi-ana, indicating a triploid nature. Flow cytometry esti-mates confirmed the presence of polyploid clones inthis species, but also revealed an important variationin 2C-values in these clones (0.71–1.07 pg). TheD. tenebrosa clones with 2C-values of 0.71 pg and0.89 pg are likely triploids, whereas the three D.tenebrosa clones with 2C-values in the range 1.00–1.07 pg are probably tetraploids because they havenuclear DNA content of 1.76- to 1.89-fold the averagediploid 2C-value. Polyploidy can be underestimated ifbased solely on limited microsatellite data. One of theputative triploid clones (clone B), with a 2C-value of0.71 pg, has only one locus out of nine with threealleles. Clone D has a 2C-value of 0.62 pg but none ofits loci presented a three allele pattern suggesting adiploid nature of this clone (screening six additionalloci also failed to reveal three alleles). The absence offour alleles in the presumed tetraploid clones couldbe due to the diploidization of these loci. Similardiploidizations are observed at allozyme loci. Manypolyploid clones show unbalanced phenotypes at onlya few loci (Dufresne & Hebert, 1995). Moreover, for allalleles to differ at one locus in a tetraploid that aroseby the chance union of gametes from the samespecies, the heterozygosity of the sexual diploidancestor must be higher than 0.7 (Lokki & Saura,1980). It is possible that none of the tetraploid clonestested here show four different alleles at a locus dueto a low level of heterozygosity in their sexual diploidancestors.

ORIGINS OF POLYPLOIDY IN THE PULICARIA LINEAGE

In North America, diploid D. pulex shows a longitu-dinal gradient in their mode of reproduction withobligately asexual populations in the eastern Canada,mixed populations in Ontario, and cyclically parthe-nogenetic populations in western Canada (Hebert& Crease, 1983; Innes, Schwartz & Hebert, 1986;Hebert et al., 1993). Transitions to obligate partheno-genesis are thought to incur due to meiosis-suppressor genes that disrupt meiosis in females butnot in males (Innes & Hebert, 1988). As a result,males carrying these genes spread the ‘asexualitygenes’ in a contagious fashion. Meiosis-suppressorgenes are thought to have arisen in an eastern D.pulex lineage and, hence, the geographical distribu-tion of obligate parthenogenesis in this species(Paland, Colbourne & Lynch, 2005). Daphnia puli-caria shows an opposite pattern with obligate asexualpopulations found in western regions and cyclicallyparthenogenetic populations in eastern regions

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(Cerný & Hebert, 1993). These two species hybridizeto form diploid apomictic clones in temperate regionsand polyploid apomictic clones in subarctic and arcticregions (Crease, Stanton & Hebert, 1989; Dufresne &Hebert, 1994, 1997). The direction of hybridization inD. middendorffiana is highly asymmetric because themajority of these clones are known to have D. puli-caria as a maternal parent (Dufresne & Hebert,1994). We report for the first time the occurrence ofthree triploid clones with D. pulex mtDNA (Disp1-21,K154, and K9). Their 2C-values are in the range0.61–0.70 pg. Two of them are from Kuujjuarapik,QC, Canada, whereas the other triploid clone wasfrom southern Ontario, a region that has been exten-sively sampled (Dufresne & Hebert, 1994, 1995, 1997;Ward et al., 1994; Weider et al., 1999a, b; Weider &Hobaek, 2003). Are there more clones in arctic orsubarctic regions with D. pulex mtDNA? Why do themajority of polyploid clones analysed so far have D.pulicaria mtDNA? These questions deserve additionalstudies (i.e. more extensive sampling in eastern sub-arctic regions and investigation of mitochondrial-nuclear interactions in both types of hybrids).

IMPORTANCE OF TRIPLOIDY IN THE

D. PULEX COMPLEX

The combined use of different techniques (microsat-ellite DNA and flow cytometry) have allowed us torectify the misleading level of ploidy (tetraploidy)previously described with allozymes and Feulgen den-sitometry (Ward et al., 1994; Dufresne & Hebert,1995, 1997; Adamowicz et al., 2002). The fact thatmost polyploids identified in the present study aretriploids suggests that asexuality is an importantprerequisite for the evolution of polyploidy inDaphnia. As most invertebrate and vertebrate trip-loid clones are closely related to diploid ancestorspecies (Otto & Whitton, 2000) that have unisexualmodes of reproduction (e.g. Thelytokous parthenogen-esis, gynogenesis, hybridogenesis; sensu Gregory &Mable, 2005), it appears that unisexuality in diploidsis an important premise to the formation of triploidunisexuals.

In plants, polyploidy has been shown to generatenovelty by genetic and epigenetic mechanisms (Levin,1983; Salmon, Ainouche & Wendel, 2005). Much lessis known about the effects of polyploidization inanimals. Members of the D. pulex complex thusprovide a useful model to study ecological and evolu-tionary implications of increase in ploidy levels inanimals.

ACKNOWLEDGEMENTS

We thank Dr M. C. Marinone for providing clonesfrom Argentina, Drs M. Cristescu and C. Caceres for

providing some eastern Daphnia pulicaria and Dr D.Innes for providing polyploid D. pulex Disp1-21 fromOntario. The flow cytometry facility at ISMER wasfunded by the Canada Economic Development agency.We are thankful to Teresa J. Crease and three anony-mous reviewers who providing useful comments onthis paper. This work was supported by a NaturalSciences and Engineering research Council (NSERC)research grants and by a Canadian Fundation forInnovation (CFI) equipment grants to France Duf-resne. Roland Vergilino acknowledges a scholarshipfrom Centre d’études nordiques.

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SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version of this article:

Table S1. Multilocus genotypes of each strains used in our study. 000 denotes alleles that we were unable todiscern.Table S2. Multilocus genotypes of each Argentinean Daphnia.

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materialssupplied by the authors. Any queries (other than missing material) should be directed to the correspondingauthor for the article.

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