Phylogenetics and evolution of a circumarctic species complex (Cladocera: Daphnia pulex)

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Biological Journal of the Linnean Society (1998), 65: 347–365. With 3 figures Article ID: bj980251

Phylogenetics and evolution of a circumarctic species complex (Cladocera: Daphnia pulex) J. K. COLBOURNE1, T. J. CREASE1, L. J. WEIDER2, P. D. N. HEBERT1, F. DUFRESNE3, AND A. HOBÆK4 1

Department of Zoology, University of Guelph, Ontario NIG 2W1, Canada; 2Max-PlanckInstitut fu¨r Limnologie, Postfach 165, D-24302 Plo¨n, Germany; 3De´partement de Biologie, Universite´ du Que´bec a` Rimouski, Que´bec G5L 3A1, Canada; 4Norwegian Institute for Water Research, Nordnesboder 5, N-5005 Bergen, Norway Received 17 December 1997; accepted for publication 23 May 1998

The evolutionary history of freshwater zooplankton is still relatively unknown. However, studies of the microcrustacean Daphnia have revealed interesting patterns; the daphniids that dominate ponds and lakes in the northern hemisphere may have recent origins, likely associated with the glacial advances and retreats during the Pleistocene. Moreover, they form species complexes that actively engage in hybridization and introgression. The present study examines the phylogenetic relationships among circumarctic members of the Daphnia pulex complex, through the analysis of sequence diversity in 498 nt of the ND5 mitochondrial gene. Our results suggest that the complex is composed of three major clades, two of which are subdivided into at least eight different lineages. Clearly, species in the complex show genetic discontinuity. Many lineages originated during the Pleistocene, but at least three lineages diverged during the Pliocene. Two taxa (D. pulex, D. pulicaria), thought to be broadly distributed in the northern hemisphere, are shown to be endemic to single continents. In general, the diversification of the pulex complex is characterized by rapidly dispersed lineages spanning enormous distances and also by endemism in temperate areas. Gene flow among lineages from the temperate region of different continents are restricted to rare intercontinental migrations across a polar bridge followed by convergent morphological evolution.  1998 The Linnean Society of London

ADDITIONAL KEY WORDS:—Arctic – speciation – cladistics – phenetics – molecular systematics – hybridization – Pleistocene glaciation – ND5 sequence – mitochondrial DNA. CONTENTS

Introduction . . . . . . . . . . . Methods . . . . . . . . . . . . Daphnia isolates . . . . . . . . DNA amplification and sequencing . . Phylogenetic analysis . . . . . . Results . . . . . . . . . . . . ND5 gene sequence diversity . . . . Phenetic analysis of sequence divergence

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348 350 350 350 352 354 354 354

∗ Correspondence to J. K. Colbourne. E-mail:[email protected] 0024–4066/98/110347+19 $30.00/0

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 1998 The Linnean Society of London

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Cladistic analysis of sequence Confidence in clades . . Discussion . . . . . . . Acknowledgements . . . . References . . . . . . .

divergence . . . . . . . . . . . . . . . .

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356 359 360 363 363

INTRODUCTION

The island-like nature of limnetic habitats would seem to favour the rapid evolution of freshwater zooplankton. Yet, their morphological similarities over vast distances suggest that speciation of microcrustaceans has been constrained by their unusual dispersal syndrome. Individuals cannot actively move beyond the boundaries of individual ponds or lakes, but their diapausing stages are capable of long-distance transport, a factor which was thought to limit opportunities for gene-pool isolation (Darwin, 1859; Mayr, 1963). However, the few efforts to probe the genetic consequences of this dispersal syndrome have revealed an unexpected and complicated pattern. Local populations of zooplankton—sampled within distances of a few kilometers—typically show large genetic differences with FST values ranging from 0.2 to 0.5 (see De Meester, 1996 for a review), indicating that gene flow is insufficient to erode divergence on this geographic scale. Yet, over large distances, populations often show both remarkable similarity in their mean gene frequencies and abrupt genotypic shifts at contact zones between different clades (Crease, Lynch & Spitze, 1990; Hebert & Finston, 1996a; Weider & Hobæk, 1997). This pattern suggests that modern populations have derived from the rapid spread of a small number of ancestral metapopulations, perhaps originally located in different glacial refugia (Crease et al., 1990; Hebert et al., 1993). However, since a similar pattern is also observed in species from areas unimpacted by glaciation (Hebert & Wilson, 1994; Hebert & Finston, 1996a), it appears that this pattern of gene-pool divergence is not restricted to a few species with an unusual biogeographic history. There is a need to further investigate the consequence of long-distance dispersal in structuring the genetics of zooplankton populations if we are to uncover the importance of geographical speciation in these organisms. No group of freshwater zooplankton has been more intensively studied than members of the Daphnia pulex complex (sensu Colbourne & Hebert, 1996). This work was motivated by their dominance in aquatic environments throughout the northern hemisphere (Hrba´cek, 1987), by their ability to hybridize, and by their breeding system and ploidy-level variation. These traits may all be epiphenomena of active speciation, as this is the sole daphniid assemblage (so far) in the northern hemisphere which shows evidence of a recent radiation. The Nearctic fauna is thought to include at least six species (Hebert, 1995). Daphnia pulex (Leydig) and D. pulicaria (Forbes) are broadly distributed in the temperate zone and their ranges also extend into the Arctic. In contrast, D. arenata (Hebert) and D. melanica (Hebert) are narrow endemics, restricted to coastal ponds in Oregon. Two other species, D. middendorffiana (Fischer) and D. tenebrosa (Sars), are restricted to the Arctic. The Palearctic fauna is thought to include the same four broadly distributed taxa from North America (Hrba´cek, 1987), but endemic species may also exist, as taxonomic studies have been less thorough in this area. Apart from the apparent overlap in species distributions, Holarctic daphniids share other significant biological similarities. Temperate-zone populations consist solely of

MOLECULAR SYSTEMATICS OF THE DAPHNIA PULEX COMPLEX

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diploid lineages, while polyploids dominate polar habitats (Beaton & Hebert, 1988; Ward et al., 1994). Polyploid lineages are invariably of hybrid origin, although several species have been involved in their formation. For example, all D. middendorffiana are polyploids that appear to be derived from hybridization events between D. pulicaria and other members of the pulex complex (Van Raay & Crease, 1995; Dufresne & Hebert, 1997). Similarly, hybridization between D. tenebrosa and D. pulex seems to always generate introgressed polyploids (Dufresne & Hebert, 1994). Although polyploids are apparently absent from the temperate zone, hybrid production is common. Diploid F1 hybrids between D. pulex and D. pulicaria comprise nearly half of the individuals sampled within temperate Canadian habitats, although they ordinarily reproduce by obligate parthenogenesis (Hebert et al., 1993). Studies of their mitochondrial DNA have so far shown that D. pulex is always the maternal parent (Crease, Stanton & Hebert, 1989; Crease et al., 1990). By contrast, hybrids from polar regions never have this species as the maternal parent. Holarctic members of the pulex complex also show breeding system diversity: some lineages reproduce by cyclic parthenogenesis and others by obligate parthenogenesis. The two North American endemics (D. arenata, D. melanica) employ only the former breeding system, but the species restricted to arctic environments are either entirely (D. middendorffiana) or largely (D. tenebrosa) asexual. Polar populations of D. pulex and D. pulicaria also reproduce asexually, but the geographic distribution of their breeding systems in the temperate zone is more complicated. For example, populations of D. pulex in the eastern half of North America reproduce by obligate parthenogenesis, while those in the west are cyclic parthenogens (Hebert et al., 1993). The asexual lineages of all four taxa show an extraordinary amount of clonal diversity, apparently linked to their recurrent generation through hybridization and sex-limited meiosis suppression (Innes & Hebert, 1988). Because these biological attributes are firmly linked to hybridization, cases of gene-pool isolation and secondary contact by subsequent dispersion (during glacial advances and retreats) seem particularly important in forging diversification within the pulex complex. In fact, phylogenetic studies using sequence divergence in the 12S rDNA have indicated that all North American members of the species complex stem from the Pleistocene (< 2 Myr ago), save D. tenebrosa, which diverged from the others during the Tertiary (Colbourne & Hebert, 1996). Unfortunately, relationships among the closely related species is uncertain, because of the relative uniformity of their sequences in this slowly evolving ribosomal gene. The present study aims to provide a thorough understanding of phylogenetic relationships among members of the D. pulex complex, especially in the polar regions of the northern hemisphere. The analysis extends prior genetic investigations of the group, in both analytical approach and geographic scale. Most preceding work has examined RFLP variation in the mitochondrial genome, but this study employs direct sequence analysis of a 498 nt fragment of the rapidly evolving ND5 gene. The present study examines sequence diversity among lineages from sites throughout Eurasia and North America, while past work provided only a local perspective on genetic affinities. Using the phylogenetic framework granted by this study, we intend to present several papers that investigate the interplay between geographic isolation and long-range transport of propagules in the evolutionary divergence of these organisms.

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Daphnia isolates Populations of the D. pulex complex were sampled from several hundred rockpools, ponds and lakes at locations throughout the Arctic (Table 1). Populations were initially surveyed for variation at six polymorphic allozyme loci. Individuals with distinct multilocus allozyme phenotypes were then chosen for mitochondrial DNA analysis in order to maximize the detection of unique haplotypes. Restriction site variation in a 2.1 kb fragment of the NADH dehydrogenase subunit 4 (ND4) and subunit 5 (ND5) genes was surveyed in isolates from 276 populations. Of the 171 unique mitochondrial DNA haplotypes that were found in the Arctic, 61 were subsequently chosen for nucleotide sequencing (Table 1) based on their higher frequency of occurrence among localities. Because the taxonomy of the D. pulex complex is paradoxical, isolates from temperate populations of D. pulex and D. pulicaria in Europe (Cerny & Hebert, 1998) and North America (Hebert et al., 1993) were included in the analysis, including the eastern, western and polar North American D. pulicaria lineages (Dufresne & Hebert, 1997). Unlike the arctic populations, the taxonomy of the temperate isolates was confirmed prior to sequencing using both morphological criteria and fixed allozyme differences (Hebert, 1995). Phylogenetic groups were later assigned species names based primarily on the placement of these temperate isolates within clades. Daphnia melanica and D. arenata from North America (Hebert, 1995) were also included in the analysis.

DNA amplification and sequencing Total genomic DNA was extracted from single animals or multiple animals from clonal cultures using the Isoquick kit (Orca Research, Bothell, WA). A 2.1 kb fragment containing part of the mitochondrial ND4 and ND5 genes was amplified from this genomic template using the primers ND4-new: 5′–ACTCTTCAGTAGCTCATATGA–3′ and ND5-new: 5′–AAGGAAGAAACCATATTAAACC–3′ in a total reaction volume of 50 ll containing 10–100 ng of genomic DNA, 0.3–0.5 lM of each primer, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 200 lM each of dGTP, dATP, dCTP and dTTP, 1% dimethyl sulfoxide and 2.5 units of Taq polymerase (Boehringer Mannheim). The amplification reaction was performed in a Perkin-Elmer-Cetus thermal cycler (Model 480) under the following conditions: one cycle of denaturation at 94°C for 1 min, and 35 cycles of denaturation at 92°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 2 min. The amplified fragment was electrophoresed on a 1% agarose gel in TAE buffer and stained with ethidium bromide. The DNA fragments were visualized under long-wave UV light, then purified from the gel using the Gene Clean II kit (Bioclean 101). The sequence of 498 nt of this fragment, from the ND5 gene, was determined using the primer DpuND5b: 5′–GGGGTGTATCTATTAATTCG–3′. Twenty to fifty nanograms of the purified fragment was sequenced using 3 pmol of the primer and the ABI Prism TaqFS dye terminator kit (Perkin-Elmer). The sequencing reactions were analysed on an ABI 377 automated sequencer.

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T 1. List of Daphnia included in the study, and their collection site Taxon

Area

Site

DOF 1a DOF 2 DOF 3 DOF 4 DOF 5a DOF 6 DOF 7 DOF 8ab DOF 9 DOF 10 DOF 11a DOF 12a DOF 13 DOF 14 DOF 15 DOF 16 DOF 17 DOF 18 DOF 19ab ESB 1 ESB 2a ESB 3 ESB 4a ESB 5 ESB 6 ESB 7 ESB 8a ESB 9 GER 1a GER 2b GRL 1 GRL 2 GRL 3a GRL 4a GRL 5 ICE 1 ICE 2 ICE 3 ICE 4a IWA 1b MAN 1 MAN 2 MAN 3a MAN 4a NOR 1 NOR 2 NOR 3 NWT 1ab NWT 2b NWT 3 ONT 1ab ONT 2a ORE 1ab ORE 2ab ORE 3b ORE 4ab ORE 5ab ORE 6b ORE 7b

District of Franklin, CAN District of Franklin, CAN District of Franklin, CAN District of Franklin, CAN District of Franklin, CAN District of Franklin, CAN District of Franklin, CAN District of Franklin, CAN District of Franklin, CAN District of Franklin, CAN District of Franklin, CAN District of Franklin, CAN District of Franklin, CAN District of Franklin, CAN District of Franklin, CAN District of Franklin, CAN District of Franklin, CAN District of Franklin, CAN District of Franklin, CAN Eastern Siberia Eastern Siberia Eastern Siberia Eastern Siberia Eastern Siberia Eastern Siberia Eastern Siberia Eastern Siberia Eastern Siberia Germany Germany Greenland Greenland Greenland Greenland Greenland Iceland Iceland Iceland Iceland Iowa, USA Manitoba, CAN Manitoba, CAN Manitoba, CAN Manitoba, CAN Norway Norway Norway Northwest Territories, CAN Northwest Territories, CAN Northwest Territories, CAN Ontario, CAN Ontario, CAN Oregon, USA Oregon, USA Oregon, USA Oregon, USA Oregon, USA Oregon, USA Oregon, USA

Summerset Island Summerset Island Summerset Island Bathurst Island Devon Island Summerset Island Bathurst Island Summerset Island Devon Island Cornwallis Island Cornwallis Island Summerset & Devon Islands Bathurst Island Summerset Island Bathurst Island Devon Island Devon Island Cornwallis Island Summerset Island Kolyma Delta Wrangel Island Kolyma Delta NW Indigirka Olenekskyi Wrangel Island Kolyma Delta Kolyma Delta Kolyma Delta Grosser Binnensee Grosser Binnensee Godhavn Sondre Stromfjord Godhavn Godhavn Uummannaq Island Western Western Western Western Amana Churchill Churchill Churchill Churchill Finmark Finmark Finmark Tuktoyaktuk Tuktoyaktuk Tuktoyaktuk Redchalk Lake Rondeau Park Florence Florence Florence; Zoil Zoil Florence Florence Lab clone from M. Lynch continued

J. K. COLBOURNE ET AL.

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T 1—continued Taxon

Area

Site

ORE 8b SAS 1 SAS 2 SVL 1a SVL 2a SVL 3 SVL 4 SVL 5 SVL 6a SWI 1b SWI 2ab SWI 3ab SWI 4ab SWI 5ab WAS 1a WSB 1 WSB 2 WSB 3 WSB 4a WSB 5 WSB 6 WSB 7a WSB 8

Oregon, USA Saskatchewan, CAN Saskatchewan, CAN Svalbard Svalbard Svalbard Svalbard Svalbard Svalbard Switzerland Switzerland Switzerland Switzerland Switzerland Washington, USA Western Siberia Western Siberia Western Siberia Western Siberia Western Siberia Western Siberia Western Siberia Western Siberia

Zoil Redberry Lake Humbolt Lake

a b

Basel Basel Basel Basel Basel Lake Washington Kachkovsky Bay Kachkovsky Bay Kola/Karelia Western Yamal Peninsula Kachkovsky Bay Western Yamal Peninsula Kolguyev Island Belyi Island

Designates taxa used for preliminary cladistic analysis. Designates taxa with no restriction site data.

Phylogenetic analysis The 498 nt sequence of the ND5 gene, coding for 166 amino acids, was aligned for all taxa by eye using the SeqApp v1.9 sequence editor (Gilbert, 1992). Estimates of sequence divergence between all pairs of unique haplotypes were calculated using the Kimura two-parameter model (Kimura, 1980) in MEGA v1.02 (Kumar, Tamura & Nei, 1993). Phenetic analysis of the resulting divergence matrix was carried out using the neighbour-joining (N-J) method of Saitou and Nei (1987) in MEGA. Brower (1994) showed that the rates of nucleotide substitution within mitochondrial genes of arthropods are relatively constant during the first few million years. However, this substitution rate is not specifically calibrated for particular genes, thus complicating historical accounts related to the origin of clades when comparing across data sets. Nonetheless, his general estimate of 2.3% pairwise sequence divergence per million years was used to approximate the times of divergence among species whose sequence divergence did not exceed 8%. This clock differs only slightly from the conventional mitochondrial sequence divergence rate of 2% per million years (Brown, George & Wilson, 1979). A cladistic analysis of the phylogenetically informative sites was performed using maximum parsimony (MP) in PAUP v3.1.1 (Swofford, 1993). Because of the large number of sequences, it was impossible to complete a search for parsimonious cladograms of all taxa. Therefore, representative taxa of all major groupings identified from the N-J tree were chosen to first verify that the branching patterns obtained via an abridged cladistic analysis were consistent with those of the N-J tree. Then, in an hierarchical fashion, relationships within monophyletic groupings were further

MOLECULAR SYSTEMATICS OF THE DAPHNIA PULEX COMPLEX

A

C

353

E D

B

F

Figure 1. A hypothetical cladogram depicting the hierarchical phylogenetic method used to obtain a stepwise agreement tree. Taxa from clades adjacent to the tail end of an arrow are used as a functional outgroup to resolve relationships among members within sister clades. The method is described in detail in the text.

analysed by successively choosing more closely related sister group taxa as functional outgroups (Fig. 1). More precisely, we selected a most parsimonious cladogram for the whole data set according to the following steps. (i) Clades which occurred in both the N-J and the abridged cladistic trees were identified, while disputed branches between the trees were collapsed to form polytomies. (ii) Representatives of the sister group of each terminal clade were selected and then used as functional outgroups to find all equally parsimonious trees describing relationships within these terminal clades (A–C, Fig. 1). (iii) A consensus tree was constructed for each set of equally parsimonious trees found in step two. (iv) Representative taxa of the next sister group to pairs of sister clades were then selected and used as functional outgroups to find the most parsimonious trees describing alliances among these clades (D–F, Fig. 1). (v) From among the most inclusive equally parsimonious trees found in step four (E & F, Fig. 1), a tree preserving the consensus topologies of internal clades obtained from step three was chosen. Thus, a most parsimonious cladogram including all taxa, with branching pattern and tree length consistent with (or better than) each functional ingroup analysis was chosen as the best tree. We call this cladogram a stepwise agreement tree. The rationale behind tree-building in this fashion is both practical and ideal; the method provides an unbiased means by which to draw a single tree from among multiple equally parsimonious cladograms, while nucleotide characters are more likely to be correctly polarized because the probability of multiple substitutions per site is reduced when taxa that are relatively recently diverged from the ingroup taxa are used to root the trees. Homoplasy was measured using both the consistency and retention indices (CI and RI: see Wiley et al., 1991), even though they were sometimes inconsistent across data sets (Archie, 1989; Naylor & Kraus, 1995). After generating a stepwise agreement tree, the confidence in each clade was assessed by (i) using PAUP to plot 100 000 trees drawn at random from all possible topologies as a function of tree length to obtain an estimate of the g1 skewness statistic (Hillis & Huelsenbeck, 1992); (ii) using AutoDecay v2.9.6 (Eriksson, 1995) to evaluate the decay index (Bremer, 1994); and

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(iii) using Random Cladistics v3.0 (Siddall, 1995a) for bootstrapping (Felsenstein, 1985) and measuring the jackknife monophyly index (Siddall, 1995b).

RESULTS

ND5 gene sequence diversity Seventy-nine unique sequences were found among the 82 isolates listed in Table 1, as isolates with different restriction site profiles for the 2.1 kb fragment had the same sequence for the 498 nt fragment. A total of 182 nt positions were polymorphic and variation at 40 of these sites led to amino acid substitutions. As many as four different amino acids occurred at some of the polymorphic sites. Further, 153 nt were informative for cladistic analysis. Corrected pairwise sequence divergence estimates ranged from 0.01% to 19%. Transitional saturation of sequences among ingroup taxa was not evident and their base composition was similar (test for homogeneity, v2=40.4, df=234, P>0.9).

Phenetic analysis of sequence divergence in the ND5 gene The N-J tree constructed from the matrix of sequence divergence shows that the pulex complex can be divided into three major groups (A–C, Fig. 2). Group A is comprised of six distinct clusters of mitochondrial DNA haplotypes. Taxa that cluster on branches one, two and three (Fig. 2) correspond to lineages that have been identified as D. pulicaria based on morphology and allozymes (Dufresne & Hebert, 1997). One lineage consists of isolates found in Greenland, Iceland and the polar regions of Europe and Canada (Fig. 2, cluster 1). Another lineage contains isolates inhabiting western temperate regions of North America (cluster 2), while the most basal D. pulicaria lineage consists of isolates with an eastern (eastern Canada, Greenland, Iceland) distribution (cluster 3). The other three clusters within Group A correspond to D. melanica haplotypes (cluster 4), D. middendorffiana haplotypes (cluster 5), and D. pulex haplotypes (cluster 6). The latter clade includes all isolates from North America that have been identified as D. pulex on the basis of morphology and allozymes, as well as isolates from Eurasia. Thus, this clade was designated ‘panarctic D. pulex’. Surprisingly, the D. arenata isolates (ORE 5, ORE 6) cluster within panarctic D. pulex. The sister group to D. pulicaria within this N-J tree is D. melanica, the only temperate Daphnia species known to possess cuticular pigmentation—a trait which is also common in populations of the arctic species, D. middendorffiana and D. tenebrosa (Hebert, 1995). Group B is subdivided into two genetically divergent clusters of haplotypes. One cluster (Fig. 2, cluster 7) consists of isolates found in western Siberia and Svalbard, Norway, but also includes isolates that have traditionally been identified as temperate European D. pulicaria (Dufresne, 1995). The other cluster (8), which is restricted to the Arctic, is very broadly distributed in both North America and Eurasia and has been identified as D. tenebrosa, based on morphology and allozymes (Dufresne & Hebert, 1995). Group C consists solely of isolates of D. pulex from Europe (SWI) and occupies the most basal position on the tree. Allozyme evidence indicated that

MOLECULAR SYSTEMATICS OF THE DAPHNIA PULEX COMPLEX

A

7

B 8

C

Sequence Divergence 0

DOF 1 DOF 2 DOF 4 DOF 3, GRL 1 DOF 5 DOF 6 DOF 7 1 DOF 8 DOF 9 ICE 1 SVL 1 GRL 2 NOR 1 DOF 10 DOF 11 SAS 1 2 SAS 2 WAS 1 ONT 1 3 ICE 2 GRL 3 ICE 3 ORE 1 4 ORE 4 ORE 2 ORE 3 DOF 13 DOF 14 DOF 12 DOF 15 5 DOF 16 DOF 17 NWT 1 NWT 2 DOF 18 DOF 19 GRL 4 NOR 2 ICE 4 NOR 3 WSB 1 ORE 5 6 ORE 6 WSB 2 GRL 5 WSB 3 ORE 7 ONT 2 ORE 8, IWA 1 NWT 3 WSB 4 WSB 5 WSB 6 GER 1 GER 2,SWI 1 SVL 2 ESB 1 MAN 4 MAN 2 MAN 3 MAN 1 WSB 7 WSB 8 ESB 2 ESB 3 SVL 3 ESB 4 ESB 5 ESB 6 ESB 7 ESB 8 SVL 4 ESB 9 SVL 6 SVL 5 SWI 2 9 SWI 3 SWI 4 SWI 5

355

polar D. pulicaria

western D. pulicaria eastern D. pulicaria D. melanica

D. middendorffiana

panarctic D. pulex

European D. pulicaria

D. tenebrosa

European D. pulex

1%

Figure 2. A neighbour-joining tree of ND5 nucleotide variation in the D. pulex complex. Letters on branches denote the three major groupings within the complex. Numbers indicate either Daphnia species or distinct mitochondrial lineages within a species. The nine lineages identified in this study are shown on the right side of the figure.

these taxa are typical European D. pulex (Hebert, Schwartz & Hrba´cek, 1989). Thus, isolates from Europe that have been identified as D. pulex based on morphology belong to two very divergent clades (see above). The branches leading to D. melanica, D. middendorffiana and D. pulicaria are very short, suggesting that these species diverged within a relatively brief period of time (Table 2). Brower’s (1994) estimate of 2.3% sequence divergence per million years suggests that panarctic D. pulex diverged from the ancestor of (D. melanica, D. middendorffiana, D. pulicaria) about 2.2 million years ago, while the split among these

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T 2. Mean sequence divergence of the ND5 mitochondrial gene between phylogenetic groups identified with the neighbour-joining method (see Fig. 2). The mean sequence divergence within major groups is on the diagonal. The mean sequence divergence between major groups is shown above the diagonal. The estimates were corrected using the Kimura (1980) two-parameter model of molecular evolution

1 2 3 4 5 6 7 8 9

1

2

3

4

5

6

7

8

9

0.010

0.024 0.007

0.025 0.035 0.005

0.036 0.047 0.035 0.015

0.037 0.044 0.029 0.037 0.009

0.051 0.060 0.049 0.054 0.044 0.016

0.158 0.168 0.154 0.158 0.160 0.155 0.014

0.148 0.160 0.147 0.145 0.156 0.158 0.075 0.028

0.178 0.176 0.166 0.182 0.180 0.172 0.165 0.175 0.012

latter three species occurred between 1.6 and 1.4 million years ago (Table 2). Using the same molecular clock, the two subgroups (clusters 7 and 8) of group B appear to have diverged from one another on the order of 3.2 million years ago. Sequence divergence between groups A and B is 15.3% while the amino acid divergence is 5.7%. So far, there is no calibrated ND5 clock to date the more ancient divergence between these two clades.

Cladistic analysis of sequence divergence in the ND5 gene The phenetic analysis suggested that European D. pulex is the most divergent group in the pulex complex. Furthermore, additional 12S rDNA sequence information confidently placed European D. pulex at the base of the species complex when included in the Colbourne and Hebert (1996) 12S phylogeny of North American members of the genus (unpublished data). Consequently, this group was used to root preliminary cladograms constructed using only a few taxa from each major lineage (see Table 1). A heuristic search using only informative characters (IC) within the ND5 sequences found three most parsimonious trees (length=275; CI=0.60; RI=0.88) that differed from each other only at nodes within the polar D. pulicaria clade. These trees (not presented) show distinct clades corresponding to all the major groups and lineages outlined by the phenetic tree, except western D. pulicaria, which grouped within polar D. pulicaria. Their topologies are similar to the phenogram, except that D. melanica was the sister clade to D. middendorffiana. The g1 statistic (0.35) of 100 000 random trees indicates strong phylogenetic signal from the total data (P
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