High genomic diversity in the bank vole at the northern apex of a range expansion: The role of multiple colonizations and end-glacial refugia.

The history of repeated northern glacial cycling and southern climatic stability has long dominated explanations for how genetic diversity is distributed within temperate species in Eurasia and North America. However, growing evidence indicates the importance of cryptic refugia for northern colonization dynamics. An important geographic region to assess this is Fennoscandia, where recolonization at the end of the last glaciation was restricted to specific routes and temporal windows. We used genomic data to analyse genetic diversity and colonization history of the bank vole (Myodes glareolus) throughout Europe (>800 samples) with Fennoscandia as the northern apex. We inferred that bank voles colonized Fennoscandia multiple times by two different routes; with three separate colonizations via a southern land-bridge route deriving from a "Carpathian" glacial refugium and one via a north-eastern route from an "Eastern" glacial refugium near the Ural Mountains. Clustering of genome-wide SNPs revealed high diversity in Fennoscandia, with eight genomic clusters: three of Carpathian origin and five Eastern. Time estimates revealed that the first of the Carpathian colonizations occurred before the Younger Dryas (YD), meaning that the first colonists survived the YD in Fennoscandia. Results also indicated that introgression between bank and northern red-backed voles (Myodes rutilus) took place in Fennoscandia just after end-glacial colonization. Therefore, multiple colonizations from the same and different cryptic refugia, temporal and spatial separations and interspecific introgression have shaped bank vole genetic variability in Fennoscandia. Together, these processes drive high genetic diversity at the apex of the northern expansion in this emerging model species.

Genomics of end-Pleistocene population replacement in a small mammal.

Current species distributions at high latitudes are the product of expansion from glacial refugia into previously uninhabitable areas at the end of the last glaciation. The traditional view of postglacial colonization is that southern populations expanded their ranges into unoccupied northern territories. Recent findings on mitochondrial DNA (mtDNA) of British small mammals have challenged this simple colonization scenario by demonstrating a more complex genetic turnover in Britain during the Pleistocene-Holocene transition where one mtDNA clade of each species was replaced by another mtDNA clade of the same species. Here, we provide evidence from one of those small mammals, the bank vole (Clethrionomys glareolus), that the replacement was genome-wide. Using more than 10 000 autosomal SNPs we found that similar to mtDNA, bank vole genomes in Britain form two (north and south) clusters which admix. Therefore, the genome of the original postglacial colonists (the northern cluster) was probably replaced by another wave of migration from a different continental European population (the southern cluster), and we gained support for this by modelling with approximate Bayesian computation. This finding emphasizes the importance of analysis of genome-wide diversity within species under changing climate in creating opportunities for sophisticated testing of population history scenarios.

Mapping 3' transcript ends in the bank vole (Clethrionomys glareolus) mitochondrial genome with RNA-Seq.

BACKGROUND: Although posttranscriptional modification of mitochondrial (mt) transcripts plays key roles in completion of the coding information and in the expression of mtDNA-encoded genes, there is little experimental evidence on the polyadenylation status and the location of mt gene poly(A) sites for non-human mammals. RESULTS: Poly(A)-enriched RNA-Seq reads collected for two wild-caught bank voles (Clethrionomys glareolus) were mapped to the complete mitochondrial genome of that species. Transcript polyadenylation was detected as unmapped adenine residues at the ends of the mapped reads. Where the tRNA punctuation model applied, there was the expected polyadenylation, except for the nad5 transcript, whose polyadenylated 3' end is at an intergenic sequence/cytochrome b boundary. As in human, two pairs of bank vole genes, nad4l/nad4 and atp8/atp6, are expressed from bicistronic transcripts. TAA stop codons of four bank vole protein-coding genes (nad1, atp6, cox3 and nad4) are incompletely encoded in the DNA and are completed by polyadenylation. This is three genes (nad2, nad3 and cob) less than in human. The bank vole nad2 gene encodes a full stop codon (TAA in one vole and TAG in the other), which is followed by a 2 bp UTR and the gene conforms to the tRNA punctuation model. In contrast, the annotations of the reference mouse and some other rodent mt genomes in GenBank include complete TAG stop codons in both nad1 and nad2, which overlap downstream trnI and trnW, respectively. Thus the RNA-Seq data of bank voles provides a model for stop codons of mt-encoded genes in mammals comparable to humans, but at odds with some of the interpretation based purely on genomic data in mouse and other rodents. CONCLUSIONS: This work demonstrates how RNA-Seq data were useful to recover mtDNA transcriptome data in a non-model rodent and to shed more light on mammalian mtDNA transcriptome and post-transcriptional modification. Even though gene content and organisation of mtDNA are strongly conserved among mammals, annotations that neglect the transcriptome may be prone to errors in relation to the stop codons.

Population genetic dynamics of an invasion reconstructed from the sediment egg bank.

Biological invasions are a global issue with far-reaching consequences for single species, communities and whole ecosystems. Our understanding of modes and mechanisms of biological invasions requires knowledge of the genetic processes associated with successful invasions. In many instances, this information is particularly difficult to obtain as the initial phases of the invasion process often pass unnoticed and we rely on inferences from contemporary population genetic data. Here, we combined historic information with the genetic analysis of resting eggs to reconstruct the invasion of Daphnia pulicaria into Lower Lake Constance (LLC) in the 1970s from the resting egg bank in the sediments. We identified the invader as 'European D. pulicaria' originating from meso- and eutrophic lowland lakes and ponds in Central Europe. The founding population was characterized by extremely low genetic variation in the resting egg bank that increased considerably over time. Furthermore, strong evidence for selfing and/or biparental inbreeding was found during the initial phase of the invasion, followed by a drop of selfing rate to low levels in subsequent decades. Moreover, the increase in genetic variation was most pronounced during early stages of the invasion, suggesting additional introductions during this period. Our study highlights that genetic data covering the entire invasion process from its beginning can be crucial to accurately reconstruct the invasion history of a species. We show that propagule banks can preserve such information enabling the study of population genetic dynamics and sources of genetic variation in successful invasive populations.

Mitogenomic phylogenetics of the bank vole Clethrionomys glareolus, a model system for studying end-glacial colonization of Europe.

We have revisited the mtDNA phylogeny of the bank vole Clethrionomys glareolus based on Sanger and next-generation Illumina sequencing of 32 complete mitochondrial genomes. The bank vole is a key study species for understanding the response of European fauna to the climate change following the Last Glacial Maximum (LGM) and one of the most convincing examples of a woodland mammal surviving in cryptic northern glacial refugia in Europe. The genomes sequenced included multiple representatives of each of the eight bank vole clades previously described based on cytochrome b (cob) sequences. All clades with the exception of the Basque - likely a misidentified pseudogene clade - were highly supported in all phylogenetic analyses and the relationships between the clades were resolved with high confidence. Our data extend the distribution of the Carpathian clade, the marker of a northern glacial refugium in the Carpathian Mountains, to include Britain and Fennoscandia (but not adjacent areas of continental Europe). The Carpathian sub-clade that colonized Britain and Fennoscandia had a somewhat different history from the sub-clade currently found in or close to the Carpathians and may have derived from a more north-westerly refugial area. The two bank vole populations that colonized Britain at the end of the last glaciation are for the first time linked with particular continental clades, the first colonists with the Carpathian clade and the second colonists with the western clade originating in a more southerly refugium in the vicinity of the Alps. We however found no evidence that a functional divergence of proteins encoded in the mitochondrial genome promoted the partial genetic replacement of the first colonists by the second colonists detected previously in southern Britain. We did identify one codon site that changed more often and more radically in the tree than expected and where the observed amino acid change may affect the reductase activity of the cytochrome bc1 complex, but the change was not specific to a particular clade. We also found an excess of radical changes to the primary protein structure for geographically restricted clades from southern Italy and Norway, respectively, possibly related to stronger selective pressure at the latitudinal extremes of the bank vole distribution. However, overall, we find little evidence of pervasive effects of deviation from neutrality on bank vole mtDNA phylogeography.

Adaptive phylogeography: functional divergence between haemoglobins derived from different glacial refugia in the bank vole.

Over the years, researchers have used presumptively neutral molecular variation to infer the origins of current species' distributions in northern latitudes (especially Europe). However, several reported examples of genic and chromosomal replacements suggest that end-glacial colonizations of particular northern areas may have involved genetic input from different source populations at different times, coupled with competition and selection. We investigate the functional consequences of differences between two bank vole (Clethrionomys glareolus) haemoglobins deriving from different glacial refugia, one of which partially replaced the other in Britain during end-glacial climate warming. This allows us to examine their adaptive divergence and hence a possible role of selection in the replacement. We determine the amino acid substitution Ser52Cys in the major expressed ß-globin gene as the allelic difference. We use structural modelling to reveal that the protein environment renders the 52Cys thiol a highly reactive functional group and we show its reactivity in vitro. We demonstrate that possessing the reactive thiol in haemoglobin increases the resistance of bank vole erythrocytes to oxidative stress. Our study thus provides striking evidence for physiological differences between products of genic variants that spread at the expense of one another during colonization of an area from different glacial refugia.

Genetic admixture drives climate adaptation in the bank vole.

Genetic admixture introduces new variants at relatively high frequencies, potentially aiding rapid responses to environmental changes. Here, we evaluate its role in adaptive variation related to climatic conditions in bank voles (Clethrionomys glareolus) in Britain, using whole-genome data. Our results reveal loci showing excess ancestry from one of the two postglacial colonist populations inconsistent with overall admixture patterns. Notably, loci associated with climate adaptation exhibit disproportionate amounts of excess ancestry, highlighting the impact of admixture between colonist populations on local adaptation. The results suggest strong and localized selection on climate-adaptive loci, as indicated by steep clines and/or shifted cline centres, during population replacement. A subset, including a haemoglobin gene, is associated with oxidative stress responses, underscoring a role of oxidative stress in local adaptation. Our study highlights the important contribution of admixture during secondary contact between populations from distinct climatic refugia enriching adaptive diversity. Understanding these dynamics is crucial for predicting future adaptive capacity to anthropogenic climate change.

Assessing the Impacts of Adaptation to Native-Range Habitats and Contemporary Founder Effects on Genetic Diversity in an Invasive Fish.

Species invading non-native habitats can cause irreversible environmental damage and economic harm. Yet, how introduced species become widespread invaders remains poorly understood. Adaptation within native-range habitats and rapid adaptation to new environments may both influence invasion success. Here, we examine these hypotheses using 7058 SNPs from 36 native, 40 introduced and 19 farmed populations of tench, a fish native to Eurasia. We examined genetic structure among these populations and accounted for long-term evolutionary history within the native range to assess whether introduced populations exhibited lower genetic diversity than native populations. Subsequent to infer genotype-environment correlations within native-range habitats, we assessed whether adaptation to native environments may have shaped the success of some introduced populations. At the broad scale, two glacial refugia contributed to the ancestry and genomic diversity of tench. However, native, introduced and farmed populations of admixed origin exhibited up to 10-fold more genetic diversity (i.e., observed heterozygosity, expected heterozygosity and allelic richness) compared to populations with predominantly single-source ancestry. The effects of introduction to a new location were also apparent as introduced populations exhibited fewer private alleles (mean = 9.9 and 18.9 private alleles in introduced and native populations, respectively) and higher population-specific Fst compared to native populations, highlighting their distinctiveness relative to the pool of allelic frequencies across tench populations. Finally, introduced populations with varying levels of genetic variation and similar genetic compositions have become established and persisted under strikingly different climatic and ecological conditions. Our results suggest that lack of prior adaptation and low genetic variation may not consistently hinder the success of introduced populations for species with a demonstrated ability to expand their native range.

Local adaptation and future climate vulnerability in a wild rodent.

As climate change continues, species pushed outside their physiological tolerance limits must adapt or face extinction. When change is rapid, adaptation will largely harness ancestral variation, making the availability and characteristics of that variation of critical importance. Here, we used whole-genome sequencing and genetic-environment association analyses to identify adaptive variation and its significance in the context of future climates in a small Palearctic mammal, the bank vole (Clethrionomys glareolus). We found that peripheral populations of bank vole in Britain are already at the extreme bounds of potential genetic adaptation and may require an influx of adaptive variation in order to respond. Analyses of adaptive loci suggest regional differences in climate variables select for variants that influence patterns of population adaptive resilience, including genes associated with antioxidant defense, and support a pattern of thermal/hypoxic cross-adaptation. Our findings indicate that understanding potential shifts in genomic composition in response to climate change may be key to predicting species' fate under future climates.

Genic distribution modelling predicts adaptation of the bank vole to climate change.

The most likely pathway for many species to survive future climate change is by pre-existing trait variation providing a fitness advantage under the new climate. Here we evaluate the potential role of haemoglobin (Hb) variation in bank voles under future climate change. We model gene-climate relationships for two functionally distinct Hb types, HbS and HbF, which have a north-south distribution in Britain presenting an unusually tractable system linking genetic variation in physiology to geographical and temporal variation in climate. Projections to future climatic conditions suggest a change in relative climatic suitability that would result in HbS being displaced by HbF in northern Britain. This would facilitate local adaptation to future climate-without Hb displacement, populations in northern Britain would likely be suboptimally adapted because their Hb would not match local climatic conditions. Our study shows how pre-existing physiological differences can influence the adaptive capacity of species to climate change.

Reticulate evolution of the Daphnia pulex complex as revealed by nuclear markers.

The study of species complexes is of particular interest to understand how evolutionary young species maintain genomic integrity. The Daphnia pulex complex has been intensively studied as it includes species that dominate freshwater environments in the Northern hemisphere and as it is the sole North American complex that shows transitions to obligate parthenogenesis. Past studies using mitochondrial markers have revealed the presence of 10 distinct lineages in the complex. This study is the first to examine genetic relationships among seven species of the complex at nuclear markers (nine microsatellite loci and one protein-coding gene). Clones belonging to the seven species of the Daphnia pulex complex were characterized at the mitochondrial NADH dehydrogenase (ND5) gene and at the Lactate dehydrogenase (LDH) locus. K-means, principal coordinate analyses and phylogenetic network analyses on the microsatellite data all separated European D. pulicaria, D. tenebrosa, North American D. pulex, D. pulicaria and their hybrids into distinct clusters. The hybrid cluster was composed of diploid and polyploid hybrids with D. pulex mitochondria and some clones with D. pulicaria mitochondria. By contrast, the phylogeny of the D. pulex complex using Rab4 was not well resolved but still showed clusters consisting mostly of D. pulex alleles and others of D. pulicaria alleles. Incomplete lineage sorting and hybridization may obscure genetic relationships at this locus. This study shows that hybridization and introgression have played an important role in the evolution of this complex.

Niche differentiation in a postglacial colonizer, the bank vole Clethrionomys glareolus.

Species-level environmental niche modeling has been crucial in efforts to understand how species respond to climate variation and change. However, species often exhibit local adaptation and intraspecific niche differences that may be important to consider in predicting responses to climate. Here, we explore whether phylogeographic lineages of the bank vole originating from different glacial refugia (Carpathian, Western, Eastern, and Southern) show niche differentiation, which would suggest a role for local adaptation in biogeography of this widespread Eurasian small mammal. We first model the environmental requirements for the bank vole using species-wide occurrences (210 filtered records) and then model each lineage separately to examine niche overlap and test for niche differentiation in geographic and environmental space. We then use the models to estimate past [Last Glacial Maximum (LGM) and mid-Holocene] habitat suitability to compare with previously hypothesized glacial refugia for this species. Environmental niches are statistically significantly different from each other for all pairs of lineages in geographic and environmental space, and these differences cannot be explained by habitat availability within their respective ranges. Together with the inability of most of the lineages to correctly predict the distributions of other lineages, these results support intraspecific ecological differentiation in the bank vole. Model projections of habitat suitability during the LGM support glacial survival of the bank vole in the Mediterranean region and in central and western Europe. Niche differences between lineages and the resulting spatial segregation of habitat suitability suggest ecological differentiation has played a role in determining the present phylogeographic patterns in the bank vole. Our study illustrates that models pooling lineages within a species may obscure the potential for different responses to climate change among populations.

A dynamic history of admixture from Mediterranean and Carpathian glacial refugia drives genomic diversity in the bank vole.

Understanding the historical contributions of differing glacial refugia is key to evaluating the roles of microevolutionary forces, such as isolation, introgression, and selection in shaping genomic diversity in present-day populations. In Europe, where both Mediterranean and extra-Mediterranean (e.g., Carpathian) refugia of the bank vole (Clethrionomys glareolus) have been identified, mtDNA indicates that extra-Mediterranean refugia were the main source of colonization across the species range, while Mediterranean peninsulas harbor isolated, endemic lineages. Here, we critically evaluate this hypothesis using previously generated genomic data (>6,000 SNPs) for over 800 voles, focusing on genomic contributions to bank voles in central Europe, a key geographic area in considering range-wide colonization. The results provide clear evidence that both extra-Mediterranean (Carpathian) and Mediterranean (Spanish, Calabrian, and Balkan) refugia contributed to the ancestry and genomic diversity of bank vole populations across Europe. Few strong barriers to dispersal and frequent admixture events in central Europe have led to a prominent mid-latitude peak in genomic diversity. Although the genomic contribution of the centrally located Carpathian refugium predominates, populations in different parts of Europe have admixed origins from Mediterranean (28%-47%) and the Carpathian (53%-72%) sources. We suggest that the admixture from Mediterranean refugia may have provisioned adaptive southern alleles to more northern populations, facilitating the end-glacial spread of the admixed populations and contributing to increased bank vole diversity in central Europe. This study adds critical details to the complex end-glacial colonization history of this well-studied organism and underscores the importance of genomic data in phylogeographic interpretation.

Relics of the Europe's warm past: phylogeography of the Aesculapian snake.

Understanding how species responded to past climate change can provide information about how they may respond to the current global warming. Here we show how a European reptile species responded to the last natural global warming event at the Pleistocene-Holocene transition that led to the Holocene climatic optimum approximately 5000-8000 years ago. The Aesculapian snake, Zamenis longissimus, is a thermophilous species whose present-day distribution in the southern half of Europe is a remnant of much wider range during the Holocene climatic optimum when populations occurred as far north as Denmark. These northern populations went extinct as the climate cooled, and presently the species is extinct from all central Europe, except few relic populations in locally suitable microhabitats in Germany and the Czech Republic. Our phylogenetic and demographic analyses identified two major clades that expanded from their respective western and eastern refugia after the last glacial maximum (18,000-23,000 years ago) and contributed approximately equally to the present range. Snakes from the relic northern populations carried the Eastern clade, showing that it was primarily the snakes from the eastern, probably Balkan, refugium that occupied the central and northern Europe during the Holocene climatic optimum. Two small, deep-branching clades were identified in near the Black Sea and in Greece. These clades provide evidence for two additional refugia, which did not successfully contribute to the colonization of Europe. If, as our results suggest, some populations responded to the mid-Holocene global warming by shifting their ranges further north than other populations of the same species, knowing what populations were able to expand in different species may provide information about what populations will be important for the species' ability to cope with the current global warming.

Nuclear and mitochondrial DNA sequence data reveal the evolutionary history of Barbus (Cyprinidae) in the ancient lake systems of the Balkans.

Freshwater fauna of ancient lakes frequently contain endemic taxa thought to have originated during the long existence of these lakes, yet uncertainties remain as to whether they represent distinct genetic lineages with respect to more widespread relatives and to the relative roles of isolation and dispersal in their evolution. Phylogenetic analyses of sequence variation at nuclear and mitochondrial genes were used to examine these issues for the freshwater fish genus Barbus in two European ancient lake systems on the Balkan Peninsula. The nuclear and mitochondrial data yielded concordant phylogeographic patterns though incomplete sorting of nuclear haplotypes between some mitochondrial clades was detected. The distributions of two currently recognized species investigated here do not match the distributions of evolutionary lineages revealed by phylogenetic analyses. The Prespa barbel, Barbus prespensis, is not endemic to the lakes Prespa as previously thought but is instead found to be widespread in the south-eastern Adriatic Sea basin, with a distribution largely corresponding to the basin of the now extinct Lake Maliq historically connected with Lake Prespa. On the other hand, a cryptic phylogenetic subdivision in a widespread species, B. rebeli, was discovered to be more distant from B. rebeli than from other Barbus species and to be endemic to the system of connected lakes Ohrid and Shkodra. The division coincides with the hydrogeographical boundary delimiting distributions of other freshwater fishes, and we suggest that this newly discovered evolutionary lineage represents a distinct species. These findings support the emerging pattern that endemic taxa have evolved not through isolation of individual lakes, but in systems of currently and historically interconnected lakes and their wider basins.

The Celtic fringe of Britain: insights from small mammal phylogeography.

Recent genetic studies have challenged the traditional view that the ancestors of British Celtic people spread from central Europe during the Iron Age and have suggested a much earlier origin for them as part of the human recolonization of Britain at the end of the last glaciation. Here we propose that small mammals provide an analogue to help resolve this controversy. Previous studies have shown that common shrews (Sorex araneus) with particular chromosomal characteristics and water voles (Arvicola terrestris) of a specific mitochondrial (mt) DNA lineage have peripheral western/northern distributions with striking similarities to that of Celtic people. We show that mtDNA lineages of three other small mammal species (bank vole Myodes glareolus, field vole Microtus agrestis and pygmy shrew Sorex minutus) also form a 'Celtic fringe'. We argue that these small mammals most reasonably colonized Britain in a two-phase process following the last glacial maximum (LGM), with climatically driven partial replacement of the first colonists by the second colonists, leaving a peripheral geographical distribution for the first colonists. We suggest that these natural Celtic fringes provide insight into the same phenomenon in humans and support its origin in processes following the end of the LGM.

Divergence with gene flow between Ponto-Caspian refugia in an anadromous cyprinid Rutilus frisii revealed by multiple gene phylogeography.

The Black and Caspian Seas have experienced alternating periods of isolation and interconnection over many Milankovitch climate oscillations and most recently became separated when the meltwater overflow from the Caspian Sea ceased at the end of the last glaciation. Climate-induced habitat changes have indisputably had profound impacts on distribution and demography of aquatic species, yet uncertainties remain about the relative roles of isolation and dispersal in the response of species shared between the Black and Caspian Sea basins. We examined these issues using phylogeographical analysis of an anadromous cyprinid fish Rutilus frisii. Bayesian coalescence analyses of sequence variation at two nuclear and one mitochondrial genes suggest that the Black and Caspian Seas supported separate populations of R. frisii during the last glaciation. Parameter estimates from the fitted isolation-with-migration model showed that their separation was not complete, however, and that the two populations continued to exchange genes in both directions. These analyses also suggested that majority of migrations occurred during the Pleistocene, showing that the variation shared between the Black and Caspian Seas is the result of ancient dispersal along the temporary natural connections between the basins, rather than of incomplete lineage sorting or recent human-mediated dispersal. Gene flow between the refugial populations was therefore an important source of genetic variation, and we suggest that it facilitated the evolutionary response of the populations to changing climate.

Playing Hide-and-Seek in Beta-Globin Genes: Gene Conversion Transferring a Beneficial Mutation between Differentially Expressed Gene Duplicates.

Increasing evidence suggests that adaptation to diverse environments often involves selection on existing variation rather than new mutations. A previous study identified a nonsynonymous single nucleotide polymorphism (SNP) in exon 2 of two paralogous ß-globin genes of the bank vole (Clethrionomys glareolus) in Britain in which the ancestral serine (Ser) and the derived cysteine (Cys) allele represent geographically partitioned functional variation affecting the erythrocyte antioxidative capacity. Here we studied the geographical pattern of the two-locus Ser/Cys polymorphism throughout Europe and tested for the geographic correlation between environmental variables and allele frequency, expected if the polymorphism was under spatially heterogeneous environment-related selection. Although bank vole population history clearly is important in shaping the dispersal of the oxidative stress protective Cys allele, analyses correcting for population structure suggest the Europe-wide pattern is affected by geographical variation in environmental conditions. The ß-globin phenotype is encoded by the major paralog HBB-T1 but we found evidence of bidirectional gene conversion of exon 2 with the low-expression paralog HBB-T2. Our data support the model where gene conversion reshuffling genotypes between high- and low- expressed paralogs enables tuning of erythrocyte thiol levels, which may help maintain intracellular redox balance under fluctuating environmental conditions. Therefore, our study suggests a possible role for gene conversion between differentially expressed gene duplicates as a mechanism of physiological adaptation of populations to new or changing environments.

The complete mitochondrial genome of the bank vole Clethrionomys glareolus (Rodentia: Arvicolinae).

We present the first complete sequence of the bank vole (Clethrionomys glareolus) mitochondrial genome (GenBank accession no. KF918859). The bank vole mitogenome is 16,353 base pairs long and shows the gene content, genome architecture and gene strand asymmetry typical for mammals. The sequence provides an important new genomic resource for the bank vole, which is a popular study species in ecological and evolutionary research.

Mitochondrial capture misleads about ecological speciation in the Daphnia pulex complex.

The North American ecological species Daphniapulicaria and Daphniapulex are thought to have diverged from a common ancestor by adaptation to sympatric but ecologically distinct lake and pond habitats respectively. Based on mtDNA relationships, European D. pulicaria is considered a different species only distantly related to its North American counterpart, but both species share a lactate dehydrogenase (Ldh) allele F supposedly involved in lake adaptation in North America, and the same allele is also carried by the related Holarctic Daphniatenebrosa. The correct inference of the species' ancestral relationships is therefore critical for understanding the origin of their adaptive divergence. Our species tree inferred from unlinked nuclear loci for D. pulicaria and D. pulex resolved the European and North American D. pulicaria as sister clades, and we argue that the discordant mtDNA gene tree is best explained by capture of D. pulex mtDNA by D. pulicaria in North America. The Ldh gene tree shows that F-class alleles in D. pulicaria and D. tenebrosa are due to common descent (as opposed to introgression), with D. tenebrosa alleles paraphyletic with respect to D. pulicaria alleles. That D. tenebrosa still segregates the ancestral and derived amino acids at the two sites distinguishing the pond and lake alleles suggests that D. pulicaria inherited the derived states from the D. tenebrosa ancestry. Our results suggest that some adaptations restricting the gene flow between D. pulicaria and D. pulex might have evolved in response to selection in ancestral environments rather than in the species' current sympatric habitats. The Arctic (D. tenebrosa) populations are likely to provide important clues about these issues.