Using dense locality sampling resolves the subtle genetic population structure of the dispersive fish species Plecoglossus altivelis

In dispersive species with continuous distributions, genetic differentiation between local populations is often absent or subtle and thus difficult to detect. To incorporate such subtle differentiation into management plans, it may be essential to analyse many samples from many localities using adequate numbers of high‐resolution genetic markers. Here, we evaluated the usefulness of dense locality sampling in resolving genetic population structure in the ayu (Plecoglossus altivelis), a dispersive fish important in Japanese inland fisheries. Genetic variability in, and differentiation between, ayu populations around the Japan–Ryukyu Archipelago were investigated in 4746 individuals collected from 120 localities by genotyping 12 microsatellite markers. These individuals represented the two subspecies of ayu, namely the Ryukyuan subspecies (Plecoglossus altivelis ryukyuensis) and both amphidromous and landlocked forms of the nominotypical subspecies (P. a. altivelis) along the archipelago. We successfully detected an absence of genetic differentiation within the landlocked form and subtle but significant differentiation and clear geographic patterns of genetic variation among populations of the amphidromous form, which had been considered genetically homogeneous. This suggests that dense locality sampling effectively resolves subtle differences in genetic population structure, reducing stochastic deviation in the detection of genetic differentiation and geographic patterns in local populations of this dispersive species. Resampling analyses based on empirical data sets clearly demonstrate the effectiveness of increasing the number of locality samples for stable and reliable estimations of genetic fixation indices. The genetic population structure observed within the amphidromous form provides useful information for identifying management or conservation units in ayu.     

福建东张水库陆封型香鱼的遗传变异

测定了福建东张水库18尾陆封型香鱼的ND4-tRNA^Ser基因533 bp序列,发现2个可变位点和2个简约信息位点,共检测到4种单倍型。基于Kimura 2-parameter模型的遗传距离在0.001 9～0.003 9之间,在NJ系统树上无明显谱系结构;单倍型多样性(Hd)和核苷酸多样性(Pi)分别为0.627和0.001 37,平均核苷酸差异数为0.732,远低于两侧洄游型日本香鱼指名亚种(Hd为0.793,Pi为0.002 94),与被列为IUCN濒危物种名录的日本琉球香鱼亚种(Hd=0.519,Pi=0.001 11)相当。东张水库香鱼遗传多样性如此贫乏到底是历史原因还是近期陆封的结果,仍需进一步研究。目前亟待采取有效措施保护东张水库陆封香鱼。     

Genetic structuring of Patagonian toothfish populations in the Southwest Atlantic Ocean: the effect of the Antarctic Polar Front and deep-water troughs as barriers to genetic exchange

The environmental and/or life history factors affecting genetic exchange in marine species with potential for high dispersal are of great interest, not only from an evolutionary standpoint but also with regard to effective management. Previous genetic studies have demonstrated substantial differentiation among populations of the Patagonian toothfish around the Southern Ocean, indicating breakdown of gene flow across large distances between inhabited shelf areas. The present study examined genetic structuring through analysis of microsatellite loci and restriction fragment length polymorphism (RFLP) of the mitochondrial ND2 gene and control region of the toothfish population in the SW Atlantic, allowing examination of the relative effects of the Antarctic Polar Front (APF), deep-water troughs and distance between sites. Mitochondrial DNA (mtDNA) data indicated a sharp genetic division between the Patagonian Shelf/North Scotia Ridge and the Shag Rocks/South Georgia samples, whereas microsatellite data showed much less distinct structuring and an intermediate position of the North Scotia Ridge samples. We suggest these data indicate that the APF, as a barrier to larval dispersal, is the major inhibitor of genetic exchange between toothfish populations, with deep-water troughs and distance between sites contributing to genetic differentiation by inhibiting migration of relatively sedentary adults. We also suggest that differences between mtDNA and nuclear DNA population patterns may reflect either genome population size effects or (putative) male-biased dispersal.     

Genetic variation and bill size dimorphism in a passerine bird, the reed bunting Emberiza schoeniclus

In passerine birds morphological differentiation in bill size within species is not commonly observed. Bill size is usually associated with a trophic niche, and strong differences in it may reflect the process of genetic differentiation and, possibly, speciation. We used both mitochondrial DNA (mtDNA) and nuclear microsatellites to study genetic variation between two subspecies of reed bunting, Emberiza schoeniclus schoeniclus and E.s. intermedia , along their distributional boundary in western Europe. These two subspecies are characterized by a high dimorphism in bill size and, although breeding populations of the two subspecies are found very close to each other in northern Italy, apparently no interbreeding occurs. The observed morphological pattern between the two subspecies may be maintained by geographically varying selective forces or, alternatively, may be the result of a long geographical separation followed by a secondary contact. MtDNA sequences of cytochrome b and ND5 (515 bp) showed little variation and did not discriminate between the two subspecies, indicating a divergence time of less than 500 000 years. The analysis of four microsatellite loci suggested a clear, although weak, degree of genetic differentiation in the large- and small-billed populations, as indicated by F _ST and R _ST values and genetic distances. The correlation between bill size and genetic distance between populations remained significant after accounting for the geographical distances between sampling localities. Altogether, these results indicate a very recent genetic differentiation between the two bill morphs and suggest that a strong selection for large bills in the southern part of the breeding range is probably involved in maintaining the geographical differentiation of this species.     

Geographical continuity and discontinuity in the meristic characteristics of ayus of the southern subspecies Plecoglossus altivelis ryukyuensis

Ichthyological Research - Two subspecies of ayu, Plecoglossus altivelis altivelis (nominotypical subspecies) and P. a. ryukyuensis (Ryukyu-ayu), differ morphologically, behaviorally, and...     

Molecular phylogeography of common garter snakes (Thamnophis sirtalis) in western North America: implications for regional historical forces

Complete ND2 and partial ND4 and cytochrome b mitochondrial DNA (mtDNA) sequences were analysed to evaluate the phylogeographic patterns of common garter snakes (Thamnophis sirtalis) in western North America. This species is widely distributed throughout North America, and exhibits extensive phenotypic variation in the westernmost part of its range. The overall phylogeographic pattern based on mtDNA sequences is concordant with results from studies of other species in this region, implicating historical vicariant processes during the Pleistocene and indicating bottleneck effects of recent dispersal into postglacial habitat. Indeed, the topology is statistically consistent with the hypothesis of both southern (Great Basin and California) and northern (Haida Gwaii) refugia. Specifically, we identified genetic breaks among three major clades: Northwest Coastal populations, Intermountain populations, and all California populations. The California clade contained the only other well-supported branching patterns detected; relationships among populations within the two northern clades were indistinguishable. These molecular splits contrast sharply with all prior geographical analyses of phenotypic variation in T. sirtalis in this region. Our results suggest that the extensive phenotypic variation in western T. sirtalis has been shaped more by local evolutionary forces than by shared common ancestry. Consequently, we consider all morphologically based subspecies designations of T. sirtalis in this region invalid because they do not reflect reciprocal monophyly of the mtDNA sequences.     

A new mitochondrial haplotype confirms the distinctiveness of the Italian wolf (Canis lupus) population

In the past century the Italian wolf has been repeatedly indicated as a distinct subspecies, Canis lupus italicus, due to its unique morphology and its distinctive mtDNA control region (CR) monomorphism. However, recent studies on wolf x dog hybridization in Italy documented the presence of a second mtDNA CR haplotype (W16), previously found only in wolves from Eastern Europe, casting doubts on the genetic uniqueness of the Italian wolves. To test whether this second haplotype belongs to the Italian wolf population, we genotyped 92 wolf DNA samples from Italy, Slovenia, Greece and Bulgaria at four mtDNA regions (control-region, ATP6, COIII and ND4 genes) and at 39 autosomal microsatellites. Results confirm the presence of two mtDNA multi-fragment haplotypes (WH14 and WH19) in the Italian wolves, distinct from all the other European wolves. Network analyses of the multi-fragment mtDNA haplotypes identified two strongly differentiated clades, with the Italian wolf WH14 and WH19 multi-fragment haplotypes rooted together. Finally, Bayesian clustering clearly assigned all the wolves sampled in Italy to the Italian population, regardless of the two different multi-fragment haplotypes. These results demonstrate that the W16 CR haplotype is part of the genetic pool of the Italian wolf population, reconfirming its distinctiveness from other European wolves. Overall, considering the presence of unique mtDNA and Y-linked haplotypes, the sharply different frequencies of genome-wide autosomal alleles and the distinct morphological features of Italian wolves, we believe that this population should be considered a distinct subspecies.     

Homogeneity at nuclear microsatellite loci masks mitochondrial haplotype diversity in the endangered fanshell pearlymussel (Cyprogenia stegaria)

We report on multiple patterns of differentiation and connectivity in the fanshell pearlymussel (Cyprogenia stegaria), based on different markers. Knowledge of genetic variation and genetic connectivity among remaining populations of this federally endangered species is needed to initiate implementation of the species recovery plan. We collected tissue samples from 96 specimens from the Green, Rolling Fork, and Licking Rivers, tributaries to the Ohio River, and the Clinch River, a tributary to the Tennessee River, providing broad coverage of the current distributional range of the species. Results from 7 nuclear DNA microsatellite markers suggested minimal population-level differentiation, whereas a mitochondrial DNA (mtDNA) marker (ND1) exhibited significant differentiation between C. stegaria in the Clinch River and the Ohio River populations. The ND1 data also confirm the existence of 2 distinct mtDNA lineages in the genus that transcends species boundaries. Further analyses suggest that the disproportionally strong signal from 2 very divergent ND1 lineages possibly masks finer-grained structure in the Ohio River population, based on one of the mtDNA lineages only. We recommend further sampling to confirm the absence of one lineage from the upper Clinch River drainage and suggest that provisional management guidelines should limit reciprocal exchanges among C. stegaria populations from the Clinch River and those in the Ohio River system.     

Ancestral polymorphisms in genetic markers obscure detection of evolutionarily distinct populations in the endangered Florida grasshopper sparrow (Ammodramus savannarum floridanus)

Genetic analyses of bird subspecies designated as conservation units can address whether they represent units with independent evolutionary histories and provide insights into the evolutionary processes that determine the degree to which they are genetically distinct. Here we use mitochondrial DNA control region sequence and six microsatellite DNA loci to examine phylogeographical structure and genetic differentiation among five North American grasshopper sparrow (Ammodramus savannarum) populations representing three subspecies, including a population of the endangered Florida subspecies (A. s. floridanus). This federally listed taxon is of particular interest because it differs phenotypically from other subspecies in plumage and behaviour and has also undergone a drastic decline in population size over the past century. Despite this designation, we observed no phylogeographical structure among populations in either marker: mtDNA haplotypes and microsatellite genotypes from floridanus samples did not form clades that were phylogenetically distinct from variants found in other subspecies. However, there was low but significant differentiation between Florida and all other populations combined in both mtDNA (FST = 0.069) and in one measure of microsatellite differentiation (theta = 0.016), while the non-Florida populations were not different from each other. Based on analyses of mtDNA variation using a coalescent-based model, the effective sizes of these populations are large (approximately 80,000 females) and they have only recently diverged from each other (< 26,000 ybp). These populations are probably far from genetic equilibrium and therefore the lack of phylogenetic distinctiveness of the floridanus subspecies and minimal genetic differentiation is due most probably to retained ancestral polymorphism. Finally, levels of variation in Florida were similar to other populations supporting the idea that the drastic reduction in population size which has occurred within the last 100 years has not yet had an impact on levels of variation in floridanus. We argue that despite the lack of phylogenetic distinctiveness of floridanus genotypes the observed genetic differentiation and previously documented phenotypic differences justify continued designation of this subspecies as a protected population segment.     

Rate of change of the phytoplankton community in Itapeva Lake (North Coast of Rio Grande do Sul,Brazil), based on the wind driven hydrodynamic regime

The mussel Mytilus trossulus is an important component of the Baltic brackish water ecosystem. The genetic structure of mussel (M. trossulus) populations was studied in sites along the Polish coast, Southern Baltic for two segments of mitochondrial DNA (mtDNA). The mode of inheritance of Mytilus mtDNA is termed doubly uniparental; two genomes are passed independently down the female (the F genome) and male (the M genome) lines of descent. The M genome has not been detected at high frequency in M. trossulus, thus the present study focuses on the F genome. PCR and RFLP analysis was used to characterise haplotypes in the coding region ND2-COIII; PCR was used to detect length variants in a major noncoding region. Significant differentiation between populations was observed in the frequency of 24 coding region haplotypes and 14 different length variants. For the three most frequent coding region haplotypes, two (I and III) are associated with the length variants, whereas the third (II) is monomorphic for a single variant of short length. It is suggested that variant II is derived by introgression from a related species, M. edulis, and may be resistant to expansion in the noncoding region. In both regions studied, the Ewens–Watterson test reveals significant deviations from neutrality with an excess of rare variants. This might be due to selection against slightly deleterious variants and is consistent with previously published results for Mytilus taxa. The present study also points towards the potential utility of mtDNA length variation in studies of population differentiation of Mytilus.     

Population differentiation in the redshank (<Emphasis Type="Italic">Tringa totanus</Emphasis>) as revealed by mitochondrial DNA and amplified fragment length polymorphism markers

The redshank (Tringa totanus) is declining throughout Europe and to implement efficient conservation measures, it is important to obtain information about the population genetic structure. The aim of the present study was two-fold. First, we analysed the genetic variation within and between populations in the Baltic region in southern Scandinavia. Evidence of genetic structure would suggest that different populations might require separate management strategies. Second, in an attempt to study large-scale genetic structure we compared the Baltic populations with redshanks from northern Scandinavia and Iceland. This analysis could reveal insights into phylogeography and long-term population history. DNA samples were collected from six breeding sites in Scandinavia presumed to include two subspecies (totanus and britannica) and a further sample from Iceland (subspecies robusta). Two methods were used to study the population genetic structure. Domain II and III of the mitochondrial control region was analysed by DNA sequencing and nuclear DNA was analysed by screening amplified fragment length polymorphism (AFLP) markers. Mitochondrial DNA showed no variation between individuals in domain II. When analysing an 481 bp fragment of domain III seven haplotypes were found among birds. On the basis of mtDNA sequences, redshanks showed some evidence of a recent expansion from a bottlenecked refugial population. Bayesian analyses of AFLP data revealed a significant genetic differentiation between suggested subspecies but not between populations within the Baltic region. Our results indicate that populations of redshanks in Europe constitute at least three separate management units corresponding to the recognised subspecies.     

Association between mitochondrial DNA and morphological evolution in Canada geese

Summary Variation within and between eight subspecies of Canada geese was assessed by restriction fragment analysis of mitochondrial DNA (mtDNA), electrophoresis of proteins encoded by nuclear DNA, and the morphometric analysis of skeletons. Estimates of mtDNA sequence divergence between Canada goose subspecies ranged from 0.04 to 2.54%. Pairwise comparisons of the three data matrices revealed that only mtDNA variation and body size are significantly correlated. Subspecies with northern breeding grounds are small-bodied and display small variations of one mtDNA clone, whereas those breeding further south are largebodied and show small differences in another mtDNA clone. Canada geese exhibit strong geographic differentiation with respect to mtDNA sequence, but weak structuring in protein-encoding nuclear DNA. This finding can be explained by a lower level of gene flow for the mitochondrial genome than for the nuclear genome, which in turn emanates from the maternal inheritance of mtDNA and male-biased dispersal in Canada geese. Despite male-mediated flow of nuclear genes, strong morphometric differentiation persists among Canada geese subspecies.     

Phylogeography of white-spotted Charr (Salvelinus leucomaenis) inferred from mitochondrial DNA sequences

The white-spotted charr (Salvelinus leucomaenis) is a coldwater-adapted fish distributed in far-eastern Asia. To assess phylogeographic patterns of this species over most of its range in the Japanese archipelago and Sakhalin Island, Russia, we examined nucleotide sequences of the mitochondrial DNA (mtDNA) cytochrome b region (557 bp) in 141 individuals from 50 populations. A total of 33 (5.5%) nucleotide positions were polymorphic and defined 29 haplotypes. Phylogenetic analysis assigned the observed haplotypes to four main clades, which were characterized by the idiosyncrasies and discontinuity of geographic distributions. The nested clade analyses revealed that the geographical distribution patterns of some haplotypes and clades were explained by historical event such as past fragmentation. Although substantial genetic differentiation was found among the four main clades, their geographic distributions overlapped extensively in several regions. Since white-spotted charr can potentially use both freshwater and marine environments, coexistence among different lineages can be attributed to secondary contact through range expansion by migratory individuals during multiple glacial periods after interglacial isolation. Finally, our data demonstrate that the current subspecies designation does not reflect the phylogeography of this species based on mtDNA analysis. Hierarchical analysis (AMOVA) also showed that genetic variation was far more pronounced within subspecies than among subspecies (i.e., among discrete regions). These results suggest that each population, rather than each subspecies, must be treated as an evolutionarily significant unit.     

Comprehensive genetic analyses reveal evolutionary distinction of a mouse (Zapus hudsonius preblei) proposed for delisting from the US Endangered Species Act

Zapus hudsonius preblei, listed as threatened under the US Endangered Species Act (ESA), is one of 12 recognized subspecies of meadow jumping mice found in North America. Recent morphometric and phylogenetic comparisons among Z. h. preblei and neighbouring conspecifics questioned the taxonomic status of selected subspecies, resulting in a proposal to delist the Z. h. preblei from the ESA. We present additional analyses of the phylogeographic structure within Z. hudsonius that calls into question previously published data (and conclusions) and confirms the original taxonomic designations. A survey of 21 microsatellite DNA loci and 1380 base pairs from two mitochondrial DNA (mtDNA) regions (control region and cytochrome b) revealed that each Z. hudsonius subspecies is genetically distinct. These data do not support the null hypothesis of a homogeneous gene pool among the five subspecies found within the southwestern portion of the species' range. The magnitude of the observed differentiation was considerable and supported by significant findings for nearly every statistical comparison made, regardless of the genome or the taxa under consideration. Structuring of nuclear multilocus genotypes and subspecies-specific mtDNA haplotypes corresponded directly with the disjunct distributions of the subspecies investigated. Given the level of correspondence between the observed genetic population structure and previously proposed taxonomic classification of subspecies (based on the geographic separation and surveys of morphological variation), we conclude that the nominal subspecies surveyed in this study do not warrant synonymy, as has been proposed for Z. h. preblei, Z. h. campestris, and Z. h. intermedius.     

Genetic diversity in the North American captive African elephant collection

One hundred and eight mitochondrial DNA (mtDNA) sequences were used in combination with multilocus genotypes generated from 19 nuclear microsatellite loci to assess the genetic diversity in the North American captive African elephant collection. The analysis of 4488 bp of mitochondrial nicotinamide adenine dinucleotide hydrogenenase subunit 4-cytochrome b (ND4-cyt b ) sequence revealed the existence of three distinct mtDNA lineages (Haplogroups I–III). The genetic distance between Haplogroups I/II and Haplogroup III was determined to be 0.046. The nucleotide diversity for the elephant mtDNA ND4-cyt b was <0.004. Analysis of multilocus genotypes indicated an average observed heterozygosity of 0.53 compared with the expected heterozygosity of 0.64. No subdivision in the data was detected including data partitioned by mtDNA haplogroup. Relatedness among the 108 individuals did not differ from that expected in random mating populations.     

Phylogeography and phylogeny of the epineritic cosmopolitan bonitos of the genus Sarda (Cuvier): inferred patterns of intra‐ and inter‐oceanic connectivity derived from nuclear and mitochondrial DNA data

Aim To reconstruct the phylogenetic relationships of the four species of the genus Sarda (Sarda sarda, Sarda orientalis, Sarda australis and Sarda chilensis) and their phylogeographic history in the context of historical and ecological biogeography. Also, to reconstruct within‐species phylogenetic relationships to test whether the North Atlantic and Mediterranean populations of Atlantic bonito (S. sarda) warrant subspecies status, and the validity of the allopatric northern and southern populations of eastern Pacific bonito (S. chiliensis), recognized as S. chiliensis lineolata and S. chiliensis chiliensis. Location Representative samples of all four Sarda species collected world‐wide were analysed. Methods Phylogenetic inference was carried out with neighbour‐joining, maximum parsimony and maximum likelihood, employing nucleotide sequences of the mitochondrial DNA (mtDNA) control region I (CR‐I) and of the single‐copy nuclear DNA (nDNA) Tmo‐4c4 gene. Analysis of molecular variance was used on the mtDNA data to estimate the extent of geographic population structuring. Results Gene trees derived from mtDNA and nDNA data yielded concordant phylogenies that support the monophyly of the genus Sarda. The following sibling pairs received strong statistical support: striped bonito (S. orientalis) with Australian bonito (S. australis), and Atlantic bonito (S. sarda) with eastern Pacific bonito (S. chiliensis). Furthermore, the origin of S. sarda mtDNA is paraphyletic with respect to S. chiliensis, and these results are indicative of introgression. The analysis of Tmo‐4c4 sequences corroborates the ancestral hybridization between these allopatric species. Comparisons of north‐west Atlantic and Mediterranean populations of S. sarda using mtDNA CR‐I data revealed substantial genetic differentiation. By contrast, no differences between the putative northern and southern allopatric subspecies of S. chiliensis were detected. Main conclusions The monophyly of the genus Sarda as indicated by morphology is corroborated using both molecular markers. However, molecular phylogenies depicted a paraphyletic relationship between S. sarda and S. chiliensis. This phylogeographical relationship is better explained by an ancestral introgression facilitated by trans‐Arctic contact during the Pleistocene. The pronounced genetic differentiation between S. sarda samples from the north‐west Atlantic and the Mediterranean is consistent with the differentiation of these two regions, but not with the amphi‐Atlantic speciation hypothesis. Finally, the S. chiliensis lineolata and S. chiliensis chiliensis subspecies status is not supported by the molecular data.     

Discrepancies in population differentiation at microsatellites, mitochondrial DNA and plumage colour in the pied flycatcher--inferring evolutionary processes

Genetic differentiation between three populations of the pied flycatcher Ficedula hypoleuca (Norway, Czech Republic and Spain, respectively) was investigated at microsatellite loci and mitochondrial DNA (mtDNA) sequences and compared with the pattern of differentiation of male plumage colour. The Czech population lives sympatrically with the closely related collared flycatcher (F. albicollis) whereas the other two are allopatric. Allopatric populations are on average more conspicuously coloured than sympatric ones, a pattern that has been explained by sexual selection for conspicuous colour in allopatry and a character displacement on breeding plumage colour in sympatry that reduces the rate of hybridization with the collared flycatcher. The Czech population was genetically indistinguishable from the Norwegian population at microsatellite loci and mtDNA sequences. Recent isolation and/or gene flow may explain the lack of genetic differentiation. Accordingly, different selection on plumage colour in the two populations is either sufficiently strong so that gene flow has little impact on the pattern of colour variation, or differentiation of plumage colour occurred so recently that the (presumably) neutral, fast evolving markers employed here are unable to reflect the differentiation. Genetically, the Spanish population was significantly differentiated from the other populations, but the divergence was much more pronounced at mtDNA compared to microsatellites. This may reflect increased rate of differentiation by genetic drift at the mitochondrial, compared with the nuclear genome, caused by the smaller effective population size of the former genome. In accordance with this interpretation, a genetic pattern consistent with effects of small population size in the Spanish population (genetic drift and inbreeding) were also apparent at the microsatellites, namely reduced allelic diversity and heterozygous deficiency.     

Inheritance of amplified fragment length polymorphism markers and their utility in population genetic analysis of Plecoglossus altivelis

The reproducibility, mode of inheritance and polymorphism of amplified fragment length polymorphism (AFLP) markers were examined in ayu Plecoglossus altivelis (Salmoniformes: Plecoglossidae). The AFLP markers were highly reproducible, their inheritance following Mendelian expectations. The number of fragments amplified (34–134), polymorphic ratio (0·15–0·78) and average heterozygosity (0·02–0·25) of the AFLP markers showed significant variation among six primer pairs and among ayu populations, including a landlocked Lake‐Biwa population, two amphidromous populations ( P. a. altivelis ) and two Ryukyu‐ayu populations ( P. a. ryukyuensis ). Although AFLP analysis provided similar results in intra‐population diversity and relationships among populations to those found by analyses of allozymes, microsatellites and mitochondrial DNA sequences, AFLPs showed higher polymorphisms and hence greater distinction between genetically close populations.     

Mitochondrial phylogeography of the Eurasian beaver Castor fiber L

Nucleotide variation in an approximately 490 bp fragment of the mitochondrial DNA control region (mtDNA CR) was used to describe the genetic variation and phylogeographical pattern in the Eurasian beaver (Castor fiber) over its entire range. The sampling effort was focused on the relict populations that survived a drastic population bottleneck, caused by overhunting, at the end of the 19th century. A total of 152 individuals grouped into eight populations representing all currently recognized subspecies were studied. Sixteen haplotypes were detected, none of them shared among populations. Intrapopulation sequence variation was very low, most likely a result of the severe bottleneck. Extreme genetic structure could result from human-mediated extinction of intermediate populations, but it could also be an effect of prior substantial structuring of the beaver populations with watersheds of major Eurasian rivers acting as barriers to gene flow. Phylogenetic analysis revealed the presence of two mtDNA lineages: eastern (Poland, Lithuania, Russia and Mongolia) and western (Germany, Norway and France), the former comprising more divergent haplotypes. The low level of sequence divergence of the entire cytochrome b gene among six individuals representing six subspecies suggests differentiation during the last glacial period and existence of multiple glacial refugia. At least two evolutionary significant units (ESU) can be identified, the western and the eastern haplogroup. The individual relict populations should be regarded as management units, the eastern subspecies possibly also as ESUs. Guidelines for future translocations and reintroductions are proposed.     

Historical introgression and the persistence of ghost alleles in the intermediate horseshoe bat (Rhinolophus affinis)

Phylogenetic conflicts between genetic markers can help to disentangle complex histories of phylogeography and introgression among taxa. We previously proposed that the Chinese mainland subspecies of the intermediate horseshoe bat Rhinolophus affinis himalayanus colonized Hainan Island to form the subspecies R. a. hainanus. Subsequent recolonization of the mainland formed a third taxon, R. a macrurus, and a secondary contact zone with the ancestral himalayanus. To test for historical and recurrent genetic exchange between these mainland subspecies, we sampled populations of each from two parapatric zones and undertook analyses using one mtDNA marker, three nuclear genes and 14 microsatellites. Nuclear DNA, echolocation call frequencies and morphological data all recovered two taxa; however, a mtDNA phylogeny revealed two himalayanus clades, of which one clustered with macrurus, as well as some shared or related mtDNA haplotypes in eastern populations. Isolation‐with‐migration (IM) models suggested some mtDNA gene flow from macrurus to himalayanus. However, strong population structure in himalayanus raises the possibility that macrurus captured mtDNA from a coastal population of himalayanus that has since become rare or extinct. To reconcile these two sets of results, we suggest that the IM estimates might reflect historical mtDNA gene flow among populations of himalayanus, before mtDNA was subsequently captured by macrurus. Finally, microsatellite‐based ABC analyses supported the island origin of macrurus; however, mtDNA‐based ABC analyses suggest this taxon might have evolved on the mainland. Our findings highlight the importance of understanding population history and structure for interpreting hybridization and introgression events.