The influence of Pleistocene glacial refugia on tawny owl genetic diversity and phylogeography in western Europe

The glacial refugia hypothesis indicates that during the height of the Pleistocene glaciations the temperate species that are today widespread in western Europe must have survived in small and climatically favourable areas located in the southern peninsulas of Iberia, Italy and Balkans. One such species is the tawny owl, a relatively sedentary, nonmigratory bird presently distributed throughout Europe. It is a tree-nesting species closely associated with deciduous and mixed coniferous woodlands. In this study I used control region mtDNA sequences from 187 individuals distributed among 14 populations to determine whether current genetic patterns in tawny owl populations were consistent with postglacial expansion from peninsular refugia. European, North African and Asian tawny owls were found to represent three distinct lineages, where North Africa is the sister clade to all European owls. Within Europe, I found three well-supported clades that correspond to each of the three allopatric refugia. Expansion patterns indicate that owls from the Balkan refugium repopulated most of northern Europe, while expansion out of Iberia and Italy had only regional effects leading to admixture in France. Estimates of population divergence times between refugia populations are roughly similar, but one order of magnitude smaller between Greece and northern Europe. Based on a wide range of mutation rates and generation times, divergence between refugia appears to date to the Pleistocene.     

Phylogeography of the brown hare (Lepus europaeus) in Europe: a legacy of south‐eastern Mediterranean refugia?

Aim We analysed the population genetics of the brown hare (Lepus europaeus) in order to test the hypothesis that this species migrated into central Europe from a number of late glacial refugia, including some in Asia Minor. Location Thirty‐three localities in Greece, Bulgaria, Italy, Croatia, Serbia, Poland, Switzerland, Austria, France, Germany, the Netherlands, Spain, the United Kingdom, Turkey and Israel. Methods In total, 926 brown hares were analysed for mitochondrial DNA (mtDNA) variation by restriction fragment length polymorphism (RFLP) performed on polymerase chain reaction‐amplified products spanning cytochrome b (cyt b)/control region (CR), cytochrome oxidase I (COI) and 12S–16S rRNA. In addition, sequence analysis of the mtDNA CR‐I region was performed on 69 individuals, and the data were compared with 137 mtDNA CR‐I sequences retrieved from GenBank. Results The 112 haplotypes detected were partitioned into five phylogeographically well‐defined major haplogroups, namely the ‘south‐eastern European type haplogroup’ (SEEh), ‘Anatolian/Middle Eastern type haplogroup’ (AMh), ‘European type haplogroup, subgroup A’ (EUh‐A), ‘European type haplogroup, subgroup B’ (EUh‐B) and ‘Intermediate haplogroup’ (INTERh). Sequence data retrieved from GenBank were consistent with the haplogroups determined in this study. In Bulgaria and north‐eastern Greece numerous haplotypes of all five haplogroups were present, forming a large overlap zone. Main conclusions The mtDNA results allow us to infer post‐glacial colonization of large parts of Europe from a late glacial/early Holocene source population in the central or south‐central Balkans. The presence of Anatolian/Middle Eastern haplotypes in the large overlap zone in Bulgaria and north‐eastern Greece reveals gene flow from Anatolia to Europe across the late Pleistocene Bosporus land‐bridge. Although various restocking operations could be partly responsible for the presence of unexpected haplotypes in certain areas, we nevertheless trace a strong phylogeographic signal throughout all regions under study. Throughout Europe, mtDNA results indicate that brown hares are not separated into discernable phyletic groups.     

The role of topography in structuring the demographic history of the pine processionary moth,Thaumetopoea pityocampa (Lepidoptera: Notodontidae)

Aim We investigated the Quaternary history of the pine processionary moth, Thaumetopoea pityocampa, an oligophagous insect currently expanding its range. We tested the potential role played by mountain ranges during the post‐glacial recolonization of western Europe. Location Western Europe, with a focus on the Pyrenees, Massif Central and western Alps. Methods Maternal genetic structure was investigated using a fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene. We analysed 412 individuals from 61 locations and performed maximum likelihood and maximum parsimony phylogenetic analyses and hierarchical analysis of molecular variance, and we investigated signs of past expansion. Results A strong phylogeographic pattern was found, with two deeply divergent clades. Surprisingly, these clades were not separated by the Pyrenees but rather were distributed from western to central Iberia and from eastern Iberia to the Italian Peninsula, respectively. This latter group consisted of three shallowly divergent lineages that exhibited strong geographic structure and independent population expansions. The three identified lineages occurred: (1) on both sides of the Pyrenean range, with more genetically diverse populations in the east, (2) from eastern Iberia to western France, with a higher genetic diversity in the south, and (3) from the western Massif Central to Italy. Admixture areas were found at the foot of the Pyrenees and Massif Central. Main conclusions The identified genetic lineages were geographically structured, but surprisingly the unsuitable high‐elevation areas of the main mountainous ranges were not responsible for the spatial separation of genetic groups. Rather than acting as barriers to dispersal, mountains appear to have served as refugia during the Pleistocene glaciations, and current distributions largely reflect expansion from these bottlenecked refugial populations. The western and central Iberian clade did not contribute to the northward post‐glacial recolonization of Europe, yet its northern limit does not correspond to the Pyrenees. The different contributions of the identified refugia to post‐glacial expansion might be explained by differences in host plant species richness. For example, the Pyrenean lineage could have been trapped elevationally by tracking montane pines, while the eastern Iberian lineage could have expanded latitudinally by tracking thermophilic lowland pine species.     

Mitochondrial gene trees support persistence of cold tolerant fairy shrimp throughout the Pleistocene glaciations in both southern and more northerly refugia

Fairy shrimp (Crustacea: Anostraca) are specialist inhabitants of temporary aquatic habitats. In many parts of the world and particularly in Western Europe, however, populations are declining while the development of adequate conservation strategies is impeded by a poor knowledge of the genetic structure and taxonomic status of remaining lineages. We reconstructed a phylogeography of the species Chirocephalus diaphanus Prévost, 1803 using partial sequences of the mitochondrial COI gene and discuss the importance of different Pleistocene refugia to explain current diversity patterns. In addition to 20 C. diaphanus populations, we also included populations of six presumably closely related chirocephalids to evaluate their taxonomic status. Based on molecular data, the Eastern European subspecies C. diaphanus romanicus deserves species status while the species status of two Italian chirocephalids, C. salinus and C. ruffoi is questionable. Results indicate European C. diaphanus lineages diverged well before the last glacial maximum and survived the Pleistocene glaciations in multiple (sub)refugia along the Iberian, Italian, and Balkan peninsula. Northern Europe was subsequently recolonized from Southern France, resulting in high levels of cryptic diversity around glacial refugia but also in more widespread haplotypes in mainland Europe.     

Phylogeography of roe deer (Capreolus capreolus) populations: the effects of historical genetic subdivisions and recent nonequilibrium dynamics

We sequenced 704 mitochondrial DNA (mtDNA) control-region nucleotides and genotyped 11 autosomal microsatellites (STR) in 617 European roe deer (Capreolus capreolus) samples, aiming to infer the species' phylogeographical structure. The mtDNA sequences were split in three distinct haplogroups, respectively, named: Clade West, sampled mainly in Iberia; Clade East, sampled mainly in Greece and in the Balkans; and Clade Central, which was widespread throughout Europe, including the eastern countries and Iberia, but not Greece. These clades might have originated in distinct Iberian and Balkanic refuges during the penultimate or the last glaciations. Clades East and West contributed little to the current postglacial mtDNA diversity in central Europe, which apparently was recolonized mainly by haplotypes belonging to Clade Central. A unique subclade within Clade Central grouped all the haplotypes sampled from populations of the Italian subspecies C. c. italicus. In contrast, haplotypes sampled in central and southern Spain joined both Clade Central and Clade West, suggesting that subspecies C. c. garganta has admixed origin. STR data support a genetic distinction of peripheral populations in north Iberia and southern Italy, and show the effects of anthropogenic disturbance in fragmented populations, which were recently reintroduced or restocked and not may be in mutation-drift equilibrium. Roe deer in central Europe are mainly admixed, while peripheral populations in north Portugal, the southern Italian Apennines and Greece represent the remains of refugial populations and should be managed accordingly.     

Phylogeography of the white-tailed eagle, a generalist with large dispersal capacity

Aim Late Pleistocene glacial changes had a major impact on many boreal and temperate taxa, and this impact can still be detected in the present‐day phylogeographic structure of these taxa. However, only minor effects are expected in species with generalist habitat requirements and high dispersal capability. One such species is the white‐tailed eagle, Haliaeetus albicilla, and we therefore tested for the expected weak population structure at a continental level in this species. This also allowed us to describe phylogeographic patterns, and to deduce Ice Age refugia and patterns of postglacial recolonization of Eurasia. Location Breeding populations from the easternmost Nearctic (Greenland) and across the Palaearctic (Iceland, continental Europe, central and eastern Asia, and Japan). Methods Sequencing of a 500 base‐pair fragment of the mitochondrial DNA control region in 237 samples from throughout the distribution range. Results Our analysis revealed pronounced phylogeographic structure. Overall, low genetic variability was observed across the entire range. Haplotypes clustered in two distinct haplogroups with a predominantly eastern or western distribution, and extensive overlap in Europe. These two major lineages diverged during the late Pleistocene. The eastern haplogroup showed a pattern of rapid population expansion and colonization of Eurasia around the end of the Pleistocene. The western haplogroup had lower diversity and was absent from the populations in eastern Asia. These results suggest survival during the last glaciation in two refugia, probably located in central and western Eurasia, followed by postglacial population expansion and admixture. Relatively high genetic diversity was observed in northern regions that were ice‐covered during the last glacial maximum. This, and phylogenetic relationships between haplotypes encountered in the north, indicates substantial population expansion at high latitudes. Areas of glacial meltwater runoff and proglacial lakes could have provided suitable habitats for such population growth. Main conclusions This study shows that glacial climate fluctuations had a substantial impact on white‐tailed eagles, both in terms of distribution and demography. These results suggest that even species with large dispersal capabilities and relatively broad habitat requirements were strongly affected by the Pleistocene climatic shifts.     

Genetic divergence and phylogeographic relationships among european perch (Perca fluviatilis) populations reflect glacial refugia and postglacial colonization

We used the widely distributed freshwater fish, perch (Perca fluviatilis), to investigate the postglacial colonization routes of freshwater fishes in Europe. Genetic variability within and among drainages was assessed using mitochondrial DNA (mtDNA) D-loop sequencing and RAPD markers from 55 populations all over Europe as well as one Siberian population. High level of structuring for both markers was observed among drainages and regions, while little differentiation was seen within drainages and regions. Phylogeographic relationships among European perch were determined from the distribution of 35 mtDNA haplotypes detected in the samples. In addition to a distinct southern European group, which includes a Greek and a southern Danubian population, three major groups of perch are observed: the western European drainages, the eastern European drainages including the Siberian population, and Norwegian populations from northern Norway, and western side of Oslofjord. Our data suggest that present perch populations in western and northern Europe were colonized from three main refugia, located in southeastern, northeastern and western Europe. In support of this, nested cladistic analysis of mtDNA clade and nested clade distances suggested historical range expansion as the main factor determining geographical distribution of haplotypes. The Baltic Sea has been colonized from all three refugia, and northeastern Europe harbours descendants from both eastern European refugia. In the upper part of the Danube lineages from the western European and the southern European refugia meet. The southern European refugium probably did not contribute to the recolonization of other western and northern European drainages after the last glaciation. However, phylogenetic analyses suggest that the southern European mtDNA lineage is the most ancient, and therefore likely to be the founder of all present perch lineages. The colonization routes used by perch probably also apply to other freshwater species with similar distribution patterns.     

Mitochondrial Haplogroup U5b3: A Distant Echo of the Epipaleolithic in Italy and the Legacy of the Early Sardinians

There are extensive data indicating that some glacial refuge zones of southern Europe (Franco-Cantabria, Balkans, and Ukraine) were major genetic sources for the human recolonization of the continent at the beginning of the Holocene. Intriguingly, there is no genetic evidence that the refuge area located in the Italian Peninsula contributed to this process. Here we show, through phylogeographic analyses of mitochondrial DNA (mtDNA) variation performed at the highest level of molecular resolution (52 entire mitochondrial genomes), that the most likely homeland for U5b3—a haplogroup present at a very low frequency across Europe—was the Italian Peninsula. In contrast to mtDNA haplogroups that expanded from other refugia, the Holocene expansion of haplogroup U5b3 toward the North was restricted by the Alps and occurred only along the Mediterranean coasts, mainly toward nearby Provence (southern France). From there, ∼7,000–9,000 years ago, a subclade of this haplogroup moved to Sardinia, possibly as a result of the obsidian trade that linked the two regions, leaving a distinctive signature in the modern people of the island. This scenario strikingly matches the age, distribution, and postulated geographic source of a Sardinian Y chromosome haplogroup (I2a2-M26), a paradigmatic case in the European context of a founder event marking both female and male lineages.     

Molecular evidence of Pleistocene bidirectional faunal exchange between Europe and the Near East: the case of the bicoloured shrew (Crocidura leucodon, Soricidae)

We sequenced 1077 bp of the mitochondrial cytochrome b gene and 511 bp of the nuclear Apolipoprotein B gene in bicoloured shrew (Crocidura leucodon, Soricidae) populations ranging from France to Georgia. The aims of the study were to identify the main genetic clades within this species and the influence of Pleistocene climatic variations on the respective clades. The mitochondrial analyses revealed a European clade distributed from France eastwards to north-western Turkey and a Near East clade distributed from Georgia to Romania; the two clades separated during the Middle Pleistocene. We clearly identified a population expansion after a bottleneck for the European clade based on mitochondrial and nuclear sequencing data; this expansion was not observed for the eastern clade. We hypothesize that the western population was confined to a small Italo-Balkanic refugium, whereas the eastern population subsisted in several refugia along the southern coast of the Black Sea.     

Phylogeography of the ant Myrmica rubra and its inquiline social parasite

Widely distributed Palearctic insects are ideal to study phylogeographic patterns owing to their high potential to survive in many Pleistocene refugia and-after the glaciation-to recolonize vast, continuous areas. Nevertheless, such species have received little phylogeographic attention. Here, we investigated the Pleistocene refugia and subsequent postglacial colonization of the common, abundant, and widely distributed ant Myrmica rubra over most of its Palearctic area, using mitochondrial DNA (mtDNA). The western and eastern populations of M. rubra belonged predominantly to separate haplogroups, which formed a broad secondary contact zone in Central Europe. The distribution of genetic diversity and haplogroups implied that M. rubra survived the last glaciation in multiple refugia located over an extensive area from Iberia in the west to Siberia in the east, and colonized its present areas of distribution along several routes. The matrilineal genetic structure of M. rubra was probably formed during the last glaciation and subsequent postglacial expansion. Additionally, because M. rubra has two queen morphs, the obligately socially parasitic microgyne and its macrogyne host, we tested the suggested speciation of the parasite. Locally, the parasite and host usually belonged to the same haplogroup but differed in haplotype frequencies. This indicates that genetic differentiation between the morphs is a universal pattern and thus incipient, sympatric speciation of the parasite from its host is possible. If speciation is taking place, however, it is not yet visible as lineage sorting of the mtDNA between the morphs.     

Postglacial colonisation patterns and the role of isolation and expansion in driving diversification in a passerine bird

Pleistocene glacial cycles play a major role in diversification and speciation, although the relative importance of isolation and expansion in driving diversification remains debated. We analysed mitochondrial DNA sequence data from 15 great reed warbler (Acrocephalus arundinaceus) populations distributed over the vast Eurasian breeding range of the species, and revealed unexpected postglacial expansion patterns from two glacial refugia. There were 58 different haplotypes forming two major clades, A and B. Clade A dominated in Western Europe with declining frequencies towards Eastern Europe and the Middle East, but showed a surprising increase in frequency in Western and Central Asia. Clade B dominated in the Middle East, with declining frequencies towards north in Central and Eastern Europe and was absent from Western Europe and Central Asia. A parsimonious explanation for these patterns is independent postglacial expansions from two isolated refugia, and mismatch distribution analyses confirmed this suggestion. Gene flow analyses showed that clade A colonised both Europe and Asia from a refugium in Europe, and that clade B expanded much later and colonised parts of Europe from a refugium in the Middle East. Great reed warblers in the eastern parts of the range have slightly paler plumage than western birds (sometimes treated as separate subspecies; A. a. zarudnyi and A. a. arundinaceus, respectively) and our results suggest that the plumage diversification took place during the easterly expansion of clade A. This supports the postglacial expansion hypothesis proposing that postglacial expansions drive diversification in comparatively short time periods. However, there is no indication of any (strong) reproductive isolation between clades and our data show that the refugia populations became separated during the last glaciation. This is in line with the Pleistocene speciation hypothesis invoking that much longer periods of time in isolation are needed for speciation to occur.     

Mitochondrial DNA variation and the phylogeography of the grey partridge (Perdix perdix) in Europe: from Pleistocene history to present day populations

Abstract For a phylogeographical analysis of European grey partridge (Perdix perdix) we sequenced 390 nucleotides of the 5′ end of the mitochondrial control region (CR) of 227 birds from several localities. The birds were divided into two major clades (western and eastern) which differed in control region 1 (CR1) by 14 nucleotide substitutions (3.6%). For estimation of the time of divergence, the whole CR of 14 specimens was sequenced. The major clades differed by 2.2%, corresponding to an estimated coalescence time of c. 1.1 million years. On CR1, 45 haplotypes were found. Western clade haplotypes were found in France, England, Germany, Poland, Italy and Austria. Eastern clade haplotypes were found in Finland, Bulgaria, Greece, and Ireland. One Finnish population and all Bulgarian and Irish populations were mixed, but only in Bulgaria was the mixing assumed to be natural. Nucleotide and haplotype diversities varied between populations, and both clades showed geographical structuring. The distribution of pairwise nucleotide differences in the eastern clade fitted the expectations of an expanding population. About 80% of the genetic structure in the grey partridge could be explained by the clades. The western clade presumably originates on the Iberian Peninsula (with related subtypes in Italy), and the eastern clade either on the Balkan or Caucasian refugia. Large‐scale hand‐rearing and releasing of western partridges have introduced very few mtDNA marks into the native eastern populations in Finland.     

Genetic differentiation of the marbled white butterfly, Melanargia galathea, accounts for glacial distribution patterns and postglacial range expansion in southeastern Europe

Isolation of Mediterranean species in the southern European peninsulas during the cold glacial phases often resulted in differentiation of several genetic lineages confined to the respective peninsulas. However, whilst there is good genetic evidence for multiple refugia in Iberia, there are only limited data available for the Balkans. Therefore, we wish to examine the hypothesis of a strong genetic structuring within southeastern Europe for the existence of multiple Balkan differentiation centres and/or several leading edges. As a model we use the marbled white butterfly, Melanargia galathea. We studied 18 allozyme loci of 564 individuals from 16 populations distributed over a large part of southeastern Europe. The single populations showed moderately high genetic diversity and no northward decline of genetic diversity was detected. The overall genetic differentiation between populations was considerable (F(ST) 7.0%). Cluster analysis discriminated three genetic groups: (i) a western flank in the former Yugoslavia, parts of eastern Austria and Hungary; (ii) an eastern flank with populations from Bulgaria and Romania (south of the southern Carpathians and eastern Carpathians); and (iii) the eastern Carpathian Basin. Hierarchical variance analysis distributed 53% of the variance among populations between these three groups. One sample from the Greek-Bulgarian border clustered within the eastern flank, but showed some tendency towards the eastern Carpathian Basin populations. Two populations from Carinthia clustered together with the eastern Carpathian Basin ones and a population from Styria showed an intermediate genetic composition between the three groups. Most probably, the eastern and the western flank groups are due to postglacial range expansion from the northeastern and the northwestern edges of the glacial differentiation centre (so-called leading edges). The eastern Carpathian Basin group may have resulted from postglacial expansion from northern Greece through valley systems of the central Balkan peninsula, maybe even expanding westwards north of the Balkan mountains reaching some parts of eastern Austria (e.g. Carinthia). Therefore, the Balkanic refugium of M. galathea may or may not have been continuous along the coastal areas of the Mediterranean, but must have been strongly genetically structured.     

Phylogeny and biogeography of the alpine newt Mesotriton alpestris (Salamandridae, Caudata), inferred from mtDNA sequences

In this paper, we performed phylogenetic analyses of Mesotriton alpestris populations from the entire range of species distribution, using fragments of two mtDNA genes, cytochrome b (309bp) and 16S rRNA ( approximately 500bp). Sequence diversity patterns and phylogenetic analyses reveal the existence of a relict lineage (Clade A) of late Miocene origin, comprising populations from south-eastern Serbia. This lineage is proposed to be ancestor to a western and an eastern lineage, which diverged during the middle Pliocene. The western lineage is further divided in two clades (Clades B, C) of middle Pliocene origin that represent populations from Italy (B) and populations from central Europe and Iberia (C). Further subdivision, dated back to the middle-late Pliocene, was found within the eastern lineage, representing southern (Clade D) and central-northern (Clade E) Balkan populations, respectively. Extensive sequence divergence, implying greater isolation in multiple refugia, is found within eastern clades, while the western clades seem to have been involved in the colonization of central, western and north-eastern Europe from a hypothetical refugium in central Europe. The extent of divergence does not support the current taxonomy indicating cryptic speciation in the Balkans, while paedomorphic lineages were found to have been evolved during early-middle Pleistocene probably as a response to the ongoing dramatic climatic oscillations.     

The shaping of mitochondrial DNA phylogeographic patterns of the brown hare (Lepus europaeus) under the combined influence of Late Pleistocene climatic fluctuations and anthropogenic translocations

The phylogeographic structure of the brown hare (Lepus europaeus) was studied by analysing mtDNA control region sequences of 98 individuals from continental and insular Greece, Bulgaria, Cyprus and northern Israel, together with 44 published sequences from Italy and central Europe. We found two distinct clades separated by an average nucleotide divergence of 6.6%, which may correspond to a Balkan and to an Asia Minor refugium. The estimated time of separation of the two clades was dated back to 105,000- 490,000 years ago. These two clades coexist in the area of northeastern Greece and Bulgaria, most likely as a result of a post-glacial northward expansion. Within the southern Balkan refugium, network analyses showed geographical structuring, which supports the hypothesis of several isolated Late Pleistocene populations. The central European and Italian populations appear to have originated from a non-detected northern Balkan population that was genetically closely related to some northern Greek populations, as a result of postglacial expansion, translocations or a combination of both. Moreover, several cases of ancient and recent translocations by humans were detected, especially for some island populations, while the eastern Aegean islands off the Asia Minor coast were most likely colonized naturally through Late Pleistocene land bridge connection. The genetic analysis presented here provides a framework for designing proper conservation and management guidelines for this species.     

European common frog (Rana temporaria) recolonized Switzerland from multiple glacial refugia in northern Italy via trans‐ and circum‐Alpine routes

The high mountain ranges of Western Europe had a profound effect on the biotic recolonization of Europe from glacial refugia. The Alps present a particularly interesting case because they form an absolute barrier to dispersal for most taxa, obstructing recolonization from multiple refugia in northern Italy. Here, we investigate the effect of the European Alps on the phylogeographic history of the European common frog Rana temporaria. Based on partial cytochrome b and COXI sequences from Switzerland, we find two mitochondrial lineages roughly north and south of the Alpine ridge, with contact zones between them in eastern and western Switzerland. The northern haplogroup falls within the previously identified Western European haplogroup, while the southern haplogroup is unique to Switzerland. We find that the lineages diverged ~110 kya, at approximately the onset of the last glacial glaciation; this indicates that they are from different glacial refugia. Phylogenetic analyses suggest that the northern and southern haplogroups colonized Switzerland via trans‐ and circum‐Alpine routes from at least two separate refugia in northern Italy. Our results illustrate how a complex recolonization history of the central European Alps can arise from the semi‐permeable barrier created by high mountains.     

A rangewide phylogeography of Hermann's tortoise, Testudo hermanni (Reptilia: Testudines: Testudinidae): implications for taxonomy

Hermann's tortoise (Testudo hermanni), the best-known western Palaearctic tortoise species, has a rare natural distribution pattern comprising the Mediterranean areas of the Iberian, Apennine, and Balkan Peninsulas, as well as Sicily, Corsica and Sardinia. The western part of this range is traditionally considered habitat for T. h. hermanni, while T. h. boettgeri occurs in the Balkans. Taxonomy of this tortoise has been challenged in recent years, with the two subspecies being considered full species and the central Dalmatian populations of T. h. boettgeri being considered a third species, T. hercegovinensis. Using an mtDNA fragment approximately 1150 bp long (cytochrome b gene and adjacent portion of tRNA-Thr gene), we investigated mtDNA diversity with regard to contrasting concepts of two subspecies or three species. Seven closely related haplotypes were identified from the western Mediterranean and 15 different, in part much-differentiated, haplotypes from the Balkans. Western Mediterranean haplotypes differ from Balkan haplotypes in 16–42 mutation steps. One to seven mutation steps occur within western Mediterranean populations. Balkan haplotypes, differing in 1−37 nucleotides, group in parsimony network analysis into three major assemblages that display, in part, a similar degree of differentiation to that of western Mediterranean haplotypes relative to Balkan haplotypes. Rates of sequence evolution are different in both regions, and low divergence, palaeogeography and the fossil record suggest a slower molecular clock in the western Mediterranean. While monophyly in western Mediterranean haplotypes is well-supported, conflicting evidence is obtained for Balkan haplotypes; maximum parsimony supports monophyly of Balkan haplotypes, but other phylogenetic analyses (Bayesian, ML, ME) indicate Balkan haplotypes could be paraphyletic with respect to the western Mediterranean clade. These results imply a process of differentiation not yet complete despite allopatry in the western Mediterranean and the Balkans, and suggest all populations of T. hermanni are conspecific. In the western Mediterranean no clear geographical pattern in haplotype distribution is found. Distribution of Balkan haplotypes is more structured. One group of similar haplotypes occurs in the eastern Balkans (Bulgaria, Republic of Macedonia, Romania and the Greek regions Evvia, Macedonia, Peloponnese, Thessaly and Thrace). Two distinct haplotypes, differing in eight to nine mutation steps from the most common haplotype of the first group, are confined to the western slope of the Taygetos Mts. in the Peloponnese. Yet another group, connected over between four and 23 mutation steps with haplotypes of the eastern Balkan group, occurs along the western slope of the Dinarid and Pindos Mts. (Istria, Dalmatia, western Greece). Taygetos haplotypes are nested within other haplotypes in all phylogenetic analyses and support for monophyly of the other Balkan groups is at best weak. We conclude that using the traditional two subspecies model should be continued for T. hermanni. Phylogeographies of T. hermanni and Emys orbicularis, another codistributed chelonian, are markedly different, but share a few similarities. Both were forced to retreat to southern refuges during Pleistocene glaciations. With the advent of Holocene warming, E. orbicularis underwent rapid range expansion and temperate regions of Europe and adjacent Asia were recolonized from refuges in the Balkans and the northern Black Sea Region. By contrast, T. hermanni remained more or less confined to refuges and nearby regions, resulting in a much smaller range, and allopatric and parapatric distribution of haplotype groups and clades. MtDNA lineages are more diverse in E. orbicularis than they are in T. hermanni on southern European peninsulas, indicating several distinct glacial refuges in close proximity and extensive intergradation during Holocene range expansion for E. orbicularis. In T. hermanni it is likely that only on the Balkan Peninsula was more than one refuge located, corresponding to the parapatric ranges of haplotype groups currently there. On the old western Mediterranean islands Corsica and Sardinia no differentiated (E. orbicularis) or only weakly differentiated haplotypes (T. hermanni) occur, even though there is evidence for the presence of both species on Corsica since at least the Middle Pleistocene. High mountain chains constitute major barriers separating distinct mtDNA clades or groups in each species.     

Mitochondrial DNA of pre‐last glacial maximum red deer from NW Spain suggests a more complex phylogeographical history for the species

The major climatic oscillations that characterized the Quaternary had a great influence on the evolution and distribution of several species. During cold periods, the distribution of temperate‐adapted species became fragmented with many surviving in southern refugia (Iberian, Italian, and Balkan Peninsulas). Red deer was one of the species that contracted its original range to southern refugia. Currently, two main lineages have been described for the species: western and eastern. We have analyzed fossils pre‐dating the last glacial maximum (LGM) from Liñares cave (NW Spain) that belongs to the peripheral range of the western clade, and fossils from the Danish Holocene belonging to the central part of the same clade. Phylogenetic analyses place our samples in the western clade. However, some specimens from Liñares represent an early split in the tree along with other pre‐LGM western samples from previous studies. Despite low bootstrap values in the Bayesian phylogenies, haplotype networks connect these foreign haplotypes to the eastern clade. We suggest a mixed phylogeographical model to explain this pattern with range expansions from the east during the expansion phase after the cold periods in marine isotope stage 3. We find slight isolation by distance in post‐LGM populations that could be a consequence of the recolonization from southern refugia after the LGM.     

Phylogeography of the Japanese giant flying squirrel, Petaurista leucogenys (Rodentia: Sciuridae): implication of glacial refugia in an arboreal small mammal in the Japanese Islands

To test the association between temperate forest dynamics and glacial refugia for arboreal small mammals, we studied the phylogeography of the Japanese giant flying squirrel ( Petaurista leucogenys ) using complete mitochondrial cytochrome b gene sequences (1140 bp). This squirrel is endemic to three of Japan's main islands: Honshu, Shikoku, and Kyushu. We examined 58 specimens of P. leucogenys collected from 40 localities in Japan. Additionally, two individuals with unknown sampling localities were included in phylogenetic analyses. There were 54 haplotypes of P. leucogenys. We found five major phylogroups (Northern, Central, South-eastern, South-western, and Southern). These phylogroups may have originated from glacial refugia during the Late Pleistocene. After the last glaciation, the Northern phylogroup, widely distributed in eastern Japan, could have extensively expanded northward from its refugia. By contrast, in western Japan, population expansion was restricted to western Japan. All members of four phylogroups existed in western Japan during glaciations. The complicated phylogeographical pattern of P. leucogenys populations originating from western Japan may have resulted from the long history.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 47–60.     

Pleistocene evolutionary history of the Clouded Apollo (Parnassius mnemosyne): genetic signatures of climate cycles and a 'time-dependent' mitochondrial substitution rate

Genetic data are currently providing a large amount of new information on past distribution of species and are contributing to a new vision of Pleistocene ice ages. Nonetheless, an increasing number of studies on the 'time dependency' of mutation rates suggest that date assessments for evolutionary events of the Pleistocene might be overestimated. We analysed mitochondrial (mt) DNA (COI) sequence variation in 225 Parnassius mnemosyne individuals sampled across central and eastern Europe in order to assess (i) the existence of genetic signatures of Pleistocene climate shifts; and (ii) the timescale of demographic and evolutionary events. Our analyses reveal a phylogeographical pattern markedly influenced by the Pleistocene/Holocene climate shifts. Eastern Alpine and Balkan populations display comparatively high mtDNA diversity, suggesting multiple glacial refugia. On the other hand, three widely distributed and spatially segregated lineages occupy most of northern and eastern Europe, indicating postglacial recolonization from different refugial areas. We show that a conventional 'phylogenetic' substitution rate cannot account for the present distribution of genetic variation in this species, and we combine phylogeographical pattern and palaeoecological information in order to determine a suitable intraspecific rate through a Bayesian coalescent approach. We argue that our calibrated 'time-dependent' rate (0.096 substitutions/ million years), offers the most convincing time frame for the evolutionary events inferred from sequence data. When scaled by the new rate, estimates of divergence between Balkan and Alpine lineages point to c. 19 000 years before present (last glacial maximum), and parameters of demographic expansion for northern lineages are consistent with postglacial warming (5-11 000 years before present).