Molecular phylogeography and the evolution and conservation of Amazonian mammals

The phylogeographic structure of 15 genera of Amazonian marsupials and rodents is summarized based on comparative sequence of the mitochondrial cytochrome b gene. The data are limited in geographical coverage, with samples widely scattered throughout Amazonia from the base of the Andes in Peru to the Guianan coast and eastern Brazil. We use this approach to define species boundaries, based minimally on the principle of reciprocal monophyly, in conjunction with morphological or other genetic discontinuities. The taxa so defined are older than previously appreciated, with many lineages dating from 1 to more than 3 Myr, and thus apparently predating the early Pleistocene. We relate patterns of concordant geographical shifts with underlying tectonic history and to current positions of major rivers. Finally, we provide comments on the utility of these data and patterns to conservation, articulating a need to incorporate phylogeographic information as part of the rationale in establishing conservation priorities at the organismal and geographical area levels.     

Inter- and intraspecific phylogeography of small mammals in the Atacama Desert and adjacent areas of northern Chile

Aim We evaluated the phylogeography of sigmodontine taxa of the genera Phyllotis and Abrothrix at the intra and interspecific level, in the Atacama desert and adjacent Andean and Puna regions of northern Chile. The major goal was to test the hypothesis that sigmodontine mice differentiated in the lowlands, most likely via peripatric speciation, dispersing from highland to lowland areas across the desert vegetated canyons, thus reaching the Pacific coast. Dispersing individuals may have found favourable habitats along these valleys, in northern Chile, which connect the high altitude Puna region with the lowlands. Location The study was conducted in northern Chile (18–22° S), in coastal pre‐Puna and Puna regions. Methods For phylogeographic analyses we analysed cytochrome b mitochondrial sequences for 29 specimens of the genera Abrothrix and Phyllotis, from the region of study. All results were analysed phylogenetically using maximum‐likelihood, Bayesian, and uncorrected median‐joining network methodology. Results In Phyllotis we recognized two major clusters of taxa: one restricted to the Puna region identified as Phyllotis xanthopygus chilensis, in close association to a pre‐Punean and lowland clade constituted by Phyllotis limatus, on the western slopes of the Andes. A similar pattern was distinguished for Abrothrix, where Abrothrix andinus was recognized in the Andean Altiplano‐pre‐Puna region and Abrothrix olivaceus in the lowlands of northern Chile. Main conclusions We found that the radiation of sigmodontine mice in the central Andes may have been facilitated by the historical events that affected high Andean elevations during Pleistocene times, as well as changes in the vegetation composition and climate that started to prevail during that time. Our results also support previous hypotheses that the major mode of evolution for small mammals in the Andes region has been based on the founder effect or the peripheral isolates model, from a central range located in the Andes.     

Long-distance gene flow and cross-Andean dispersal of lowland rainforest bees (Apidae: Euglossini) revealed by comparative mitochondrial DNA phylogeography

Euglossine bees (Apidae; Euglossini) exclusively pollinate hundreds of orchid species and comprise up to 25% of bee species richness in neotropical rainforests. As one of the first studies of comparative phylogeography in a neotropical insect group, we performed a mitochondrial DNA (mtDNA)-based analysis of 14 euglossine species represented by populations sampled across the Andes and/or across the Amazon basin. The mtDNA divergences within species were consistently low; across the 12 monophyletic species the mean intraspecific divergence among haplotypes was 0.9% (range of means, 0-1.9%). The cytochrome oxidase 1 (CO1) divergence among populations separated by the Andes (N = 11 species) averaged 1.1% (range 0.0-2.0%). The mtDNA CO1 data set displayed homogeneous rates of nucleotide substitution, permitting us to infer dispersal across the cordillera long after the final Andean uplift based on arthropod molecular clocks of 1.2-1.5% divergence per million years. Gene flow across the 3000-km breadth of the Amazon basin was inferred from identical cross-Amazon haplotypes found in five species. Although mtDNA haplotypes for 12 of the 14 euglossine species were monophyletic, a reticulate CO1 phylogeny was recovered in Euglossa cognata and E. mixta, suggesting large ancestral populations and recent speciation. Reference to closely related outgroups suggested recent speciation for the majority of species. Phylogeographical structure across a broad spatial scale is weaker in euglossine bees than in any neotropical group previously examined, and may derive from a combination of Quaternary speciation, population expansion and/or long-distance gene flow.     

Genetic variability and phylogeography of the cyprinid Telestes muticellus within the Italian peninsula as revealed by mitochondrial DNA

A phylogeographic analysis of 12 populations of Telestes muticellus was performed on the basis of mitochondrial DNA sequences of the cytochrome b gene. The main aims were to determine phylogenetic relationships from congener species and to highlight the patterns of genetic differentiation within the Italian peninsula. The results obtained showed a significant divergence with the congener species, indicating a possible Miocene origin for the genus. Divergence was found to be low for T. muticellus , yet distinct lineages resulted as being allopatric within the two main Italian biogeographic districts, the Padano-Venetian and the Tuscano-Latium, suggesting the existence of recent vicariance coupled with past demographic regression. The high level of genetic differentiation found between populations highlighted the low dispersal ability of this rheophilic Cyprinid, whose migration was mainly related to connections between the upper parts of river basins. The distribution of sequence characteristics found suggests that particular attention should be paid to the conservation management of this species.     

The historical bridge between the Amazon and the Atlantic Forest of Brazil: a study of molecular phylogeography with small mammals

Abstract Aim To examine how the genetic diversity of selected taxa of forest‐dwelling small mammals is distributed between and within the major rain forest domains of Amazonia and Atlantic Forest and the intervening interior forests of Brazil, as inferred by the relationships between gene genealogies and geography. I also addressed the historical importance of the central Brazilian forests in connecting Amazon and Atlantic Forest populations of rodents and marsupials. Methods I evaluated variation in the mitochondrial cytochrome b gene to estimate the levels of sequence divergence between those taxa occurring throughout the Amazon, Atlantic Forest, and forests in the Cerrado and Caatinga regions. I inferred the hierarchical relationships between haplotypes, populations and formal taxa using the cladistic approach of maximum parsimony. I compared areas and the clades identified by superimposing cladograms on the geographical distribution of samples. The degree of concordance both in phylogeny and the depth of the nodes in these phylogenies, in addition to patterns of geographical distribution of clades, permitted me to make inferences on how, when and where the taxa differentiated. Results Sequence similarity is often greater between samples from the Atlantic Forest and either Amazon or central Brazilian forests than it is within each of the two rain forest domains. The Atlantic Forest clades are either not reciprocally monophyletic or are the sister group to all the other clades. There is some indication of northern and southern components in the Atlantic Forest. Given the geographical distribution of clades and the relatively deep levels of divergence, the central Brazilian area does not behave as a separate region but is complementary to either Amazon or Atlantic Forest. Patterns of area relationships differ across taxa, suggesting that different processes and/or historic events affected the diversification within each lineage. Main conclusions The Amazon and the Atlantic forests are not exclusive in terms of their small mammal faunas; both overlap broadly with taxa occurring in gallery forests and dry forests in central Brazil. Central Brazilian forests are an integral part of the evolutionary scenario of lowland small mammals, playing an important role as present and past habitats for rain forest species. Therefore, representatives from this area should always be included in analyses of the evolutionary history of lowland rain forest faunas. The incongruence of branching patterns among areas is in agreement with recent results presented for Neotropical passerine birds and indicates that a single hypothesis of Neotropical area relationships is unlikely. These findings reinforce the idea that speciation in the Neotropics will not be explained by any single model of vicariance or climatic changes.     

Post‐glacial colonization of eastern Europe from the Carpathian refugium: evidence from mitochondrial DNA of the common vole Microtus arvalis

There is now considerable evidence for the survival of temperate species within glacial refugia that were situated at relatively high latitudes, notably the Carpathian Basin and Dordogne region in Europe. However, the prevalence of fossil remains in such locations is rarely matched by molecular evidence for their contribution to subsequent geographical and demographic expansion of the species in question. One obstacle to this has been insufficient analysis of modern samples from the relevant areas, in particular the parts of eastern Europe that surround the Carpathian refugium. In the present study, we examine the patterns of variation in mitochondrial DNA of the common vole (Microtus arvalis), obtained from existing museum specimens and from newly‐collected samples obtained in this area. We show that common voles from one of six extant mitochondrial DNA lineages have colonized most of the species' range in eastern Europe. We contend that the post‐glacial dispersal of this lineage most likely originated from the Carpathian refugium, adding support to the argument that such northern refugia made an important contribution to existing genetic diversity in Europe. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, ●● , ●●–●●.     

Subterranean archipelago in the Australian arid zone: mitochondrial DNA phylogeography of amphipods from central Western Australia

In 1998, a unique subterranean ecosystem was discovered in numerous isolated calcrete (carbonate) aquifers in the arid Yilgarn region of Western Australia. Previous morphological and genetic analyses of a subterranean water beetle fauna suggest that calcrete aquifers are equivalent to closed island habitats that have been isolated for millions of years. We tested this hypothesis further by phylogeographic analyses of subterranean amphipods (Crangonyctoidea: Paramelitidae and Hyalidae) using mitochondrial DNA sequence data derived from the cytochrome oxidase I gene. Phylogenetic analyses and population genetic analyses (samova) provided strong evidence for the existence of at least 16 crangonyctoid and six hyalid divergent mitochondrial lineages, each restricted in their distribution to a single calcrete aquifer, in support of the 'subterranean island (archipelago) hypothesis' and extending its scope to include entirely water respiring invertebrates. Sequence divergence estimates between proximate calcrete populations suggest that calcretes have been isolated at least since the Pliocene, coinciding with a major aridity phase that led to the intermittent drying of surface water. The distribution of calcretes along palaeodrainage channels and on either side of drainage divides, have had less influence on the overall phylogeographic structure of populations, with evidence that ancestral crangonyctoid and hyalid species moved between catchments multiple times prior to their isolation within calcretes. At least two potential modes of evolution may account for the diversity of subterranean amphipod populations: dispersal/vicariance of stygobitic species or colonization of calcretes by surface species and independent evolution of stygobitic characteristics.     

Phylogeny and phylogeography of the Liolaemus darwinii complex (Squamata: Liolaemidae): evidence for introgression and incomplete lineage sorting

Although mitochondrial DNA markers have several properties that make them suitable for phylogeographic studies, they are not free of difficulties. Phylogeographic inferences within and between closely related species can be mislead by introgression and retention of ancestral polymorphism. Here we combine different phylogenetic, phylogeographic, and population genetic methods to extract the maximum information from the Liolaemus darwinii complex. We estimate the phylogeographic structure of L. darwinii across most of its distributional range, and we then estimate relationships between L. darwinii and the syntopic species L. laurenti and L. grosseorum. Our results suggest that range expansion of these lineages brought them into secondary contact in areas where they are presently in syntopy. Here we present the first evidence for introgression in lizards from temperate South America (of L. danwinii mitochondrial DNA into L. laurenti and L. grosseorum), and for incomplete lineage sorting (between L. darwinii and L. laurenti). We show that a combination of methods can provide additional support for inferences derived from any single method and thus provide more robust interpretations and narrow the range of plausible hypotheses about mechanisms and processes of divergence. Additional studies are needed in this group of lizards and in other codistributed groups to determine if Pleistocene climatic changes could be a general factor influencing the evolutionary history of a regional biota.     

Phylogenetic evidence of mitochondrial DNA introgression among pocket gophers in New Mexico (family Geomyidae)

Mitochondrial DNA (mtDNA) variation in the cytochrome b gene was determined for two divergent taxa of pocket gophers, Thomomys bottae actuosus and T. b. ruidosae . These two taxa hybridize in a narrow contact zone, but introgression of nuclear markers such as allozymes or chromosomes does not extend much beyond the hybrid zone (Patton et al. 1979). We found that despite their distinctness, the two subspecies shared very similar mtDNA haplotypes. By a comparison of phylogenetic histories derived from nuclear markers (allozymes) and from mtDNA haplotypes sampled in different populations of T. bottae from New Mexico, we show that apparent similarity is due to an introgression of T. b. ruidosae mtDNA into T. b. actuosus nuclear background. Evidence of introgression is not limited to the present-day contact zone between these two taxa, but extends at least 75 km away from it. The actuosus haplotype coexists along with the ruidosae mtDNA in the Gallinas Mts., which are inhabited by otherwise pure T. b. actuosus , while further north only typical actuosus haplotypes were detected. Of several potential mechanisms which could lead to such a geographical pattern of variation, we argue that a combination of range shifts due to climatic fluctuations, and genetic drift are most likely. Horizontal gene transfers due to hybridization are historical events which seem rather common among pocket gophers. Although they can be identified with careful phylogenetic study using independent data sets, the potential for misinterpreting a gene tree as an organismal tree is great in this and other groups of animals.     

A molecular perspective on the phylogeny of placental mammals based on mitochondrial 12S rDNA sequences,with special reference to the problem of the Paenungulata

Part of the 12S rDNA gene was amplified and sequenced for 11 placental mammals, 3 marsupials, and 2 monotremes. Multiple alignments for these sequences and nine additional placental sequences taken from GenBank were obtained using CLUSTAL. Phylogenetic analyses were performed using standard parsimony, transversion parsimony, and Lake's method of invariants. All of our analyses uniteLoxodontia withDugong. Procavia, in turn, is a sister group to these taxa, thus supporting the monophyly of the Paenungulata. Perissodactyls are a sister group to paenungulates when transitions and transversions are both included but not when transitions are omitted. Likewise, cetaceans are a sister group to artiodactyls on minimum length trees under standard parsimony but not under transversion parsimony. Rodent monophyly and bat monophyly also receive mixed support, as does a putative alliance between primates and lagomorphs. Interestingly, the percentage divergence between the echidna and the platypus is less than for the rat and mouse.     

Intraspecific phylogeography of the Cape galaxias from South Africa: evidence from mitochondrial DNA sequences

Mitochondrial cytochrome b gene sequences were obtained from representatives of five populations of the Cape galaxias Galaxias zebratus . Four highly distinct genotypes were revealed, with sequence divergence values ranging from 5.8 to 13.8%. Some of the pairwise divergences are the highest yet reported for cytochrome b within a fish species, and are more typical of interspecific and intergeneric comparisons. In contrast, the low genetic divergence (0.3%) between two recently recorded populations from the Krom and Gamtoos (Kouga) Rivers indicates recent gene flow. Parsimony analysis consistently produced a tree with the population from the Olifants River on the west coast as ancestral and eastern populations as progressively more derived. Estimated divergence times for the Olifants population range from 4.4 to 6.6 million years ago. The findings suggest that another detailed revision is required to determine whether these divergent populations represent a single species or a species complex.     

Use of mitochondrial DNA sequences to test the Ceratomorpha (Perissodactyla:Mammalia) hypothesis

Wood (J. Mamm. 18, 106–118; 1937) united the superfamilies Tapiroidea sensu stricto and Rhinocerotoidea in the suborder Ceratomorpha and aligned the Ceratomorpha with the suborder Hippomorpha within the order Perissodactyla. Although the monophyly of the Ceratomorpha appears now well-supported in paleontological and morphological analyses, the molecular relationship among the three extant superfamilies Tapiroidea, Rhinocerotoidea, and Equoidea has not yet been examined due to the limited amount of molecular information on tapirs. In the present study, we examined the phylogenetic position of Tapiroidea, represented by the complete mitochondrial cytochrome b gene (1140 bp) of a lowland tapir ( Tapirus terrestris ), and a Indian tapir ( Tapirus indicus ), relative to modern horses, zebras, donkeys, and rhinoceroses. The phylogenetic analyses using standard parsimony, neighbour-joining and maximum likelihood algorithms revealed monophyly of the Perissodactyla and three clearly distinct lineages: the modern horses, tapirs, and rhinoceroses. However, the sister-taxon relationship of the tapirs to either the rhinoceroses or the horses was not resolved conclusively in the bootstrap analysis. Spectral analysis, in which phylogenetic information is displayed independently of any selected tree, revealed that the DNA sequences available do not contain enough phylogenetic signal for any of the alternative hypotheses on the basal diversification of perissodactyls. The short branch lengths among the three perissodactyl lineages suggest that they diverged within a relatively short period, a finding consistent with molecular divergence datings and the fossil evidence that indicates a major radiation of the early perissodactyls approximately 54–50 million years ago.     

Comparative phylogeography of endemic cyprinids in the south-west Iberian Peninsula: evidence for a new ichthyogeographic area

The comparative phylogeography and evolutionary history of three native cyprinid genera, Squalius (subfamily Leuciscinae), Chondrostoma (subfamily Leuciscinae) and Barbus (subgenus Luciobarbus ; subfamily Cyprininae), were examined focusing mainly in the South-Western region of the Iberian Peninsula, where recently described endemic species are present with considerably restricted distribution areas. In order to accomplish that the variation at the complete cytochrome b gene (1140 bp) was analysed for specimens from the South-Western region, and also for representatives of the three genera from all over the Iberian Peninsula. Data indicate different evolutionary histories, with distinct time periods of colonization between the two cyprinid subfamilies in the Iberian Peninsula. Four new Iberian ichthyogeographic areas are accordingly proposed based on congruent phylogeographic and geological evidences: the South-Western, the South-Eastern, the Atlantic and the Mediterranean. Evidence was provided for the older isolation of the South-Western area in the Miocene during the Endorheic Drainages phase, designating a clearly defined and distinct ichthyogeographic area. A new molecular clock calibration is proposed for the subgenus Luciobarbus .     

Phylogenetic relationships among species of the Neotropical genus Graomys (Rodentia: Cricetidae): contrasting patterns of skull morphometric variation and genetic divergence

Subtle differences of external traits characterize species of rodents in the Neotropical genus Graomys. On the other hand, the species differ markedly in chromosome number. In the present study, we evaluate the possible evolutionary forces involved in the evolution of the genus by assessing the degree of intra‐ and interspecific genetic and morphological variation. A phylogenetic analysis demonstrates the existence of at least three species with high levels of genetic distance (10%), which diverged between 1 and 1.5 Mya. Neither Graomys griseoflavus, nor Graomys chacoensis present marked phylogeographical structure. Regarding morphological characters, these species show shape differences in the skull that could be attributable to differences in the local conditions they inhabit, being more marked in G. griseoflavus than in G. chacoensis. The skull shape of G. chacoensis could have evolved under genetic drift, whereas evidence reported in the present study indicates that this character could be under selective pressures in G. griseoflavus. Reconstruction of the ancestral area suggests that G. griseoflavus originated in the central Monte desert, whereas G. chacoensis originated in the Chaco ecoregion surrounding the austral extreme of the Yungas rainforest. Subsequently, both species would have undergone demographic and geographical expansions almost simultaneously, starting approximately 150 000–175 000 years ago. The complex evolutionary history of the genus could be partly explained by the decoupling of morphological, karyological and molecular traits.     

The molecular phylogenetics of the genus Oligoryzomys (Rodentia: Cricetidae) clarifies rodent host–hantavirus associations

Several species of the genus Oligoryzomys are natural hosts of different hantavirus genotypes affecting humans. The systematics of the genus is confusing, which complicates the identification of the rodent host and hence the potential endemic areas of hantavirus pulmonary syndrome. In this study, we analyse molecular data to infer phylogenetic relationships among Central and South American specimens of Oligoryzomys, and compare our results with previously published data on karyotypic, geographic distribution and host–virus associations to solve contradictory taxonomic reports. We identified 25 clades, each one corresponding to a different putative species. The phylogenetic trees show that Oligoryzomys longicaudatus is strongly related to the Oligoryzomys flavescens complex, which comprises four clades; Oligoryzomys nigripes is related to Oligoryzomys stramineus, Oligoryzomys vegetus is related to Oligoryzomys fulvescens from Central America, and Oligoryzomys brendae is the sister species of Oligoryzomys aff. destructor. We identified the following rodent host–hantavirus genotype relationships: O. longicaudatus–Andes; O. flavescens ‘West'–Bermejo; O. flavescens ‘East'–Lechiguanas; O. nigripes–Juquitiba; Oligoryzomys microtis–Rio Mamore and Rio Mamore‐3; Oligoryzomys chacoensis–Oran; Oligoryzomys costaricencis–Choclo; Oligoryzomys delicatus–Maporal; Oligoryzomys utiaritensis–Castelo dos Sonhos; Oligoryzomys sp. RT2012–Rio Mamore‐4; Oligoryzomys sp. (and not Oligoryzomys fornesi)–Anajatuba. This work, besides contributing to the development of prevention programmes for hantavirus epidemiology in Latin America, represents a comprehensive update of the systematics of the genus Oligoryzomys. © 2014 The Linnean Society of London     

Population genetic structure and comparative diversity of smallmouth bass Micropterus dolomieu: congruent patterns from two genomes

Genetic diversity and divergence patterns of smallmouth bass Micropterus dolomieu spawning groups are analysed across its northern native range with mtDNA cytochrome b gene sequences and eight unlinked nuclear DNA microsatellite loci. Results reveal high levels of genetic variability and significant differences in allelic representation among populations (mtDNA: mean ± s.e ., H_D = 0·50 ± 0·06, mean ± s.e ., θ_ST = 0·41 ± 0·02 and microsatellites: mean ± s.e . H_O = 0·46 ± 0·03, mean ± s.e . θ_ST = 0·25 ± 0·01). The distributions of 28 variant mtDNA haplotypes, which differ by an average of 3·94 nucleotides (range = 1–8), denote divergent representation among geographic areas. Microsatellite data support nine primary population groups, whose high self‐assignment probabilities likewise display marked divergence. Genetic patterns demonstrate: (1) high genetic diversity in both genomes, (2) significant divergence among populations, probably resulting from natal site homing and low lifetime migration, (3) support for three post‐glacial refugia that variously contributed to the current northern populations, which remain evident today despite waterway connectivity and (4) a weak yet significant genetic isolation by geographic distance pattern, indicating that other processes affect the differences among populations, such as territoriality and site fidelity.     

神农架自然保护区非飞行哺乳动物的物种丰富度:沿海拔梯度的分布格局

于1999～2001年调查了神农架自然保护区6个地点不同栖息地的非飞行哺乳动物的物种丰富度。栖息地分为8类：原始林、择伐林（采伐枯立木）、次生林、灌木林、草地、常年性河流水溪、农田和人居住区。小型非飞行哺乳动物调查用捕鼠夹;大型非飞行哺乳动物调查主要根据皮张收购资料以及样线法和痕迹法;用10 m×10 m的样方调查林地树种丰富度。调查发现,神农架自然保护区有非飞行哺乳动物59种［不包括引进种梅花鹿（Cervus nippon）］。在同一海拔高度,原始林通常比择伐林和次生林的物种丰富度高,说明采伐严重降低了物种丰富度。对比同一栖息地不同海拔高度的物种丰富度,我们发现,在中海拔地段（800～1700 m）物种丰富度最高：如在原始林和次生林,海拔1700 m的东溪物种丰富度最高;在择伐林,海拔800 m的九冲物种丰富度最高。聚类分析显示,6个地点的哺乳动物物种组成可以分为两组：高海拔组（2100 m以上）和中低海拔组（1700 m以下）。各地点的哺乳动物物种组成与植被的垂直分布是一致的。各地点的物种丰富度与单位面积（100 m²）树种平均丰富度、栖息地类型数和海拔高度相关。3个环境变量间也是相关的：海拔高度对单位面积树种平均丰富度和栖息地类型数有重要影响。根据研究结果提出两点保护建议：第一,保护区的移民迁出和退耕还林工程应首先在物种丰富度最高的九冲进行,而后是东溪和下谷;第二,为了增加个体流和基因流,保护区东西两片相间的非保护区地带应划入保护区,建立栖息地廊道。     

The evolutionary history of a mammal species with a highly fragmented range: the phylogeography of the European snow vole

The European snow vole Chionomys nivalis has a patchy distribution restricted to rocky habitats across southern Europe and the Near and Middle East. We carried out a phylogeographic study to provide a biogeographic scenario, based on molecular data, outlining the major processes that determined the current distribution of the species. The samples include 26 snow voles from 14 different populations across the entire species range from Spain to Anatolia and Israel. Nearly complete sequences (1037 bp) of the mitochondrial gene for cytochrome b were sequenced. Relationships among haplotypes were inferred with neighbour-joining, maximum likelihood, maximum parsimony analyses and minimum spanning network. An analysis of mismatch distribution was used to cast light on past demographic expansion. We found 22 different haplotypes that fall into six distinct lineages, all but one is supported by high bootstrap values with all methods. Four lineages are allopatric (Tatra Mts., Iberia, Balkans and Middle East) while divergent haplotypes from two lineages show sympatry in the Alps and the Apennines. The basal relationships of these lineages could not be established by any tree. The mean pairwise genetic distance between lineages ranges from 2.4 to 4.2%. The shape of the mismatch distribution indicated a past expansion event dating back to between 158 000 and 84 000 years ago. These data can be interpreted with the existence of southern glacial refugia (Iberia, Balkans, Middle East and Italy) and one additional northern glacial refugium. The lack of phylogenetic resolution among lineages and the shape of mismatch distribution are indicative of a simultaneous and rapid splitting due to a relatively fast initial expansion of populations. Moreover, the analysis supports the hypothesis of the European origin of C. nivalis and its subsequent eastward dispersion during the Middle Pleistocene.