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.     

Morphological discrimination of two genetic groups of a Japanese salamander, Hynobius naevius (Amphibia, Caudata)

Hynobius naevius, distributed on western Honshu, Shikoku, and Kyushu Islands of Japan, includes two genetically distinct groups (Groups A and B) that have never been delimited morphologically. Using specimens from the entire species range, we investigated the possibility of distinguishing these groups morphologically. Multivariate analyses of morphometric characters resulted in recognition of two groups that corresponded well to the two genetic groups. One (Group A) was characterized by larger body, compressed tail, shallower vomerine tooth series, bluish- or reddish-purple ground color, and pale-white lateral markings. In contrast, another (Group B) was characterized by smaller body, cylindrical tail, longer vomerine tooth series, reddish-brown ground color, and white lateral markings. Group A was composed of populations from the Chugoku District of Honshu and northern Kyushu, and could not be divided into subgroups, while Group B encompassed populations from the Chubu and Kinki Districts of Honshu, Shikoku and Kyushu, and was subdivided into three local subgroups that are geographically separated by marine straits. Morphometric differentiation in Group A is presumed to have been less affected by genetic factors than by other factors, such as ecological relationships with other, coexisting species. Differentiation in Group B is assumed to have been enhanced not only by genetic but also by climatological factors.     

Color polymorphism and historical biogeography in the Japanese patellogastropod limpet Cellana nigrolineata (Reeve) (Patellogastropoda: Nacellidae)

Cellana nigrolineata is amongst the most common and largest patellogastropod limpets in Japan, and has two color morphs. Analyses of anatomical and morphological characters, shell structure, and mitochondrial COI data (658bp) of these color morphs suggested that they represent intraspecific genetic variation. Molecular analysis demonstrated that the species can be subdivided into three genetically distinct groups: (Clade 1) Honshu, Shikoku to Eastern Kyushu, (Clade 2) Western Kyushu and (Clade 3) Southern Kyushu. Clade 1 and Clade 2 + 3 are distributed on the coastlines adjacent to two warm-water currents, the Kuroshio and Tsushima Currents, respectively. The southern population (Clade 3) is currently isolated by inhospitable sandy shores. The subdivision of these groups likely dates from the glacial period of Plio-Pleistocene time.     

Phylogenetic relationships and biogeography of the Japanese clawed salamander, Onychodactylus japonicus (Amphibia: Caudata: Hynobiidae), and its congener inferred from the mitochondrial cytochrome b gene

Using the mitochondrial cytochrome b gene, we investigated phylogenetic relationships between and within the Japanese clawed salamander, Onychodactylus japonicus, and its close continental relative O. fischeri. Monophyly of O. japonicus was well supported, and O. japonicus was clearly distinguished from O. fischeri. However, O. fischeri comprises genetically distinct populations from Russia, NE China, and Korea that do not form a monophyletic group. Within O. japonicus, four major clades were clearly recognized: Clade I from northern Tohoku district, Clade II from southern Tohoku district and the Tsukuba Mountains, Clade III from southwestern Honshu, and Clade IV from Kinki and Chugoku districts in Honshu and from Shikoku. Although genetic distances among these clades were large (5.5-9.6%), relationships among the clades were unresolved. All clades except Clade I contained two or three distinct subclades. In several localities in Kinki and Chugoku, Clades III and IV were sympatric. The estimated divergence times and available geohistorical data suggest that O. japonicus began to differentiate in the Upper Late Miocene and that the pattern of genetic differentiation of this species has been affected strongly by climate changes and geohistorical events such as volcanic activity and mountain formation. Our results suggest that both O. fischeri and O. japonicus comprise multiple cryptic species.     

Morphological and allozymic variation in Hynobius boulengeri and H. stejnegeri (Amphibia: Urodela: Hynobiidae)

We studied morphological and allozymic variation in populations of Japanese salamanders, Hynobius boulengeri and H. stejnegeri. Adult H. boulengeri showed sexual dimorphism, and juveniles differed greatly from adults in many morphological characters. From the results of multivariate analyses of morphological characters, the populations were divided into four groups: (I) H. boulengeri from Honshu, (II) H. boulengeri from Shikoku, (III) H. boulengeri from the Sobo-Katamuki Mountains of Kyushu and H. stejnegeri, and (IV) H. boulengeri from the Amakusa Islands and the Osumi Peninsula. Phenotypic relationships among the four groups were identical to relationships clarified by allozymic analyses, except for group IV, which was included in group III in the allozyme tree. Some morphometric characters were significantly correlated with environmental variables. We consider H. stejnegeri to be a valid species based on its unique color pattern, morphometric characters, and allelic composition, even though it was nested within group III of H. boulengeri by both morphological and allozymic analyses. We propose that group I from Honshu and group II from Shikoku should be treated as H. boulengeri sensu stricto and H. hirosei, respectively. Resolving the taxonomic status of the remaining populations of groups III and IV from Kyushu requires further study.     

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.     

Phylogenetic relationships of the North American chorus frogs (Pseudacris: Hylidae)

We examined phylogenetic relationships of the North American chorus frogs (Pseudacris: Hylidae) from 38 populations using 2.4 kb of 12S and 16S mtDNA to elucidate species relationships and examine congruence of previous phylogenetic hypotheses. Parsimony, maximum likelihood, and Bayesian phylogenies are consistent and reveal four strongly supported clades within Pseudacris: (1). A West Coast Clade containing regilla and cadaverina, (2). a Fat Frog Clade including ornata, streckeri, and illinoensis, (3). a Crucifer Clade consisting of crucifer and ocularis, and (4). a Trilling Frog Clade containing all other Pseudacris. Explicit hypothesis testing using parametric bootstrapping indicates that previous phylogenetic hypotheses are rejected by our sequence dataset. Within the Trilling Frog Clade, brimleyi and brachyphona form the sister group to the Nigrita Clade: nigrita, feriarum, triseriata, kalmi, clarkii, and maculata. The Nigrita Clade shows geographic division into three clades: (1). populations of maculata and triseriata west of the Mississippi River and Canadian populations, (2). southeastern US populations of feriarum and nigrita, and (3). northeastern US populations of feriarum, kalmi, and triseriata. We find that subspecific epithets for crucifer (crucifer and bartramiana) and nigrita (nigrita and verrucosa) are uninformative, therefore we discourage recognition of these subspecies. Pseudacris regilla, cadaverina, ocularis, and crucifer are maintained in Pseudacris.     

Phylogeography of the canyon treefrog, Hyla arenicolor (Cope) based on mitochondrial DNA sequence data

Patterns of phylogeography and gene flow were examined in the canyon treefrog, Hyla arenicolor. A total of 973 bp of mitochondrial cytochrome b sequence data were obtained for 65 individuals from 53 populations, yielding 50 unique haplotypes. Interpopulation sequence variation ranged from 0 to 13.7%. Phylogenetic analysis revealed three deeply divergent mtDNA lineages. These three Clades were mapped onto geography and found to represent completely concordant, nonoverlapping, geographical regions. Levels of sequence divergence between the three Clades were equal to or greater than published levels of divergence found in other vertebrate species and genera. Furthermore, one Clade of H. arenicolor was found to be more closely related to the outgroup H. eximia than to other H. arenicolor, suggesting that the taxonomy of this species may require revision.     

New insights into the origin and the genetic status of the Balkan donkey from Serbia

The Balkan donkey (Equus asinus L.) is commonly regarded as a large‐sized, unselected, unstructured and traditionally managed donkey breed. We assessed the current genetic status of the three largest E. asinus populations in the central Balkans (Serbia) by analysing the variability of nuclear microsatellites and the mitochondrial (mtDNA) control region of 77 and 49 individuals respectively. We further analysed our mtDNA dataset along with 209 published mtDNA sequences of ancient and modern individuals from 19 European and African populations to provide new insights into the origin and the history of the Balkan donkey. Serbian donkey populations are highly genetically diverse at both the nuclear and mtDNA levels despite severe population decline. Traditional Balkan donkeys in Serbia are rather heterogeneous; we found two groups of individuals with similar phenotypic features, somewhat distinct nuclear backgrounds and different proportions of mtDNA haplotypes belonging to matrilineal Clades 1 and 2. Another group, characterized by larger body size, different coat colour, distinct nuclear gene pool and predominantly Clade 2 haplotypes, was delineated as the Banat donkey breed. The maternal landscape of the large Balkan donkey population is highly heterogeneous and more complex than previously thought. Given the two independent domestication events in donkeys, multiple waves of introductions into the Balkans from Greece are hypothesized. Clade 2 donkeys probably appeared in Greece prior to those belonging to Clade 1, whereas expansion and diversification of Clade 1 donkeys within the Balkans predated that of Clade 2 donkeys.     

Phylogenetic relationships of the Chinese brown frogs (genus Rana) inferred from partial mitochondrial 12S and 16S rRNA gene sequences

Based on partial sequences of the 12S and 16S ribosomal RNA genes, we estimated phylogenetic relationships among brown frogs of the Rana temporaria group from China. From the phylogenetic trees obtained, we propose to include Rana zhengi in the brown frogs. Monophyly of the brown frogs was not unambiguously supported, but four well-supported clades (A, B, C, and D) always emerged, although relationships among them remained unresolved. Clade A contained brown frogs with 24 chromosomes and was split into two distinct subclades (Subclade A-1: R. chensinensis and R. huanrenensis; Subclade A-2: R. dybowskii). Polytomous relationships among populations of R. chensinensis and R. huanrenensis suggested the necessity of further taxonomic assessment. Rana kunyuensis proved to be the sister group to R. amurensis, and these two species formed Clade B. Clade C was composed of R. omeimontis and R. chaochiaoensis, and Clade D included R. sauteri, which has been placed in other ranid genera. These relationships did not change after adding published data, and monophyly of Subclade A-1, A-2, and other East Asian brown frogs with 24 chromosomes (R. pirica and R. ornativentris) was ascertained, though their relationships were unresolved. Clade C, together with R. japonica and R. longicrus, also formed a monophyletic group. Brown frogs related to Clades A and C were estimated to have dispersed from continental Asia to adjacent regions through multiple events.     

A genetic method to distinguish crossbred Inobuta from Japanese wild boars

To distinguish pig-wild boar crossbred Inobuta from Japanese wild boar populations, a genetic method by using mitochondrial DNA (mtDNA) haplotypes and the nuclear glucosephosphate isomerase-processed pseudogene (GPIP) was developed. Sixteen mtDNA haplotypes from 152 wild boars from Kyushu, Shikoku and Honshu islands of Japan were distinct from those from Asian and European domestic pigs. Five alleles of GPIP were classified into two groups: 1). alleles GPIP*1, GPIP*3 and GPIP*3a from Japanese wild boars, Asian wild boars and domestic pigs; 2). alleles GPIP*4 and GPIP*4a from European wild boars and domestic pigs. An extensive genetic survey was done to distinguish the crossbred Inobuta from 60 wild boars hunted on Tsushima Island, Goto Islands, and Nagasaki and Ooita Prefectures. The mtDNA haplotypes from the 60 samples showed Japanese wild boars, but four wild boar samples from Nagasaki Prefecture had the European GPIP allele, GPIP*4. These results showed that nuclear DNA polymorphism analysis is useful, in addition to mtDNA haplotype assay, to detect "Inobuta" having the European genotype from Japanese wild boar populations.     

鳇亚科似鳇属鱼类的物种界定和系统发育关系

逗亚科似逗属鱼类的物种界定保持争议, 系统发育关系尚待解决。研究取样似逗属鱼类所有种, 使用核基因多位点序列重建似逗属鱼类的系统发育关系, 运用分子的物种界定方法并结合形态特征分析厘定我国似逗属鱼类的分类。贝叶斯系统发育树结果表明: 桂林似逗与平江似逗是单系种; 似逗与扁嘴似逗是多系种, 前者包括五个谱系A至E, 后者包括两个谱系A与B。POFAD距离分析和Structurama分析的结果表明似逗和扁嘴似逗的每个谱系是独立遗传种群, BP & P分析结果强烈支持它们是不同种。*Beast物种树结果揭示: 扁嘴似逗谱系B位于似逗属鱼类的基部位置; 似逗谱系A与B是姊妹群关系, 似逗谱系C是扁嘴似逗谱系A的姊妹群, 它们一起与桂林似逗形成姊妹群关系; 似逗谱系D与E是姊妹群关系, 它们一起是平江似逗的姊妹群。结合形态证据, 对我国似逗属鱼类分类厘定如下: 限定严格意义似逗包括似逗谱系A+B; 恢复长吻似逗(Pseudogobio longirostris Mori, 1934)给予似逗谱系C分类名; 似逗谱系D与E是隐存种, 桂林似逗与平江似逗是有效种。     

One Species or Several? Discordant Patterns of Geographic Variation between Allozymes and mtDNA Sequences among Spiders in the Genus Metepeira (Araneae: Araneidae)

Paradoxically, an allozyme study of Metepeira “spinipes” (sensu lato) demonstrated extensive gene flow among four populations whose members are nevertheless morphologically and behaviorally distinct. Initially, the authors tentatively concluded that the populations exhibited panmixis and suggested that local environmental effects accounted for the apparent morphological and behavioral differences. However, they later concluded that such differences were too great to be accounted for by the environment alone and that the four populations actually represented three different species. To confirm that the allozyme results were, in fact, artifactual, we reexamined the relationships among these populations by sequencing a portion of the 12S mtDNA ribosomal subunit. In contrast to the allozyme result, our results demonstrate good agreement between patterns of genetic and morphological/behavioral variation. We suggest (1) that the allozyme allele frequencies are homogenized by balancing selection, not gene flow as was previously concluded, and therefore (2) that this study provides another instance in which inferences about population structures from allozyme data are misleading.     

Occurrence of two types of Hynobius naevius in Northern Kyushu, Japan (Amphibia: Urodela)

A survey to examine genetic variation among Hynobius naevius from four localities of Fukuoka Pref., northern Kyushu, Japan, resulted in the detection of two, sympatric, genetic types (A and B) that are clearly different in the allelic frequencies of four loci (ACOH-A, ACOH-B, ADH-A, and SOD-A) in each locality. Morphological investigations between the two genetic types also proved that they are clearly discriminated; the type A is about 75 mm in SVL, lacks mottling pattern on bluish purple dorsum, and possesses relatively short vomerine teeth series, while the type B is about 60 mm in SVL, and has light mottling on reddish purple ground color. These results strongly suggest that reproductive isolation occurs between these two types, and that they could be regarded as separate species. Populations from Toyota-cho, western Honshu, and Yabe-machi, central Kyushu, both close to Fukuoka Pref., were very similar to the types A and B, respectively. From these results, we consider that two evolutionary lineages that first evolved allopatrically in western Honshu and southern Kyushu secondarily contacted and became sympatric in the region of northern Kyushu.     

Differential geographic patterns of mitochondrial DNA variation in two sympatric species of Japanese wood mice, Apodemus speciosus and A. argenteus

We examined the gene sequences of mitochondrial cytochrome b (cyt b) in two Japanese wood mouse species, Apodemus speciosus (n = 89) and A. argenteus (n = 46), which are distributed on the four main islands of Japan (Hokkaido, Honshu, Shikoku, and Kyushu) and on the small islands surrounding them. Apodemus speciosus, the larger of the two species, showed substantial genetic variation, with a maximum of 3% sequence divergence, and remarkable phylogenetic subdivision with two major clades. One clade represents haplotypes from a central region, including Honshu, Shikoku, Kyushu, and their adjacent islands; the other clade includes haplotypes from Hokkaido and the peripheral islands, forming four subclades: a) Hokkaido, b) Sado Island, c) Satsunan Islands, and d) the Izu Islands. Sequence divergence among the four subclades was 1.0 to 1.5%, implying that A. speciosus colonized these geographic regions 0.2 to 0.3 million years ago, assuming a substitution rate of 2.4% per million years. The population on the Izu Islands has preserved haplotypes that are distinct from those in any other region, providing good evidence for the natural colonization of the volcanic islands of the Izu Islands. The cyt b sequence variation had no relation to the karyotypic dimorphism for the eastern (2n = 48) and western (2n = 46) geographic groups, between which a strict border exists at central Honshu. On the other hand, Apodemus argenteus, the smaller of the two species, showed a similar level of sequence divergence (maximum of 3%) but no substantial geographic differentiation: populations in Hokkaido, Sado, and Yakushima shared similar haplotypes with each of the central populations, suggesting that genetic exchanges occurred among the localities in the last 0.15 million years. The apparent genetic structure of the mitochondrial DNA found in the A. speciosus population might be caused solely by long-term existence in insular regions, presumably due to ecological superiority relative to A. argenteus.     

Spatial framework of nine distinct local populations of the Japanese dormouse Glirulus japonicus based on matrilineal cytochrome b and patrilineal SRY gene sequences

We previously revealed the presence of six genetically distinct matrilineal populations of the Japanese dormouse Glirulus japonicus in the distribution range of Honshu, Shikoku, and Kyushu islands. In this study, we extended this analysis using mitochondrial cytochrome b gene sequences (n = 96) and Y-chromosome-specific SRY gene sequences (n = 22) from individuals collected from Honshu, Shikoku, Kyushu, and Oki Dogo I. The cytochrome b sequence data allowed us to define precise geographic ranges of the six previously known and three newly found distinct matrilineal lineages: northeastern Honshu (I), east-central Honshu (II), west-central Honshu and the Kii Peninsula (III), the western part of Honshu (IV), Shikoku (V), westernmost Honshu and Kyushu (VI), the northern part of central Honshu (VII), the southern part of central Honshu (VIII), and Oki Dogo I. (IX). Our inference of geographic borders suggests that regions of lower and higher altitudes in the mountain systems played important roles in driving the hosting and separation of lineages, respectively. Six matrilineal lineages (I, II, V, VI, VIII, and XI) were shown to possess their own SRY haplotypes, while lineages III and IV shared one haplotype. These data together with our previous observation of nuclear ribosomal RNA gene variation indicate advanced populational subdivision in this species. It is thus evident that each of the populations, including those living at high latitudes and in limited geographic spaces, have survived for several million years. A specific ability to tolerate cold may have permitted G. japonicus to preserve anciently diverged lineages in each locality.     

Molecular phylogeographic analysis of East Asian cryptoperlan stoneflies (Insecta: Plecoptera, Peltoperlidae)

Cryptoperlan stoneflies inhabit the headwaters or upper stream areas of rivers. A molecular phylogeographic study of cryptoperlans in the Japanese archipelago and on Taiwan Island has been conducted. Altogether the mtDNA 16S rRNA region of 71 individuals from 61 populations, the mtDNA COI region of 76 individuals from 41 populations, and the nDNA Histone 3 region of 56 individuals from 52 populations were sequenced and analyzed. The respective ML, NJ, MP and Bayesian dendrograms were proposed from the sequencing data for the 16S rRNA region (362-bp), the COI region (540-bp), and the Histone 3 region (322-bp), estimated using Yoraperla uenoi as an outgroup. Based upon those data and the resulting dendrograms, it has become clear that the cryptoperlan stoneflies of the Japanese archipelago and those of Taiwan Island comprise two major clades. The first of these two major clades consists of a number of OTUs [operational taxonomic units: Cryptoperla japonica (Honshu, Shikoku and Kyushu Islands) + C. ishigakiensis (Ishigaki-jima Island) + Cryptoperla spp. (Okinawa-jima and Taiwan Islands)]. The other clade consists of the species Cryptoperla kawasawai inhabiting only Shikoku Island. Of particular note, C. kawasawai was observed to be significantly genetically differentiated from all other cryptoperlans examined. Yet, despite the fact that the specimens of C. japonica were taken from a very broad range of populations, their genetic diversity was relatively low, similar to that of C. kawasawai, which inhabits only a limited region within Shikoku Island. Furthermore, even the species C. kawasawai was revealed to be composed of two significantly genetically differentiated subclades. It is considered that this genetic structure among cryptoperlans largely reflects the geological history from the middle to upper Miocene Epoch (i.e., Tortonian stage) of the Japanese archipelago and Taiwan Island.     

GENETIC DIVERSITY AND POPULATION STRUCTURE IN CAMELLIA JAPONICA L. (THEACEAE)

Camellia japonica is a widespread and morphologically diverse tree native to parts of Japan and adjacent islands. Starch gel electrophoresis was used to score allelic variation at 20 loci in seeds collected from 60 populations distributed throughout the species range. In comparison with other plant species, the level of genetic diversity within C. japonica populations is very high: 66.2% of loci were polymorphic on average per population, with a mean number of 2.16 alleles per locus; the mean observed and panmictic heterozygosities were 0.230 and 0.265, respectively. Genotypic proportions at most loci in most populations fit Hardy-Weinberg expectations. However, small heterozygote deficiencies were commonly observed (mean population fixation index = 0.129). It is suggested that the most likely cause of the observed deficiencies is population subdivision into genetically divergent subpopulations. The overall level of population differentiation is greater than is typically observed in out-breeders: The mean genetic distance and identity (Nei's D and I) between pairs of populations were 0.073 and 0.930, respectively, and Wright's Fst was 0.144. Differences among populations appeared to be manifested as variation in gene frequencies at many loci rather than variation in allelic composition per se. However, the patterns of variation were not random. Reciprocal clinal variation of gene frequencies was observed for allele pairs at six loci. In addition, principal components analysis revealed that populations tended to genetically cluster into four regions representing the geographic areas Kyushu, Shikoku, western Honshu, and eastern Honshu. There was a significant relationship between genetic and geographic distance (r = 0.61; P < 0.01). Analysis of variance on allozyme frequencies showed that there was approximately four times as much differentiation among populations within regions, as among regions. It is likely that the observed patterns of population relationships result from the balance between genetic drift in small subpopulations and gene flow between them.     

Ecological and morphological differentiation among cryptic evolutionary lineages in freshwater limpets of the nominal form-group Ancylus fluviatilis (O.F. Müller, 1774)

The phylogeny and potential mode of speciation of the river limpet Ancylus fluviatilis (Basommatophora) was examined using mitochondrial DNA sequences from 16S ribosomal RNA, cytochrome c oxidase subunit I (COI) and nuclear DNA from internal transcribed spacer (ITS-1) regions from 103 populations across Europe. Four highly divergent lineages were observed within Ancylus. Clade 1, representing the nominal taxon Ancylus fluviatilis (O.F. Müller, 1774), is mainly found in central and northern Europe, Clade 2 is present in a single Portuguese population, Clade 3 is distributed on the Canary islands, North Africa and the eastern Mediterranean region, whereas Clade 4 inhabits the Northern Mediterranean coasts. Phylogenetic analyses revealed an overall consistent topology of nuclear and mitochondrial gene trees. Based on a molecular clock, we estimated that the basic radiation occurred in the late Pliocene. Although clades differ significantly in size independent shell shape, morphological differentiation of lineages is not feasible without genetic data. Environmental data related to climate (precipitation, temperature, etc.) showed a significant differentiation of clades. Clade 1 dwells in relatively colder and more stable habitats than Clades 3 and 4, whose habitats in turn differ in a low or high amount of precipitation during spring and autumn, respectively. Based on the combined data sets on mitochondrial DNA, nuclear DNA, morphological and ecological differentiation, we conclude that Ancylus represents a cryptic species complex of reproductively and genetically isolated lineages. In addition, the joint analysis suggests that ecological speciation is probable to explain current patterns.