Toward the phylogeny of Caucasian rock lizards: implications from mitochondrial DNA gene sequences (Reptilia: Lacertidae)

A maximum parsimony phylogeny of 14 Caucasian species of rock lizards, genus Ijicerta , subgenus Archaeolacerta , was constructed from mitochondrial cytochrome b and ATPase 6 partial gene sequences. Congruence analyses were carried out between the two genes. A synthesis of the data sets reveals three well supported monophyletic groups: (1) the caucasica group including ( Lacerta derjugini (( Lalpina, L. clarkorum ) ( L. caucasica, L. daghestanica ))); (2) the rudis group including ( L. parvula ( L. portschinskii ( L. vakntini, L. rudis ))); and (3) the saxicola group including ( L. mixta ( L. nairensis ( L. saxicola, L. raddeijj ). Despite the diagnosis of three groups, the placement of L. praticola as a basal taxon is uncertain, as are the relationships among the three groups. The mitochondrial DNA sequence data suggested prior hybridization between L. mixta and L. alpina and possibly between L. saxicola and L. nairensis. Lacerta raddei was resolved as a paraphyletic species on the mtDNA tree; this may result from either hybridization or random gene sorting.     

Molecular evolution of satellite DNA repeats and speciation of lizards of the genus Darevskia (Sauria: Lacertidae).

Satellite DNA repeats were studied in Caucasian populations of 18 rock lizard species of the genus Darevskia. Four subfamilies (Caucasian Lacerta satellites (CLsat)I-IV) were identified, which shared 70%-75% sequence similarity. The distribution of CLsat subfamilies among the species was studied. All the species could be divided into at least 3 clades, depending on the content of CLsat subfamilies in each genome: "saxicola", "rudis", and "mixta" lizards. CLsatI was found in all studied species, but in very different quantities; the "saxicola" group contained this subfamily predominantly. The "rudis" group also contained CLsatIII, and the "mixta" group carried considerable amounts of CLsatII. The highest concentrations of CLsatI and CLsatII were detected in 2 ground lizards--D. derjugini and D. praticola, respectively. D. parvula predominantly carried CLsatIII. CLsatIV was found only in the Crimean species D. lindholmi. The distribution patterns of satellite subfamilies show possible postglacial speciation within the genus Darevskia. A hybrid origin of species that possess 2 or 3 CLsat subfamilies and important clarifications to the systematics of the genus are proposed.     

A molecular phylogenetic analysis of invasive and ornamental brooms and their relationships within the Genistoid legumes

The Cytisus-Genista complex includes species that have become invasive following introduction into new geographic ranges as ornamental shrubs. Despite their impacts, the evolutionary relationships among invasives, ornamentals, and native-range species have never been investigated. Our objective was to examine relationships within the Cytisus-Genista complex to determine (1) the taxonomic identity of invasive "French broom" and ornamental "sweet broom" and (2) whether "sweet broom" contributes to "French broom" populations directly or via hybridization. We used sequence data from chloroplast and nuclear regions to gain insight into evolutionary origins and to confirm taxonomic status. Our phylogenetic analyses suggest a complex evolutionary history that includes hybridization events. Placement of invasive and ornamental individuals within the Cytisus-Genista complex resolves taxonomic uncertainty in these groups, as our phylogenetic analyses recovered separate "French broom" and "sweet broom" clades within the G. monspessulana clade in the genus Genista. Extensive cloning and sequencing of the ITS region revealed that, although the majority of invasive "French broom" in California is Genista monspessulana, hybridization with individuals from the ornamental "sweet broom" clade likely occurs in populations throughout the state.     

Speciation in Caucasian lizards: climatic dissimilarity of the habitats is more important than isolation time

Given sufficient time and limited gene flow, evolutionary lineages tend to transform into separate species. Mechanisms preventing assimilation during repeated gene‐flow events include divergent adaptations and the development of pre‐ or postzygotic isolation. We analysed the morphological and genetic boundaries of three species of the rock lizard clade Darevskia ‘rudis’ (Darevskia rudis, Darevskia valentini, and Darevskia portschinskii) in relation to the environment, and tried to reconstruct evolutionary pathways underlying the observed separation among the species. We studied the geographic distribution of the scalation traits, microsatellite genotypes, and mitochondrial haplotypes. Our analyses showed consistent morphological and genetic patterns at the centre of the ranges for each species, but asymmetric distribution of alleles and scalation characters within the current contact zones among the species. The genetic and morphological diversification of the clade has been shaped during glacial isolation in an area of Southern Caucasus, away from the Black Sea Coast. The ancestral lineage of D. portschinskii separated from the common D. rudis–D. valentini lineage in the middle Pleistocene, and the two latter lineages separated in relatively recent geological time. Neither of the lineages attained complete lineage sorting; moreover, isolation and migration modelling have helped to detect recombinant gene flow from D. rudis to D. portschinskii (but not to D. valentini). This is most likely linked with climatically more similar suitable habitats between D. rudis and D. portschinskii than between D. valentini and the other two species. In itself, the isolation period was insufficient for the development of intrinsic isolation mechanisms in the system studied. Thus, differential landscape‐dependent selection within the contact zones is likely to have triggered the rapid development of isolation mechanisms. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 876–892.     

Pleistocene effects on the European freshwater fish fauna: double origin of the cobitid genus Sabanejewia in the Danube basin (Osteichthyes: Cobitidae)

Biogeographical hypotheses of European freshwater fishes were inferred using phylogeographic analysis of the complete cytochrome b and ATP synthase 8 and 6 mitochondrial genes (1982bp). To test the relative importance of drainage origin versus Pleistocene glaciations in the origin of primary freshwater fishes in Europe, we reconstructed the phylogenetic relationships of the genus Sabanejewia which is distributed in European waters. The phylogenetic relationships recovered for the genus Sabanejewia (n=75) provide support for the monophyly of six main evolutionary mtDNA lineages: Sabanejewia larvata, Sabanejewia romanica, Sabanejewia aurata/Sabanejewia caucasica, Sabanejewia kubanica, Sabanejewia baltica, and the Danubian-Balkanian complex. The Caucasian-Caspian mtDNA lineages, S. kubanica, S. aurata/S. caucasica, and the Northern European S. baltica represents the sister group of the Danubian-Balkanian complex mtDNAclade, supporting a Caucasian-Northern European origin of most of mtDNA lineages of the Central European freshwater fish fauna. The mtDNA divergence observed between the Danubian Sabanejewia species is too dissimilar to support their contemporary origin. Rather, the mtDNA data suggest that the Danubian Sabanejewia lineages most likely have a double origin, indicating that the European Sabanejewia lineages have experienced different historical processes for the following reasons. First, the origin of the S. larvata and S. romanica mtDNA clades predates the origin of the Danubian-Balkanian complex, and our results showed that the completion of the Alps and the origin of the Danube drainage seem to have promoted the speciation of the earliest Sabanejewia clades in the Miocene. Second, small genetic distances and the geographical pattern found within the Danubian-Balkanian complex clade indicate that the lineages included in this clade spread recently across the Danube and Greek river drainages. The inclusion of the S. balcanica species within all mtDNA lineages suggests that cyclical cold periods during the Pleistocene glaciations have favoured its rapid expansion and genetic homogenisation across Central European and Greek waters.     

Molecular-genetic diversification of lizard complex D. raddei (Sauria: Lacertidae): early stages of speciation

A number of populations of genus Darevskia lizards were studied using inter-SINE-PCR (IS-PCR). The number and size of PCR-amplified spacers of genomic DNA flanked by SINE-type repeats were compared in 17 populations of the D. raddei species complex along with several other species from the same genus. Nei and Li's (D(NL)) genetic distances between populations were unequivocal. Individual differences among D. r. nairensis sample specimens and between the samples fall into a range of less than 0.1. Individual variability intra each of the D. r. raddei samples--into a range of 0.1-0.2, but the inter samples differences in this subspecies depends on its geographic localizations. Thus the differences between 10 samples from Armenia and Karabach fall into a range of 0.2-0.3, but between all these and two samples from Talysh (Azerbajdzhan)--of 0.3-0.4. At the same time, the variability between both known subspecies (D. r. raddei and D. r. nairensis) reaches the same values. The differences between "good" species D. raddei and D. rudis were about 0.60-0.7. It may be supposed that D. r. raddei is currently undergoing the speciation process. The population of lizards from Turkey which is considered to be close to D. raddei by some researchers and to D. rudis by others is, according to our molecular data, more closely related to the latter species. The difference of homogeneity between other populations was revealed by comparison of the DNL values. Possible phylogeographic pathways of Darevskia species distribution based on the molecular data were proposed.     

Molecular evolution of satellite DNA CLsat in lizards of the Darevskia species (Sauria: Lacertidae): correlation with species diversity

The structure and evolution of a satellite DNA family was examined in lizards from the genus Darevskia (family Lacertidae). Comparison of tandem units of repeated DNA (satDNA), CLsat, in all species from the genus Darevskia has shown that their variability is largely based on single-nucleotide substitutions, which constitute about 50 diagnostic positions underlying classification of the family into three subfamilies. Maximum differences between the subfamilies reached 25%. At this level of tandem unit divergence between the subfamilies, no cross-hybridization between them was observed (at 65 degrees C). The individual variability of one subfamily within the species was on average 5% while the variability between species consensuses within a subfamily was 10%. The presence of highly conserved regions in all monomers and some features of their organization show that satellites of all Darevskia species belong to one satDNA family. The organization of unit sequences of satellites CLsat and Agi also detected by us in another lizard genus, Lacerts s. str. was compared. Similarity that was found between these satellites suggests their relatedness and common origin. A possible pathway of evolution of these two satDNA families is proposed. The distribution and content of CLsat repeat subfamilies in all species of the genus was examined by Southern blotting hybridization. Seven species had mainly CLsatI (83 to 96%); three species, approximately equal amounts of CLsatI and CLsatIII (the admixture of CLsatII was 2-3%); and five species, a combination of all three subfamilies in highly varying proportions. Based on these results as well as on zoogeographic views on phylogeny and taxonomy of the Darevskia species, hypotheses on the evolution of molecular-genetic relationships within this genus are advanced.     

Molecular evidence for the hybrid origin of species in the soft coral genus Alcyonium (Cnidaria: Anthozoa: Octocorallia)

Several recent studies have suggested that hybridization may play a previously unrecognized and important role in the evolution of corals. Our observations of polymorphic and recombinant sequences in the multicopy ribosomal internal transcribed spacer (ITS) region suggested the possible hybrid origin of two European soft coral species, Alcyonium hibernicum and Bellonella bocagei. To examine this possibility further we cloned and sequenced ITS-1 from multiple individuals and populations of these two species as well as two sympatric congeners, A. coralloides and A. sp. M2. Phylogenetic analyses separated the observed sequence variants into two distinct clades. All A. coralloides sequences belonged to clade A, while A. sp. M2 had only clade B sequences. A majority of A. hibernicum individuals, however, contained both clade A and B sequences that were identical to the predominant sequence variants found in A. coralloides and A. sp. M2, respectively. This pattern of additivity suggests that A. hibernicum originated from a hybrid cross between A. coralloides and A. sp. M2, a hypothesis that is supported by its unusual mode of reproduction (meiotic parthenogenesis). The predominant sequence variant found in B. bocagei was a unique, derived clade B sequence; in addition, however, most individuals of this species also had copies of a sequence identified as a recombinant between clade A and clade B sequence types. The presence of this recombinant sequence in the B. bocagei genome suggests that this species may also be the product of past hybridization events within the clade. Reticulate evolution may explain the failure of several previous studies to resolve the phylogeny of these four species.     

Phylogenetic divergence in leatherside chub (Gila copei) inferred from mitochondrial cytochrome b sequences

We examined intra-specific phylogenetic relationships in leatherside chub, Gila copei. The complete mitochondrial (mt) cytochrome b gene (1140 bp) was sequenced for 30 individuals from 10 populations that span the geographical distribution of this species. Traditional phylogenetic analyses revealed two deeply divergent and evolutionarily distinct mtDNA clades that are geographically separated in northern and southern drainage basins. Interpopulation sequence variation between clades ranged from 7.7 to 8.1%. The northern clade was genetically more similar and phylogenetically more closely related to the selected out-group Lepidomeda m. mollispinus than to the southern clade, suggesting that the taxonomy of this species may require revision. Sequence variation among populations within clades ranged from 0 to 0.3% in the north and from 0 to 0.7% in the south. Statistical parsimony was used to construct phylogenetic networks of haplotypes within clades. Nested clade analysis revealed that geographical fragmentation has played an important role in genetic structuring within northern and southern clades.     

Allopolyploidy in the Wintergreen Group of tribe Gaultherieae (Ericaceae) inferred from low‐copy nuclear genes

DNA sequence data from the low‐copy nuclear genes waxy (GBSSI) and leafy were compared with plastid and ITS sequence data from prior studies to reconstruct phylogenetic relationships in the Wintergreen Group of tribe Gaultherieae (Ericaceae). We conducted phylogenetic analysis with 102 species that includes representatives of all 15 major clades previously identified within the Wintergreen Group and that together span its circum‐Pacific distribution. Results yielded two distinct homeologous copies of waxy for two of the clades, each in widely separated parts of the tree. It also yielded two copies of leafy for one of the clades; only one copy of leafy was found for the other clade, but it was placed in the same major clade as its waxy counterpart and well away from its placement in a prior plastid analysis. A combined four‐locus (waxy, leafy, ITS and plastid data) phylogenetic analysis of all available relevant data placed the copies of each of the clades in two distinct positions in the phylogeny with strong overall statistical support. In combination with evidence from morphology, reproductive biology and cytology, the results suggest that these clades arose through allopolyploid hybridization between lineages deep in the phylogeny but relatively close geographically. This finding confirms previous assumptions that hybridization has played an important role in the evolution of the Gaultherieae.     

New satellite DNA in Lacerta s. str. lizards (Sauria: Lacertidae): evolutionary pathways and phylogenetic impact

A new tandemly repeated (satellite) DNA family namely Agi160, from Lacerta agilis and Lacerta strigata (Lacerta sensu stricto (s. str.), Linnaeus 1758) have been cloned and sequenced. Agi160 is found in the above two species, as well as two other representatives of the same genus, L. viridis and L. media. DNA hybridization did not reveal it in Darevskia, Podarcis, Zootoca, Eremias, Ophisops, and Gallotia - the other genera of the family Lacertidae. The results suggest that Agi160 is a Lacerta s. str. specific family of tandem DNA repeats. However, a comparison between sequences of Agi160 and CLsat repeat units revealed 60 bp regions 62-74% identical. The latter is a satellite DNA family typical for Darevskia (syn. "L. saxicola complex") (Grechko et al., Molecular-genetic classification and phylogenetic relatedness of some species of Lacertidae lizards by taxonoprint data. Mol Biol 32:172-183, 1988.). Both Agi160 and CLsat tandem repeats share several common features (e.g., the same AT content and distribution of multiple short A-T runs, internal structure of repeated units, the presence of conservative regions). These data are indicative of their common origin and a possibly strong selective pressure upon conserving both satellites. A comparative analysis of structure, organization, and abundance of these two families of satDNA reveals evolutionary pathways that led to their formation and divergence. The data are consistent with the hypotheses of the concerted evolution of satellite DNA families. The possibility of use of Agi160 as a phylogenetic tool, defining relationships within Lacerta s. str., as well as within the whole family of Lacertidae is discussed.     

Integration and dissociation of limb elements in flying vertebrates: a comparison of pterosaurs, birds and bats

Flapping flight has evolved independently in three vertebrate clades: pterosaurs, birds and bats. Each clade has a unique flight mechanism involving different elements of the forelimb. Here, patterns of limb integration are examined using partial correlation analysis within species and matrix correlation analysis across species to test whether the evolution of flapping flight has involved developmental dissociation of the serial homologues in the fore- and hind limb in each clade. Our sample included seven species of birds, six species of bats, and three species of pterosaurs for which sufficient sample sizes were available. Our results showed that, in contrast to results previously reported for quadrupedal mammals, none of the three clades demonstrated significant integration between serial homologues in the fore- and hind limb. Unexpectedly, there were few consistent patterns of within-forelimb correlations across each clade, suggesting that wing integration is not strongly constrained by functional relationships. However, there was significant integration within the hind limbs of pterosaurs and birds, but not bats, possibly reflecting the differing functions of hind limbs (e.g. upright support vs. suspension) in these clades.     

Species status and phylogeography of two closely related Coptolabrus species (Coleoptera: Carabidae) in South Korea inferred from mitochondrial and nuclear gene sequences

We investigated the species status and intraspecific phylogeography in South Korea of two ground beetle species, Coptolabrus jankowskii and Coptolabrus smaragdinus (Coleoptera: Carabidae), using statistical parsimony networks and nested clade analyses based on sequences from the mitochondrial cytochrome oxidase subunit I (COI) and nuclear phosphoenolpyruvate carboxykinase (PepCK) and wingless (Wg) genes. Although traditional parsimony tree construction generally failed to resolve interspecific relationships and construct biologically meaningful genealogies, analysis using statistical parsimony networks yielded statistically significant inter- and intraspecific genealogical structures. We found that although these two species represent a notable case of trans-species polymorphisms in both mitochondrial and nuclear gene sequences, their status as separate species was evidenced by the nonrandom association between species and nested clades at various nesting levels. The exceptional occurrence of shared identical or very similar COI sequences was considered to be the result of introgressive hybridization. In addition, range expansion and fragmentation events across the Korean Peninsula and adjacent islands were inferred from nested clade phylogeographical analyses. The COI gene revealed the geographical divergence of major eastern and western clades and historical biogeographical events within each major clade, whereas the nuclear PepCK gene, which did not reveal corresponding east-west clades, indicated past fragmentation and range expansion across wide areas that may have been the result of older biogeographical events. Thus, phylogeographical inferences drawn from analyses of mitochondrial and nuclear genes can reveal different and potentially complementary information about phylogeographical processes.     

Cryptic diversity and unexpected evolutionary patterns in the meadow lizard,Darevskia praticola (Eversmann, 1834)

Darevskia praticola differs from the other species of the genus in having a large but disjunct distribution, covering the Balkan and the Caucasus regions. Furthermore, most Darevskia species occupy saxicolous habitats, whereas D. praticola inhabits meadows and forest environments. Here we determine the phylogeographic and phylogenetic relationships of Darevskia praticola sensu lato and evaluate the current, morphology-based taxonomy. We sequenced two mtDNA genes (Cyt-b and ND4) and two nuclear loci (MC1R and RELN) for samples collected across the species range. Because our sequences amplified with the Cyt-b primers appear to represent a nuclear pseudogene we excluded this marker from the final analysis. Our results support monophyly of D. praticola and show its division into three clades. The first divergence, dated to the Late Pliocene, is between the Balkans and the Caucasus. The Caucasus lineage is further subdivided in a western Greater Caucasus and a Transcaucasia clade, likely due to subsequent differentiation during the Pleistocene. Our findings do not support the current taxonomic arrangement within D. praticola. The main geographic divergence likely happened due to a vicariance event associated with Plio-Pleistocene climatic and vegetation oscillations.     

Phylogeny and taxonomy of endangered South and South-east Asian freshwater turtles elucidated by mtDNA sequence variation (Testudines: Geoemydidae: Batagur, Callagur, Hardella, Kachuga, Pangshura)

Using DNA sequences of the mitochondrial cytochrome b gene, we investigated phylogeny and taxonomy of South and South-east Asian turtles of all species and subspecies of the genera Batagur, Callagur, Hardella, Kachuga and Pangshura. We found three major clades: (i) a moderately to well-supported clade containing all large riverine species assigned so far to Batagur, Callagur and Kachuga ; (ii) a well-supported monophylum comprising the four Pangshura species; and (iii) Hardella that could constitute either the sister-taxon of Pangshura or of a clade comprising Batagur, Callagur, Kachuga and Pangshura. The genus Kachuga is clearly polyphyletic. Therefore, we recommend placing all Batagur, Callagur and Kachuga species in one genus. According to the International Code of Zoological Nomenclature Batagur Gray, 1856, being originally erected at higher rank, takes precedence over the simultaneously published name Kachuga Gray, 1856, and the younger name Callagur Gray, 1870, resulting in an expanded genus Batagur. Indonesian and Malaysian Batagur baska proved to be highly distinct from our sequences of this species from the Sundarbans (Bangladesh, adjacent India), suggesting that a previously unidentified species is involved. This finding is of high conservation relevance in the critically endangered B. baska. The currently recognized subspecies within Hardella thurjii , Pangshura smithii and P. tentoria do not correspond well with mtDNA clades. Considering that the two subspecies of H. thurjii are likely to be based only on individual ontogenetic differences, we propose abandoning the usage of subspecies within H. thurjii . In the Ghaghra River, Uttar Pradesh (India) we detected shared haplotypes in P. smithii and P. tentoria, implying that the unusual morphological characters of the Ghaghra River population of P. tentoria could be the result of interspecific hybridization.     

The taxonomy of the Littorina saxatilis speicies-complex, with particular reference to the systemaitc status of Littorina patula Jeffrys

Variation in shell shape and penis morphology of Littorina rudis Maton is examined using data from all parts of Britain. The shell shape variation within populations of L. rudis is shown to account for Liltorina patula Jeffrys at the only site where the latter species was recorded. In addition, the shell shape of I,. rudis varies with exposure, individuals on exposed shores having a relatively larger aperture than those on sheltered shores. Wave action and desiccation are considered the most likely factors maintaining this variation. The penis morphology of L. rudis varies within and between shores to an extent that renders the use of this character invalid for distinguishing L. patula from L. rudis. The radulae of adults of L. rudis, L. patula and Littorina nigrolineata (Gray) are similar in structure having blunt cusps, whilst adult Littorina neglecta Bean and juvenile L. rudis have pointed cusps. The possibility of a neotenous origin of L. neglecta from L. rudis is discussed. On the evidence presented here it is suggested that L. patula must be regarded as a synonym of L. rudis.     

Comparative Phyloclimatic Analysis and Evolution of Ecological Niches in the Scimitar Babblers (Aves: Timaliidae: Pomatorhinus)

We present the first extensive and integrative analysis of niche evolution based on climatic variables and a dated molecular phylogeny of a heterogeneous avian group of Southeast Asian scimitar babblers of the genus Pomatorhinus. The four main clades of scimitar babblers have species that co-occur in similar areas across southern Asia but some have diverged at different timeframes, with the most recently evolved clade harboring the highest number of species. Ecological niche models and analysis of contributing variables within a phylogenetic framework indicate instances of convergent evolution of members of different clades onto similar ecological parameter space, as well as divergent evolution of members from within clades. Pomatorhinus species from different clades occupying Himalayan foothills show convergence towards similar climatic tolerances, whereas within a clade, allopatric sister-species occurring in the Himalayas have diverged to occupy different climatic parameter spaces. Comparisons of climatic tolerances of Himalayan foothills taxa with species distributed further south in Assam/Burma and Burma/Thailand indicate convergence towards similar parameter spaces in several climatic variables. Niche overlap was observed to be lower among species of the youngest clade (ruficollis) and higher among species of older clades (ferruginosus). Analysis of accumulation of ecological disparity through time indicates rapid divergence within recent time frames. As a result, Himalayan taxa originating at different temporal scales within the four main scimitar babbler clades have differentiated ecologically only in recently diverged taxa. Our study suggests that the repeated orogenic and climatic fluctuations of the Pliocene and Pleistocene within mainland Southeast Asia served as an important ecological speciation driver within scimitar babblers, by providing opportunities for rapid geographic expansion and filling of novel environmental niches.     

EVOLUTIONARY CONSEQUENCES OF THE LOSS OF PHOTOSYNTHESIS IN CHLAMYDOMONADACEAE: PHYLOGENETIC ANALYSIS OF Rrn18 (18S rDNA) IN 13 POLYTOMA STRAINS (CHLOROPHYTA)1

Complete sequences of the Rrn 18 genes were obtained from 13 strains of the nonphotosynthetic algal genus Polytoma. Phylogenetic analyses showed that these strains formed two clades. One clade shows only modest sequence diversity but is represented by strains collected at widely dispersed sites in Europe and America. The other clade consists of a single isolate from the Canary Islands. Both clades lie well within the extended clade that includes all species of Chlamydomonas for which sequence data are available. The two Polytoma clades are separated from each other by several green species, suggesting that the extant nonphotosynthetic Chlamydomonadaceae arose from photosynthetic ancestors at least twice. These results suggest that nonphotosynthetic mutants are capable of establishing lineages that can spread widely but have a higher probability of extinction than their photosynthetic congeners.     

Geographical divergence in the Japanese land snail Euhadra herklotsi inferred from its molecular phylogeny and genital characters

We studied genetic variation within the Japanese land snail Euhadra herklotsi, which occurs on Kyushu and the surrounding islands, using partial sequences of the mitochondrial COI gene and nuclear ITS2 genes. The phylogenetic analysis revealed the existence of two major clades: clade N in the north and clade S in the south. These clades were parapatric and overlapped in southern Kyushu. Genetic divergence was high in clade N, whereas it was much lower in clade S. In addition, isolation-by-distance within each clade was implied. Since no current geographical barriers separate these clades, the genetic structure of clade S might have been influenced by historical events, such as volcanic activity, and a resulting population bottleneck followed by range expansion. The genital characteristics of clade-S snails were distinct from those of clade-N snails, and snails in both clades were sympatric at one locality. The shells of clade-N snails were generally larger than those of clade-S snails, but the shell-size variation within each clade could not be explained simply by environmental variables. Our study suggests that E. herklotsi likely consists of two sibling species. The taxonomic status of the previously proposed subspecies of E. herklotsi and related species requires reassessment.     

Phylogeography and introgressive hybridization of the ground beetle Carabus yamato in Japan based on mitochondrial gene sequences

To study the phylogeography of the ground beetle Carabus yamato in Japan, we compared 1,020-bp sequences of the mitochondrial NADH dehydrogenase subunit 5 (ND5) gene from 373 specimens from 37 localities with those of three parapatric species (C. albrechti, C. kimurai, and C. japonicus) that might share mitochondrial lineages with C. yamato through introgressive hybridization. We found 81 haplotypes from C. yamato. Of these, 17 haplotypes were considered to be of an introgressed lineage from C. albrechti, based on the phylogeny and geographic distribution. In addition, one haplotype of C. kimurai was likely an introgressant from C. yamato. Putative introgression events among the four species were restricted to these two directional cases. We analyzed the phylogeography of C. yamato using nested clade phylogeographical analysis and population genetic parameters. The mitochondrial lineages of C. yamato were estimated to have diverged no more than approximately 1.12 million years ago, implying that the estimated historical events occurred after the Early Pleistocene. Carabus yamato was inferred to have experienced a contraction of its distribution range, followed by recent range expansion. Populations in the western and eastern regions, segregated by Ise Bay and the Nobi and Okazaki Plains, diverged in the mitochondrial clades. The northern and most western populations possessed one clade only (except an introgressed lineage), whereas eastern and some southwestern populations possessed several diverged clades, which were considered to be ancestral; these populations may have been associated with refugia during glacial periods.