Genetic diversity and phylogenetic relationship among the western and the Asian honey bees based on two mitochondrial gene segments (COI and ND5)

The Asian honey bee species i.e., Apis cerana (the eastern honey bee), A. dorsata (the giant honey bee), and the western or European honey bee (A. mellifera) collected from Pakistan were studied using partial sequences from two mitochondrial genes (i) the Cytochrome c oxidase I (COI) and (ii) the mitochondrially encoded NADH dehydrogenase 5 (ND5) and then compared with other honey bees sequences (already submitted from different countries around the globe) obtained after the national center for biotechnology information (NCBI). DNA sequences were analyzed employing molecular evolutionary genetics analysis and Kimura 2-parameter model, neighbor-joining method was applied to investigate phylogenetic relationships, and DNA sequence polymorphism was applied to measure the genetic diversity within the genus Apis. The phylogenetic analyses yielded consistent results. Based on COI gene fragment in two Asian and European honey bee species from Pakistan and from other countries showed considerable genetic diversity levels and deviation among the species. While in contrast the phylogenetic analyses based on ND5 gene fragment in Asian and European honey bee species from Pakistan and other countries showed comparatively higher genetic diversity indices and variations than the COI gene. So, in the genus Apis, the mitochondrial ND5 region has shown the possibility to answer the interactions among species. A further detailed work (by linking the analysis of other genomic and mitochondrial genes) is required for good quality solution to establish the concise genetic diversity and interaction among the Apis species. The objective of this study was to explore the extent of genetic differences and phylogenetic links among the three kinds of honey bee species from Pakistan and comparing them with other bee species around the globe.     

Morphological and genetic analyses of xeniid soft coral diversity (Octocorallia; Alcyonacea)

Studies on the biodiversity and evolution of octocorals are hindered by the incomplete knowledge of their taxonomy, which is due to few reliable morphological characters. Therefore, assessment of true species diversity within abundant and ecologically important families such as Xeniidae is difficult. Mitochondrial genes provide a reliable solution to this problem for a wide range of taxa. However, low mutation rates of the mitochondrial DNA in octocorals result in insufficient variability for species discrimination. We compared the variation of a fragment of the Signal Recognition Particle 54 gene (SRP54, proposed for octocorals) and the mitochondrial ND6/ND3 marker among members of the xeniid genera Ovabunda, Xenia, Heteroxenia and Bayerxenia. The mean uncorrected pairwise sequence divergence was 39 % for SRP54 compared to 2 % for ND6/ND3. Morphological assignments were not always supported by genetics: Species diversity was underestimated (one case) or overestimated, probably reflecting intraspecific polymorphisms or hinting at recent speciations. ND6/ND3 is informative for some species-level assignments, whereas SRP54 shows the variability needed for species delimitations within this understudied taxon. Our results on both genes show their potential for evolutionary and biodiversity studies in Xeniidae.     

Sibling species in the Chrysis ignita complex: molecular,morphological and trophic differentiation of Baltic species,with a description of two new cryptic species (Hymenoptera: Chrysididae)

Cryptic species complexes cause major challenges for taxonomists and alter understanding of species diversity. In Northern Europe, the Chrysis ignita species group is one such complex with numerous sympatric sibling species. The objective of this paper is to assess the taxonomy of 15 species from this group using three different approaches: molecular, morphological and trophic differentiation. The analysed set of molecular markers included a 7400‐bp‐long sequence of the mitochondrial genome covering complete sequences of CO1, CO2, ATP8, ATP6, CO3, ND3, 16S and 12S rRNA, nine tRNAs and a partial sequence of CytB, as well as a 3880‐bp‐long sequence of the nuclear DNA covering a part of 18S rRNA, the ITS1, 5.8S rRNA, ITS2 and a part of 28S rRNA. Discrete diagnostic characters of each species sequence were retrieved using the Characteristic Attribute Organisation System algorithm and a molecular identification key was compiled. The study revealed a higher evolutionary rate of the genes ATP8, ATP6, CO3, ND3 and CytB compared to that of CO1, CO2 and 16S; the studied nuclear markers demonstrated a lower evolutionary rate than the mitochondrial markers. A consensus tree compiled based on the combined mtDNA and nuclear markers with a strongly supported topology resolved the position of the C. schencki – C. parietis sp.n. clade as sister to the C. ignita – C. impressa clade and supported the monophyly of the C. angustula – C. longula clade. We compiled a morphometric species identification key applying linear discriminant equations. The trophic differentiation was assessed using data on host preferences of ten Chrysis species reared from trap‐nests; the analysis demonstrated that most of them are specialists exploiting a single or a few taxonomically related host species. In most cases, all three approaches supported the distinct status of the included species. Moreover, two previously undescribed species were consistently supported by the molecular methods. Therefore, we describe these as new, namely C. horridula sp.n. and C. parietis sp.n. Only C. mediata and C. solida were not clearly distinguished using the molecular phylogeny reconstruction methods. However, based on distinctive niche divergence, the presence of molecular characters and morphometric differences, we consider them as phylogenetically young but distinct species. In view of the weak morphological and molecular differentiation, the widely overlapping distribution areas and often similar habitat preferences and the trophic specialization, the C. ignita complex presents a possible model for studies of sympatric cryptic speciation. This published work has been registered in ZooBank, http://zoobank.org/urn:lsid:zoobank.org:pub:1EBAF0E1‐5FB7‐4CF4‐A595‐C11982448360 .     

Uncovering cryptic diversity in Aspidomorphus (Serpentes: Elapidae): Evidence from mitochondrial and nuclear markers

The Papuan region, comprising New Guinea and nearby islands, has a complex geological history that has fostered high levels of biodiversity and endemism. Unfortunately, much of this diversity remains undocumented. We examine the evolutionary relationships of the venomous snake genus Aspidomorphus (Elapidae: Hydrophiinae), a Papuan endemic, and document extensive cryptic lineage diversification. Between Aspidomorphus species we find 22.2–27.9% corrected cyt-b sequence divergence. Within species we find 17.7–23.7% maximum sequence divergence. These high levels of genetic divergence may have complicated previous phylogenetic studies, which have had difficulty placing Aspidomorphus within the subfamily Hydrophiinae. Compared to previous studies, we increase sampling within Hydrophiinae to include all currently recognized species of Aspidomorphus and increase species representation for the genera Demansia and Toxicocalamus. We confirm monophyly of Aspidomorphus and resolve placement of the genus utilizing a set of seven molecular markers (12S, 16S, cyt-b, ND4, c-mos, MyHC-2, and RAG-1); we find strong support for a sister-group relationship between Aspidomorphus and a Demansia/Toxicocalamus preussi clade. We also use one mitochondrial (cyt-b) and one nuclear marker (SPTBN1) to document deep genetic divergence within all currently recognized species of Aspidomorphus and discuss the Solomon Island Arc as a potential center of divergence in this species. Lastly, we find high levels of concordance between the mtDNA and nuDNA markers used for inter-species phylogenetic reconstruction.     

Comparative Analysis of Mitochondrial DNA Diversity in Smelts

The polymerase chain reaction (PCR) was used to amplify a segment of the mitochondrial DNA coding for NADH-dehydrogenase subunits ND5/ND6 in five smelt species (family Osmeridae). Amplified DNA was screened for restriction fragment length polymorphism (RFLP). Nucleotide sequence divergence of mitochondrial DNA between species ranges from 11.9 (between Hypomesus nipponensis and H. japonicus) to 24.7% (between Osmerus mordax dentex and Mallotus villosus catervarius). The genetic divergence between populations of H. nipponensis, H. japonicus, and Osmerus mordax dentex was 0.32, 0.08 to 0.15, and 0.025%, respectively. The absence of common haplotypes enables differentiation of closely related smelt species and, therefore, can be used for solving current problems in the taxonomy and biogeography of this family.     

Parasitism rates and parasitoid complexes of the wheat midges, <Emphasis Type="Italic">Sitodiplosis mosellana</Emphasis>, <Emphasis Type="Italic">Contarinia tritici</Emphasis> and <Emphasis Type="Italic">Haplodiplosis marginata</Emphasis>

Three species of cecidomyiid midges (Diptera: Cecidomyiidae), whose larvae overwinter in the soil, can cause significant yield losses on wheat in Europe: the orange wheat blossom midge, Sitodiplosis mosellana (Géhin), the yellow wheat blossom midge, Contarinia tritici (Kirby), and the saddle gall midge, Haplodiplosis marginata (von Roser). The biological control of wheat midges by their parasitoids can contribute to reduce the midge populations. Soil samples were collected in several fields in Belgium in 2012–2014 in order to characterize the parasitism rates and parasitoid complexes in overwintering larvae. The parasitism rates varied greatly between the sampled fields: 3–100, 0–100 and 2% for S. mosellana, H. marginata and C. tritici, respectively. The parasitism rate was not related to the larval density of wheat midge. The three wheat midges have totally distinct parasitoid complexes in Belgium. Eight species (Hymenoptera: Pteromalidae and Platygastridae) were found as parasitoid of S. mosellana: Macroglenes penetrans (Kirby), Amblypasis tritici (Walker), Euxestonotus error (Fitch), Euxestonutus sp. Fouts, Leptacis sp. Foerster, Platygaster gracilipes (Huggert), Platygaster nisus Walker, and Platygaster tuberosula (Kieffer). According to their abundance, M. penetrans, E. error and P. tuberosula appeared as the main parasitoids of S. mosellana in Belgium. For the two other wheat midges, only one species of the family Platygastridae was found for each midge: Platygaster equestris (Spittler) for H. marginata and Synopeas myles (Walker) for C. tritici.     

Phylogeographic evidence links the threatened ‘Grampians’ Mountain Dragon (Rankinia diemensis Grampians) with Tasmanian populations: conservation implications in south-eastern Australia

The importance of protecting genetic diversity within a species is increasingly being recognised by conservation management authorities. However, discrepancies in conservation policy between authorities, such as state versus national bodies, can have significant implications for species management when they cross state boundaries. We conducted a phylogeographic study of the south-eastern Australian lizard Rankinia diemensis to identify evolutionary significant units (ESUs), including the endangered population from the Grampians National Park in western Victoria. Phylogenetic analyses of two gene regions (mtDNA: ND2; nuclear: RAG1) revealed high levels of genetic divergence between populations, indicating isolation over long evolutionary time frames. Based on criteria of genetic divergence and isolation, R. diemensis contains at least two ESUs that require specific management. We found that R. diemensis from the Grampians are closely related to Tasmanian populations, but that the divergence between these regions is great enough (3.7 % mtDNA) that they should be considered separate ESUs. However, we believe the close evolutionary ties between these two regions needs to be taken into account; yet under current practises, conservation management of subspecific ESUs relies on state-level efforts. We argue that another population that occurs on the Victorian coast also qualifies as an ESU and requires targeted conservation action. Rankinia diemensis provides a case-in-point of the discrepancy between the state-level approach of maintaining genetic variation within a species and the more conservative Commonwealth focus on conserving biodiversity at the species level.     

Genetic Differentiation of Three Sympatric Charr Species from the Genus SalvelinusInferred from PCR–RFLP Analysis of Mitochondrial DNA

We examined differentiation of three sympatric charr species Dolly Varden charr Salvelinus malmaWalbaum, white-spotted charrS.leucomaenisPallas, and Levanidov charrS. levanidovi Chereshnev, Scopetz, Gudkov. Using restriction fragment length polymorphism analysis (RFLP), three mitochondrial DNA fragments (ND1/ND2, ND5/ND6, and Cytb/D-loop) amplified in PCR were compared. The divergence of mtDNA nucleotide sequences betweenS. levanidovi and S. leucomaenis was about 9.7%, between S. levanidovi and S. malma, 8.7%, and between S. malma andS. laecomaenis, 7.5%. These results indicate genetic isolation of the species examined and support the earlier suggestion on closeness of Levanidov charr to the common ancestor of the genus Salvelinus.     

Examining the genetic diversity of the orange blossom wheat midge,Sitodiplosis mosellana (Géhin), across North America

The orange blossom wheat midge, Sitodiplosis mosellana (Géhin) (Diptera: Cecidomyiidae), is a significant pest of wheat (Triticum spp.) grown in the Northern Hemisphere. It was accidently introduced to North America over 200 years ago and has subsequently spread throughout the northern Great Plains. Since 2010, several Canadian spring wheat varieties containing the resistance gene Sm1 have been released. Due to the potential of wheat midge populations to evolve virulent biotypes to Sm1, cultivars containing Sm1 are grown with a susceptible cultivar in an interspersed refuge. An understanding of the genetic diversity of wheat midge populations could provide important information on the potential development of resistance to Sm1. In the current study, we used two mitochondrial genes (CO1 and ND4) from wheat midge collected across the northern Great Plains and Québec in North America to assess population structure and genetic diversity. We found limited genetic diversity and population structure across the sampled North American populations. We also assessed North American haplotype similarity to wheat midge collected from Europe and China and found high similarity between North American and European populations, although sampling in Europe was limited. This supports the hypothesis that North American populations originated from Europe.     

Differentiation of the Dolly Varden Char (Salvelinus malma) and the Taranetz Char (Salvelinus taranetzi) Inferred from the PCR–RFLP Analysis of Mitochondrial DNA

Genetic differentiation of two sympatric char species, the Dolly Varden char (Salvelinus malma Walbaum) and the Taranetz char (S. taranetzi Kaganovski), has been studied. Restriction fragment length polymorphism (RFLP) analysis has been used to compare three mitochondrial DNA (mtDNA) fragments (ND1/ND2, ND5/ND6, and Cytb/D-loop) amplified via polymerase chain reaction (PCR). The divergence between S. malma and S. taranetzi inferred from mtDNA nucleotide sequences is 2.8%; between S. leucomaenis and S. taranetzi, 7.1%; and between S. malma and S. leucomaenis, 7.5%. The absence of common haplotypes and the degree of divergence indicate that the Dolly Varden char and the Taranetz char are genetically isolated and confirm that S. taranetzimay be regarded as a separate species.     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Molecular signature of the D-loop in the brown pencilfish Nannostomus eques (Characiformes, Lebiasinidae) reveals at least two evolutionary units in the Rio Negro basin, Brazil

The genetic variability of the brown pencilfish Nannostomus eques was studied, based on an analysis of sequences from the control region (1084 bp) of mitochondrial (mt)DNA in 125 individuals collected from eight tributaries along the upper (Açaituba, Miuá, Jaradi and Arixanã), middle (Demini), and lower (Jacundá, Maguari and Catalão) Rio Negro (Brazil). Phylogenetic inferences using mtDNA data from N. eques revealed two evolutionary units. Genetic distance between them ranged from 5·5 to 8·3% and differed by 8·5–11·8% from the sister species pencilfish Nannostomus unifasciatus. The time of divergence between the two evolutionary units was estimated to be the Middle Pliocene (c. 2·99 million years before present). Population genetic analysis (DNA polymorphism, AMOVA and Mantel test) showed high haplotype diversity (HD, >0·90) in each evolutionary unit, a strong population genetic structure in the Demini River that formed a monophyletic group and a correlation between genetic divergence and geographical distance in only one of these units (evolutionary unit 1). On the basis of molecular data, the rapids and waterfalls near São Gabriel da Cachoeira (Upper Rio Negro) were the main barriers to gene flow within evolutionary unit 1 in some localities. The emergences of the Branco River and the Anavilhanas Archipelago were apparently responsible for the discrepancy in distribution of the two evolutionary units, except at Jacundá, where the evolutionary units were sympatric. In view of the differences between the evolutionary units, N. eques cannot be treated as a single stock in the Rio Negro basin. These results may have important implications for the fishery management of this ornamental fish.     

The Role of Integrative Taxonomy in the Conservation Management of Cryptic Species: The Taxonomic Status of Endangered Earless Dragons (Agamidae: Tympanocryptis) in the Grasslands of Queensland,Australia

Molecular phylogenetics is increasingly highlighting the prevalence of cryptic species, where morphologically similar organisms have long independent evolutionary histories. When such cryptic species are known to be declining in numbers and are at risk of extinction due to a range of threatening processes, the disjunction between molecular systematics research and conservation policy becomes a significant problem. We investigate the taxonomic status of Tympanocryptis populations in Queensland, which have previously been assigned to T. tetraporophora, using three species delimitation approaches. The taxonomic uncertainties in this species-group are of particular importance in the Darling Downs Earless Dragon (T. cf. tetraporophora), which is ranked as an endangered ‘species’ of high priority for conservation by the Queensland Department of Environment and Heritage Protection. We undertook a morphological study, integrated with a comprehensive genetic study and species delimitation analyses, to investigate the species status of populations in the region. Phylogenetic analyses of two gene regions (mtDNA: ND2; nuclear: RAG1) revealed high levels of genetic divergence between populations, indicating isolation over long evolutionary time frames, and strongly supporting two independent evolutionary lineages in southeastern Queensland, from the Darling Downs, and a third in the Gulf Region of northern Queensland. Of the three species delimitation protocols used, we found integrative taxonomy the most applicable to this cryptic species complex. Our study demonstrates the utility of integrative taxonomy as a species delimitation approach in cryptic complexes of species with conservation significance, where limited numbers of specimens are available.     

Genetic divergence in the tribe Electronini (Myctophidae)

To reveal evolutionary relationships and the rate of divergence of lanternfishes in the tribe Electronini (Myctophidae), analysis of sequence of cox 1 mtDNA fragment in three genera (12 species) of the tribe and in a closely related genus Myctophum (9 species) was performed. The results support the tribe monophyly; however, the divergence within it appeared to be not profound: genetic distances between genera and subgenera are smaller than between species within Myctophum and other genera of myctophids. In subgenera of Protomyctophum, species formation has obviously not completed since interspecies distances in them (0.5–4.7%) are comparable to estimates of intraspecific variation in other lanternfishes. Minimal genetic differences were found between species P. (Hierops) arcticum and P. (H.) subparallelum, whose ranges at the present time are isolated in the northern and southern hemispheres. After calibration of the molecular clock, the time of isolation of these two species can be dated to the Middle Pleistocene. Weak tribe diversification indicates its evolutionary youth, which can be related to the absence of efficient hydrographic barriers for isolation. The data obtained do not permit to consider tribe members as the most primitive in the family Myctophidae.     

Divergence history of the Carpathian and smooth newts modelled in space and time

Information about demographic history is essential for the understanding of the processes of divergence and speciation. Patterns of genetic variation within and between closely related species provide insights into the history of their interactions. Here, we investigated historical demography and genetic exchange between the Carpathian (Lissotriton montandoni, Lm) and smooth (L. vulgaris, Lv) newts. We combine an extensive geographical sampling and multilocus nuclear sequence data with the approximate Bayesian computation framework to test alternative scenarios of divergence and reconstruct the temporal and spatial pattern of gene flow between species. A model of recent (last glacial period) interspecific gene flow was favoured over alternative models. Thus, despite the relatively old divergence (4–6 mya) and presumably long periods of isolation, the species have retained the ability to exchange genes. Nevertheless, the low migration rates (ca. 10^?6 per gene copy per generation) are consistent with strong reproductive isolation between the species. Models allowing demographic changes were favoured, suggesting that the effective population sizes of both species at least doubled as divergence reaching the current ca. 0.2 million in Lm and 1 million in Lv. We found asymmetry in rates of interspecific gene flow between Lm and one evolutionary lineage of Lv. We suggest that intraspecific polymorphism for hybrid incompatibilities segregating within Lv could explain this pattern and propose further tests to distinguish between alternative explanations. Our study highlights the importance of incorporating intraspecific genetic structure into the models investigating the history of divergence.     

Long-Distance Wind-Borne Dispersal of Sitodiplosis mosellana Géhin (Diptera:Cecidomyiidae) in Northern China

Surveys based on the density of the adult wheat midge, Sitodiplosis mosellana (Géhin) (Diptera: Cecidomyiidae), were conducted at ground level and at heights between 10 and 75 m in Xushui county (39.02 °N, 115.65 °E), Hebei province, China, in 2011. The HYSPLIT-4 model was used to analyze the possible dispersion trajectories and distances of S. mosellana depending on the experimental location. The wheat midges were trapped at altitudes from 10 to 75 m a.g.l. strongly suggest that they might also be present at much higher altitudes, where potentially, they could take advantage of the fast moving air currents. 6-h backward-trajectories indicate that adult S. mosellana could have come from both north and south, and the trajectory distances were 25–107 Km. 6-h forward-trajectories indicate that adults most likely migrated to an area located in Tianjin, Beijing and northern Hebei province, north china, and the trajectory distances were 28–195 Km. In this study, no male midges were captured at 10 m or above, which suggest male midges don’t engage in wind borne long distance migration, suggesting S. mosellana mated before dispersal. The information presented here is helpful to alter the way we view their migration over limited distances and add to our ability in outbreak forecasts.     

Population genetic structure of northern Dolly Varden char <Emphasis Type="Italic">Salvelinus malma malma</Emphasis> in Asia and North America

The level of genetic differentiation of northern Dolly Varden char Salvelinus malma malma from Asia and North America was evaluated using the data on mtDNA variation (regions ND1/ND2, ND5/ND6, and Cytb/D-loop) obtained by means of PCR-RFLP analysis. For S. m. malma, the mean values of haplo-type and nucleotide diversity were 0.5261 ± 0.00388 and 0.001558, respectively. The mean estimate of the population nucleotide divergence constituted 0.055%. It was demonstrated that S. m. malma on the most part of the species range examined (drainages of the Beaufort Sea, Chukotka Sea, Bering Sea, and the Sea of Okhotsk) was characterized by the population genetic structure with the low level of genetic differentiation and divergence. At the same time, populations from the Pacific Ocean Gulf of Alaska demonstrated marked genetic differentiation, supported by the high pairwise G4ST values (from 0.4198 to 0.5211) and nucleotide divergence estimates (mean divergence, 0.129%), from Asian and North American populations. Analysis of molecular variance (AMOVA) showed that most of the mtDNA variation in S. m. malma fell in the intrapopulation component (72.5%). At the same time, the differences between the populations (21.1%) and between the regions (6.4%) made lower contribution to the total variation.     

Intraspecific mitochondrial genome comparison identified CYTB as a high-resolution population marker in a new pest Athetis lepigone

A new outbreak pest, Athetis lepigone (Möschler) (Lepidoptera: Noctuidae), has caused severe economic loss in maize crops in China. In order to conduct population genetics study with a more polymorphic and scientific mitochondrial marker, we sequenced the complete mitochondrial genomes of 13 different A. lepigone individuals. Intraspecific comparison of all PCGs showed that the NADH dehydrogenase and cytochrome b genes had the highest nucleotide diversity. We also found evidence of episodic positive selection on two amino acids, which are encoded by NADH dehydrogenase genes (ND3 and ND4L), against a background of widespread neutral selection of all other mitochondrial PCGs. The genetic divergence observed in this study indicated that the cytochrome b gene (CYTB) is better than COI at recovering population structure. The preliminary population genetic analysis illustrated strong gene flow among A. lepigone populations in China. Our study provides basic information for further research on population genetics of A. lepigone.     

Do Cryptic Species Exist in Hoplobatrachus rugulosus? An Examination Using Four Nuclear Genes,the Cyt b Gene and the Complete MT Genome

he Chinese tiger frog Hoplobatrachus rugulosus is widely distributed in southern China, Malaysia, Myanmar, Thailand, and Vietnam. It is listed in Appendix II of CITES as the only Class II nationally-protected frog in China. The bred tiger frog known as the Thailand tiger frog, is also identified as H. rugulosus. Our analysis of the Cyt b gene showed high genetic divergence (13.8%) between wild and bred samples of tiger frog. Unexpected genetic divergence of the complete mt genome (14.0%) was also observed between wild and bred samples of tiger frog. Yet, the nuclear genes (NCX1, Rag1, Rhod, Tyr) showed little divergence between them. Despite this and their very similar morphology, the features of the mitochondrial genome including genetic divergence of other genes, different three-dimensional structures of ND5 proteins, and gene rearrangements indicate that H. rugulosus may be a cryptic species complex. Using Bayesian inference, maximum likelihood, and maximum parsimony analyses, Hoplobatrachus was resolved as a sister clade to Euphlyctis, and H. rugulosus (BT) as a sister clade to H. rugulosus (WT). We suggest that we should prevent Thailand tiger frogs (bred type) from escaping into wild environments lest they produce hybrids with Chinese tiger frogs (wild type).     

Genetic divergence of sympatric charrs of the genus <Emphasis Type="Italic">Salvelinus</Emphasis> from Nachikinskoe Lake (Kamchatka)

We studied genetic differentiation of two charr species, Dolly Varden Salvelinus malma malma Walbaum and resident lacustrine charr Salvelinus sp., which sympatrically inhabit Nachikinskoe Lake (the Bol’shaya River basin) in southwestern Kamchatka Peninsula. Using restriction analysis (RFLP), three mitochondrial DNA fragments (ND1/ND2, ND5/ND6, and Cyt b/D-loop) amplified in polymerase chain reaction (PCR) were compared. The divergence of the mtDNA sequences between Salvelinus sp. and S. malma malma was 2.8%; Salvelinus sp. and S. taranetzi, 0.36%; Salvelinus sp. and S. krogiusae, 0.21%; Salvelinus sp. and S. alpinus, 3.0%. These results point to reproductive isolation of charrs in Nachikinskoe Lake and support the earlier suggestion on a close relationship between Salvelinus sp., S. taranetzi, and S. krogiusae.