Systematics of the freshwater leech genus Hirudinaria Whitman, 1886 (Arhynchobdellida,Hirudinidae) from northeastern Thailand

In total, 435 specimens of the Southeast Asian freshwater leech species within the Hirudinidae family were collected from 17 locations of various types of aquatic habitats in northeastern Thailand. They were all morphologically placed within the genus Hirudinaria Whitman, 1886 and there were three distinct species: the common Hirudinaria manillensis, 78.2% of all collected specimens and at all 17 locations, Hirudinaria javanica at 20.3% of collected samples and from five locations and a rarer unidentified morphospecies (Hirudinaria sp.) with six samples from only two locations. The karyotypes of these three species were examined across their range in this study area for 38, 11 and 6 adult specimens of Hirudinaria manillensis, Hirudinaria javanica and Hirudinaria sp., respectively. This revealed different chromosome numbers among all three species, with Hirudinaria javanica having n = 13, 2n = 26, Hirudinaria manillensis lacked one small chromosome pair with n = 12, 2n = 24, and the unknown Hirudinaria sp. differed from any known Hirudinaria karyotypes in exhibiting a higher chromosome number (n = 14, 2n = 28) and a gradual change in size from large to small chromosomes. This suggests that the unknown Hirudinaria sp. is a new biological species. However, phylogenetic analysis based upon a 658 bp fragment of the cytochrome oxidase subunit I gene placed this unknown morphospecies within the Hirudinaria manillensis clade, perhaps then suggesting a recent sympatric speciation, although this requires further confirmation. Regardless, the chromosomes of all three species were asymmetric, most with telocentric elements. A distinct bi-armed chromosome marker was present on the first chromosome pair in Hirudinaria javanica, whilst it was on pairs 1, 2, 3 and 5 in Hirudinaria manillensis, and on pairs 3 and 5 for the unknown Hirudinaria sp.     

Genetic study reveals close link between Irish and Northern Spanish specimens of the protected Lusitanian slug Geomalacus maculosus

The slug Geomalacus maculosus is a prominent member of the Lusitanian fauna. As its global distribution is restricted to western Ireland and northern Iberia, it is protected under EU legislation. Nothing is known about the genetic variability and population structure of this species, so, with a special view to shedding light on the origin of the Irish G. maculosus, tissue samples from 78 specimens were collected from 13 locations within Ireland and ten locations within Iberia and partial sequences of the mitochondrial 16S rRNA and cytochrome oxidase subunit 1 (COI) and from the nuclear internal transcribed spacer 1 region (ITS‐1) were compared. The genetic diversity of the Irish G. maculosus was found to be greatly reduced compared with the Iberian populations, with only one (16S rRNA) and two (COI) mitochondrial haplotypes identified respectively. No private Irish ITS‐1 haplotype was found. Based on the COI sequences, the Irish specimens clustered closest to Spanish specimens from Northern Asturias and Cantabria, and the bGMYC analysis identified five further Iberian clades that were highly genetically differentiated suggesting long‐term allopatric divergence.     

DNA barcoding reveals cryptic diversity in the peanut worm Sipunculus nudus

Peanut worm (Sipunculus nudus) is a cosmopolitan species mainly distributed in tropical and subtropical coastal waters. Analysis of the mitochondrial cytochrome c oxidase subunit I (COI) gene sequences among S. nudus from GenBank revealed high genetic variation (p‐distance, 0.115–0.235; k2p, 0.128–0.297) and paraphyletic relationships. These indicated misidentification and/or cryptic diversity may be present in the genus Sipunculus. To understand the genetic diversity and to manage the recourse of S. nudus, we collected specimens from coastal waters of southern China and Taiwan. In the phylogenetic topology, specimens can be separated into four distinct clades; three of these clades (clade A, B and C) were only represented from this region (southern China and Taiwan), but the clade D grouped with individuals from Central America (Atlantic coast). Furthermore, individuals of clades A and D were collected at the same location, which does not support the hypothesis that this genetic break reflects contemporary geographical isolation. The four distinct clades observed among coastal waters of southern China and Taiwan indicated underestimated diversity. It is noteworthy that the cryptic diversity is vulnerable under high pressure of human activity.     

Molecular identification of a cryptic species in the Amazonian predatory catfish genus Pseudoplatystoma (Bleeker, 1962) from Peru

Pseudoplatystoma species are highly prized South American Pimelodid migratory catfishes. Until recently, their taxonomy was not clearly established, with discrepancies between morphological and molecular analyses. Here, Pseudoplatystoma species from the Peruvian Amazon were characterized at the molecular level from a sample representing the observed range of their color pattern variations in the study area. Analyses were performed using seven microsatellite loci for 103 specimens and, for part of them (52), using sequences of two regions of their mitochondrial genome [Cytochrome Oxidase subunit I (COI) and Control Region (CR)]. Factorial correspondence analysis and assignment tests based on microsatellite polymorphism showed that the specimens originally identified as P. punctifer belonged to two different gene pools highly differentiated from P. tigrinum. Morphological examination identified two different morphotypes (with and without black stripes), suggesting the existence of two distinct taxa within P. punctifer. This result was corroborated by the ML tree based on CR sequences, where all individuals but four clustered in a similar way as in the FCA and Bayesian assignment tests. For these four individuals, mitochondrial introgression or retention of ancestral polymorphism was likely. In contrast, the ML tree based on COI sequences showed that reciprocal monophyly was not yet achieved for this marker for the two P. punctifer taxa. The existence of three sympatric species of Pseudoplatystoma in the Peruvian Amazon is discussed in relation to their molecular characteristics, color patterns and ecology. Evolutionary scenarios regarding their divergence are hypothesized.     

DNA barcode sheds light on systematics and evolution of neotropical freshwater trahiras

We assessed the presence of independent evolving lineages of the trahira, Hoplias malabaricus, one of the few freshwater fish species having wide distribution in the Neotropics which is the region with the highest global diversity of freshwater fish. To achieve that goal, 58 mitochondrial sequences of cytochrome c oxidase subunit I (COI; DNA barcoding) were generated from collected samples and 85 obtained from public databases, which were analyzed in comparison to chromosomal and geological data. The magnitude of genetic diversity found among different sampling sites was greater than 2%. Molecular species delimitation methods indicated the existence of a least four distinct lineages. The recognised cytotypes did not form monophyletic groups, suggesting that the karyotypic macrostructure could be a homoplastic character. The haplotype relationships suggested secondary contacts between the ecoregions of Northern and Northeastern Brazil that were shaped by coastal routes between adjacent watersheds during the Pleistocene epoch and probable exchanges of their ichthyofaunas. Our results indicated that multiple factors have driven the diversification of H. malabaricus, from ancient geological events linked to the reactivation of tectonic faults to more recent occurrences related to eustatic changes in ocean levels. Ultimately, the magnitude of its genetic diversity suggests the necessity of revising its taxonomic status.     

Cytochrome c oxydase I phylogenetic analysis of Haemogregarina parasites (Apicomplexa,Coccidia, Eucoccidiorida,Haemogregarinidae) confirms the presence of three distinct species within the freshwater turtles of Tunisia

Species of Haemogregarina are apicomplexan blood parasites that use vertebrates as intermediate hosts. Due to limited interspecific morphological characters within the genus during the last decade, 18S rRNA gene sequences were widely used for species identification. As coinfection patterns were recently reported from nuclear molecular data for two sympatric freshwater turtles Mauremys leprosa and Emys orbicularis from Tunisia, our objectives were to design COI specific primers to confirm the presence of three distinct species in both host species. Blood samples were collected from 22 turtles, from which DNAs were extracted and used as templates for amplification. Following different rounds of PCR and nested PCR, we designed specific Haemogregarina COI primers that allowed the sequencing of nine distinct haplotypes. Phylogenetic Bayesian analysis revealed the occurrence of three well-differentiated sublineages that clustered together into a single clade. Based on pairwise genetic distances (p-distance), we confirmed the occurrence of three distinct but phylogenetically closely related species coinfecting M. leprosa and E. orbicularis in the same aquatic environments. Our results demonstrate that the use of fast evolving genes within Haemogregarina will help to investigate the parasite diversity within both intermediate vertebrate and definitive invertebrate hosts, and to assess the evolution, historical biogeography and specificity of haemogregarines.     

Genetic Diversity and Evolution of Chinese Traditional Medicinal Fungus Polyporus umbellatus (Polyporales,Basidiomycota)

Background

Polyporus umbellatus is an important medicinal fungus distributed throughout most area of China. Its wide distribution may have resulted in substantial intraspecific genetic diversity for the fungus, potentially creating variation in its medical value. To date, we know little about the intraspecific genetic diversity of P. umbellatus.

Methodology/Principal Findings

The objective of this research was to assess genetic differences of P. umbellatus from geographically diverse regions of China based on nrDNA ITS and 28S rRNA (LSU, large subunit) sequences. Significant sequence variations in the ITS and LSU sequences were detected. All sclerotial samples were clustered into four clades based on phylogenetic analysis of ITS, LSU and a combined data set of both regions. Heterogeneity of ITS and LSU sequences was detected in 5 and 7 samples respectively. All clone sequences clustered into the same clade except for one LSU clone sequences (from Henan province) which clustered into two clades (Clade I and Clade II). Significant genetic divergence in P. umbellatus was observed and the genetic diversification was greater among sclerotial samples from Shaanxi, Henan and Gansu provinces than among other provinces. Polymorphism of ITS and LSU sequences indicated that in China, P. umbellatus may spread from a center (Shaanxi, Henan and Gansu province) to other regions.

Conclusions/Significance

We found sclerotial samples of P. umbellatus contained levels of intraspecific genetic diversity. These findings suggested that P. umbellatus populations in Shaanxi, Henan and Gansu are important resources of genetic diversity and should be conserved accordingly.     

Genetic and morphological divergence within the Sebastes pachycephalus complex (Scorpaeniformes: Scorpaenidae)

Genetic and morphological divergence among the four subspecies in the Sebastes pachycephalus complex (S. pachycephalus pachycephalus, S. p. nigricans, S. p. nudus and S. p. chalcogrammus) was clarified. Principal coordinate analysis (PCoA) based on AFLP clearly divided 55 specimens of the complex into two groups, the S. p. pachycephalus?CS. p. nigricans group (P-Ni group) and the S. p. nudus?CS. p. chalcogrammus group (Nu-C group), although three specimens occupied intermediate positions. The minimum spanning network (MSN) based on partial sequences of the mitochondrial control region (mtCR) failed to separate either the P-Ni and Nu-C groups or the four subspecies into distinct clades, although restricted gene flow and genetic differentiation between the former were indicated by the F _ST estimation. Differences in morphological characters, including counts of pectoral fin rays and counts of dorsal fin spines lacking basal scales, were also evident between the two groups. However, little or no genetic or morphological difference was found between the two subspecies within each group. It was concluded that the P-Ni and Nu-C groups of the S. pachycephalus complex actually represent two different species, which is further supported by their sympatric distribution. Differences in dorsal body coloration and the presence or absence of brown spots on the ventral surface, which were formerly used to discriminate between four ??subspecies,?? may simply represent intraspecific variation. The three specimens occupying intermediate positions in the AFLP PCoA also occupied equivocal positions between the two species in the principal component analysis (PCA) based on morphometric characters, suggesting that they were hybrids between the two species. The star-shaped MSN of mtCR, which lacks distinct clades representing the two species, may be due to not only interspecific hybridization but also the sharing of ancestral haplotypes.     

Reassessment of species distribution and occurrence of mud crab (Scylla spp., Portunidae) in Malaysia through morphological and molecular identification

This study utilized genetic and morphometric approaches to assess the molecular and morphometric differentiation among commercially important species of mud crab. Molecular investigations were derived from 542 bp mitochondrial DNA COI on 249 individuals within genus Scylla from nine states in Malaysia represents four marine regions; South China Sea, Sulu Sea, Straits of Singapore and Straits of Malacca. Four specimens were obtained from Indonesia to give a robust analysis in this study. For species delimitation, Automatic Barcode Gap Discovery (ABGD) method on a web interface was employed. Analysis on phylogenetics was implemented utilizing Neighbour joining (NJ) and Maximum Parsimony (MP) methods. The inter- and intraspecies genetic distances (D_s) was computed using Kimura 2-parameter distance and executed in MEGA version 5.05. All samples were genetically and morphologically identified and clustered into four distinct species. Among the species, S. olivacea was the most abundant (n = 111), on the other hand the occurrence of S. paramamosain in Malaysia was very low (n = 29). No single individual of S. serrata from Malaysia was recorded in this study. Both genetic distance and phylogenetic approaches exhibited a correlative monophyletic association among all specimens analysed. This present study is crucial as it reports the reassessment of all species within genus Scylla in Malaysia, eventually could be employed as a reference source for subsequent research mainly on mariculture and other conservation efforts for the species.     

A possible circulation of a dominant Dibothriocephalus nihonkaiensis haplotype in Japan revealed by molecular analysis of clinical tapeworm samples

Human diphyllobothriasis, caused by Dibothriocephalus nihonkaiensis, is prevalent globally, especially in regions where raw fish is consumed. Recent molecular diagnostic techniques have made species identification of tapeworm parasites and the determination of genetic variations among parasite populations possible. However, only a few studies done over a decade ago, have reported on the genetic variation among D. nihonkaiensis in Japan. The present study employed PCR-based mitochondrial DNA analysis to specifically detect D. nihonkaiensis from archived clinical samples, and to determine any genetic variation that may exist among the Japanese broad tapeworms from patients of Kanagawa Prefecture, Japan. Target genes were amplified from DNA extracted from the ethanol- or formaldehyde-fixed samples by PCR. Further sequencing and comparative phylogenetic analyses based on mitochondrial COI and ND1 sequences were also performed. In our results, all PCR-amplified and sequenced samples were identified as D. nihonkaiensis. Analysis of COI sequences revealed two haplotype lineages. However, clustering of almost all COI (and ND1) sample sequences into one of the two haplotype clades, together with reference sequences from different countries worldwide, revealed a common haplotype among D. nihonkaiensis samples in our study. Our results suggest a possible presence of a dominant D. nihonkaiensis haplotype, with a global distribution circulating in Japan. Results from this study have the potential to improve the management of clinical cases and establish robust control measures to reduce the burden of human diphyllobothriasis in Japan.     

Genetic diversity of genus Vespa including an invaded species of V. velutina (Hymenoptera: Vespidae) in Korea inferred from DNA barcoding data

With about 5000 known species, the Vespidae is a large family belongs to order Hymenoptera. The genus Vespa with 22 species is one of the four genera of the subfamily Vespinae. In Korea, 10 species and subspecies are recognized. Because of their social behavior, their treat to human health and their impact in apiculture, the reliable and sometimes automated identification of these insects to species level are important. To test the efficacy of DNA barcoding method for identification of species of the genus Vespa in Korea, 30 samples of eight Korean species of genus Vespa were collected and mitochondrial DNAs of 658 bp fragment cytochrome oxidase subunit 1 (CO1) region were sequenced. A Bayesian Inference based on COI gene of the Korean Vespa species was constructed. The phylogenetic tree shoed that identification of all specimens is possible based on COI gene and we found strong relation between the sequences of the collected species from different localities in South Korea which clustered together with 100% support with sequences of the same species in GenBank. The results demonstrate that DNA barcoding is a useful technique for rapid and accurate species recognition in Korean Vespa species. The DNA barcode part of COI for V. binghami is provided for the first time that can help for identification of this species through DNA barcoding. Also, the genetic diversity among Korean Vespa velutina was zero suggests that the invasion might have occurred in a single event with small number of founders.     

High Diversity of mtDNA Haplotypes Confirms Syntopic Occurrence of Two Field Mouse Species <Emphasis Type="Italic">Apodemus uralensis</Emphasis> and <Emphasis Type="Italic">A. witherbyi</Emphasis> (Muridae: <Emphasis Type="Italic">Apodemus</Emphasis>) in Armenia

Wood mice of the genus Apodemus belong to the most frequent and epidemiologically important rodents of Europe and adjacent regions. Previous studies showed that in the Middle East region species of this genus exhibit extraordinary morphological similarity precluding their proper determination without application of molecular characters. In order to determine the species of the studied populations and to obtain an insight into their phylogeographic history, we analyzed their genetic variation. We sequenced 1139 bp fragment of the mitochondrial DNA control region and flanking tRNA genes in samples collected from six localities. Phylogenetic analyses revealed presence of distinct clades corresponding to species A. uralensis and A. witherbyi. In most localities we confirmed presence of both species which suggests their large sympatric and syntopic occurrence. We recognized an extensive genetic variability, 38 specimens of A. uralensis belong to 32 distinct haplotypes, while 19 specimens of A. witherbyi to 14 haplotypes. We confirmed presence of several distinct haplotypes that may originate from multiple wood mouse colonization waves from distinct geographic regions.     

Molecular Analysis of the Freshwater Prawn Macrobrachium olfersii (Decapoda,Palaemonidae) Supports the Existence of a Single Species throughout Its Distribution

Macrobrachium olfersii is an amphidromous freshwater prawn, widespread along the eastern coasts of the Americas. This species shows great morphological modifications during ontogenesis, and several studies have verified the existence of a wide intraspecific variation. Because of this condition, the species is often misidentified, and several synonyms have been documented. To elucidate these aspects, individuals of M. olfersii from different populations along its range of distribution were investigated. The taxonomic limit was established, and the degree of genetic variability of this species was described. We extracted DNA from 53 specimens of M. olfersii, M. americanum, M. digueti and M. faustinum, which resulted in 84 new sequences (22 of 16S mtDNA, 45 of Cythocrome Oxidase I (COI) mtDNA, and 17 of Histone (H3) nDNA). Sequences of three genes (single and concatenated) from these species were used in the Maximum Likelihood and Bayesian Inference phylogenetic analyses and COI sequences from M. olfersii were used in population analysis. The genetic variation was evaluated through the alignment of 554 bp from the 16S, 638 bp from the COI, and 338 bp from the H3. The rates of genetic divergence among populations were lower at the intraspecific level. This was confirmed by the haplotype net, which showed a continuous gene flow among populations. Although a wide distribution and high morphological intraspecific variation often suggest the existence of more than one species, genetic similarity of Caribbean and Brazilian populations of M. olfersii supported them as a single species.     

Molecular analysis of mitochondrial cytochrome oxidase I gene of Aedes aegypti L. mosquitoes

Aedes aegypti is the most important arboviral vector worldwide. Recent studies reported that genetic variations and gene flow among same mosquito species is responsible for different disease transmission rate. Hence, to understand the relationship between genetic diversity and disease transmission potential, study on genetic variations among mosquito populations is essential. The aim of present study was to investigate the genetic variations of Ae. aegypti targeting COI gene from nine villages of Jalna District, Maharashtra and three laboratory strains originated from Aurangabad, Delhi and transgenic OX513A strain imported from OXITEC, UK. OX513A strain consists of a self-limiting dominant lethal gene construct intended for its use in suppression of Ae. aegypti population by sustained male adult releases in the environment. Mosquito eggs from field and laboratory strains were reared to adults and identified on the basis of morphological characteristics followed by COI gene sequence. Result of MSA and haplotype analysis revealed low genetic variations among field samples and Aurangabad strain, belonged to two haplotypes (H1 and H2) except Ramkheda village represented by separate haplotype H3. Other laboratory DEL strain and transgenic OX513A have great genetic variability to all isolates and have a separate haplotypes H4 and H5. Similar results were observed in phylogenetic analysis. Our observation of phylogenies revealed close relationship among the DEL and transgenic strain OX513A with few Indian and worldwide isolates. The information on genetic variability of mosquito population could help to understand and design the strategies for risk mitigation and effective implementation of new vector control tools like genetically modified mosquitoes.     

Resolving genetic diversity in Australasian Culex mosquitoes: Incongruence between the mitochondrial cytochrome c oxidase I and nuclear acetylcholine esterase 2

Insects that vector pathogens are under constant surveillance in Australasia although the repertoire of genetic markers to distinguish what are often cryptic mosquito species remains limited. We present a comparative assessment of the second exon–intron region of the acetylcholine esterase 2 gene (ace-2) and the mitochondrial DNA cytochrome c oxidase I (COI) using two closely related Australasia mosquitoes Culex annulirostris and Culex palpalis. The COI revealed eight divergent lineages of which four were confirmed with the ace-2. We dissect out the nuclear chromosomal haplotypes of the ace-2 as well as the exon–intron regions by assessing the protein’s tertiary structure to reveal a hypervariable 5’-exon that forms part of an external protein loop and displays a higher polymorphic rate than the intron. We retrace the evolutionary history of these mosquitoes by phylogenetic inference and by testing different evolutionary hypotheses. We conclude that DNA barcoding using COI may overestimate the diversity of Culex mosquitoes in Australasia and should be applied cautiously with support from the nuclear DNA such as the ace-2. Together the COI and ace-2 provide robust evidence for distinct cryptic Culex lineages—one of which correlates exactly with the southern limit of Japanese encephalitis virus activity in Australasia.     

Morphological and Molecular Characterization of a New Trichuris Species (Nematoda- Trichuridae), and Phylogenetic Relationships of Trichuris Species of Cricetid Rodents from Argentina

Populations of Trichuris spp. isolated from six species of sigmodontine rodents from Argentina were analyzed based on morphological characteristics and ITS2 (rDNA) region sequences. Molecular data provided an opportunity to discuss the phylogenetic relationships among the Trichuris spp. from Noth and South America (mainly from Argentina). Trichuris specimens were identified morphologically as Trichuris pardinasi, T. navonae, Trichuris sp. and Trichuris new species, described in this paper. Sequences analyzed by Maximum Parsimony, Maximum Likelihood and Bayesian inference methods showed four main clades corresponding with the four different species regardless of geographical origin and host species. These four species from sigmodontine rodents clustered together and separated from Trichuris species isolated from murine and arvicoline rodents (outgroup). Different genetic lineages observed among Trichuris species from sigmodontine rodents which supported the proposal of a new species. Moreover, host distribution showed correspondence with the different tribes within the subfamily Sigmodontinae.     

Multigene Phylogeography of Bactrocera caudata (Insecta: Tephritidae): Distinct Genetic Lineages in Northern and Southern Hemispheres

Bactrocera caudata is a pest of pumpkin flower. Specimens of B. caudata from the northern hemisphere (mainland Asia) and southern hemisphere (Indonesia) were analysed using the partial DNA sequences of the nuclear 28S rRNA and internal transcribed spacer region 2 (ITS-2) genes, and the mitochondrial cytochrome c oxidase subunit I (COI), cytochrome c oxidase subunit II (COII) and 16S rRNA genes. The COI, COII, 16S rDNA and concatenated COI+COII+16S and COI+COII+16S+28S+ITS-2 nucleotide sequences revealed that B. caudata from the northern hemisphere (Peninsular Malaysia, East Malaysia, Thailand) was distinctly different from the southern hemisphere (Indonesia: Java, Bali and Lombok), without common haplotype between them. Phylogenetic analysis revealed two distinct clades (northern and southern hemispheres), indicating distinct genetic lineage. The uncorrected ‘p’ distance for the concatenated COI+COII+16S nucleotide sequences between the taxa from the northern and southern hemispheres (‘p’ = 4.46-4.94%) was several folds higher than the ‘p’ distance for the taxa in the northern hemisphere (‘p’ = 0.00-0.77%) and the southern hemisphere (‘p’ = 0.00%). This distinct difference was also reflected by concatenated COI+COII+16S+28S+ITS-2 nucleotide sequences with an uncorrected ''p'' distance of 2.34-2.69% between the taxa of northern and southern hemispheres. In accordance with the type locality the Indonesian taxa belong to the nominal species. Thus the taxa from the northern hemisphere, if they were to constitute a cryptic species of the B. caudata species complex based on molecular data, need to be formally described as a new species. The Thailand and Malaysian B. caudata populations in the northern hemisphere showed distinct genetic structure and phylogeographic pattern.     

Glacial-driven vicariance in the amphipod Gammarus duebeni

We have examined the genetic diversity using mitochondrial COI and ND2 sequence data from 306 specimens of the amphi-Atlantic-distributed amphipod Gammarus duebeni. Marine populations from the Atlantic Ocean, the Baltic and North Sea, as well as freshwater populations from Ireland, Cornwall and Brittany were analysed.G. duebeni is a complex of five allopatric lineages. Freshwater populations result from multiple invasions of marine ancestors, represented by distinct lineages. We interpret the recent distribution of lineages as the outcome of a series of spatio-temporal vicariant events caused by Pleistocene glaciations and sea level changes. The freshwater lineages are therefore regarded as ‘glacial relicts’. Furthermore, inter-specific competition with, for example, Gammarus pulex (which is absent in Ireland and western Brittany) may be another important determinant in the distribution of freshwater G. duebeni. In Ireland and Brittany, three freshwater refugia are suggested. The significantly limited gene flow detected among marine populations is more likely due to inter-specific competition than to salinity. The G. duebeni-complex represents a model system for the study of allopatric speciation accompanied by major habitat shifts. The pattern of spatio-temporal origins of the freshwater entities we describe here provides an excellent system for investigating evolutionary adaptations to the freshwater environment. Our data did not confirm the presently used subspecies classification but are only preliminary in the absence of nuclear genetic analyses.     

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.