The phylogenetic relationships of introduced Aphelinus (Hymenoptera: Aphelinidae), biological control agents of the Russian wheat aphid (Homoptera: Aphididae)

Abstract  Several species of Aphelinus have been introduced to the US from the Old World for biological control of the Russian wheat aphid, Diuraphis noxia (Modvilko). Reproductive incompatibility has been observed among populations collected from different geographic areas. We examined whether or not the reproductive incompatibility between strains of A. asychis was caused by distant phylogenetic relationships. Ribosomal DNA sequences of internal transcribed spacers 2 (ITS2) were analyzed in several species of Aphelinus collected from multiple sites of Europe and Asia. The phylogenetic analysis showed that strains within the species A. albipodus and A. asychis are not monophyletic, and two clearly divergent clades were revealed among sequenced samples. Our results suggest that the reproductive incompatibility between three exotic strains of A. asychis was more likely caused by divergence of phylogeny than by symbiotic bacteria.     

Beringian origins and cryptic speciation events in the fly agaric (Amanita muscaria)

Amanita muscaria sensu lato has a wide geographic distribution, occurring in Europe, Asia, Africa, Australia, New Zealand, and North, Central and South America. Previous phylogenetic work by others indicates three geographic clades (i.e. 'Eurasian', 'Eurasian-alpine' and 'North American' groups) within A. muscaria. However, the historical dispersal patterns of A. muscaria remained unclear. In our project, we collected specimens from arctic, boreal and humid temperate regions in Alaska, and generated DNA sequence data from the protein-coding beta-tubulin gene and the internal transcribed spacer (ITS) and large subunit (LSU) regions of the ribosomal DNA repeat. Homologous sequences from additional A. muscaria isolates were downloaded from GenBank. We conducted phylogenetic and nested clade analyses (NCA) to reveal the phylogeographic history of the species complex. Although phylogenetic analyses confirmed the existence of the three above-mentioned clades, representatives of all three groups were found to occur sympatrically in Alaska, suggesting that they represent cryptic phylogenetic species with partially overlapping geographic distributions rather than being allopatric populations. All phylogenetic species share at least two morphological varieties with other species, suggesting ancestral polymorphism in pileus and wart colour pre-dating their speciations. The ancestral population of A. muscaria likely evolved in the Siberian-Beringian region and underwent fragmentation as inferred from NCA and the coalescent analyses. The data suggest that these populations later evolved into species, expanded their range in North America and Eurasia. In addition to range expansions, populations of all three species remained in Beringia and adapted to the cooling climate.     

Two in one: cryptic species discovered in biological control agent populations using molecular data and crossbreeding experiments

There are many examples of cryptic species that have been identified through DNA‐barcoding or other genetic techniques. There are, however, very few confirmations of cryptic species being reproductively isolated. This study presents one of the few cases of cryptic species that has been confirmed to be reproductively isolated and therefore true species according to the biological species concept. The cryptic species are of special interest because they were discovered within biological control agent populations. Two geographically isolated populations of Eccritotarsus catarinensis (Carvalho) [Hemiptera: Miridae], a biological control agent for the invasive aquatic macrophyte, water hyacinth, Eichhornia crassipes (Mart.) Solms [Pontederiaceae], in South Africa, were sampled from the native range of the species in South America. Morphological characteristics indicated that both populations were the same species according to the current taxonomy, but subsequent DNA analysis and breeding experiments revealed that the two populations are reproductively isolated. Crossbreeding experiments resulted in very few hybrid offspring when individuals were forced to interbreed with individuals of the other population, and no hybrid offspring were recorded when a choice of mate from either population was offered. The data indicate that the two populations are cryptic species that are reproductively incompatible. Subtle but reliable diagnostic characteristics were then identified to distinguish between the two species which would have been considered intraspecific variation without the data from the genetics and interbreeding experiments. These findings suggest that all consignments of biological control agents from allopatric populations should be screened for cryptic species using genetic techniques and that the importation of multiple consignments of the same species for biological control should be conducted with caution.     

Cryptic speciation on the high seas; global phylogenetics of the copepod family Eucalanidae

Few genetic data are currently available to assess patterns of population differentiation and speciation in planktonic taxa that inhabit the open ocean. A phylogenetic study of the oceanic copepod family Eucalanidae was undertaken to develop a model zooplankton taxon in which speciation events can be confidently identified. A global survey of 20 described species (526 individuals) sampled from 88 locations worldwide found high levels of cryptic diversity at the species level. Mitochondrial (16S rRNA, CO1) and nuclear (ITS2) DNA sequence data support 12 new genetic lineages as highly distinct from other populations with which they are currently considered conspecific. Out of these 12, at least four are new species. The circumglobal, boundary current species Rhincalanus nasutus was found to be a cryptic species complex, with genetic divergence between populations unrelated to geographic distance. 'Conspecific' populations of seven species exhibited varying levels of genetic differentiation between Atlantic and Pacific basins, suggesting that continental landmasses form barriers to dispersal for a subset of circumglobal species. A molecular phylogeny of the family based on both mitochondrial (16S rRNA) and nuclear (ITS2, 18S rRNA) gene loci supports monophyly of the family Eucalanidae, all four eucalanid genera and the 'pileatus' and 'subtenuis' species groups.     

Development and parasitism by Aphelinus certus (Hymenoptera: Aphelinidae), a parasitoid of Aphis glycines (Hemiptera: Aphididae)

Since its introduction in 2000, the soybean aphid (Aphis glycines Matsumura) has been a serious pest of soybean in North America. Currently, insecticide application is the only recommended control method. However, a number of natural enemies have the potential to regulate soybean aphid populations. In 2007, Aphelinus certus Yasnosh, a soybean aphid parasitoid native to Asia, was found in commercial soybean fields in Ontario. This is the first record of this species in North America. To evaluate the potential biological control services provided by A. certus for soybean aphid management, temperature-dependent developmental parameters and functional response to soybean aphid were determined. A. certus is capable of completing its development between temperatures of 15.3 and 30.2°C. The lower thresholds of development for the egg-mummy and mummy-adult life stages were determined to be 9.1 and 11.6°C, respectively. The lethal temperature of development for the egg-mummy and mummy-adult life stages were 29.5 and 31.0°C, respectively. In this temperature range, A. certus did not exhibit temperature-dependent mortality; however, parasitism rate increased with temperature. A. certus exhibited a type II functional response to the soybean aphid.     

Cryptic ecological diversification of a planktonic estuarine copepod, Acartia tonsa

The recent discovery of cryptic species in marine holoplankton, organisms that 'drift' in oceanic currents throughout their life cycle, contrasts with their potential for long-distance passive dispersal and presumably high gene flow. These observations suggest that holoplankton species are adapting to surprisingly small-scale oceanographic features and imply either limited dispersal or strong selection gradients. Acartia tonsa is a widespread and numerically dominant estuarine copepod containing deep mitochondrial lineages within and among populations along the northwestern Atlantic coast. In this study, we intensively investigated A. tonsa populations in Chesapeake Bay with the goals of testing species status for the deep lineages and testing for their association with environmental features over space and time. Phylogenetic analyses of DNA sequences from mitochondrial cytochrome c oxidase I (mtCOI) and the nuclear ribosomal internal transcribed spacer (nITS) resolved two concordant monophyletic clades. Deep divergence between the two clades (13.7% uncorrected sequence divergence for mtCOI and 32.2% for nITS) and genealogical concordance within sympatric populations strongly suggest that the two clades represent reproductively isolated cryptic species. Based on restriction fragment length polymorphisms of mtCOI, representatives from the two clades were found consistently associated with contrasting salinity regimes (oligohaline vs. meso-polyhaline) with an overlap between 2 and 12 PSU in samples from 1995 to 2005. Finding these patterns in one of the best-known estuarine copepods reinforces the conclusion that marine biodiversity is underestimated, not only in terms of species numbers, but also with respect to niche partitioning and the potential importance of ecological divergence in marine holoplankton.     

Phylogeny and biogeography of Rhododendron subsection Pontica, a group with a tertiary relict distribution

Rhododendron subgenus Hymenanthes subsection Pontica is exceptional among Tertiary relict groups in having a high proportion of species (4 of 11) native to SW Eurasia. A phylogeny based on cpDNA matK and trnL-F indicated that multiple Pontica lineages colonised each of SW Eurasia, SE North America, and NE Asia, with little or no speciation within regions thereafter. Therefore, multiple (3-4) Pontica lineages survived the Quaternary in SW Eurasia, in contrast to other Tertiary relict genera. Pontica comprises two major clades, one of which is wholly Eurasian, and paraphyletic with respect to at least some of the remaining 200 species of subgenus Hymenanthes, which are all distributed in SE Asia. The other clade has species from W and SE North America, SW Eurasia, and NE Asia. According to synonymous matK substitution data, the two clades diverged 9-6 million years ago (mya), whereas most divergence within them happened 5-3 mya. Although the phylogeny indicates probable trans-Atlantic migration for one of two America-Eurasia disjunctions in Pontica, the timing supports migration via Beringia for both.     

DNA barcoding reveals 24 distinct lineages as cryptic bird species candidates in and around the Japanese Archipelago

DNA barcoding using a partial region (648 bp) of the cytochrome c oxidase I (COI) gene is a powerful tool for species identification and has revealed many cryptic species in various animal taxa. In birds, cryptic species are likely to occur in insular regions like the Japanese Archipelago due to the prevention of gene flow by sea barriers. Using COI sequences of 234 of the 251 Japanese‐breeding bird species, we established a DNA barcoding library for species identification and estimated the number of cryptic species candidates. A total of 226 species (96.6%) had unique COI sequences with large genetic divergence among the closest species based on neighbour‐joining clusters, genetic distance criterion and diagnostic substitutions. Eleven cryptic species candidates were detected, with distinct intraspecific deep genetic divergences, nine lineages of which were geographically separated by islands and straits within the Japanese Archipelago. To identify Japan‐specific cryptic species from trans‐Paleartic birds, we investigated the genetic structure of 142 shared species over an extended region covering Japan and Eurasia; 19 of these species formed two or more clades with high bootstrap values. Excluding six duplicated species from the total of 11 species within the Japanese Archipelago and 19 trans‐Paleartic species, we identified 24 species that were cryptic species candidates within and surrounding the Japanese Archipelago. Repeated sea level changes during the glacial and interglacial periods may be responsible for the deep genetic divergences of Japanese birds in this insular region, which has led to inconsistencies in traditional taxonomies based on morphology.     

Evidence for subdivision of the root-endophyte Phialocephala fortinii into cryptic species and recombination within species

The genetic structure of the root-endophyte Phialocephala fortinii was analyzed in three study sites using 11 single-copy RFLP probes. A total of 541 strains isolated from surface-sterilized, fine roots (diameter 0.5-3 mm) of Norway spruce (Picea abies) were examined. The average gene diversity (H) was high in all three study sites. Cluster analysis showed that up to four well-separated clusters of multi-locus haplotypes were present within the sites. Significant population subdivision was detected among these clusters, suggesting that groups of multi-locus haplotypes were reproductively isolated and that P. fortinii is a species complex composed of several cryptic species. This hypothesis was supported by ISSR-PCR which showed clusters consistent with those of the multi-locus haplotypes identified by RFLP analysis. In contrast, ITS sequence analysis did not allow to separate the species as clearly. The index of association (IA) did not deviate significantly from zero within any cryptic species, suggesting that recombination occurs within these species. Cryptic species occurred sympatrically. Thalli of two cryptic species were detected in the same 5-mm-long root segment in one instance. No significant differentiation was observed among populations of the same cryptic species in forest stands located approximately 5 km from each other. This finding is consistent with significant gene flow over this spatial scale. In addition, several isolates with both identical multi-locus haplotype and identical ISSR fingerprint were found at each study site indicating genotype flow or a recent common history between study sites.     

Out of the Qinghai-Tibet Plateau: evidence for the origin and dispersal of Eurasian temperate plants from a phylogeographic study of Hippophaë rhamnoides (Elaeagnaceae)

Numerous temperate plants now distributed across Eurasia are hypothesized to have originated and migrated from the Qinghai-Tibet Plateau (QTP) and adjacent regions. However, this hypothesis has never been tested through a phylogeographic analysis of a widely distributed species. Here, we use Hippopha? rhamnoides as a model to test this hypothesis. We collected 635 individuals from 63 populations of the nine subspecies of H. rhamnoides. We sequenced two maternally inherited chloroplast (cp) DNA fragments and also the bi-paternally inherited nuclear ribosomal ITS. We recovered five major clades in phylogenetic trees constructed from cpDNA and internal transcribed spacer (ITS) sequence variation. Most sampled individuals of six subspecies that are distributed in northern China, central Asia and Asia Minor/Europe, respectively, comprised monophyletic clades (or subclades) nested within those found in the QTP. Two subspecies in the QTP were paraphyletic, while the placement of another subspecies from the Mongolian Plateau differed between the ITS and cpDNA phylogenetic trees. Our phylogeographic analyses supported an 'out-of-QTP' hypothesis for H. rhamnoides followed by allopatric divergence, hybridization and introgression. These findings highlight the complexity of intraspecific evolutions and the importance of the QTP as a center of origin for many temperate plants.     

Morphologically cryptic biological species within the liverwort Frullania asagrayana

? Premise of the study: The Frullania tamarisci complex includes eight Holarctic liverwort species. One of these, F. asagrayana, is distributed broadly throughout eastern North America from Canada to the Gulf Coast. Preliminary genetic data suggested that the species includes two groups of populations. This study was designed to test whether the two groups are reproductively isolated biological species. ? Methods: Eighty-eight samples from across the range of F. asagrayana, plus 73 samples from one population, were genotyped for 13 microsatellite loci. Sequences for two plastid loci and nrITS were obtained from 13 accessions. Genetic data were analyzed using coalescent models and Bayesian inference. ? Key results: Frullania asagrayana is sequence-invariant at the two plastid loci and ITS2, but two clear groups were resolved by microsatellites. The two groups are largely reproductively isolated, but there is a low level of gene flow from the southern to the northern group. No gene flow was detected in the other direction. A local population was heterogeneous but displayed strong genetic structure. ? Conclusions: The genetic structure of F. asagrayana in eastern North America reflects morphologically cryptic differentiation between reproductively isolated groups of populations, near-panmixis within groups, and clonal propagation at local scales. Reproductive isolation between groups that are invariant at the level of nucleotide sequences shows that caution must be exercised in making taxonomic and evolutionary inferences from reciprocal monophyly (or lack thereof) between putative species.     

Phylogeographic Analysis of the Firefly, Luciola lateralis, in Japan and Korea Based on Mitochondrial Cytochrome Oxidase II Gene Sequences (Coleoptera: Lampyridae)

Luciola lateralis is widely distributed throughout the Korean Peninsula, northeast China, Sakhalin, and Japan. Two ecological types are recognized in Japan based on flash and hatching time characteristics. The mitochondrial cytochrome oxidase II gene was surveyed by restriction fragment length polymorphism analysis for Japan (46 populations) and Korea (two populations). Eleven haplotypes were detected. Gene trees revealed that haplotypes between Japan and Korea are much more differentiated in nucleotide sequences (8.1%) than those within Japan (0.3-1.4%) and Korea (0.7%). Haplotypes between Honshu and Hokkaido are not separated as clades, and the two ecological types cannot be segregated from each other phylogenetically. We suggest that the Japanese populations of this species may have dispersed within one million years ago and that ecological differences may be the result of physiological adaptation to cold climates.     

Genetic divergence of Platycerus hongwonpyoi (Coleoptera: Lucanidae) in South Korea

We examined the genetic divergence of Platycerus hongwonpyoi Imura & Choe, 1989 in South Korea using the nuclear wingless (Wg) gene, internal transcribed spacer (ITS) region and mitochondrial cytochrome oxidase subunit I (COI) gene. We found no variation in Wg or ITS. Based on COI, P. hongwonpyoi was split into four well defined and one weakly supported clades, which were inferred to have diverged 2.11–1.33 Ma. The Platycerus hongwonpyoi population size seems to have decreased during the past several tens of thousands of years. The divergence times of major clades of P. hongwonpyoi were comparable with those involved in the speciation of certain Japanese species. Frequent overlapping of different clades at the same sites suggests the occurrence of secondary gene flow following differentiation in South Korea. In conclusion, the genus Platycerus underwent strikingly different divergence patterns in South Korea compared with Japan according to the disparate topographies of these two geographical areas.     

Species identity of geographically distinct populations of the glassy-winged sharpshooter parasitoid Gonatocerus ashmeadi: morphology, DNA sequences, and reproductive compatibility

Gonatocerus ashmeadi is a common and seemingly widespread egg parasitoid of Homalodisca coagulata, the glassy-winged sharpshooter (GWSS). Location records for G. ashmeadi indicate its natural range to be the southeastern USA and northeastern Mexico (which coincides with the presumed native range of GWSS), and possibly southern and central California (CA) (the adventive range of GWSS). The purpose of our work was to determine whether G. ashmeadi in the USA and northeastern Mexico is one species or a complex of reproductively incompatible sibling species. We used three approaches to determine the species identity of different G. ashmeadi populations: (1) reassessment of key morphological features using scanning electron microscopy (SEM) to determine if subtle morphological differences exist between G. ashmeadi populations which could indicate species differences; (2) to determine if molecular differences exist between G. ashmeadi populations collected from different regions by comparing mitochondrial and ribosomal DNA sequences; (3) mating compatibility studies to determine if different populations of G. ashmeadi are reproductively isolated, or if mating occurs, whether offspring are viable thereby defining species groups on the basis of successful interbreeding. Results from these three areas (morphology, DNA sequences, and reproductive compatibility) have been evaluated collectively; leading us to the conclusion that G. ashmeadi as it is currently viewed is a valid species and not an aggregate of morphologically indistinguishable cryptic species.     

A wide geographical survey of mitochondrial DNA variation in the great spotted woodpecker complex,Dendrocopos major (Aves: Picidae)

One of the fundamental goals of phylogeographical studies should be to achieve a comprehensive geographical sampling of any investigated group. In this study, we conducted the most comprehensive geographical investigation to date for the great spotted woodpecker complex (Dendrocopos major), including populations from North Africa and Eurasia [including specimens from China, Japan and southern Caucasia (Anatolia, Azerbaijan and Iran)], in order to evaluate its genetic structure and population history. At the same time, we tested species limits within the D. major complex, which currently includes 14 recognized subspecies based on morphology and coloration. We based our study on haplotypes for the mitochondrial gene NADH dehydrogenase subunit 2 (ND2). Most haplotypes were obtained from museum toe pads, although we also used some previously published data. We also tested gene flow through MDIV, and estimated divergence dates among lineages using BEAST. The analysis of 352 base pairs of the ND2 gene from 155 individuals sampled from 33 populations showed significant phylogeographical structure across the breeding range. Our results found four distinct and reciprocally monophyletic clades: China, Japan, Iran–Azerbaijan and Eurasia–North Africa, with no phylogeographical structure within them. Coalescent‐based gene flow analysis showed restricted gene flow between China and Japan and between Japan and Eurasia. On the basis of the gene flow and phylogenetic analysis results, we propose the recognition of at least four different species within the complex. We also propose that, within the Eurasia–North Africa clade, a rapid population expansion through ‘leading edge expansion’ from refugia in Iberia, Kursk and North Africa, as well as irruptive and loop migration, can explain the lack of phylogeographical structure. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ?? , ??–??.     

Inter- and intraspecific evolutionary relationships of the rice frog Rana limnocharis and the allied species R. cancrivora inferred from crossing experiments and mitochondrial DNA sequences of the 12S and 16S rRNA genes

The rice frog Rana limnocharis is widely distributed in Southeast Asia and the rest of the Asian region extending from India to Japan. In Japan, the Sakishima-island populations of this species were regarded as a distinct species based on morphological and genetic divergences. The main purposes of this study were to confirm the presence of intraspecific reproductively isolating mechanisms in the Sakishima-island populations of R. limnocharis, and to clarify molecular inter- and intraspecific relationships of R. limnocharis and an allied species, Rana cancrivora. The hybridization experiments revealed that there were no reproductively isolating mechanisms between the Sakishima-island populations and other populations of R. limnocharis. The molecular evolutionary relationships were investigated by analyzing nucleotide sequences of the mitochondrial 12S and 16S rRNA genes using 12 populations of R. limnocharis from Japan and Taiwan, and two populations of R. cancrivora from Thailand and the Philippines. The phylogenetic trees constructed by the NJ method showed that the two populations of R. cancrivora were clearly separated from the 12 populations of R. limnocharis, and that the 12 populations of R. limnocharis were broadly divided into three clades; the first comprising eight populations from the main islands of Japan, the second comprising the Sakishima-island populations, and the third comprising the Okinawa-island and Taiwan populations. Interestingly, the Okinawa-island and Taiwan populations of R. limnocharis showed a close relationship that possibly reflected a secondary contact between the two populations. Based on the present crossing experiments and molecular data, it seems reasonable to regard the Sakishima-island populations as a single subspecies of R. limnocharis.     

Phylogenetics and genetic diversity of the Cotesia flavipes complex of parasitoid wasps (Hymenoptera: Braconidae), biological control agents of lepidopteran stemborers

The Cotesia flavipes complex of parasitoid wasps (Hymenoptera: Braconidae) are economically important for the biological control of lepidopteran stemboring pests associated with gramineous crops. Some members of the complex successfully parasitize numerous stemborer pest species, however certain geographic populations have demonstrated variation in the range of hosts that they parasitize. In addition, the morphology of the complex is highly conserved and considerable confusion surrounds the identity of species and host-associated biotypes. We generated nucleotide sequence data for two mtDNA genes (COI, 16S) and three anonymous nuclear loci (CfBN, CfCN, CfEN) for the C. flavipes complex. To analyze genetic variation and relationships among populations we used (1) concatenated mtDNA and nDNA data, (2) a nDNA multilocus network approach, and (3) two species tree inference methods, i.e. Bayesian estimation of species trees (BEST) and Bayesian inference of species trees from multilocus data with (*)BEAST. All phylogenetic analyses provide strong support for monophyly of the complex and the presence of at least four species, C. chilonis (from China and Japan), C. sesamiae (from Africa), C. flavipes (originating from the Indo-Asia region but introduced into Africa and the New World), and C. nonagriae (from Australia and Papua New Guinea). Haplotype diversity of geographic populations relates to historical biogeographic barriers and biological control introductions, and reflects previous reports of ecological variation in these species. Strong discordance was found between the mitochondrial and nuclear markers in the Papua New Guinea haplotypes, which may be an outcome of hybridization and introgression of C. flavipes and C. nonagriae. The position of Cotesia flavipes from Japan was not well supported in any analysis and was the sister taxon to C. nonagriae (mtDNA, (*)BEAST), C. flavipes (nDNA) or C. flavipes+C. nonagriae (BEST) and, may represent a cryptic species. The concatenated five gene phylogenetic analyses did not support the overall separation and monophyly of clades associated with different host species, although some clades did show specific host associations, possibly due to localized host availability, rather than host specificity. Our results provide a framework for assessing whether distinct lineages represent cryptic species, and for examining parasitoid-host evolution and compatibility more generally. Given the limitations of morphological based identification for members of this complex, molecular identification is recommended prior to any biological control introductions.     

MOLECULAR PHYLOGEOGRAPHY, RETICULATION, AND LINEAGE SORTING IN MEDITERRANEAN SENECIO SECT. SENECIO (ASTERACEAE)

Abstract The Mediterranean species complex of Senecio serves to illustrate evolutionary processes that are likely to confound phylogenetic inference, including rapid diversification, gene tree‐species tree discordance, reticulation, interlocus concerted evolution, and lack of complete lineage sorting. Phylogeographic patterns of chloroplast DNA (cpDNA) haplotype variation were studied by sampling 156 populations (502 individuals) across 18 species of the complex, and a species phylogeny was reconstructed based on sequences from the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. For a subset of species, randomly amplified polymorphic DNAs (RAPDs) provided reference points for comparison with the cpDNA and ITS datasets. Two classes of cpDNA haplotypes were identified, with each predominating in certain parts of the Mediterranean region. However, with the exception of S. gallicus, intraspecific phylogeographic structure is limited, and only a few haplotypes detected were species‐specific. Nuclear sequence divergence is low, and several unresolved phylogenetic groupings are suggestive of near simultaneous diversification. Two well‐supported ITS clades contain the majority of species, amongst which there is a pronounced sharing of cpDNA haplotypes. Our data are not capable of diagnosing the relative impact of reticulation versus insufficient lineage sorting for the entire complex. However, there is firm evidence that S. flavus subsp. breviflorus and S. rupestris have acquired cpDNA haplotypes and ITS sequences from co‐occurring species by reticulation. In contrast, insufficient lineage sorting is a viable hypothesis for cpDNA haplotypes shared between S. gallicus and its close relatives. We estimated the minimum coalescent times for these haplotypes by utilizing the inferred species phylogeny and associated divergence times. Our data suggest that ancestral cpDNA polymorphisms may have survived for ca. 0.4–1.0 million years, depending on molecular clock calibrations.     

Phylogenetic relationships and genetic diversity of badgers from the Korean Peninsula: Implications for the taxonomic status of the Korean badger

Accurate taxonomic classification of wildlife species is crucial for guiding biological research and for developing effective management and conservation programs. The taxonomic status of Eurasian badgers from South Korea remains poorly resolved. Here we assessed the phylogenetic relationships and genetic variation of Eurasia badgers using partial mitochondrial fragments to elucidate the evolutionary history and taxonomic status of badgers from the Korean Peninsula. Forty-eight unique haplotypes from 125 individuals were observed. Phylogenetic reconstructions and reduced median networks indicate that Eurasian badgers consisted of four geographic clades (Japan, Eastern Eurasia, Western Eurasia, and Caucasus) with a relatively weak split observed within Eastern Eurasia. Estimated divergence time between the Japanese and Eastern Eurasian clades, including the Korean population, was 467,100 years (69,200–1,085,500 years). The results of this study support the hypothesis that the Japanese badger migrated from the Eurasian continent over the Korea-Japan land bridge and that the Korean Peninsula was an important refugia during the Pleistocene. Our study confirmed that the South Korean badger, Meles meles, belongs to the Eastern Eurasian clade. Based on these results and those of previous studies, we recommend that the scientific name of the Korean badger be changed from M. meles to Meles leucurus (Asian badger).     

Evolutionary relationships and reproductive isolating mechanisms in the rice frog (Fejervarya limnocharis) species complex from Sri Lanka, Thailand, Taiwan and Japan, inferred from mtDNA gene sequences, allozymes, and crossing experiments

The rice frog (Fejervarya limnocharis) species complex is widely distributed, from India to Japan, and most prevalently in Southeast Asia. Conspicuous morphological variation has been reported for this species complex throughout its distribution range. In the present study, we used mtDNA gene sequence and allozyme analyses to infer evolutionary affinities within this species complex using eight populations (Sri Lanka; Bangkok and Ranong in Thailand; Taiwan; and Hiroshima, Okinawa, Ishigaki and Iriomote in Japan). We also conducted crossing experiments among four populations from Japan, Thailand, and Sri Lanka in order to find out more about the reproductive isolating mechanisms that might exist among the East, Southeast, and South Asian populations of this species complex. The crossing experiments revealed that the Sri Lanka population is reproductively isolated from the Hiroshima, Bangkok, and Ranong populations by complete hybrid inviability, and that the Bangkok population may be reproductively isolated from the Hiroshima population by partial hybrid inviability. Thus, it is not unreasonable to regard the Sri Lanka population as a species separated from F. limnocharis. The mtDNA and allozyme data showed that the Ranong population is most closely related to the Bangkok population in nuclear genome, but more similar to the Okinawa and Taiwan populations in mtDNA genome. The present, preliminary survey may raise questions about the species status of these particular populations and also about the nature of the biological species concept.