Phylogeography of a northeast Asian spruce, Picea jezoensis, inferred from genetic variation observed in organelle DNA markers

Range-wide genetic variation of the widespread cold-temperate spruce Picea jezoensis was studied throughout northeast Asia using maternally inherited mitochondrial DNA and paternally inherited chloroplast DNA markers. This study assessed 33 natural populations including three varieties of the species in Japan, Russia, China, and South Korea. We depicted sharp suture zones in straits around Japan in the geographical distribution pattern of mitochondrial haplotypes (GST=0.901; NST=0.934). In contrast, we detected possible extensive pollen flow without seed flow across the straits around Japan during the past population history in the distribution pattern of chloroplast haplotypes (GST=0.233; NST=0.333). The analysis of isolation by distance of the species implied that by acting as a barrier for the movement of seeds and pollen, the sharp suture zones contributed considerably to the level of genetic differentiation between populations. Constructed networks of mitochondrial haplotypes allowed inference of the phylogeographical history of the species. We deduced that the disjunction with Kamchatka populations reflects range expansion and contraction to the north of the current distribution. Within Japan, we detected phylogeographically different types of P. jezoensis between Hokkaido and Honshu islands; P. jezoensis in Honshu Island may have colonized this region from the Asian continent via the Korean peninsula and the species in Hokkaido Island is likely to have spread from the Asian continent via Sakhalin through land bridges. Japanese endemism of mitochondrial haplotypes in Hokkaido and Honshu islands might have been promoted by separation of these islands from each other and from the Asian continent by the straits during the late Quaternary.     

Allozyme diversity in Japanese populations of Hemerocallis thunbergii, H. middendorffii, and H. exaltata (Liliaceae)

Eight populations of Hemerocallis thunbergii, H. middendorffii , and H. exaltata from Japan were analyzed by starch gel electrophoresis to measure genetic variation at ten allozyme loci. The former two species are widely distributed in eastern Asia, whereas H. exaltata is restricted to the Tobishima and Sado Islands in western Japan. Populations of the three species maintain high levels of allozyme variation: mean expected heterozygosities were 0.288, 0.209, and 0.169, respectively. The UPGMA phenogram obtained from Nei's genetic distance values indicated that H. exaltata is allozymically the most distinct species, whereas populations of H. thunbergii and H. middendorffii are closely related. However, based on gross morphology H. exaltata and H. middendorffii are closely related. This discrepancy and the observed high levels of allozyme variation within populations of the three Hemerocallis species in Japan may in part be due to genetic change among populations of the two Japanese archipelago species.     

Phylogeography of the Korean pine (Pinus koraiensis) in northeast Asia: inferences from organelle gene sequences

Range-wide genetic variation of Korean pine (Pinus koraiensis) was assessed using maternally inherited mtDNA and paternally inherited cpDNA for 16 natural populations throughout northeast Asia in order to study its phylogeographical history during the Quaternary. The cpDNA variation indicated that there was no difference between populations on the Asian continent and those in the Japanese archipelago. In contrast, the mtDNA variation indicated that there was significant difference between the populations from the two regions, with each region having a different lineage. The continental populations exhibited no diversity in the mtDNA examined despite the species’ current extensive range and large populations. Conversely, while the Korean pine is rare in Japan, the Japanese populations exhibited greater levels of mtDNA diversity (H _T?=?0.502). The higher mtDNA diversity and evidence from numerous Korean pine macrofossil remains dated to the Pleistocene and recovered various sites in Japan suggest that the Japanese archipelago once served as a refugium to a much larger Korean pine population with a more extensive range than is the case today. The presence of the single mtDNA haplotype across the Asian continent suggests that the present widespread populations could have expanded from a single refugium population after the last glacial periods.     

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.     

Genetic structure of the asiatic black bear in Japan using mitochondrial DNA analysis

The genetic structure of the Asiatic black bear (Ursus thibetanus) in Japan was studied to understand the events that occurred during its evolution. The left domain of the mitochondrial control region (about 240 bp) was sequenced, defining 27 haplotypes that consisted of 23 haplotypes from 333 bears in Japan and 22 bears in the Asian continent. The network tree of the control region indicated that the Japanese population formed a distinct clade from the continental population. The phylogeographic analysis of the haplotypes indicated that the Shikoku and Kii Hanto populations had diverged during the initial phase from the ancestral population. After the 3 dominant haplotypes were rapidly distributed throughout Japan in the early stage of the population dispersal, the Japanese population diverged into eastern and western populations. Using the entire mitochondrial cytochrome b sequence, divergence time between the Japanese and the Continental populations suggested that the Japanese population might have colonized into Japan through the land bridge from the Korean Peninsula around 500 ka, which is consistent with paleontological evidence. Our finding that bears in western Japan exhibit lower genetic diversity and higher levels of genetic differentiation than bears in eastern Japan provides a vital contribution to conservation policy for these isolated populations.     

Molecular phylogeny and geographic distribution of wood-feeding cockroaches in East Asian Islands

Molecular phylogenetic relationships of the wood-feeding cockroach genera Salganea and Panesthia (Blaberidae; Panesthiinae) in East Asian Islands (Ryukyu archipelago and Taiwan Island) were analyzed based on the DNA sequence of the complete mitochondrial cytochrome oxidase II gene. Unweighted parsimony analysis resulted in high bootstrap support for relationships within Panesthia taxa; however, some nodes were unresolved between members of Salganea. Comparison of the number of transitions and transversions with genetic distance at each codon position suggested that saturation of third-codon substitutions has occurred between certain pairs of taxa. Consequently, differential weighting of substitutions at these sites was performed, which resulted in a substantial increase in resolution of Salganea relationships. The inferred phylogenies for both genera displayed good correspondence to the geographical locations of populations but however did not agree with previous subspecies designations based on morphological characters. It appears that both cockroach genera invaded the Ryukyu archipelago from the Taiwan region via a land-bridge present in the Miocene period. Invasion of the main islands of Japan by these cockroaches most likely occurred before the formation of the Tokara Tectonic Strait. Our study suggests that several barriers to gene flow have arisen and persisted over the past approximately 10 million years, which have caused segregation and vicariant speciation of the cockroach taxa of this region.     

Phylogeography of the Coastal Mosquito Aedes togoi across Climatic Zones: Testing an Anthropogenic Dispersal Hypothesis

The coastal mosquito Aedes togoi occurs more or less continuously from subarctic to subtropic zones along the coasts of the Japanese islands and the East Asian mainland. It occurs also in tropical Southeast Asia and the North American Pacific coast, and the populations there are thought to have been introduced from Japan by ship. To test this hypothesis, the genetic divergence among geographic populations of A. togoi was studied using one mitochondrial and three nuclear gene sequences. We detected 71 mitochondrial haplotypes forming four lineages, with high nucleotide diversity around temperate Japan and declining towards peripheral ranges. The major lineage (L1) comprised 57 haplotypes from temperate and subarctic zones in Japan and Southeast Asia including southern China and Taiwan. Two other lineages were found from subtropical islands (L3) and a subarctic area (L4) of Japan. The Canadian population showed one unique haplotype (L2) diverged from the other lineages. In the combined nuclear gene tree, individuals with mitochondrial L4 haplotypes diverged from those with the other mitochondrial haplotypes L1—L3; although individuals with L1—L3 haplotypes showed shallow divergences in the nuclear gene sequences, individuals from Southeast Asia and Canada each formed a monophyletic group. Overall, the genetic composition of the Southeast Asian populations was closely related to that of temperate Japanese populations, suggesting recent gene flow between these regions. The Canadian population might have originated from anthropogenic introduction from somewhere in Asia, but the possibility that it could have spread across the Beringian land bridge cannot be ruled out.     

西南地区6种魔芋属植物基于cpDNA序列的遗传多样性研究

研究野生作物资源的遗传变异及分化机制对种质资源的收集与改良具有重要意义。魔芋是我国西南地区的特色经济作物,但由于受到人为活动干扰,野生种群不断衰退。为评估西南地区魔芋属(Amorphophallus)野生群体的遗传多样性,探究代表性物种的系统发育地位,该研究利用3个叶绿体DNA(cpDNA)片段,分析了魔芋6个物种的遗传多样性,重建了种间系统发育关系。结果表明:(1)西南地区野生魔芋群体的遗传多样性普遍较低,虽然单倍型多样性(H_d)均值为0.428,但近一半群体只有1个单倍型,6个物种整体水平上的单倍型多样性在0.704到0.983之间。(2)在6个物种间检测到高水平的遗传分化,遗传分化系数(F_ST)值在0.481到0.967之间。(3)系统发育分析表明,选取的27个魔芋种主要聚成3个分支:非洲分支、东南亚分支和东亚大陆分支。疣柄魔芋(A. paeoniifolius)隶属于东南亚分支,而东亚大陆分支A包含花魔芋(A. konjac)和西盟魔芋(A. krausei),东亚大陆分支B由东亚魔芋(A. kiusianus)、滇魔芋(A. yunnanensis)和东京魔芋(A. tonkinensis)构成。生境隔离与人为干扰造成了西南地区野生魔芋群体较低的遗传多样性,魔芋属东亚大陆分支的分化可能与早期的快速扩张和生态适应有关。该研究为西南地区魔芋资源的合理保护、可持续利用和杂交育种提供了参考资料。     

Multicytotypic Populations of Scilla scilloides Polyploid Complex Found in Jiangxi,China

Two populations ( located at Sanhu town , Xingan co., Jiangxi province ) of Scilla scilloides polyploid complex were surveyed cytologically , and four cytotypes, BB, BBB, BBBB, and ABBB were observed among two hundred and sixty three individuals . BBBB and ABBB were new records in East Asian continent . Until now, nine of the 12 euploid cytotypes of this polyploid complex have been found in the East Asian continent , which suggests the continent is one of the important locations where the cytotypic differentiation of the complex takes place . The occurrence of ABBB and BBB, but not ABB and aneuploids maybe demonstrates the distribution range of BB have contracted on the continent and Korean peninsula, AABB populations of plants with subglobose bulb have degenerated on the continent since Tertiary , and the mixed populations of BB and AABB are the relicts of populations on the continent , Korean peninsula and Cheju island . The cytotype component similarity in these populations to that in populations on the Japanese archipelago may show some historical relations between them, but this fact is not able to manifest the hypothesis that this species on the Japanese islands is one of“prehistoric naturalized”plants introduced from China .     

Landscape genetics of alpine-snowbed plants: comparisons along geographic and snowmelt gradients

The genetic structure of three snowbed-herb species (Peucedanum multivittatum, Veronica stelleri, and Gentiana nipponica) was analyzed using allozymes across nine populations arranged as a matrix of three snowmelt gradients x three geographic locations within 3 km in the Taisetsu Mountains, northern Japan. Phenologically asynchronous populations are packed within a local area in alpine snowbeds, because flowering season of alpine plants depends strongly on the timing of snowmelt. Moderate genetic differentiation was detected among local populations in every species (FST=0.03-0.07). There was a significant correlation between the geographic distance and genetic distance in the P. multivittatum populations, but not in the V. stelleri and G. nipponica populations. On the other hand, a significant correlation between the phenological distance caused by snowmelt timing and genetic distance was detected in the V. stelleri and G. nipponica populations, but not in the P. multivittatum populations. The snowmelt gradient or geographic separation influenced hierarchical genetic structure of these species moderately (FRT <0.04). Restriction of gene flow due to phenological separation and possible differential selection along the snowmelt gradient may produce genetic clines at microgeographic scale in these species.     

ANALYSIS OF MITOCHONDRIAL DNA DIVERSITY WITHIN AND BETWEEN NORTH AND SOUTH ATLANTIC RIGHT WHALES

DNA sequences of the mitochondrial control region of 180 North Atlantic right whales ( Euhalaena glacialis ) and 16 South Atlantic right whales ( E. australis ) have been determined using a combination of direct DNA sequencing and single stranded conformation polymorphism (SSCP) analysis. Five haplotypes were found in E. glacialis , and 10 in E. australis , but none were shared, supporting the reproductive isolation and separate species status of the North and South Atlantic right whales. One haplotype in E, glacialis was found in only three males born before 1982 and this matriline will likely be lost soon. The nucleotide diversity estimates for the five North Atlantic right whale haplotypes was 0.6% and 2.0% for the 10 haplotypes found in the South Atlantic right whales. The average haplotypic diversity was 0.87 in E. glacialis and 0.96 in E. australis , which is consistent with other studies showing a lower level of genetic variation in the North Atlantic right whale. Phylogenetic analysis identified two major assemblages of haplotypes in E. australis from the samples collected from Peninsula Valdes, suggesting a mixing of two historically divergent populations. Using genetic distance measurements with a divergence rate of 0.5%–1.0%/myr, we estimate E. glacialis diverged from E. australis 3–12.5 mya.     

Phylogeographic structure and late Quaternary population history of the Japanese oak Quercus mongolica var. crispula and related species revealed by chloroplast DNA variation

Generally, oaks dominate the broadleaf deciduous forests in Japan. The genetic variation in 6 cpDNA regions (trnT-trnL, trnL-trnF, atpB-rbcL, and trnH-psbA speacers, trnL intron, and matK gene) with regard to the Japanese oak (Quercus mongolica var. crispula) and 3 related species in the section Prinus (Q. serrata, Q. dentata and Q. aliena) was investigated in 598 trees belonging to 44 populations distributed throughout the Japanese archipelago. Additional samples were collected from Korea, China, and Russia (Sakhalin). Thirteen haplotypes (I to XIII) were identified on the bases of 15 nucleotide substitutions and 3 indels. Haplotypes I and II were discovered in northeastern Japan, whereas haplotypes III to IX were distributed in southwestern Japan. The boundary distinguishing these 2 groups was located in central Japan coincident with the Itoigawa-Shzuoka tectonic line. Haplotype I was also found in Sakhalin, whereas haplotypes VI, VII, VIII, X, XI, XII, and XIII were found in Korea and China. Four oak species in the same location shared identical haplotypes, suggesting cpDNA introgression by occasional hybridization. Both the values of total haplotype diversity (HT) and haplotype diversity within populations (HS) in Q. mongolica var. crispula were higher in the southwestern populations than in the northeastern populations. A haplotype network indicated that haplotype VI is the ancestral haplotype. The presence of identical haplotypes in Korea, China, and Japan suggested that the haplotypes diversified on the Eurasian continent before the last glacial period. The difference in genetic structure between the northeastern and southwestern regions indicates a difference in the history of migration and recolonization in Japan during the last glacial period.     

江西发现绵枣儿多倍体复合体多细胞型混合居群

对来自江西省新干县三湖镇绵枣儿(Scilla scilloides)多倍体复合体两个居群263个植株进行了细胞学调查,发现4个整倍体细胞型,即BB、BBB、BBBB和ABBB。其中BBBB和ABBB为大陆上首次记录。至此在东亚大陆上已经发现该多倍体复合体12个整倍体细胞型中的9个。这表明东亚大陆也是该复合体细胞型分化的重要地区之一。此地出现ABBB和BBB,而未出现ABB以及非整倍体可能是说明自第四纪以来BB细胞型在大陆和朝鲜半岛的分布区正在收缩;大陆上AABB已经接近消失;而大陆、朝鲜半岛和济州岛(韩国)的BB和AABB混合居群只是残遗。大陆与日本列岛居群细胞型组成的相似性可能暗示着它们的某种历史关系,但是并不能支持日本分布的绵枣儿是从中国引入的“史前归化”植物。     

Recent colonization and expansion through the Lesser Sundas by seven amphibian and reptile species

The Lesser Sundas Archipelago is comprised of two parallel chains of islands that extend between the Asian continental shelf (Sundaland) and Australo‐Papuan continental shelf (Sahul). These islands have served as stepping stones for taxa dispersing between the Asian and Australo‐Papuan biogeographical realms. While the oceanic barriers have prevented many species from colonizing the archipelago, a number of terrestrial vertebrate species have colonized the islands either by rafting/swimming or by human introduction. Here, we examine phylogeographic structure within the Lesser Sundas for three snake, two lizard and two frog species that each has a Sunda Shelf origin. These species are suspected to have recently colonized the archipelago, though all have inhabited the Lesser Sundas for over 100 years. We sequenced mtDNA from 231 samples to test whether there is sufficiently deep genetic structure within any of these taxa to reject human‐mediated introduction. Additionally, we tested for genetic signatures of population expansion consistent with recent introduction and estimated the ages of Lesser Sundas clades, if any exist. Our results show little to no genetic structure between populations on different islands in five species and moderate structure in two species. Nucleotide diversity is low for all species, and the ages of the most recent common ancestor for species with monophyletic Lesser Sundas lineages date to the Holocene or late Pleistocene. These results support the hypothesis that these species entered the archipelago relatively recently and either naturally colonized or were introduced by humans to most of the larger islands in the archipelago within a short time span.     

Evolution and biogeography of talpid moles from continental East Asia and the Japanese islands inferred from mitochondrial and nuclear gene sequences

We sequenced the cytochrome b gene from two little-studied mammal species from the highlands of Southwest China, the long-tailed mole Scaptonyx fusicaudus and the gracile shrew-like mole Uropsilus gracilis. This data was used to examine the phylogenetic relationships among 19 talpid species within the family Talpidae (Mammalia: Eulipotyphla). Cytochrome b gene trees supported a basal placement of shrew-like moles (Uropsilus) within the Talpidae, and suggested that fossorial specializations arose twice during talpid evolution. To assess the evolutionary relationships of moles endemic to this region, we additionally sequenced the 12S rRNA gene and the nuclear recombination-activating gene-1 from eight and ten East Asian taxa, respectively. Analyses of these single and concatenated data sets suggested that East Asian shrew moles diverged prior to the evolution of fossorial Eurasian moles. However, we were unable to determine whether semi-fossorial shrew moles are monophyletic. In contrast, fossorial Eurasian genera (Talpa, Mogera and Euroscaptor) were consistently found to form a monophyletic clade, with Mogera and Euroscaptor representing sister taxa. Furthermore, this fossorial clade grouped with the semi-aquatic Desmana, although with fairly low (35-62%) bootstrap support. Mogera imaizumii was found to be more closely related to M. wogura than to M. tokudae. This implies that the ancestors of these three species entered Japan from the Asian continent in this order via a series of migration events, suggesting that the Japanese Islands have played an important role in preserving mole lineages from ancient to recent times.     

Post-glacial range fragmentation is responsible for the current distribution of Potentilla matsumurae Th. Wolf (Rosaceae) in the Japanese archipelago

Aim We aimed to elucidate how the current geographic distribution of alpine plants in the Japanese archipelago was shaped during Quaternary climatic oscillations, using Potentilla matsumurae as a case study. According to previous phylogeographic studies, post‐glacial range fragmentation (vicariance scenario) and stepwise migration (dispersal scenario) are both possible. We thus aimed to assess which scenario is more probable for the distribution changes of alpine plants in the Japanese archipelago. Location The alpine zone in the Japanese archipelago. Methods Using amplified fragment length polymorphism we determined the genotype of 161 individuals of P. matsumurae from 22 populations. Relationships among individuals and populations were examined using principal coordinates analysis and a neighbour‐joining (NJ) tree, respectively. To examine the genetic population structure, we performed analysis of molecular variance (amova ) and structure analysis. Results Differentiation between central Honshu and northern Japan was not very strong based on the principal coordinates analysis among individuals, the NJ tree of populations (59% bootstrap support), or amova (12% of genetic variation). Moreover, structure analysis did not detect clear geographic differentiation across populations. Although the populations in central Honshu were structured geographically (Mantel test: r = 0.45, P < 0.005; NJ tree), those in northern Japan did not exhibit geographic structure regardless of geographic distance (Mantel test: r = 0.26, P = 0.03; NJ tree). Population relationships in the NJ tree did not always reflect the geographic location. Main conclusions The current geographic structure of P. matsumurae could not be explained by stepwise migration. This suggests that a single continuous distribution during the last glacial period was later fragmented, perhaps by recovering forest, during the post‐glacial period, resulting in the current distribution and phylogeographic structure of P. matsumurae. Our data support the vicariance scenario.     

Genetic origins of the Japanese: A partial support for the dual structure hypothesis

Based on the morphological characteristics of the skull and teeth, Hanihara ([1991] Japan Review 2:1–33) proposed the “dual structure model” for the formation of modern Japanese populations. We examine this model by dividing it into two independent hypotheses: 1) the Upper Paleolithic population of Japan that gave rise to the Neolithic Jomon people was of southeast Asian origin, and 2) modern Ainu and Ryukyuan (Okinawa) populations are direct descendants of the Jomon people, while Hondo (Main Island)-Japanese are mainly derived from the migrants from the northeast Asian continent after the Aeneolithic Yayoi period. Our aim is to examine the extent to which the model is supported by genetic evidence from modern populations, particularly from Japan and other Asian areas. Based on genetic distance analyses using data from up to 25 “classic” genetic markers, we find first that the three Japanese populations including Ainu and Ryukyuan clearly belong to a northeast Asian cluster group. This negates the first hypothesis of the model. Then, we find that Ainu and Ryukyuans share a group contrasting with Hondo-Japanese and Korean, supporting the second hypothesis of the model. Based on these results, we propose a modified version of the dual structure model which may explain the genetic, morphological, and archaeological evidence concerning the formation of modern Japanese populations. Am J Phys Anthropol 102:437–446, 1997. © 1997 Wiley-Liss, Inc.     

Genetic diversity and genetic structure of populations of Ranunculus japonicus Thunb. (Ranunculaceae)

Abstract In order to clarify the genetic diversity and population structure of Ranunculus japonicus , allozymic analysis was conducted on 60 populations in southwestern Japan. Considerable genetic variati ons were detected among the populations of R. japonicus . The genetic diversities within species ( H _es = 0.215) and within populations ( H _ep = 0.172) were slightly higher than those of other perennial herbs with widespread distribution and outcrossing plants. Significantly higher values of fixation index were detected in some populations, which might have arisen from restricted mating partners. The majority of genetic variation (approx. 80%) resided within a population and a moderate level of genetic differentiation ( G _ST = 0.203) was observed among populations. The F _ST value (0.203) suggests the existence of a substantial population structure in this species. The highly significant correlation between geographic distance and F _ST values indicates that isolation by distance has played an important role in the construction of the genetic structure of this species.     

Deep genetic divergences in Aoraki denticulata (Arachnida, Opiliones, Cyphophthalmi): a widespread 'mite harvestman' defies DNA taxonomy

Aoraki denticulata (Arachnida, Opiliones, Cyphophthalmi, Pettalidae), a widespread 'mite harvestman' endemic to the South Island of New Zealand, is found in leaf littler habitats throughout Nelson and Marlborough, and as far south as Arthur's Pass. We investigated the phylogeography and demographic history of A. denticulata in the first genetic population-level study within Opiliones. A total of 119 individuals from 17 localities were sequenced for 785 bp of the gene cytochrome c oxidase subunit I; 102 of these individuals were from the Aoraki subspecies A. denticulata denticulata and the remaining 17 were from the subspecies A. denticulata major. An extraordinarily high degree of genetic diversity was discovered in A. denticulata denticulata, with average uncorrected p-distances between populations as high as 19.2%. AMOVA, average numbers of pairwise differences, and pairwise F(ST) values demonstrated a significant amount of genetic diversity both within and between populations of this subspecies. Phylogenetic analysis of the data set revealed many well-supported groups within A. denticulata denticulata, generally corresponding to clusters of specimens from single populations with short internal branches, but separated by long branches from individuals from other populations. No haplotypes were shared between populations of the widespread small subspecies, A. denticulata denticulata. These results indicate a subspecies within which very little genetic exchange occurs between populations, a result consistent with the idea that Cyphophthalmi are poor dispersers. The highly structured populations and deep genetic divergences observed in A. denticulata denticulata may indicate the presence of cryptic species. However, we find a highly conserved morphology across sampling localities and large genetic divergences within populations from certain localities, equivalent to those typically found between populations from different localities. Past geological events may have contributed to the deep genetic divergences observed between sampling localities; additionally, the high divergence within populations of A. denticulata denticulata suggests that the rate of COI evolution may be accelerated in this taxon. In contrast, the larger subspecies A. denticulata major shows much less differentiation between and within sampling localities, suggesting that it may disperse more easily than its smaller counterpart. The fact that the remarkable genetic divergences within populations of A. denticulata denticulata from certain localities are equivalent to divergences between localities poses a challenge to the rapidly spreading practice of DNA taxonomy.     

Evidence of a secondary interspecific mitochondrial DNA introgression in the pond loach Misgurnus dabryanus (Teleostei: Cobitidae) population introduced in Japan

The pond loach Misgurnus dabryanus is a freshwater fish with a distribution range spanning the eastern part of the Asian continent, the Korean Peninsula, and Taiwan. The pond loach was transplanted to the Japanese archipelago through the co-inclusion with dojo loach (Misgurnus anguillicaudatus species complex) populations, which were imported live from China for food materials, and it is currently distributed widely across Japan. A previous mitochondrial DNA (mtDNA) analysis revealed that a pond loach population in Ehime Prefecture (Shikoku Island, Japan) included two highly diverged mtDNA groups (Groups I and II). To examine the origin of these two distinct forms of mtDNA within the Japanese pond loach population, we performed phylogenetic analyses using sequences based on the mtDNA of cytochrome oxidase b (cyt b) and the nuclear DNA recombination activating gene 1 (RAG-1). We also conducted a random amplified polymorphic DNA (RAPD) analysis to examine the establishment of reproductive isolation between sympatric pond loaches with two different mtDNA groups. Our mtDNA phylogenetic results indicated that the two diverged pond loach mtDNA sequences showed polyphyletic relationships among Misgurnus species and its related genus Cobitis. In contrast, there were no clear divergence in nuclear DNA among the pond loaches irrespective of their mtDNA groups, and they all formed monomorphic clades in the phylogenetic relationships among the species. The discrepancy between the mtDNA and nuclear DNA genes support that the existence of two diverged forms of DNA within the pond loach population could be attributed to past mtDNA introgressions from other species rather than convergent evolution. Previous mtDNA phylogenetic studies among Cobitidae revealed that the dojo loach also consisted of two genetically diverged polyphyletic clades: an original Misgurnus mtDNA and an introgressed mtDNA from Cobitis species. In our mtDNA result, the Group II haplotype of the pond loach was included in the mtDNA from the introgressed dojo loach. This suggested that the Group II haplotype was derived from introgressed dojo loach mtDNA. The close relationships between the introgressed dojo loach and the pond loach mtDNA indicated that this secondary introgression had recently occurred via hybridization in a recent artificial aquaculture or transportation process. Common RAG-1 alleles and RAPD bands were shared between the sympatric pond loaches with original and introgressed mtDNAs. This indicates that the introgressed mtDNA haplotype is included as one of the polymorphic genotypes within the pond loach populations, and does not represent existence of different cryptic species.