Genetic structure in chloroplast and nuclear microsatellites in Rosa rugosa around sea straits in northern Japan

Sea‐strait geological history and seawater dispersal patterns can affect the genetic structure of coastal plants. We investigated microsatellite (simple sequence repeat, SSR) variations in chloroplast (cp) and nuclear (n) DNA of the coastal shrub R osa rugosa, native to northeastern Asia, and three species closely related to it. In northern Japan, we sampled R. rugosa along coasts around the Soya and Tsugaru straits with different histories of the Quaternary land formation as a result of different water depths. Both cpSSR and nSSR variations suggest rare hybridization between R . rugosa and the closely related species. Variations in one of two cpSSRs showed genetic differentiation between eastern and western Hokkaido. This genetic structure may result from introgression from R. davurica in eastern Hokkaido and/or isolation between the eastern and western coastlines of Hokkaido as a result of the Quaternary land formation in the Soya Strait. On the other hand, variations in 10 nSSRs were geographically homogeneous with weak isolation by distance along coastlines. These results suggest that extensive gene flow has been homogenizing the genetic structure of R . rugosa in northern Japan.     

Evolutionary and biogeographical implications of variation in skull morphology of raccoon dogs (Nyctereutes procyonoides,Mammalia: Carnivora)

Morphological characteristics reflect geographical variation resulting from adaptation to varying environmental conditions. Carnivore species distributed over a wide geographical range generally have highly polymorphic morphological variation. The raccoon dog (Nyctereutes procyonoides) has a longitudinal distribution restricted to East Asia and the northern Indochina Peninsula. Its unique geographical range makes it an appropriate model to examine how morphological differences are influenced by geography. To demonstrate morphological evolution of Russian, Chinese, Korean and Japanese raccoon dogs predicted by geographical differences, we tested the island rule and Bergmann's rule. We compared craniodental variation among populations and examined morphological implications for intraspecific taxonomic status. Insular raccoon dogs possessed substantially smaller body size than those from the mainland. Moreover, different island effects among Japanese islands were demonstrated by markedly larger occipital condyle breath in the Hokkaido population. Larger skull size in Russian and Hokkaido raccoon dogs could be explained by Bergmann's rule. Based on previous chromosomal and molecular studies and results of our morphological analyses, we suggest Japanese raccoon dogs are a distinct species from the mainland N. procyonoides.     

Genetic variation and differentiation of the two river sculpins,Cottus nozawae andC. amblystomopsis,deduced from allozyme and restriction enzyme-digested mtDNA fragment length polymorphism analyses

Genetic differentiation of the two sibling species,Cottus nozawae andC. amblystomopsis, from the northern part of Japan (Hokkaido Island and the Tohoku District) was investigated using allozyme variations and restriction fragment length polymorphisms of mitochondrial DNA. Although the two species are morphologically very similar, previously being thought to be a single species, they have different life-cycles;C. nozawae has a fluvial life-cycle with a small number of large-sized eggs, whereasC. amblystomopsis is an amphidromous species with a large number of small-sized eggs. Four populations ofC. amblystomopsis from Hokkaido Island and 24 populations ofC. nozawae (22 from Hokkaido Island and 2 from the Tohoku District) were sampled and examined Intrapopulational differentiation in the two species was measured by examining several indexes, including proportion of polymorphic loci (P), mean heterozygosity (H) and nucleotide diversity (π). All measurements were higher in theC. amblystomopsis populations, suggesting that intrapopulational variation inC. nozawae was less than inC. amblystomopsis and reflecting the difference in effective population sizes between them. Cluster analyses were performed using the UPGMA method, based on the data matrices of genetic distance (D) and the net nucleotide difference (δ) between populations. TheC. nozawae andC. amblystomopsis populations from Hokkaido Island composed a large cluster (Hokkaido group), while theC. nozawae populations from the Tohoku District composed a different cluster (Tohoku group). Bootstrap probabilities deduced from 1000 bootstrap replications for presence or absence of restriction sites showed that the mtDNA haplotypes detected within the Tohoku Group occurred in 99.9% of the bootstrap replicates outside the mtDNA haplotypes of the Hokkaido group, while those within the Hokkaido group occurred in 3.5–64.9% of bootstrap replicates. Consequently, the Hokkaido populations of the two species (Hokkaido group) were genetically close to each other, whileC. nozawae from the Tohoku District (Tohoku group) were distant from the Hokkaido group. These results suggest that the ancestral populations of the two species on Hokkaido Island shared the same gene pool, even after becoming geographically isolated from the ancestral population ofC. nozawae in the Tohoku District by the formation of the Tsugaru Straits.     

Prasiola (Prasiolales,Trebouxiophyceae) in Japan: a survey of freshwater populations and new records of marine taxa

Recent collections from marine and freshwater locations have enabled the investigation of diversity of Prasiola in Japan. Sequence data from the rbc L and tuf A markers revealed the presence of three marine species and one freshwater species. Prasiola delicata was confirmed to occur on Daikokujima, Prasiola calophylla was found for the first time in Japan from Hokkaido, and a species within the P. meridionalis/linearis/stipitata complex was found on both Hokkaido and Daikokujima. Collections from a range of populations of freshwater Prasiola, identified here as P. japonica, were found to be conspecific and identical in rbc L and tuf A sequences to freshwater collections from Nepal, Korea, and China.     

Further Analysis of Intraspecific Sequence Variation of Chloroplast DNA in Primula cuneifolia Ledeb. (Primulaceae): Implications for Biogeography of the Japanese Alpine Flora

Primula cuneifolia Ledeb. (Primulaceae), we analyzed intraspecific variation of the nucleotide sequences of non-coding regions of chloroplast DNA: the intergenic spacers between trnT (UGU) and the trnL (UAA) 5′exon, the trnL (UAA) 3′exon and trnF (GAA), and atpB and rbcL. In 20 populations of P. cuneifolia, 22 nucleotide substitutions and five insertions/deletions were inferred, and their genetic distances ranged from 0.001 to 0.008. Eight distinct haplotypes could be recognized and each haplotype was found to be geographically structured. Three major clades (the Northern, Hokkaido and Southern clades) were revealed in phylogenetic analyses of the haplotypes. The haplotypes of the Northern clade had a wider distribution area in the populations of Mt. Rausu and Rishiri Island of eastern and northern Hokkaido in Japan, northward to Unalaska Island in the Aleutians, and those of the Hokkaido clade were distributed in the populations of central Hokkaido and Mt. Iwaki of the northern Honshu in Japan; in addition, those of the Southern clade were observed only in the populations of the central Honshu. It was shown that the genetic diversifications of the Southern clade were higher than those of the Northern and Hokkaido clades. Furthermore, it was shown that the topology within the Southern clade was hierarchical, and the haplotypes of the Southern populations in the clade were derivative. From these results, we concluded that the cpDNA haplotypes of the three clades in P. cuneifolia arose and assumed the present distribution areas through several cycles of glacial advance and retreat in the Pleistocene. Received 24 June 1998/ Accepted in revised form 28 December 1998     

The population dynamics of the voleClethrionomys rufocanus in Hokkaido, Japan

Population dynamics of the gray sided-vole,Clethrionomys rufocanus, in Hokkaido, Japan were described on the basis of 225 time series (being from 12 to 31 years long); 194 of the time series have a length of 23 years or longer. The time series were classified into 11 groups according to geographic proximity and topographic characteristics of the island of Hokkaido. Mean abundance varied among populations from 1.07 to 21.07 individuals per 150 trap-nights. The index of variability for population fluctuation (s-index) ranged from 0.204 to 0.629. Another index for population variability (amplitude on log-10 scale) ranged from 0.811 to 2.743. Mean abundance and variability of populations were higher in the more northern and eastern regions of the island. Most populations, except for the southernmost populations, exhibited significant direct density-dependence in population growth. Detection rate for delayed density-dependence varied among groups from 0% to 22.6%. Both direct and delayed density-dependence tended to be stronger in the more northern and eastern populations. The proportion of cyclic populations was higher in the northern-eastern areas than that in the southern-western areas. There was a clear gradient from the asynchronous populations in southwest, to the highly synchronized populations in the northeast.     

Characterization of six microsatellite markers in Trillium camschatcense using a dual‐suppression‐polymerase chain reaction technique

Trillium camschatcense is a herbaceous perennial plant distributed in Hokkaido and northern Honshu, Japan. Geographical variations in the breeding system (partial selfing or obligate outcrossing) are reported in the populations of Hokkaido. We isolated six polymorphic microsatellite loci from this species. The number of allele per locus ranged from four to 12, whereas the expected heterozygosities ranged from 0.69 to 0.83. These markers may allow further investigations to reveal the evolutionary and ecological processes of mating system in T. camschatcense.     

Ground crickets singing in volcanic warm “islets” in snowy winter: Their seasonal life cycles,photoperiodic responses and origin

The ground cricket Dianemobius nigrofasciatus overwinters as an egg in Japan, being univoltine in Hokkaido and northern Honshu and bivoltine farther south. In Hokkaido, however, this cricket is heard singing in winter in several fumarolic fields covered with moss and grasses locally known as “bokke”. In such warm “islets” the adult density was high in early summer and again in autumn, indicating that the cricket is bivoltine in contrast to the univoltine life cycle outside the bokke habitats in Hokkaido. Eggs laid by females collected at regular intervals from a bokke habitat showed a clear seasonal cycle of diapause incidence. At 26°C, the bokke strains produced non‐diapause eggs under long days and diapause eggs under short days as in the southern bivoltine populations, although the critical day‐length was longer than in the south. Several strains derived from non‐bokke habitats in Hokkaido and northern Honshu produced high percentages of diapause eggs under long days as well as short days as expected for the univoltine life cycle. Winter adults singing in bokke habitats could be either survivors of the autumn generation or individuals derived from eggs laid in autumn and then matured in response to the high soil temperature. In the laboratory, the proportion of egg diapause in short days was decreased by selection only for several generations. Phylogenetic trees of bokke and non‐bokke populations, based on both the nucleotide sequence of the mitochondrial COI gene and four allozyme loci, suggest that bokke populations have not been isolated from non‐bokke populations for an evolutionarily significant time.     

Variation of sterility and fertility in Utricularia australis f. australis in Hokkaido, northern Japan

Seed-production ability was studied in Utricularia australis R. Br. f. australis Komiya and Shibata through field observation, pollination experiment, pollen culture, and isozyme analysis for populations in various regions of Hokkaido, northern Japan. Utricularia australis f. australis has previously been postulated to be sterile in Japan, however, in the present study, fertile populations were found in eastern Hokkaido. Bisexual sterility and male sterility were suspected to be present in populations in the western part of Hokkaido. There was also a strain that seemed to produce no seed because of intra-clonal inbreeding depression or self-incompatibility. Isozyme analysis of three enzyme systems indicated a uniformity of multienzymatic phenotype (MEP) in each population or region. The MEP grouping of populations corresponded to the type of sterility and fertility revealed by pollination and pollen culture experiments. These experiments suggested that each regional strain had its own sterility or fertility type.     

A taxonomic study of thePoa macrocalyx complex,with particular reference to the populations in eastern Hokkaido

An attempt was made to investigate some populations of thePoa macrocalyx complex in eastern Hokkaido in relation to the taxonomy of this complex. Individual specimens preserved in TI, KYO and TNS were also studied. Chromosome counts of 2n=42 were obtained in the majority of a total of 84 collections, and counts of 2n=63 (incl. ca. 63), ca. 70 and 84 were also made in a few collections. High percentages of viable pollen were observed. The morphological features which were designated by previous authors to be significant in separatingPoa sachalinensis fromP. macrocalyx and in discriminating between the varieties ofP. macrocalyx were critically examined with emphasis on the observations of infra-population variation. It was confirmed that (1) the populations of thePoa macrocalyx complex in eastern Hokkaido are greatly variable morphologically and are also in a dynamic state cytogenetically, (2) plants of this complex in eastern Hokkaido belong to one and the same species,P. macrocalyx, and the varietiesfallax, scabriflora andtatewakiana may be merely morphs, and (3) some plants which are somewhat different from the plants in eastern Hokkaido with respect to glume features are distributed in Sakhalin, and northern and central Hokkaido. Some problems which should be subjected to further studies are pointed out.     

Inter-populational difference in microsatellite–centromere map distances in the loach, <Emphasis Type="Italic">Misgurnus anguillicaudatus</Emphasis>

Microsatellite–centromere recombination rates were estimated at 21 loci in relation to centromere of chromosomes in gynogenetic diploid lines induced from loaches Misgurnus anguillicaudatus of two different populations in Japan. All the microsatellite loci gave allelic segregation according to the Mendelian manner of inheritance in normal diploid families. Since loaches from the Kita population in the southern area of Hokkaido Island and those from the Memanbetsu population in the northern area, Hokkaido, Japan, were reported by previous genetic studies to be genetically diversified, map distances were compared between loaches from the two different populations. Three (Mado7, Mac3 and Mac49) of five loci, which could be compared inter-populationally, gave significantly different recombination rates, i.e., map distances. The results support the presence of genetic difference between the two populations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Geographical variation in the cranial and external characters of the little tube-nosed bat,Murina silvatica in the Japanese archipelago

The distribution ofMurina silvatica (Yoshiyuki, 1983) in the Japanese archipelago extends over about 2000 km from north to south. Specimens were obtained from populations in Hokkaido and Yakushima, which are at the northern and southern ends of the range, and from two intermediate locations in Honshu. Measurements of cranial and external morphology were examined for evidence of geographical variation. The results of both multivariate analysis of variance and cluster analysis showed that there was no distinct cline in skull morphology among the Hokkaido, Tohoku and Chubu populations. However, the results of multivariate analysis of variance showed that all measures were significantly greater for the Yakushima population than for the others. Similarly, in a dendrogram of cluster analysis, the Yakushima population formed a cluster that was distinct from the other populations. However, as the difference between the Yakushima population and the other populations was less than the variation found within the Hokkaido, Tohoku and Chubu populations, morphological divergence of the Yakushima population was attributable to intraspecific variation. The island of Yakushima is the most isolated of the four locations and the morphological divergence of this population may be associated with its relative geographic isolation.     

Influence of northern limit range on genetic diversity and structure in a widespread North American tree,sugar maple (Acer saccharum Marshall)

Due to climate change, the ranges of many North American tree species are expected to shift northward. Sugar maple (Acer saccharum Marshall) reaches its northern continuous distributional limit in northeastern North America at the transition between boreal mixed‐wood and temperate deciduous forests. We hypothesized that marginal fragmented northern populations from the boreal mixed wood would have a distinct pattern of genetic structure and diversity. We analyzed variation at 18 microsatellite loci from 23 populations distributed along three latitudinal transects (west, central, and east) that encompass the continuous–discontinuous species range. Each transect was divided into two zones, continuous (temperate deciduous) and discontinuous (boreal mixed wood), based on sugar maple stand abundance. Respective positive and negative relationships were found between the distance of each population to the northern limit (D_north), and allelic richness (A_R) and population differentiation (F_ST). These relations were tested for each transect separately; the pattern (discontinuous–continuous) remained significant only for the western transect. structure analysis revealed the presence of four clusters. The most northern populations of each transect were assigned to a distinct group. Asymmetrical gene flow occurred from the southern into the four northernmost populations. Southern populations in Québec may have originated from two different postglacial migration routes. No evidence was found to validate the hypothesis that northern populations were remnants of a larger population that had migrated further north of the species range after the retreat of the ice sheet. The northernmost sugar maple populations possibly originated from long‐distance dispersal.     

Concordant clines and significant correlation between floral and pollen characters in Asarum heterotropoides var. heterotropoides (Aristolochiaceae)

To examine geographic differentiation in Asarum heterotropoides var. heterotropoides in Hokkaido Is. in the northern Japan, in which two putative cryptic species have been suspected to exist, extensive and detailed morphological research on 794 individuals from 44 populations throughout Hokkaido Is. was performed. Among the characters examined, the angle between and tip shape of the calyx lobes and the supratecta of the pollen grains were significantly correlated and were found in similar geographic clines. Among them, the pollen showed two discrete states in almost distinct distribution. Multidimensional scaling analysis showed that individuals within each of the two pollen types had different trends in flower characters. Consequently, we assumed a cline from south to north on Hokkaido Is. For the causes of the cline, the two hypotheses were proposed, primary geographic differentiation or extensive introgressive hybridization between two distinct geographical species existing in the past.     

Contrasting patterns of nuclear microsatellite genetic structure of Fraxinus mandshurica var. japonica between northern and southern populations in Japan

Aim The aim of this study is to detect extant patterns of population genetic structure of Fraxinus mandshurica var. japonica in Japan, and to provide insights into the post‐glacial history of this species during the Holocene. Location Hokkaido and Honshu islands, Japan (including the Oshima and Shimokita peninsulas). Methods We examined nine polymorphic nuclear microsatellite loci to assess genetic variation within and among 15 populations across almost the entire range of the species in Japan. Extant patterns of geographical structure were analysed using Bayesian clustering, Monmonier’s algorithm, analysis of molecular variance, Mantel tests and principal coordinates analysis. Recent bottlenecks within populations and regional genetic variation were also assessed. Results Northern populations (Hokkaido Island and the Shimokita Peninsula) formed a single homogeneous deme, maintaining the highest level of allelic diversity on the Oshima Peninsula. By contrast, southern populations (Honshu Island) demonstrated strong substructure on both coasts. Specifically, populations on the Pacific side of Honshu exhibited significant bottlenecks and erosion of allelic diversity but preserved distinct subclusters diverging from widespread subclusters on the Japan Sea side of this island. Main conclusions Genetic evidence and life history traits suggest that F. mandshurica occupied cryptic northern refugia on the Oshima Peninsula during the Last Glacial Maximum, which is reflected in the species’ extant northern distribution. Strong geographical structure in southern populations, in agreement with fossil pollen records, suggests geographical isolation by mountain ranges running north–south along Honshu. Given that this tree species is cold‐adapted and found in riparian habitats, populations on the Pacific side of Honshu probably contracted into higher‐elevation swamps during warm post‐glacial periods, leading to a reduction of effective population sizes and rare allelic richness.     

Glacial refugia and postglacial expansion of the alpine–prealpine plant species Polygala chamaebuxus

The shrubby milkwort (Polygala chamaebuxus L.) is widely distributed in the Alps, but occurs also in the lower mountain ranges of Central Europe such as the Franconian Jura or the Bohemian uplands. Populations in these regions may either originate from glacial survival or from postglacial recolonization. In this study, we analyzed 30 populations of P. chamaebuxus from the whole distribution range using AFLP (Amplified Fragment Length Polymorphism) analysis to identify glacial refugia and to illuminate the origin of P. chamaebuxus in the lower mountain ranges of Central Europe. Genetic variation and the number of rare fragments within populations were highest in populations from the central part of the distribution range, especially in the Southern Alps (from the Tessin Alps and the Prealps of Lugano to the Triglav Massiv) and in the middle part of the northern Alps. These regions may have served, in accordance with previous studies, as long‐term refugia for the glacial survival of the species. The geographic pattern of genetic variation, as revealed by analysis of molecular variance, Bayesian cluster analysis and a PopGraph genetic network was, however, only weak. Instead of postglacial recolonization from only few long‐term refugia, which would have resulted in deeper genetic splits within the data set, broad waves of postglacial expansion from several short‐term isolated populations in the center to the actual periphery of the distribution range seem to be the scenario explaining the observed pattern of genetic variation most likely. The populations from the lower mountain ranges in Central Europe were more closely related to the populations from the southwestern and northern than from the nearby eastern Alps. Although glacial survival in the Bohemian uplands cannot fully be excluded, P. chamaebuxus seems to have immigrated postglacially from the southwestern or central‐northern parts of the Alps into these regions during the expansion of the pine forests in the early Holocene.     

Genetic structure of Japanese populations of an ambrosia beetle,Xylosandrus germanus (Curculionidae: Scolytinae)

We examined the genetic structures of 13 Japanese populations of an ambrosia beetle, Xylosandrus germanus (Curculionidae: Scolytinae), to understand the effects of geographical barriers on the colonization dynamics of this species. The genetic structure was studied using portions of the mitochondrial cytochrome oxidase I (COI) gene. A phylogenetic analysis revealed three distinct lineages (clades A, B and C) within X. germanus. Clade A contained 21 haplotypes from all 13 populations; whereas clade B contained eight haplotypes from Hokkaido (Sapporo and Furano), Iwate and Nagano populations; and clade C contained only a single a haplotype from the Hokkaido (Furano) population. In the analysis of molecular variance (amova ), the greatest amount of genetic variation was detected between populations in Hokkaido and those in Honshu and other southern islands. Between these two groups of populations, all the values of the coefficient of gene differentiation were significantly larger than zero, except for the Hokkaido (Sapporo) versus Nagano comparison. Our results confirm that for X. germanus, gene flow has been interrupted between Hokkaido and Honshu since the last glacial maximum.     

Phylogeography of a freshwater sculpin, Cottus nozawae, from the northeastern part of Honshu Island, Japan

 The fluvial sculpin, Cottus nozawae, is a coldwater-adapted fish distributed in Hokkaido Island and the northeastern part of Honshu Island (Tohoku District), Japan. Mitochondrial DNA (mtDNA) control region sequencing was used to investigate the geographic distribution of genetic variation and phylogeography of C. nozawae. Most populations possessed unique haplotypes, few being shared across river systems. Phylogenetic analysis of the sequences of the mtDNA control region and adjacent regions of C. nozawae revealed three distinct phylogenetic groups that differed by 3.05% to 3.11%, corresponding to distinct geographic regions, Hokkaido Island, northern Tohoku District, and Yamagata Prefecture (southwestern Tohoku District), respectively. The divergence times of three groups were estimated to be about 1.5 million years ago by applying a general rate for mtDNA, suggesting that the divergence among them might have occurred in the early Pleistocene. Divergence among the haplotypes within the group from the northern Tohoku District was also high (1.84%), no haplotypes being shared by local populations in different river systems in this region. Local populations from a single river system in this region comprise a distinct lineage that differed from other river systems. Such genetically divergent population structures among the different regions and river systems are considered to have resulted mainly from long-term isolation and restricted gene flow among river systems, probably promoted by the fluvial benthic life history and low dispersal ability of this species. Received: April 12, 2001 / Revised: December 1, 2001 / Accepted: December 19, 2001     

The occurrence and run timing of naturally spawning chum salmon in northern Japan

Since the late 20th century, the biomass of Pacific salmon Oncorhynchus spp. has increased. Hokkaido, northern Japan, is one of the main areas of chum salmon O. keta production in the North Pacific and intensive hatchery programs support the recent high abundance. However, proper management of naturally spawning populations is necessary to conserve healthy stocks of this species. In 2008, we started a program to assess the naturally spawning chum salmon populations in Hokkaido. Of the total of approximately 1,500 rivers in Hokkaido, 238 rivers with lengths of longer than 8 km (excluding those rivers used for hatchery broodstock collection) were surveyed in 2008 and 2009. The number of non-enhanced rivers found to contain naturally reproducing chum salmon was 59 (31.4% of surveyed rivers) and 50 (37.6% of surveyed rivers) rivers in 2008 and 2009, respectively. Including the rivers where hatchery broodstock were collected and rivers shorter than 8 km that contain naturally spawning chum salmon, chum salmon ascended at least 191 and 175 rivers in Hokkaido in 2008 and 2009, respectively. Repeated foot surveys indicated that the run timings of naturally spawning chum salmon may be affected by coastal commercial fisheries. This study showed that naturally spawning chum salmon remain in many rivers in Hokkaido where hatchery programs have been intensively conducted.     

The Arctic Warbler Phylloscopus borealis– three anciently separated cryptic species revealed

The Arctic Warbler Phylloscopus borealis breeds across the northern Palaearctic and northwestern‐most Nearctic, from northern Scandinavia to Alaska, extending south to southern Japan, and winters in Southeast Asia, the Philippines and Indonesia. Several subspecies have been described based on subtle morphological characteristics, although the taxonomy varies considerably among different authors. A recent study (T. Saitoh et al. (2010) BMC Evol. Biol. 10 : 35) identified three main mitochondrial DNA clades, corresponding to: (1) continental Eurasia and Alaska, (2) south Kamchatka, Sakhalin and northeast Hokkaido, and (3) most of Japan (Honshu, Shikoku, Kyushu). These three clades were estimated to have diverged during the late Pliocene to early Pleistocene (border at c. 2.6 million years ago). Differences in morphometrics have also been reported among members of the three clades (T. Saitoh et al. (2008) Ornithol. Sci. 7 : 135–142). Here we analyse songs and calls from throughout the range of the Arctic Warbler, and conclude that these differ markedly and consistently among the populations representing the three mitochondrial clades. Kurile populations, for which no sequence data are available, are shown to belong to the second clade. To determine the correct application of available scientific names, mitochondrial DNA was sequenced from three name‐bearing type specimens collected on migration or in the winter quarters. Based on the congruent variation in mitochondrial DNA, morphology and vocalizations, we propose that three species be recognized: Arctic Warbler Phylloscopus borealis (sensu stricto) (continental Eurasia and Alaska), Kamchatka Leaf Warbler Phylloscopus examinandus (Kamchatka (at least the southern part), Sakhalin, Hokkaido and Kurile Islands), and Japanese Leaf Warbler Phylloscopus xanthodryas (Japan except Hokkaido).