House mouse Mus musculus dispersal in East Eurasia inferred from 98 newly determined complete mitochondrial genome sequences.

The Eurasian house mouse Mus musculus is useful for tracing prehistorical human movement related to the spread of farming. We determined whole mitochondrial DNA (mtDNA) sequences (ca. 16,000 bp) of 98 wild-derived individuals of two subspecies, M. m. musculus (MUS) and M. m. castaneus (CAS). We revealed directional dispersals reaching as far as the Japanese Archipelago from their homelands. Our phylogenetic analysis indicated that the eastward movement of MUS was characterised by five step-wise regional extension events: (1) broad spatial expansion into eastern Europe and the western part of western China, (2) dispersal to the eastern part of western China, (3) dispersal to northern China, (4) dispersal to the Korean Peninsula and (5) colonisation and expansion in the Japanese Archipelago. These events were estimated to have occurred during the last 2000-18,000 years. The dispersal of CAS was characterised by three events: initial divergences (ca. 7000-9000 years ago) of haplogroups in northernmost China and the eastern coast of India, followed by two population expansion events that likely originated from the Yangtze River basin to broad areas of South and Southeast Asia, including Sri Lanka, Bangladesh and Indonesia (ca. 4000-6000 years ago) and to Yunnan, southern China and the Japanese Archipelago (ca. 2000-3500). This study provides a solid framework for the spatiotemporal movement of the human-associated organisms in Holocene Eastern Eurasia using whole mtDNA sequences, reliable evolutionary rates and accurate branching patterns. The information obtained here contributes to the analysis of a variety of animals and plants associated with prehistoric human migration.

Cryptic Speciation of the Oriental Greenfinch Chloris sinica on Oceanic Islands.

The Oriental greenfinch, Chloris sinica, is a small seed-eating finch that breeds in the eastern Palearctic region, an area that spans from Russia in the east to China, Korea, and Japan in the south and southwest. Several subspecies have been described based on subtle morphological characteristics, although the taxonomy varies among different authors. Although many ecological studies have been performed, there has been no phylogenetic study that encompasses the species' entire geographical range. We used four regions of mitochondrial DNA to analyze the intraspecies genetic phylogeny and diversity of the Oriental greenfinch. In addition, we performed morphometric analyses using museum specimens. Genetic analysis identified two clades that diverged approximately 1.06 million years ago. These were a population from the Ogasawara Islands, Japan (subspecies kittlitzi, Clade B), and the other populations (Clade A, which could not be subdivided according to geographic context). Morphometric analyses showed that the population on the Kuril Islands (subspecies kawarahiba) had the longest mean wing length, whereas C. s. kittlitzi had the shortest wings. Chloris s. kittlitzi also had the longest mean bill length, probably because it has adapted to feeding on the Ogasawara Islands. Based on molecular phylogeny and morphology analyses, we recommend that C. s. kittlitzi should be treated as a completely distinct species, called the Ogasawara greenfinch, Chloris kittlitzi. It is critically endangered and needs to be specially protected.

Contrasting phylogeographic histories between the continent and islands of East Asia: Massive mitochondrial introgression and long-term isolation of hares (Lagomorpha: Lepus).

Hares of the genus Lepus are distributed worldwide, and introgressive hybridization is thought to be pervasive among species, leading to reticulate evolution and taxonomic confusion. Here, we performed phylogeographic analyses of the following species of hare across East Asia: L. timidus, L. mandshuricus, L. coreanus, and L. brachyurus collected from far-eastern Russia, South Korea, and Japan. Nucleotide sequences of one mitochondrial DNA and eight nuclear gene loci were examined, adding sequences of hares in China from databases. All nuclear DNA analyses supported the clear separation of three phylogroups: L. timidus, L. brachyurus, and the L. mandshuricus complex containing L. coreanus. On the other hand, massive mitochondrial introgression from two L. timidus lineages to the L. mandshuricus complex was suggested in continental East Asia. The northern population of the L. mandshuricus complex was mainly associated with introgression from the continental lineage of L. timidus, possibly since the last glacial period, whereas the southern population of the L. mandshuricus complex experienced introgression from another L. timidus lineage related to the Hokkaido population, possibly before the last glacial period. In contrast to continental hares, no evidence of introgression was found in L. brachyurus in the Japanese Archipelago, which showed the oldest divergence amongst East Asian hare lineages. Our findings suggest that glacial-interglacial climate changes in the circum-Japan Sea region promoted distribution shifts and introgressive hybridization among continental hare species, while the geographic structure of the region contributed to long-term isolation of hares on the islands, preventing inter-species gene flow.

Evolution of MHC class I genes in Eurasian badgers, genus Meles (Carnivora, Mustelidae).

Because of their role in immune defense against pathogens, major histocompatibility complex (MHC) genes are useful in evolutionary studies on how wild vertebrates adapt to their environments. We investigated the molecular evolution of MHC class I (MHCI) genes in four closely related species of Eurasian badgers, genus Meles. All four species of badgers showed similarly high variation in MHCI sequences compared to other Carnivora. We identified 7-21 putatively functional MHCI sequences in each of the badger species, and 2-7 sequences per individual, indicating the existence of 1-4 loci. MHCI exon 2 and 3 sequences encoding domains α1 and α2 exhibited different clade topologies in phylogenetic networks. Non-synonymous nucleotide substitutions at codons for antigen-binding sites exceeded synonymous substitutions for domain α1 but not for domain α2, suggesting that the domains α1 and α2 likely had different evolutionary histories in these species. Positive selection and recombination seem to have shaped the variation in domain α2, whereas positive selection was dominant in shaping the variation in domain α1. In the separate phylogenetic analyses for exon 2, exon 3, and intron 2, each showed three clades of Meles alleles, with rampant trans-species polymorphism, indicative of the long-term maintenance of ancestral MHCI polymorphism by balancing selection.

Deep Phylogeographic Breaks in Magpie Pica pica Across the Holarctic: Concordance with Bioacoustics and Phenotypes.

We examined sequences of the mitochondrial control region in magpies (Pica pica) from the entire distribution range and found deep genetic splits into four major lineages: (1) group West (Europe-Siberia), (2) group East (southern Far East), (3) P. p. mauritanica (North Africa), and (4) P. p. hudsonia (North America). These lineages show a geographic pattern corresponding to known subspecies or subspecies groups. Genetic variation within the widely-distributed group West is low and neutrality tests supported a recent expansion scenario. The haplotypes from Kamchatka, representing a separated sublineage with clear affinity to the European-Siberian group, are almost identical, implying a recent bottleneck. Group East contained two subclades without clear geographic pattern, presumably due to admixing of populations that had diverged in Pleistocene refuges. The homogeneity of the Kyushu population supports historical reports of introduction of the species from Korea. In contrast, the high variation in the recently established Hokkaido population may reflect an ongoing invasion from several populations of the Far Eastern mainland. Bioacoustic data based on chatter call differentiate groups of subspecies and reflect phylogeographic patterns, i.e., mitochondrial lineages. Furthermore, we report the fast spreading of P. p. jankowskii towards the west along the upper Amur River, and a slower shifting of P. p. leucoptera in the opposite direction thus yielding a new contact zone. Overall, our data support a scenario of divergence in geographic isolation, but the ongoing expansion of distribution ranges may lead to major changes in phylogeographic patterns.

Spatial and temporal aspects of occurrence of Mogera species in the Japanese islands inferred from mitochondrial and nuclear gene sequences.

We assessed dispersal and vicariant events in four species of Japanese moles in the genera Mogera and Euroscaptor to better understand the factors shaping intra- and interspecific differentiation in Japanese moles. We used the combined viewpoints of molecular phylogeny and historical geology using nucleotide sequences of mitochondrial (cytochrome b; Cytb) and nuclear (A2ab, Bmp4, Tcf25, vWf) genes. The divergence times estimated from the molecular data were verified with available geological data on the chronology of fluctuations in sea level in the Korea Strait, assuming sequential migration and speciation events. This produced possible migration times of 5.6, 3.5, 2.4, and 1.3 million years ago for four species of Japanese moles, Euroscaptor mizura, Mogera tokudae, M. imaizumii, and M. wogura, respectively. For the western Japanese mole M. wogura, Cytb sequences revealed four major phylogroups with strong geographic affinities in southwestern Central Honshu (I), western Honshu/Shikoku (II), Kyushu/westernmost Honshu (III), and Korea/Russian Primorye (IV). The nuclear gene sequences supported the distinctiveness of phylogroups I and IV, indicating long, independent evolutionary histories. In contrast, phylogroups II and III were merged into a single geographic group based on the nuclear gene data. Intraspecific divergences in M. imaizumii and M. tokudae were rather apparent in Cytb but not in nuclear gene sequences. The results suggest that repeated dispersal events have occurred between the Asian continent and the Japanese Islands, and intensive vicariant events associated with abiotic and biotic factors have created higher levels of species and genetic diversities in moles occurring on the Japanese Islands.

Insights into Korean red fox (Vulpes vulpes) based on mitochondrial cytochrome b sequence variation in East Asia.

The red fox (Vulpes vulpes) is the most widely distributed terrestrial carnivore in the world, occurring throughout most of North America, Europe, Asia, and North Africa. In South Korea, however, this species has been drastically reduced due to habitat loss and poaching. Consequently, it is classified as an endangered species in Korea. As a first step of a planned red fox restoration project, preserved red fox museum specimens were used to determine the genetic status of red foxes that had previously inhabited South Korea against red foxes from neighboring countries. Total eighty three mtDNA cytochrome b sequences, including 22 newly obtained East Asian red fox sequences and worldwide red fox sequences from NCBI, were clustered into three clades (i.e., I, II, and III) based on haplotype network and neighbor-joining trees. The mean genetic distance between clades was 2.0%. Clade III contained South Korean and other East Asian samples in addition to Eurasian and North Pacific individuals. In clade III, South Korean individuals were separated into two lineages of Eurasian and North Pacific groups, showing unclear phylogeographic structuring and admixture. This suggests that South Korean red fox populations may have been composed of individuals from these two different genetic lineages.

Speciation and population divergence in a mutualistic seed dispersing bird.

Bird-mediated seed dispersal is crucial for the regeneration and viability of ecosystems, often resulting in complex mutualistic species networks. Yet, how this mutualism drives the evolution of seed dispersing birds is still poorly understood. In the present study we combine whole genome re-sequencing analyses and morphometric data to assess the evolutionary processes that shaped the diversification of the Eurasian nutcracker (Nucifraga), a seed disperser known for its mutualism with pines (Pinus). Our results show that the divergence and phylogeographic patterns of nutcrackers resemble those of other non-mutualistic passerine birds and suggest that their early diversification was shaped by similar biogeographic and climatic processes. The limited variation in foraging traits indicates that local adaptation to pines likely played a minor role. Our study shows that close mutualistic relationships between bird and plant species might not necessarily act as a primary driver of evolution and diversification in resource-specialized birds.

Phylogeography of ancient and modern brown bears from eastern Eurasia.

The brown bear (Ursus arctos) is an iconic carnivoran species of the Northern Hemisphere. Its population history has been studied extensively using mitochondrial markers, which demonstrated signatures of multiple waves of migration, arguably connected with glaciation periods. Among Eurasian brown bears, Siberian populations remain understudied. We have sequenced complete mitochondrial genomes of four ancient (~4.5-40 kya) bears from South Siberia and 19 modern bears from South Siberia and the Russian Far East. Reconstruction of phylogenetic relationships between haplotypes and evaluation of modern population structure have demonstrated that all the studied samples belong to the most widespread Eurasian clade 3. One of the ancient haplotypes takes a basal position relative to the whole of clade 3; the second is basal to the haplogroup 3a (the most common subclade), and two others belong to clades 3a1 and 3b. Modern Siberian bears retain at least some of this diversity; apart from the most common haplogroup 3a, we demonstrate the presence of clade 3b, which was previously found mainly in mainland Eurasia and Northern Japan. Our findings highlight the importance of South Siberia as a refugium for northern Eurasian brown bears and further corroborate the hypothesis of several waves of migration in the Pleistocene.

Migration-tracking integrated phylogeography supports long-distance dispersal-driven divergence for a migratory bird species in the Japanese archipelago.

Long-distance dispersal (LDD) outside a species' breeding range contributes to genetic divergence. Previous phylogeographic studies of migratory bird species have not discriminated LDD from vicariant speciation in their diversification process. We conducted an integrative phylogeographic approach to test the LDD hypothesis, which predicts that a Japanese migratory bird subspecies diverged from a population in the coastal region of the East China Sea (CRECS) via LDD over the East China Sea (ECS). Haplotype networks of both mitochondrial and nuclear genes of its three subspecies were reconstructed to examine whether the Japanese subspecies of the Brown Shrike (Lanius cristatus superciliosus) diverged from an ancestral CRECS population. A species distribution model (SDM) for the Japanese subspecies was constructed using bioclimatic variables under the maximum entropy algorithm. It was projected backwards to the climate of the last glacial maximum (LGM) to infer the candidate source area of colonization. A migratory route of L. c. superciliosus, which possibly reflects a candidate past colonization route, was tracked by light-level geolocators. Molecular phylogenetic networks suggest that the Japanese subspecies diverged from a population in the CRECS and maintained anciently diverged haplotypes. The SDM inferred that the emerged continental shelf of the ECS and the present CRECS were suitable breeding areas for the Japanese subspecies during the LGM. A major migratory route for L. c. superciliosus was inferred between the CRECS and the Japanese archipelago across the ECS. Our integrative approach supported the LDD hypothesis for divergence of the Japanese subspecies of the Brown Shrike. Shrinkage of the ECS may have been responsible for successful population establishment, due to a sufficient number of migrants overshooting to the Japanese archipelago from the CRECS. Our framework provides a new phylogeographic scenario for this region. Discriminating LDD and vicariance models helps improve our understanding of the phylogeographic histories of migratory species.

Influence of Quaternary environmental changes on mole populations inferred from mitochondrial sequences and evolutionary rate estimation.

Quaternary environmental changes fundamentally influenced the genetic diversity of temperate-zone terrestrial animals, including those in the Japanese Archipelago. The genetic diversity of present-day populations is taxon- and region-specific, but its determinants are poorly understood. Here, we analyzed cytochrome b gene (Cytb) sequences (1140 bp) of mitochondrial DNA (mtDNA) to elucidate the factors determining the genetic variation in three species of large moles: Mogera imaizumii and Mogera wogura, which occur in central and southern mainland Japan (Honshu, Shikoku, and Kyushu), and Mogera robusta, which occurs on the nearby Asian continent. Network construction with the Cytb sequences revealed 10 star-shaped clusters with apparent geographic affinity. Mismatch distribution analysis showed that modes of pairwise nucleotide differences (τ values) were grouped into five classes in terms of the level, implying the occurrence of five stages for rapid expansion. It is conceivable that severe cold periods and subsequent warm periods during the late Quaternary were responsible for the population expansion events. The first and third oldest events included island-derived haplotypes, indicative of the involvement of land bridge formation between remote islands, hence suggesting an association of the ends of the penultimate (PGM, ca. 130,000 years ago) and last (LGM, ca. 15,000 years ago) glacial maxima, respectively. Since the third event was followed by the fourth, it is plausible that the termination of the Younger Dryas and subsequent abrupt warming ca. 11,500 years ago facilitated the fourth expansion event. The second event most likely corresponded to early marine isotope stage (MIS) 3 (ca. 53,000 years ago) when the glaciation and subsequent warming period were predicted to have influenced biodiversity. Utilization of the critical times of 130,000, 53,000, 15,000, and 11,500 years ago as calibration points yielded evolutionary rates of 0.03, 0.045, 0.10 and 0.10 substitutions/site/million years, respectively, showing a time-dependent manner whose pattern was similar to that seen in small rodents reported in our previous studies. The age of the fifth expansion event was calculated to be 5800 years ago with a rate of 0.10 substitutions/site/million years ago during the mid-Holocene, suggestive of the influence of humans or other unspecified reasons, such as the Jomon marine transgression.

Complete mitochondrial genomes of five subspecies of the Eurasian magpie Pica pica, obtained with Oxford Nanopore MinION, and their interpretation regarding intraspecific taxonomy.

The complete mitochondrial (mt) genomes of five subspecies of the Eurasian (Common) magpie Pica pica were determined for the first time. Lengths of the circular genomes comprise 13 protein-coding genes, two rRNA genes (for 12S rRNA and 16S rRNA), 22 tRNA genes, and the non-coding control region (CR). Gene content and lengths of the genomes (16,936-16,945 bp) are similar to typical vertebrate mt genomes. The subspecies studied differs by several single substitutions and indels, especially in the CR. The phylogenetic tree based on complete mt genomes shows a deep divergence of the two groups of subspecies which supports the proposed division into two distinct species: P. pica and P. serica.

Evolution of heterogeneous genome differentiation across multiple contact zones in a crow species complex.

Uncovering the genetic basis of species diversification is a central goal in evolutionary biology. Yet, the link between the accumulation of genomic changes during population divergence and the evolutionary forces promoting reproductive isolation is poorly understood. Here, we analysed 124 genomes of crow populations with various degrees of genome-wide differentiation, with parallelism of a sexually selected plumage phenotype, and ongoing hybridization. Overall, heterogeneity in genetic differentiation along the genome was best explained by linked selection exposed on a shared genome architecture. Superimposed on this common background, we identified genomic regions with signatures of selection specific to independent phenotypic contact zones. Candidate pigmentation genes with evidence for divergent selection were only partly shared, suggesting context-dependent selection on a multigenic trait architecture and parallelism by pathway rather than by repeated single-gene effects. This study provides insight into how various forms of selection shape genome-wide patterns of genomic differentiation as populations diverge.