Origin and diversification of pheretimoid megascolecid earthworms in the Japanese Archipelago as revealed by mitogenomic phylogenetics.

Megascolecid earthworms of the pheretimoid group are dominant detritivores of soil ecosystems in the Japanese Archipelago and East Asia. However, their diversity and phylogenetic relationships are poorly understood. We assembled whole mitogenome sequences for 197 megascolecid earthworms collected throughout Japan to study the phylogenetic relationships, phylogeography, divergence times, and diversification of important morphological characteristics among pheretimoid earthworms. Using 197 mitogenome sequences and 24 published mitogenome sequences from the East Asian mainland (221 sequences in total), we constructed a maximum likelihood tree and found that the pheretimoid earthworms currently assigned to Amynthas, Metaphire, Duplodicodrilus, and Manus are involved in the most senior genus Amynthas; thus, Amynthas can be treated as the sole genus encompassing all of the above genera. Within the Amynthas group, we identified three major lineages that led to four groups of endemic species in Japan. These lineages originated from different lineages on the East Asian mainland and Taiwan Island, indicating multiple colonization events from the East Asian mainland by different ancestral lineages, possibly after the Miocene. We also assembled nuclear ribosomal DNA sequences encompassing the 18S to 28S rRNA genes. The nuclear gene tree showed major groups consistent with the mitogenome tree except for different (and not well-resolved) relationships among major clades. Our molecular data covered 115-158 native and 7 non-native Amynthas group species in Japan in terms of DNA-based species delimitation. Our findings provide a basis for understanding the evolutionary relationships among diversified megascolecid earthworms in the Amynthas group in Japan and adjacent regions.

Reproductive isolation via divergent genital morphology due to cascade reinforcement in Ohomopterus ground beetles.

Secondary contact between incipient species and selection against maladaptive hybridization can drive reinforcement between populations in contact and result in reproductive character displacement (RCD). Resultant divergence in mating traits within a species may generate downstream reproductive isolation between populations with displaced and non-displaced traits, referred to as the cascade reinforcement hypothesis. We examined this hypothesis using three allopatric populations of the ground beetle Carabus maiyasanus with a genital lock-and-key system. This species shows RCD in male and female genital morphologies in populations in contact with the sister species C. iwawakianus. In a reciprocal mating experiment using three allopatric populations with differences in male and female genital sizes, insemination failure increased as the difference in genital size increased. Based on the reproductive isolation index, insemination failure was the major postmating-prezygotic isolation barrier, at least in one population pair with comparable total isolation to those of other species pairs. By contrast, there was only incomplete premating isolation among populations. These results suggest that RCD in genital morphologies drives incipient allopatric speciation, supporting the cascade reinforcement hypothesis. These findings provide insight into the roles of interspecific interactions and subsequent trait diversification in speciation processes.

Reproductive Character Displacement in Genital Morphology in Ohomopterus Ground Beetles.

AbstractGenital morphology reveals rapid diversification among species, and species-specific divergence in genital morphology may result in reproductive isolation and promote speciation. Natural selection against maladaptive hybridization may cause species-specific genital divergence. In this context, divergence in mating traits is expected to be greater between sympatric populations than between allopatric populations in a pair of species, known as reproductive character displacement (RCD). However, there are few examples of RCD in the genital morphology of closely related species. Additionally, processes leading to RCD have rarely been inferred. In this study, we examined RCD and its underlying mechanisms by focusing on species-specific genital morphologies of closely related Ohomopterus ground beetle species. A morphological analysis showed patterns of RCD in species-specific genital parts in both sexes. Interspecific hybridization was confirmed by a mate choice experiment and by a population genetic analysis indicating extensive interspecific gene flow, suggesting that reinforcement is the most plausible process underlying the observed RCD. We found variation in the degree of displacement in contact zones, which may correspond with the ongoing process of genital evolution and speciation. Our results provide support for the lock-and-key hypothesis of genital evolution in closely related Ohomopterus species.

The complete mitochondrial genomes of two Japanese endemic Satyrinae butterflies, Neope goschkevitschii and Lethe sicelis (Lepidoptera, Nymphalidae).

Neope goschkevitschii and Lethe sicelis are endemic Satyrinae butterflies in mainland Japan, which belongs to the Palearctic realm. In this study, we determined the mitochondrial genomes of these two species. The total length of the mitochondrial genome was 15,286 bp and 15,196 bp for N. goschkevitschii and L. sicelis, respectively, and both mitochondrial genomes were extremely AT-rich. Phylogenetic analysis revealed each of these species was closely related to a member of the same genus, respectively.

Complementary molecular methods reveal comprehensive phylogenetic diversity integrating inconspicuous lineages of early-diverged wood-decaying mushrooms.

Among terrestrial microorganisms, mushroom-forming fungi have been relatively well investigated, however the inconspicuous strains may be overlooked by conventional visual investigations causing underestimation of their phylogenetic diversity. Herein, we sought to obtain a comprehensive phylogenetic diversity profile for the early-diverging wood-decaying mushrooms Dacrymycetes, using an approach that combines fruiting-body collection, culture isolation, and environmental DNA (eDNA) metabarcoding of decaying branches. Among the 28 operational taxonomic units (OTUs) detected during a three-year investigation, 10 each were from fruiting bodies and cultured mycelia and 27 were detected as eDNA sequences. eDNA metabarcoding revealed various lineages across the Dacrymycetes phylogeny. Alternatively, fruiting-body and culture surveys uncovered only ~50% of the OTUs detected through eDNA metabarcoding, suggesting that several inconspicuous or difficult-to-isolate strains are latent in the environment. Further, eDNA and culture surveys revealed early-diverging clades that were not identified in the fruiting-body survey. Thus, eDNA and culture-based techniques can uncover inconspicuous yet phylogenetically important mushroom lineages that may otherwise be overlooked via typical visual investigations.

Genetic basis of species-specific genitalia reveals role in species diversification.

The diversity of genital morphology among closely related animals with internal fertilization is well known, but the genetic backgrounds are unclear. Here, we show that, in Carabus (Ohomopterus) beetles showing correlated evolution of male and female genital parts, only a few major quantitative trait loci (QTLs) determine differences in genital dimensions between sister species, and sequence divergence is pronounced in the genomic regions containing genital QTLs. The major QTLs for male and female genital dimensions reside in different locations within the same linkage group, implying that coevolution between the sexes is only loosely constrained and can respond to sexually antagonistic selection. The same genomic regions containing the major QTLs show elevated divergence between three pairs of parapatric species with marked differences in genital parts. Our study demonstrates that species diversification can follow coevolution of genitalia between the sexes, even without tight linkage of loci affecting male and female genital dimensions.

The complete mitochondrial genome of the critically endangered diving beetle Dytiscus sharpi (Coleoptera: Dytiscidae).

The diving beetles Dytiscus sharpi is one of the most critically endangered species in Japan, caused by excessive capture and destruction of their habitats. Here, the complete mitochondrial genome sequence (mitogenome) of D. sharpi is described. The entire mitogenome sequence was 19,476 bp long, containing 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and 1 control region. The average A + T content of mitogenome was 81.3%. This is the first description of a complete mitochondrial genome of Dytiscus, and the second of Dytiscinae. The primary genetic data obtained in this study is expected to contribute to conservation genetic studies using various genetic analysis methods, including environmental DNA.

The complete mitochondrial genomes of two endangered bitterling Acheilognathus tabira tohokuensis and A. tabira erythropterus (Cyprinidae, Acheilognathinae).

Acheilognathus tabira (tabira bitterling) comprises of 5 subspecies, all of which are endangered. In this study, the mitochondrial genome (mitogenome) of the 2 subspecies, A. tabira tohokuensis and A. tabira erythropterus, whose mitogenomes have not been reported previously, was determined. The total lengths of A. tabira tohokuensis and A. tabira erythropterus mitogenomes were 16,774 bp and 16,770 bp, respectively, and were noted as slightly AT-rich. Phylogenetic analysis revealed that these 2 subspecies of A. tabira were the most closely related, out of the 5 subspecies. The deciphered mitogenomes would be useful for conservation and evolutionary studies.

Phylogeography of Endangered Bitterling Acheilognathus melanogaster Endemic to Eastern Japan.

The bitterling Acheilognathus melanogaster is a critically endangered primary freshwater fish endemic to the Pacific side of eastern Japan. To elucidate A. melanogaster genetic structure, we investigated phylogeography in nine populations, using gene sequences of mitochondrial Cytochrome b (Cytb), as well as nuclear Rhodopsin (Rho) and glycosyltransferase (Glyt). We found four Cytb-based geographical clusters unevenly divided between the northern and southern regions, with smaller groups in the south. Of the nuclear genes, Glyt did not show geographical differentiation, whereas Rho formed two clusters: one widely occurring and another restricted to central regions. Genetic diversity was generally higher in southern than in northern populations. Our results suggest that conservation of southern local populations is particularly important in maintaining the genetic diversity of this endangered fish.

Factors affecting interspecific differences in genetic divergence among populations of Anolis lizards in Cuba.

BACKGROUND: Geographical patterns and degrees of genetic divergence among populations differ between species, reflecting relative potentials for speciation or cladogenesis and differing capacities for environmental adaptation. Identification of factors that contribute to genetic divergence among populations is important to the understanding of why some species exhibit greater interpopulation genetic divergence. In this study, we calculated the mean pairwise genetic distances among populations as species' average genetic divergence by a phylogeny using nuclear and mitochondrial genes of 303 individuals from 33 Cuban Anolis species and estimated species ages by another phylogeny using nuclear and mitochondrial genes of 51 Cuban and 47 non-Cuban Anolis species. We identified factors that influence species' differences in genetic divergence among 26 species of Anolis lizards from Cuba. Species ages, environmental heterogeneity within species ranges, and ecomorph types were considered as factors affecting average genetic divergences among populations. RESULTS: The phylogenies presented in this study provide the most comprehensive sampling of Cuban Anolis species to date. The phylogeny showed more conservative evolution of Anolis ecomorphs within Cuba and identified twig anoles as a monophyletic group. Subsequent Phylogenetic Generalized Least Squares (PGLS) analyses showed that species age was positively correlated with species' average genetic divergence among populations. CONCLUSION: Although previous studies have focused on factors affecting genetic divergence within species, the present study showed for the first time that species differences in genetic divergence could be largely affected by species age.

Genetic Structure of Dytiscus sharpi in North and South Hokuriku in Japan Inferred from Mitochondrial and Nuclear Gene Sequence.

Dytiscus sharpi is a critically endangered diving beetle endemic to Japan that is distributed in five distant areas today. Information on the population genetics of this species is crucial for its conservation. We investigated the genetic differentiation and divergence of the D. sharpi in seven populations in North and South Hokuriku on the Sea of Japan side, using mitochondrial cytochrome oxidase subunit I (COI) and nuclear Histone 3 and 28S rRNA gene sequences. Although the nuclear markers showed little geographic genetic differentiation, nine COI haplotypes were identified from 31 individuals, and these haplotypes were divided into two distinct networks A and B. The network A consisted of a single haplotype, and network B consisted of eight haplotypes. The populations in North Hokuriku possessed the haplotypes of both networks, whereas those in South Hokuriku possessed only the haplotypes of network B. The genetic diversity was relatively high in South and North Hokuriku. However, populations in South Hokuriku showed low genetic diversity and the isolation-by-distance pattern was observed among the populations, suggesting a restricted gene flow. Phylogenetic analysis including the data from a previous study revealed that the network A was closely related to a haplotype from Kanto, which is on the Pacific side. These results suggested that in the past this species was distributed widely across the Hokuriku area and the fragmentation of its distribution areas is a recent event. These results will contribute to strategies for the preservation of the current genetic diversity of D. sharpi.

Factors restricting the range expansion of the invasive green anole Anolis carolinensis on Okinawa Island, Japan.

The green anole Anolis carolinensis invaded the Ogasawara Islands in Japan, drove various native species to extinction, and its distribution expanded 14 years after initial establishment. A. carolinensis invaded Okinawa Island, but it has not expanded its distribution in more than 25 years, although its density is extremely high in the southern region. To determine whether A. carolinensis has the potential to expand its distribution on Okinawa Island, we performed phylogenetic analysis of mitochondrial ND2 DNA sequences to study the origin of A. carolinensis that invaded Okinawa Island. We further used a species distribution model (MaxEnt) based on the distribution of native populations in North America to identify ecologically suitable areas on Okinawa Island. Nucleotide sequence analysis shows that the invader A. carolinensis originated in the western part of the Gulf Coast and inland areas of the United States and that a portion of the anoles on Okinawa was not introduced via the Ogasawara Islands. The MaxEnt predictions indicate that most areas in Okinawa Island are suitable for A. carolinensis. Therefore, A. carolinensis may have the potential to expand its distribution in Okinawa Island. The predictions indicate that habitat suitability is high in areas of high annual mean temperature and urbanized areas. The values of precipitation in summer in the northern region of Okinawa Island were higher compared with those of North America, which reduced the habitat suitability in Okinawa Island. Adaptation to low temperatures, an increase in the mean temperature through global warming, and an increase in open environments through land development will likely expand the distribution of A. carolinensis in Okinawa Island. Therefore, we must continue to monitor the introduced populations and be alert to the possibility that city planning that increases open environments may cause their range to expand.

Application of RAD-based phylogenetics to complex relationships among variously related taxa in a species flock.

Restriction site-associated DNA (RAD) sequences from entire genomes can be used to resolve complex phylogenetic problems. However, the processed data matrix varies depending on the strategies used to determine orthologous loci and to filter loci according to the number of taxa with sequence data for the loci, and often contains plenty of missing data. To explore the utility of RAD sequences for elucidating the phylogenetics of variously related species, we conducted RAD sequencing for the Ohomopterus ground beetles and attempted maximum-likelihood phylogenetic analyses using 42 data matrices ranging from 1.6×10(4) to 8.1×10(6) base pairs, with 11-72% missing data. We demonstrate that robust phylogenetic trees, in terms of bootstrap values, do not necessarily result from larger data matrices, as previously suggested. Robust trees for distantly related and closely related taxa resulted from different data matrices, and topologically different robust trees for distantly related taxa resulted from various data matrices. For closely related taxa, moderately large data matrices strongly supported a topology that is incompatible with morphological evidence, possibly due to the effect of introgressive hybridization. Practically, exploring variously prepared data matrices is an effective way to propose important alternative phylogenetic hypotheses for this study group.

Factors determining the direction of ecological specialization in snail-feeding carabid beetles.

A stout-slender dimorphism in body shape is observed among carabid beetles of the subtribe Carabina, which feed on land snails. We hypothesized that this dimorphism has resulted from divergent ecological specialization for feeding on different-sized land snails. Therefore, we examined whether the geographic variation in the body shape of Damaster blaptoides, a representative snail-feeding species in Japan, is correlated with the size of Euhadra, a genus of land snails frequently consumed by D. blaptoides. An analysis of beetle specimens from the whole distribution area of D. blaptoides determined that more slender beetle populations occurred in localities harboring larger snails, whereas more stout beetles inhabited localities harboring smaller snails. This pattern could be adaptive because slender beetles exhibit high feeding performance for large snails by inserting their heads into the shells, whereas stout beetles do so for small snails by crushing the shells. The D. blaptoides populations showed a clear genetic isolation-by-distance pattern, which could be effective in promoting such local adaptation. Thus, food resources as well as geographic isolation may have promoted adaptive divergence of external morphology in the snail-feeding carabid beetles.

Historical divergence of mechanical isolation agents in the ground beetle Carabus arrowianus as revealed by phylogeographical analyses.

In the carabid genus Carabus subgenus Ohomopterus, diverged body size and genital morphology serve as mechanical reproductive barriers. To elucidate the diverging process of body and genital sizes in Carabus arrowianus, which exhibits marked morphological diversity among geographical populations and may represent an early stage of speciation, we analysed a mitochondrial gene sequence for 1051 individuals from 63 populations and male morphology for 359 individuals from 47 populations. Two discrete morphological groups segregated by geographical barriers were distinguished, one of which possessed smaller bodies and shorter genitalia (S group) than the other (L group), which exhibited larger bodies and exaggerated genitalia. Genetic divergence between the two groups was significant but not large. Phylogeographical and population genetic analyses indicated that the L group was derived from the S group, and a coalescent simulation revealed that the two groups diverged during the latest middle Pleistocene (0.13 million years ago), with a much larger effective population size in the L group than the S group. Because the body size divergence could not be explained by adaptation to climatic conditions and genital morphology is considered to be subject to sexual selection, we postulated that a population division and colonization in favourable habitats caused by the Pleistocene climatic and geographical change might facilitate natural and sexual selection for enlarged body and genital sizes in the L group.

Diversification in a fluctuating island setting: rapid radiation of Ohomopterus ground beetles in the Japanese Islands.

The Japanese Islands have been largely isolated from the East Asian mainland since the Early Pleistocene, allowing the diversification of endemic lineages. Here, we explore speciation rates and historical biogeography of the ground beetles of the subgenus Ohomopterus (genus Carabus) based on nuclear and mitochondrial gene sequences. Ohomopterus diverged into 15 species during the Pleistocene. The speciation rate was 1.92 Ma(-1) and was particularly fast (2.37 Ma(-1)) in a group with highly divergent genitalia. Speciation occurred almost solely within Honshu, the largest island with complex geography. Species diversity is highest in central Honshu, where closely related species occur parapatrically and different-sized species co-occur. Range expansion of some species in the past has resulted in such species assemblages. Introgressive hybridization, at least for mitochondrial DNA, has occurred repeatedly between species in contact, but has not greatly disturbed species distinctness. Small-island populations of some species were separated from main-island populations only after the last glacial (or the last interglacial) period, indicating that island isolation had little role in speciation. Thus, the speciation and formation of the Ohomopterus assemblage occurred despite frequent opportunities for secondary contact and hybridization and the lack of persistent isolation. This radiation was achieved without substantial ecological differentiation, but with marked differentiation in mechanical agents of reproductive isolation (body size and genital morphology).

Mechanical barriers to introgressive hybridization revealed by mitochondrial introgression patterns in Ohomopterus ground beetle assemblages.

To reveal the role of diverged body size and genital morphology in reproductive isolation among closely related species, we examined patterns of, and factors limiting, introgressive hybridization between sympatric Ohomopterus ground beetles in central Japan using mitochondrial NADH dehydrogenase subunit 5 (ND5) gene sequences. We sampled 17 local assemblages that consisted of two to five species and estimated levels of interspecific gene flow using the genetic distance, D(A), and maximum-likelihood estimates of gene flow. Sharing of haplotypes or haplotype lineages was detected between six of seven species that occurred in the study areas, indicating mitochondrial introgression. The intensity and direction of mitochondrial gene flow were variable among species pairs. To determine the factors affecting introgression patterns, we tested the relationships between interspecific D(A) and five independent variables: difference in body size, difference in genital size, phylogenetic relatedness (nuclear gene sequence divergence), habitat difference, and species richness of the assemblage. Body and genital size differences contributed significantly to preventing gene flow. Thus, mechanical isolation mechanisms reduce the chance of introgressive hybridization between closely related species. Our results highlight the role of morphological divergence in speciation and assemblage formation processes through mechanical isolation.

Phylogeography and introgressive hybridization of the ground beetle Carabus yamato in Japan based on mitochondrial gene sequences.

To study the phylogeography of the ground beetle Carabus yamato in Japan, we compared 1,020-bp sequences of the mitochondrial NADH dehydrogenase subunit 5 (ND5) gene from 373 specimens from 37 localities with those of three parapatric species (C. albrechti, C. kimurai, and C. japonicus) that might share mitochondrial lineages with C. yamato through introgressive hybridization. We found 81 haplotypes from C. yamato. Of these, 17 haplotypes were considered to be of an introgressed lineage from C. albrechti, based on the phylogeny and geographic distribution. In addition, one haplotype of C. kimurai was likely an introgressant from C. yamato. Putative introgression events among the four species were restricted to these two directional cases. We analyzed the phylogeography of C. yamato using nested clade phylogeographical analysis and population genetic parameters. The mitochondrial lineages of C. yamato were estimated to have diverged no more than approximately 1.12 million years ago, implying that the estimated historical events occurred after the Early Pleistocene. Carabus yamato was inferred to have experienced a contraction of its distribution range, followed by recent range expansion. Populations in the western and eastern regions, segregated by Ise Bay and the Nobi and Okazaki Plains, diverged in the mitochondrial clades. The northern and most western populations possessed one clade only (except an introgressed lineage), whereas eastern and some southwestern populations possessed several diverged clades, which were considered to be ancestral; these populations may have been associated with refugia during glacial periods.