Shohei Komaki

Species diversity of insects in Japan: Their origins and diversification processes

Japan is considered a global hot spot of biodiversity. With regard to species diversity, insects are no exception. To date, more than 32,000 insect species have been identified in Japan, while around 100,000 species of insects are estimated to inhabit this country. In this paper, we outline background factors having contributed to diversification of Japanese insects. Of course, the high degree of Japanese insect diversity is the result of many complex factors. In addition to the humid Asian monsoon climate and the extensive latitudinal gradient of habitats, the extremely complex geological history has contributed as an important factor to generate and maintain the high species diversity and endemism. In particular, the independent origins of northeastern and southwestern Japan from the Eurasian continent have greatly contributed to the diverse composition of Japanese insect fauna. To highlight the importance of this process, we introduce some case studies and previously published papers focusing on several insect groups with low dispersal ability. Those cases indicate that the geological history of Japan has played an important role in the differentiation of Japanese insect species. Besides such geological factors, climatic and ecological factors in combination have contributed to the formation of Japanese insect fauna in complicated ways and produced its particularly high degree of biodiversity. The knowledge compiled here will provide useful information for future studies aiming to understand more deeply the processes of speciation and faunal formation of Japanese insects.

Characterisation of major histocompatibility complex class I genes in Japanese Ranidae frogs

The major histocompatibility complex (MHC) is a key component of adaptive immunity in all jawed vertebrates, and understanding the evolutionary mechanisms that have shaped these genes in amphibians, one of the earliest terrestrial tetrapods, is important. We characterised MHC class I variation in three common Japanese Rana species (Rana japonica, Rana ornativentris and Rana tagoi tagoi) and identified a total of 60 variants from 21 individuals. We also found evolutionary signatures of gene duplication, recombination and balancing selection (including trans-species polymorphism), all of which drive increased MHC diversity. A unique feature of MHC class I from these three Ranidae species includes low synonymous differences per site (dS) within species, which we attribute to a more recent diversification of these sequences or recent gene duplication. The resulting higher dN/dS ratio relative to other anurans studied could be related to stronger selection pressure at peptide binding sites. This is one of the first studies to investigate MHC in Japanese amphibians and permits further exploration of the polygenetic factors associated with resistance to infectious diseases.

Distributional Change and Epidemic Introgression in Overlapping Areas of Japanese Pond Frog Species Over 30 Years

Pelophylax nigromaculatus, P. porosus porosus, and P. p. brevipoda are three pond frog species distributed in Japan. Their distributions overlap at two basins in central Japan (P. nigromaculatus and P. p. porosus in the Matsumoto basin, and P. nigromaculatus and P. p. brevipoda in the Ina basin), and hybrid descendants have been found in these areas. To clarify the distribution areas and hybrid zones of the frogs, and to understand the mode of introgressive hybridization and its impact on the frog populations, we conducted exhaustive sampling at each basin and performed allozyme and mtDNA analyses of 233 individuals. Analysis using genetic markers clearly detected nine F1 hybrids and 94 hybrid descendants of P. nigromaculatus and P. porosus from the overlapping areas of both basins. Allozyme and mtDNA data suggest directional hybridization between female P. p. porosus and male P. nigromaculatus in the Matsumoto basin. Over the past 30 years, the distribution of P. p. porosus has been narrowed and fragmented by the invasion of P. nigromaculatus, seemingly because of directional hybridization in the Matsumoto basin. In the Ina basin, the “pure” P. p. brevipoda (n = 8) population was extremely reduced by gene introgression from P. nigromaculatus, yet its distribution was barely changed compared to the Matsumoto basin. Consequently, this study shows that P. porosus populations are threatened by interspecific hybridization with P. nigromaculatus, and that introgressive hybridization damaged P. porosus populations by different means in each basin.

Microsatellite Marker Development by Multiplex Ion Torrent PGM Sequencing: a Case Study of the Endangered Odorrana narina Complex of Frogs

The endangered Ryukyu tip-nosed frog Odorrana narina and its related species, Odorrana amamiensis, Odorrana supranarina, and Odorrana utsunomiyaorum, belong to the family Ranidae and are endemically distributed in Okinawa (O. narina), Amami and Tokunoshima (O. amamiensis), and Ishigaki and Iriomote (O. supranarina and O. utsunomiyaorum) Islands. Because of varying distribution patterns, this species complex is an intrinsic model for speciation and adaptation. For effective conservation and molecular ecological studies, further genetic information is needed. For rapid, cost-effective development of several microsatellite markers for these and 2 other species, we used next-generation sequencing technology of Ion Torrent PGM™. Distribution patterns of repeat motifs of microsatellite loci in these modern frog species (Neobatrachia) were similarly skewed. We isolated and characterized 20 new microsatellite loci of O. narina and validated cross-amplification in the three-related species. Seventeen, 16, and 13 loci were cross-amplified in O. amamiensis, O. supranarina, and O. utsunomiyaorum, respectively, reflecting close genetic relationships between them. Mean number of alleles and expected heterozygosity of newly isolated loci varied depending on the size of each inhabited island. Our findings suggested the suitability of Ion Torrent PGM™ for microsatellite marker development. The new markers developed for the O. narina complex will be applicable in conservation genetics and molecular ecological studies.

Development and Validation of PCR-RFLP Assay to Identify Three Japanese Brown Frogs of the True Frog Genus Rana

Abstract: Two species of the true frog genus Rana (R. japonica and R. ornativentris) are widely distributed on the main islands of Japan with some degree of habitat overlap. These species could be key indicators for environmental conditions and accurate identification is important. However, morphological characters in early stages of development are often insufficient to distinguish the two species, although their presence is often inferred from egg masses. Therefore, we developed a PCR-RFLP protocol for efficient and cost-effective identification of the species. We used a short partial 16S rRNA fragment (approx. 550 bp) and two diagnostic restriction enzymes (Spe I and Hph I) to identify species from DNA material. This approach allowed us to accurately assign all samples even without locality data. We also confirmed the performance of our method using another brown frog, R. t. tagoi.

Genome-wide meta-analysis in Japanese populations identifies novel variants at the TMC6 – TMC8 and SIX3 – SIX2 loci associated with HbA 1c

Glycated haemoglobin (HbA1c) is widely used as a biomarker for the diagnosis of diabetes, for population-level screening, and for monitoring the glycaemic status during medical treatment. Although the heritability of HbA1c has been estimated at ~55–75%, a much smaller proportion of phenotypic variance is explained by the HbA1c-associated variants identified so far. To search for novel loci influencing the HbA1c levels, we conducted a genome-wide meta-analysis of 2 non-diabetic Japanese populations (n = 7,704 subjects in total). We identified 2 novel loci that achieved genome-wide significance: TMC6–TMC8 (P = 5.3 × 10−20) and SIX3–SIX2 (P = 8.6 × 10−9). Data from the largest-scale European GWAS conducted for HbA1c supported an association between the novel TMC6–TMC8 locus and HbA1c (P = 2.7 × 10−3). The association analysis with glycated albumin and glycation gap conducted using our Japanese population indicated that the TMC6–TMC8 and SIX3–SIX2 loci may influence the HbA1c level through non-glycaemic and glycaemic pathways, respectively. In addition, the pathway-based analysis suggested that the linoleic acid metabolic and 14-3-3-mediated signalling pathways were associated with HbA1c. These findings provide novel insights into the molecular mechanisms that modulate the HbA1c level in non-diabetic subjects.

iMETHYL: an integrative database of human DNA methylation, gene expression, and genomic variation

We launched an integrative multi-omics database, iMETHYL (http://imethyl.iwate-megabank.org). iMETHYL provides whole-DNA methylation (~24 million autosomal CpG sites), whole-genome (~9 million single-nucleotide variants), and whole-transcriptome (>14 000 genes) data for CD4+ T-lymphocytes, monocytes, and neutrophils collected from approximately 100 subjects. These data were obtained from whole-genome bisulfite sequencing, whole-genome sequencing, and whole-transcriptome sequencing, making iMETHYL a comprehensive database.

Living in a Japanese onsen: field observations and physiological measurements of hot spring amphibian tadpoles, Buergeria japonica

The Japanese stream tree frog, Buergeria japonica , is widely distributed across the southern islands of Japan and Taiwan. While the species is known to inhabit hot springs, this has only been reported in Taiwan. To further understand the utilization of hot springs by B. japonica , we conducted field observations of tadpoles from a hot spring on Kuchinoshima Island, a tiny volcanic island of southwestern Japan. We found that tadpoles on Kuchinoshima Island inhabited hot spring pools with extremely high temperatures that exceeded temperatures in which any other amphibians have been found. In addition, we conducted thermal tolerance measurements and found that the thermal tolerance of B. japonica tadpoles was high. These findings suggest that high thermal tolerance of B. japonica is maintained even at the northern tip of its distribution, and this has allowed them to widen their available niche and inhabit a hot spring on the tiny island of Kuchinoshima.

Genome-wide analysis of polymorphism × sodium interaction effect on blood pressure identifies a novel 3′- BCL11B gene desert locus

Excessive sodium intake is a global risk factor for hypertension. Sodium effects on blood pressure vary from person to person; hence, high-risk group targeting based on personal genetic information can play a complementary role to ongoing population preventive approaches to reduce sodium consumption. To identify genetic factors that modulate sodium effects on blood pressure, we conducted a population-based genome-wide interaction analysis in 8,768 Japanese subjects, which was >3 times larger than a similar previous study. We tested 7,135,436 polymorphisms in the discovery cohort, and loci that met suggestive significance were further examined in an independent replication cohort. We found that an interaction between a novel 3′-BCL11B gene desert locus and daily sodium consumption was significantly associated with systolic blood pressure in both discovery and replication cohorts under the recessive model. Further statistical analysis of rs8022678, the sentinel variant of the 3′-BCL11B gene desert locus, showed that differences in mean systolic blood pressure between high and low sodium consumption subgroups were 5.9 mm Hg (P = 8.8 × 10−12) in rs8022678 A carriers and −0.3 mm Hg (P = 0.27) in rs8022678 A non-carriers, suggesting that the rs8022678 genotype can classify persons into sodium-sensitive (A carriers) and sodium-insensitive (A non-carriers) subgroups. Our results implied that rs8022678 A carriers may receive a greater benefit from sodium-lowering interventions than non-carriers.

The widespread misconception about the Japanese major biogeographic boundary, the Watase line (Tokara gap), revealed by bibliographic and beta diversity analyses

The biota of the Japanese Archipelago is divided into the Palearctic and Oriental realms by the Watase line (Tokara gap), a major biogeographic boundary of Japan. This boundary is generally placed between Akusekijima and Kodakarajima Islands of the Tokara Archipelago, and has been the subject of many biogeographic debates. However, despite being widely accepted, the position of the boundary is doubtful because of a lack of clear evidence. Here, to verify the definition and existence of the biogeographic boundary, we performed a documentary search and beta diversity analysis of multiple taxa. Our documentary search suggested that the Watase line (Tokara gap) should be put between Yakushima/Tanegashima and Amamioshima Islands, but recent references to it clearly deviate from its original definition, and that the placement of the boundary line between Akusekijima and Kodakarajima Islands is based on limited and biased evidence. Our beta diversity analyses found no common biogeographic boundary dividing the Tokara Archipelago into two realms, and showed that the beta diversity pattern of this region is explained by the areas and geographic distances of the islands in agreement with the general principles of island biogeography. The widespread misunderstanding of biogeography in this region could have been perpetuated by preconception and the citation of references without verification. Our study proposes that revision of the biogeography in the Tokara Archipelago, a gap region between the Palearctic and Oriental realms, is necessary and demonstrates the negative influence of preconception in biogeographic debate.