Variations in the phenological patterns of a caddisfly inhabiting the same mountain massifs: Life-history differences in different altitudinal zones.

Organisms inhabiting mountainous regions can experience large vertical environmental changes, and show different ecological characteristics between altitudes, thus facilitating allopatric fragmentation even in geographically close populations. This study compared the life-history patterns of a species of limnephilid caddisfly, Asynarchus sachalinensis, in several genetically differentiated populations between alpine and sub-alpine zones in a temperate mountainous region. We showed that in the sub-alpine populations, larval development started earlier with increasing water temperature in spring, and adult emergence was also earlier. The occurrence of adults was extremely low in mid-summer, probably due to summer diapause, followed by a larger number of ovary-developed females in autumn. On the other hand, in the alpine zone, increasing water temperature was delayed compared to the sub-alpine zone, and larval development occurred from early to mid-summer. Adult emergence and ovary-developed individuals were concentrated in mid-summer. Hence, summer diapause was not observed. These results indicated life-history differences between genetically differentiated populations at different altitudes. As the timing of adult occurrence and ovarian developmental patterns differ between populations at different altitudes, it is possible that reproductive isolation is facilitated or maintained between populations.

Evolutionary history of a cold-adapted limnephilid caddisfly: Effects of climate change and topography on genetic structure.

The distribution of organisms is influenced by complex factors such as the phylogenetic evolutionary histories of species, the physiological and ecological characteristics of organisms, climate, and geographical and geohistorical features. In this study, we focused on a caddisfly, Asynarchus sachalinensis (Trichoptera: Limnephilidae), which has adapted to cold habitats. From phylogeographic analyses based on the mitochondrial DNA (mtDNA) cytochrome c oxidase subunit I (COI) and 16S rRNA regions and the nuclear DNA (nDNA) 18S rRNA, 28S rRNA, carbamoyl-phosphate synthetase (CAD), elongation factor-1 alpha (EF1-α), and RNA polymerase II (POLII) regions, two distinct genetic clades were detected. Clade I was shown to be widely distributed from Sakhalin to Honshu, whereas Clade II was only distributed within Honshu. The distributions of these clades overlapped in Honshu. The habitats were located at relatively lower altitudes for Clade I and higher altitudes for Clade II. The divergence time of these clades was estimated to be during the Pleistocene, indicating that repeated climatic changes facilitated distributional shifts. Haplotype network and demographic analyses based on the mtDNA COI region showed contrasting genetic structures in the two clades. It was indicated that the population sizes of Clade I had expanded rapidly in a recent period, whereas Clade II had maintained stable population sizes. The habitats of Clade II were typically isolated and scattered at high altitudes, resulting in restricted migration and dispersal because of their discontinuous "Sky Island" habitats. The habitats of Clade I were located at relatively low altitudes, and it was assumed that the populations were continuous, which resulted in a higher frequency of migration and dispersal between populations. Thus, differences in the spatial scale of the adapted habitats of each clade may have resulted in different patterns of population connectivity and fragmentation associated with repeated climatic changes during the Pleistocene. Our study provided new insight into the distributional patterns of cold-adapted aquatic insects in the Japanese Archipelago. Furthermore, the distributional shifts predicted by ecological niche modeling under future climatic change conditions were different for each clade. Therefore, different principles are required in the assessment of each clade to predict temporal changes in their distributions.

New species of the genus Trichosetodes Ulmer, 1915 (Trichoptera, Leptoceridae) from Ratanakiri province, Cambodia, based on morphological and molecular data.

Three new species of Trichosetodes, namely T.carmelaesp. nov., T.katiengensissp. nov. and T.ratanakiriensissp. nov. are described and illustrated by male specimens. The male genitalia of T.carmelaesp. nov. can be distinguished from the other 16 species of the genus found in Southeast Asia by the shape of the phallicata. The phallicata of T.carmelaesp. nov. bears a tuft of long hairs in the middle of the dorsal edge. Trichosetodeskatiengensissp. nov. can be distinguished from the other species in Southeast Asia by the shape of the phallicata which is divided into dorsal and ventral branches in lateral view, and T.ratanakiriensissp. nov. by the characters of the left inferior appendage and the shape of segment IX. The posterior end of the left inferior appendage of T.ratanakiriensissp. nov. is not forked and the ventral and lateral views of the posteroventral lobes of segment IX are rounded. Illustrations of male genitalia of Trichosetodeskampongspeuensis Malicky & Kong, 2020 are provided for comparison. The molecular diversity of new Trichosetodes species was analyzed using the mitochondrial large subunit ribosomal rRNA gene region (16S rRNA). In terms of their genetic divergence, T.ratanakiriensissp. nov. and T.kampongspeuensis exhibited remarkable proximity, with only a 1.4% distance. On the contrary, T.carmelaesp. nov. displayed genetic disparity exceeding 6.3% when compared to both T.ratanakiriensissp. nov. and T.kampongspeuensis.

Phylogeography of the true freshwater crab, Geothelphusa dehaani: Detected dual dispersal routes via land and sea.

Dispersal is an important factor that determines the potential for colonization to pioneer sites. Although most decapods employ seaward migration for reproduction with a planktonic larval phase, true freshwater crabs spend their entire life cycle in freshwater. Therefore, it is expected that genetic regionality can be easily detected. In this study, we focused on true freshwater crabs, Geothelphusa Stimpson, 1858. Herein, we reveal the evolutionary history and dispersal patterns of freshwater crustaceans. We collected and genetically analyzed 283 specimens at 138 localities across the Japanese Islands. Phylogenetic analyses were conducted on the combined dataset (mtDNA COI, 16S, and nDNA ITS1, histone H3 regions) and the data set based on the mtDNA COI region. The phylogenetic relationships detected 10 clades that were highly monophyletic. The highlights of this study were the discovery of several cryptic species or undescribed species, and the completely different heterogeneous dual dispersal pathways within a single species; i.e., both land and ocean routes. Although it was concluded that Japanese crabs are basically genetically divided by straits, strong evidence for dispersion via ocean currents was also detected (i.e., a "sweepstake"). It was also confirmed that Geothelphusa dehaani (White, 1847) could survive in seawater.

A major flood caused by a typhoon did not affect the population genetic structure of a river mayfly metapopulation.

Floods affect the population structure of organisms that inhabit streams. In recent decades, the scale of floods has become larger due to climate change. Under these circumstances, on 12 October 2019, the largest typhoon in the history of observation in Japan struck the Japanese Archipelago. This typhoon caused heavy rainfall in various places, and the Chikuma-Shinano River System (Japan's largest) suffered great damage. Eight years before the large-scale disturbance in the river system, the population structure of the mayfly Isonychia japonica was studied in detail using quantitative sampling (population numbers and biomass) and by sequencing the mtDNA cytochrome c oxidase subunit I. To understand the impact of the flood on the population and genetic structures, we repeated the same research approximately 1 year after the flood. Direct comparison of sites before and after the flood revealed no significant changes between pre- and post-flood population genetic structure. This indicates high in situ resistance and/or resilience recovery of the populations to the disturbance. We hypothesize that this high resistance/resilience to flood disturbance is a result of strong selection for such traits in the rivers of the Japanese Archipelago, which are short, steep, flow rapidly and violently, and are strongly affected by floods.

Behavior of snow monkeys hunting fish to survive winter.

Japanese macaques, Macaca fuscata, of Kamikochi in the Japanese Alps endure one of the coldest and harshest environments during winter when scarcity of food puts them at risk. However, various behaviors have evolved to mitigate potential mortality. These macaques typically eat bamboo leaves and the bark of woody plants in winter, but our previous study using the feces of Japanese macaques collected in the winter and DNA metabarcoding analysis revealed conclusively for the first time consumption of riverine benthos and brown trout. In this paper, we investigate how Japanese macaques hunt fish and collect these riverine biota by extensively observing their behavior, including the use of infrared sensor cameras. Many researchers have tracked Japanese macaques as part of behavioral and ecological studies, but previously the techniques by which Japanese macaques capture swimming fish has not been documented. Herein, for the first time we consider how novel macaque foraging behavior traits have evolved to secure valuable animal protein for winter survival when food resources are scarce.

Seasonal modifications of longitudinal distribution patterns within a stream: Interspecific interactions in the niche overlap zones of two Ephemera mayflies.

Niche differentiation between closely related species leads to differentiation of their habitats. Segregation based on slight differences in environmental factors, that is niche differentiation on the microhabitat scale, allows more species to inhabit a certain geographic space. Therefore, such fine scale niche differentiation is an important factor in the support of species diversity. In addition, niche differentiation on the microhabitat scale and/or the differentiation of breeding seasons can be considered typical mechanisms that facilitate multispecies' co-existence. In this study, sister species (Commonly, Ephemera japonica inhabit at upstream region and Ephemera strigata inhabit at middle stream region), which often coexist in the upper to middle reaches of river systems of the Japanese Islands, were targeted and the following aspects were investigated. First, differences in habitat preference and interspecific differences in flow distribution patterns on a geographically fine scale were tracked in detail. Subsequently, the temporal transitions of their distribution patterns were investigated in detail and seasonal changes were investigated. Finally, we thoroughly investigated the disappearance of nymphs of each species from the river due to emergence affected the distribution of each species (by conducting daily emergence surveys). Combining results of these multiple studies also suggested that there may be spatiotemporal interspecial interaction between these two species within/around their overlapping regions. Traditionally, the longitudinal distribution pattern of these two Ephemera mayflies has been thought to be established based on a difference in habitat preferences, but this study revealed that the interspecific interaction between the two species also plays an important role. This study provides new insights into species diversity and distribution pattern formation in river-dwelling species.

Long-read HiFi sequencing correctly assembles repetitive heavy fibroin silk genes in new moth and caddisfly genomes.

Insect silk is a versatile biomaterial. Lepidoptera and Trichoptera display some of the most diverse uses of silk, with varying strength, adhesive qualities, and elastic properties. Silk fibroin genes are long (>20 Kbp), with many repetitive motifs that make them challenging to sequence. Most research thus far has focused on conserved N- and C-terminal regions of fibroin genes because a full comparison of repetitive regions across taxa has not been possible. Using the PacBio Sequel II system and SMRT sequencing, we generated high fidelity (HiFi) long-read genomic and transcriptomic sequences for the Indianmeal moth (Plodia interpunctella) and genomic sequences for the caddisfly Eubasilissa regina. Both genomes were highly contiguous (N50  = 9.7 Mbp/32.4 Mbp, L50  = 13/11) and complete (BUSCO complete  = 99.3%/95.2%), with complete and contiguous recovery of silk heavy fibroin gene sequences. We show that HiFi long-read sequencing is helpful for understanding genes with long, repetitive regions.

Lateral Transmission of Yeast Symbionts Among Lucanid Beetle Taxa.

To deepen understanding the evolutionary process of lucanid-yeast association, the lateral transmission process of yeast symbionts among stag beetle genera Platycerus and Prismognathus around the border between Japan and South Korea was estimated based on molecular analyses and species distribution modelings. Phylogenetic analyses were based on yeast ITS and IGS sequences and beetle COI sequences using Prismognathus dauricus from the Tsushima Islands and Pr. angularis from Kyushu, Japan, as well as other sequence data from our previous studies. The range overlap based on the species distribution model (SDM) and differentiation in ecological space were analyzed. Based on the IGS sequences, Clade II yeast symbionts were shared by Platycerus hongwonpyoi and Pr. dauricus in South Korea and the Tsushima Islands, and Platycerus viridicuprus in Japan. Clade III yeasts were shared by Pr. dauricus from the Tsushima Islands and Pr. angularis in Japan. During the Last Interglacial period when the land bridge between Japan and the Korean Peninsula existed, range overlap was predicted to occur between Pl. viridicuprus and Pr. dauricus in Kyushu and between Pr. dauricus and Pr. angularis in Kyushu and the Tsushima Islands. The ecological space of Pl. hongwonpyoi was differentiated from that of Pl. viridicuprus and Pr. angularis. We demonstrated the paleogeographical lateral transmission process of Scheffersomyces yeast symbionts among lucanid genera and species: putative transmission of yeasts from Pr. dauricus to Pl. viridicuprus in Kyushu and from Pr. angularis to Pr. dauricus in Kyushu or the Tsushima Islands. We also found that the yeast symbionts are likely being replaced in Pr. dauricus on the Tsushima Islands. We present novel estimates of the lateral transmission process of microbial symbionts based on phylogenetic, SDM and environmental analyses among lucanid beetles.

Winter diet of Japanese macaques from Chubu Sangaku National Park, Japan incorporates freshwater biota.

The Japanese macaque (Macaca fuscata) is native to the main islands of Japan, except Hokkaido, and is the most northerly living non-human primate. In the Chubu Sangaku National Park of the Japanese Alps, macaques live in one of the coldest areas of the world, with snow cover limiting the availability of preferred food sources. Winter is typically a bottleneck for food availability potentially resulting in marked energy deficits, and mortality may result from famine. However, streams with groundwater upwelling flow during the winter with a constant water temperature of about 5 °C are easily accessible for Japanese macaques to search for riverine biota. We used metabarcoding (Cytochrome c oxidase I) of fecal samples from Japanese macaques to determine their wintertime diet. Here we provide the first robust evidence that Japanese macaques feed on freshwater biota, including brown trout, riverine insects and molluscs, in Chubu Sangaku National Park. These additional food sources likely aid their winter survival.

Genome-wide molecular phylogenetic analyses and mating experiments which reveal the evolutionary history and an intermediate stage of speciation of a giant water bug.

The intermediate stages of speciation are important for understanding the processes involved in the creation of biodiversity, and also comprise a number of interesting phenomena. However, difficulties are associated with dividing clear speciation stages because speciation is a continuous process. Therefore, the elucidation of speciation is an interesting and important task in evolutionary biology. We herein present an example of a species in an intermediate stage of speciation using the giant water bug Appasus japonicus (Heteroptera, Belostomatidae) that was investigated using mating experiments and phylogenetic analyses of the mtDNA COI (658 bp) and 16S rRNA (435 bp) regions, and nDNA SSR (13 loci) and its genome-wide SNPs (11,241 SNPs). The results of our phylogenetic analyses based on their mtDNA data set and the genome-wide SNPs data set strongly supported the paraphyly of the Japanese populations. Therefore, it is suggested that their ancestral lineage which being distributed in the Japanese Archipelago subsequently migrated to the Eurasian Continent (i.e., back-dispersal occurred). Furthermore, the results of the mating experiments suggested that among A. japonicus, even between closely related lineages, premating reproductive isolation has been established by the differentiation of copulatory organ morphologies. In contrast, premating reproductive isolation is not established in the absence of the differentiation of copulatory organ morphologies, even if genetic differentiation is prominent. These results suggested that their phylogenetic distance does not predict premating reproductive isolation. Furthermore, in the present study, we present a clear example of premating reproductive isolation driving speciation between closely related lineages.

A description of the second species of the genus Bleptus Eaton, 1885 (Ephemeroptera: Heptageniidae) from Japan, and phylogenetic relationships of two Bleptus mayflies inferred from mitochondrial and nuclear gene sequences.

A new mayfly species, Bleptus michinokuensis sp. nov. (Ephemeroptera: Heptageniidae) is described on the basis of specimens of male and female adults and mature nymphs collected at a seepage zone of a small freshwater branch of the 'Tachiya-zawa-gawa' River located amongst the northern foothills of Mt. Gassan (Shonai-machi Town, Yamagata Prefecture, Japan). This new Bleptus species is characterized by its clear fore and hind wings. That is, they neither exhibit the distinct black band on the fore wings, nor the characteristic darkened margins along the edges of both the fore and hind wings. Rather it has a blackish colored terminal half of its fore legs (i.e., tibial, tarsal and pretarsal segments). These features differ clearly when comparing them to the other known species, Bleptus fasciatus Eaton. The information and data describing the habitat and distribution range of this new species are also noted. We also examined and discussed the genetic relationship of two Bleptus mayflies to settle the taxonomic status, inferred from the partially sequenced cytochrome c oxidase subunit I (COI) and large mitochondrial ribosomal subunit (16S rRNA) genes, and also the nuclear internal transcribed spacer 2 (ITS2) gene sequences. Consequently, phenetic and molecular phylogenetic analyses agreed well in terms of clustering.

Phylogeography of the northernmost distributed Anisocentropus caddisflies and their comparative genetic structures based on habitat preferences.

Knowledge of the factors that determine the distribution ranges of organisms is necessary to understand their evolutionary and ecological significance and contribution to biodiversity. A very effective mean of studying such factors is to compare the distribution characteristics and genetic structures of closely related species with differing habitat preferences. Freshwater aquatic insects are relatively easy to observe and the basis of their corresponding niche differentiation easier to identify. Freshwater habitats are categorized lotic or lentic water according to flow regime. In Japanese Islands, the genus Anisocentropus of the calamoceratid caddisfly, the target group in this study, was morphologically reconfirmed that three species, that is, Anisocentropus kawamurai, A. pallidus, and A. magnificus. Among these, A. kawamurai prefers lotic environments and A. pallidus is adapted to lentic water habitats. The distribution range of these sister species overlaps within the Japanese Islands. We estimated the phylogeny and the evolutionary history of Anisocentropus caddisflies worldwide. We estimated divergence periods by two methods, a single locus with various specimens and multiple loci with reduced numbers of the specimens. As a result, we elucidated the phylogenetic position of Japanese species within the cosmopolitan genus Anisocentropus, and also revealed their dual origin. In addition, we demonstrated that the contrasting genetic structures between the sister species distributed in widely overlapping areas were due to differentiation in their respective adapted environmental preferences. Although, in general, it is known that species adapted to lentic water have greater dispersal potential and so are associated with wider distribution areas by means of examining their comparative genetic structures, we revealed a new pattern of genetic locality existing in the genetic structures of the species adapted to lentic water. We then present evidence that suggests the ecological preferences of a species are an important factor in understanding the evolutionary history of that species.

Wide-Scale Gene Flow, Even in Insects that have Lost their Flight Ability: Presence of Dispersion Due to a Unique Parasitic Ecological Strategy of Piggybacking Hosts.

We focused on Meloe beetles that have lost all flight ability, and conducted molecular phylogeographic analyses based on their mitochondrial DNA COI and nuclear DNA EF1- α regions. Meloe beetles infiltrate bumblebee nests by attaching to bumblebees as they pollinate flowers and thereafter have a unique and specific life history as they complete their life-cycle within the host nest; flight-based dispersal is achieved by piggybacking on bumblebees. In fact, Meloe beetles, which cannot fly, even inhabit remote islands (i.e., "Oceanic Islands"). Regarding four species, i.e., Meloe coarctatus, Meloe proscarabaeus, Meloe violaceus and Meloe corvinus, the conventional morphological classification system based on morphological characteristics was strongly supported by the molecular markers. On the other hand, for two species, Meloe menoko and Meloe auriculatus, it was found that M. menoko may be evaluated as having a paraphyletic relationship with M. auriculatus. Furthermore, two other cryptic, undescribed species were also discovered in this study. One was collected in the Nikko Highland, and inhabited the area sympatrically with M. coarctatus. The other was collected from Hachijo-jima Island. These cryptic species were highly differentiated, independent lineages in terms of mitochondrial and nuclear gene regions. That is to say, a new level of species diversity was revealed among the Meloe beetle species, known for their unique and strange ecological and ethological characteristics.

Possibility of Undeveloped Egg Absorption during Embryogenesis: A Unique Phenomenon Observed in the Ovoviviparous Mayfly Cloeon dipterum.

Oviparous, ovoviviparous and viviparous reproduction are interesting subjects for understanding animals' evolutionary pathways and adaptation to their life history and habitat conditions. In this study, we examined the reproductive mode of the ovoviviparous mayfly Cloeon dipterum, particularly comparing embryogenesis between hand-pairing and unmated females' common oviduct. Our study suggested that the high developmental rate of C. dipterum observed in a recent study could be ascribed to their absorption of unfertilized eggs. The developmental rates of hand-paired females were almost 100%, while their egg-bearing numbers were lower than those of virgin females. Thus, such reduced egg numbers suggest the maternal absorption of unfertilized eggs. This trait is thought to have evolved with the ovoviviparous characteristics of C. dipterum. We identified the basis of the irregularity of this species exhibiting such a high (i.e., 100%) developmental rate in our previous recent study.

Development of microsatellite markers for a giant water bug, Appasus japonicus, distributed in East Asia.

We developed microsatellite markers for Appasus japonicus (Heteroptera: Belostomatidae). This belostomatid bug is distributed in East Asia (Japanese Archipelago, Korean Peninsula and mainland China) and often listed as an endangered species in the Red List or the Red Data Book at the national and local level in Japan. Here, we describe twenty novel polymorphic microsatellite loci developed for A. japonicus, and marker suitability was evaluated using 56 individuals from four A. japonicus populations (Nagano, Hiroshima and Yamaguchi prefectures, Japan, and Chungcheongnam-do, Korea). The number of alleles per locus ranged from 1 to 12 (mean = 2.5), and the average observed and expected heterozygosity and fixation index per locus were 0.270, 0.323 and 0.153, respectively. In addition, a population structure analysis was conducted using the software STRUCTURE, and its result suggested that the 20 markers described here will be useful for investigating the genetic structure of A. japonicus populations, which should contribute to population genetics studies of this species.

Development of microsatellite markers for a soricid water shrew, Chimarrogale platycephalus, and their successful use for individual identification.

The soricid water shrew Chimarrogale platycephalus is a mammalian species endemic to the Japanese Islands. The animals inhabit the islands of Honshu and Kyushu, and are considered to be extinct in Shikoku. Information on this water shrew from Honshu and Kyushu is scarce, and C. platycephalus is registered on many local governments' red lists as an endangered species. There are very few studies on their ethology, ecology or phylogenetics, due to difficulties related to the shrews being both nocturnal and aquatic: to study C. platycephalus, field research must be conducted in mountain streams at night. To overcome these challenges, we previously established a genetic analysis method using the feces of C. platycephalus, as a result of which the amount of phylogenetic and phylogeographic data has increased and our understanding of the species has improved. In this study, microsatellite markers were developed, and analyses using markers for 21 loci were performed. Moreover, to confirm the ability of these 21 microsatellite markers to differentiate individuals, all markers were tested using fecal and tissue specimens from 12 individuals reared separately in an aquarium. Using as few as 12 of these loci, individual differentiation with 100% accuracy should be achievable. The development of microsatellite markers in this study and the establishment of individual identification methods should greatly contribute to future ecological, ethological, population genetics and biogeographical research on C. platycephalus.

Macroinvertebrate communities in streams with contrasting water sources in the Japanese Alps.

Alpine streams are typically fed from a range of water sources including glacial meltwater, snowmelt, groundwater flow, and surface rainfall runoff. These contributions are projected to shift with climate change, particularly in the Japanese Alps where snow is expected to decrease, but rainfall events increase. The overarching aim of the study was to understand the key variables driving macroinvertebrate community composition in groundwater and snowmelt-fed streams (n = 6) in the Kamikochi region of the northern Japanese Alps (April-December 2017). Macroinvertebrate abundance, species richness, and diversity were not significantly different between the two stream types. Community structure, however, was different between groundwater and snowmelt-fed streams with macroinvertebrate taxa specialized for the environmental conditions present in each system. Temporal variation in the abundance, species richness, and diversity of macroinvertebrate communities was also significantly different between groundwater and snowmelt streams over the study period, with snowmelt streams exhibiting far higher levels of variation. Two snowmelt streams considered perennial proved to be intermittent with periodic drying of the streambed, but the macroinvertebrates in these systems rebounded rapidly after flows resumed with no reduction in taxonomic diversity. These same streams, nevertheless, showed a major reduction in diversity and abundance following periods of high flow, indicating floods rather than periodic drying was a major driver of community structure. This conclusion was also supported from functional analyses, which showed that the more variable snowmelt streams were characterized by taxa with resistant, rather than resilient, life-history traits. The findings demonstrate the potential for significant turnover in species composition with changing environmental conditions in Japanese alpine stream systems, with groundwater-fed streams potentially more resilient to future changes in comparison to snowmelt-fed streams.

Community Structure of Aquatic Insects Adapted to Lentic Water Environments, and Fine-Scale Analyses of Local Population Structures and the Genetic Structures of an Endangered Giant Water Bug Appasus japonicus.

Environments such as floodplains and the marshlands of rivers, lakes and ponds, are important habitats for aquatic insects adapted to lentic water conditions. In addition, ponds and paddy fields artificially created for agriculture are also important alternative habitats for lotic water-dependent wildlife. In this study, we focused on aquatic insects in ponds in the Matsumoto Basin, located in the center of Japan. Although this is an urbanized area, aquatic animals adapted to floodplains inhabit it at a relatively high density for Japan. We conducted a multifaceted evaluation of the environments of the 33 ponds in this region and conducted a survey of the aquatic insect fauna inhabiting them. In this study, we conducted quantitative sampling, focusing on two insect orders adapted to large-scale lentic water environments (i.e., Heteroptera and Coleoptera), and observed five species of three families and 16 species of five families from the Matsumoto Basin, respectively. Within these species, eight endangered species were included. Furthermore, we carried out a genetic structure analysis for the giant water bug, Appasus japonicus, inhabiting these ponds in high density, and conducted a comparative evaluation of their genetic diversity between these ponds. A total of 530 specimens of A. japonicus were genetically analyzed for the mitochondrial DNA COI region, and 26 haplotypes were observed. The degree of genetic diversity between the ponds was clearly demonstrated. In addition, we discussed the wintering possibilities for the giant water bugs based on their corresponding surrounding environmental factors, and comprehensively discussed their "source-sink" relationships in this region. Therefore, this is a comprehensive study focused on the relevant environmental factors, diversification of their community structures, their population structures, and their genetic structure at a fine scale.

A Phylogenetically Distinct Group of Glandirana rugosa Found in Kyushu, Japan.

The Japanese wrinkled frog Glandirana rugosa is separated into five genetically different groups. One group in western Japan is further divided into three subgroups, found in Kyushu, Shikoku, and western Honshu. We collected G. rugosa frogs at 39 sites in Kyushu and determined nucleotide sequences of the mitochondrial 12S and 16S rRNA genes for phylogenetic analysis. Unexpectedly, we found a group of frogs in southeastern Kyushu that did not cluster with any of the pre-existing five groups of G. rugosa on the phylogenetic trees. The frogs in the new group and G. rugosa in Kyushu were externally similar, but there were a few significant differences in morphological features between the two populations. In addition, we observed significant differences in the frogs' calls . Thus, the group of the frogs in southeastern Kyushu may represent a new candidate species in the genus Glandirana. We discuss the possibility of a new species.