Taxonomic insights and evolutionary history in East Asian terrestrial slugs of the genus Meghimatium.

East Asia, specifically the Japanese Archipelago, is a biodiversity hotspot of both vertebrates and invertebrates. Mollusks represent a burst of species diversity in this region due to the effects of biotic and abiotic factors on their morphological traits, such as shell shape and size. However, the evolutionary history of terrestrial slugs in East Asia remains unknown. In the present study, we investigated the molecular phylogeny of terrestrial slugs of the genus Meghimatium. This genus includes three described and eight undescribed species, and our study used all except for two. Based on phylogeny and the species delimitation tests, the genus Meghimatium was split into many putative species, suggesting higher species diversity than previously thought based on morphological and anatomical studies and that almost undescribed species may be inappropriate. Therefore, morphological traits, such as body size and colour, conventionally considered for classification may easily vary or be similar across geographic region. Moreover, the divergence time of this genus is almost concordant with the geographical time scale of the formation of the Japanese mainland. Our findings suggest that molecular phylogenetics helps classify Japanese Meghimatium slugs, but comprehensive taxonomic revisions using multi-locus analyses are needed.

Resolving species-level diversity of Beringiana and Sinanodonta mussels (Bivalvia: Unionidae) in the Japanese archipelago using genome-wide data.

Accurate species identification is of primary importance in ecology and evolutionary biology. For a long time, the unionid mussels Beringiana and Sinanodonta have puzzled researchers trying to unravel their diversity because of their poorly discernible morphologies. A recent study conducted species delineation of unionid mussels based on mitochondrial DNA variation, opening up a new avenue to grasp species diversity of the mussels. However, mtDNA-based classification may not align with species boundaries because mtDNA is prone to introgression and incomplete lineage sorting that cause discordance between species affiliation and gene phylogeny. In this study, we evaluated the validity of the mtDNA-based classification of unionid mussels Beringiana and Sinanodonta in Japan using mitochondrial sequence data, double digest restriction site-associated DNA library (ddRAD) sequencing, and morphological data. We found significant inconsistencies in the mitochondrial and nuclear DNA phylogenies, casting doubt on the reliability of the mtDNA-based classification in this group. In addition, nuclear DNA phylogeny revealed that there are at least two unionid lineages hidden in the mtDNA phylogeny. Although molecular dating technique indicates that Beringiana and Sinanodonta diverged >35 million years ago, their shell morphologies are often indistinguishable. Specifically, morphological analyses exhibited the parallel appearance of nearly identical ball-like shell forms in the two genera in Lake Biwa, which further complicates species identification and the morphological evolution of unionid mussels. Our study adds to a growing body of literature that accurate species identification of unionid mussels is difficult when using morphological characters alone. Although mtDNA-based classification is a simple and convenient way to classify unionid mussels, considerable caution is warranted for its application in ecological and evolutionary studies.

Uncovering overlooked diversity using molecular phylogenetic approach: A case of Japanese sphaeriid clams (Bivalvia: Sphaeriidae).

Revealing the species and lineage diversity of a taxon is important for many biological studies of wildlife. In recent decades, DNA-based approaches have been widely utilised to elucidate the diversity of taxa, especially those that are difficult to distinguish based on morphological traits. This study focused on freshwater clams (Sphaeriidae) in Japan, a biodiversity hotspot of freshwater molluscs. Molecular phylogenetic approaches, including divergence time estimation, species delimitation, rarefaction, and biogeographic area estimation, were used to reveal the nature of the species diversity and its formation process, which are largely unknown. Our delimitation and rarefaction analyses suggest that Japanese sphaeriid clams consist of at least 18 delimitated lineages. This lineage diversity is relatively high compared to other Japanese freshwater molluscs, and in addition, the majority of the Japanese lineage appears to have high endemicity despite the possibility of long-distance dispersal in sphaeriid clams. Our biogeographical analyses suggest that this diversity may be due to the combination of colonisation, during the period when Japan was connected to the continent, and the relatively recent dispersal. Our results highlight the overlooked biodiversity of Japan and provide a basis for further Japanese sphaeriid research, including conservation perspectives.

Species identification of introduced veronicellid slugs in Japan.

Reliable identification of species is important for protecting native ecosystems against the invasion of non-native species. DNA barcoding using molecular markers, such as the mitochondrial cytochrome oxidase subunit 1 (COI) gene, helps researchers distinguish species. In this study, we focused on introduced veronicellid slugs in the Ryukyu Islands and some greenhouses on mainland Japan. Some veronicellids are medium-to-high risk pest species for humans. Identifying veronicellid species by their external morphology is difficult and unreliable because there is substantial overlap between intraspecific variation and interspecific differentiation. Therefore, internal morphologies such as male genitalia have been the primary traits used to distinguish veronicellids. To identify introduced veronicellid slugs in Japan to the species level, we used morphological assessment of male genitalia and DNA barcoding of the standard COI gene fragment. We also conducted species-delimitation analyses based on the genetic data. The results showed that five evolutionarily significant units, corresponding to four nominal species inhabit the Ryukyu Islands, of which two species were also found in the greenhouses of mainland Japan, including the first record of Sarasinula plebeia in Japan. The presence of non-native slug species could increase the transmission of parasites in Japan.

High-throughput SNPs dataset reveal restricted population connectivity of marine gastropod within the narrow distribution range of peripheral oceanic islands.

Molecular studies based on the high resolution genetic markers help us to grasp the factor shaping the genetic structure of marine organisms. Ecological factors linking to life history traits have often explained the process of genetic structuring in open and connectable oceanic environments. Besides, population genetic divergence can be affected by fragmented habitat, oceanic current, and past geographical events. In the present study, we demonstrated the genetic differentiation of marine gastropod Monodonta sp. within a narrow range of peripheral oceanic islands, the Ogasawara Islands. Genetic analyses were performed not only with a mitochondrial DNA marker but also with a high-throughput SNPs dataset obtained by ddRAD-seq. The results of the mtDNA analyses did not show genetic divergence among populations, while the SNPs dataset detected population genetic differentiation. Population demographic analyses and gene flow estimation suggested that the genetic structure was formed by sea level fluctuation associated with the past climatic change and regulated by temporal oceanographic conditions. These findings provide important insights into population genetic patterns in open and connectable environments.

Patterns of diversification of the operculate land snail genus Cyclophorus (Caenogastropoda: Cyclophoridae) on the Ryukyu Islands, Japan.

The Ryukyu Islands, an island chain in southwestern Japan, originated from land masses that separated from the Eurasian continent due to the formation of sea barriers about 1.55 million years ago. In this study, we investigated the phylogenetic relationships of the operculate land snail genus Cyclophorus (Caenogastropoda: Cyclophoridae) in the Ryukyu Archipelago and surrounding regions based on DNA sequence data. According to our results, all studied Cyclophorus specimens from Japan form a monophyletic group containing eight subclades. Six of these subclades were found only on the Ryukyu Islands. On most islands of the Ryukyu Archipelago, no more than one Cyclophorus subclade was recorded, which may be due to limited ecological niche space and competition. No subclade was found to occur on both sides of the Watase Line, a regional zoogeographical boundary. Divergence times were estimated based on a time-calibrated phylogeny. We found that multiple splits among the Japanese Cyclophorus subclades predate the emergence of major sea barriers in the Ryukyu Archipelago. Vicariance due to sea barrier formation, as assumed for many other taxa from the region, was thus likely not the main driver for subclade divergence in these snails. Instead, certain geographical features might have shaped the diversification of subclades prior to sea barrier formation. Given that Cyclophorus populations were also present on islands that have never been connected to other land masses, the snails must have colonized them via oversea dispersal. As not all nominal taxa corresponded to monophyletic groups, our molecular phylogenetic approach revealed that a taxonomic revision of the Japanese Cyclophorus fauna is necessary. The eight subclades may be regarded as potential species-level groups based on COI p-distances. A canonical discriminant analysis using shell morphological data revealed slight differences among the subclades.

Comparing the genetic diversity and population structure of sister marine snails having contrasting habitat specificity.

BACKGROUND: To grasp the processes of spatial genetic structuring in open and connectable marine environments is the principal study goal in molecular biological studies. Comparative seascape genetics using multiple species are a powerful approach to understand the physical geographic and oceanographic effects on genetic variation. Besides, species-specific ecological traits such as dispersal abilities and habitat specificity are important factors for spatial genetic structuring. METHODS AND RESULTS: We focused on the sister marine snail species Tegula kusairo and T. xanthostigma around the Japanese mainland, which have contrasting habitat specificities for wave strength. Tegula kusairo only inhabits sheltered coastal environments, while T. xanthostigma is found mainly on wave-exposed rocky shores facing the open sea. We estimated their genetic diversity indices and levels of population differentiation based on mtDNA. We found that the genetic diversity of T. kusairo was lower than that of T. xanthostigma, while their level of population genetic differentiation was higher than that of T. xanthostigma. Namely, the species specific to weak wave environments had a higher level of population genetic differentiation than the species specific to strong wave action. CONCLUSION: Ecological traits linked not only to dispersal abilities but also to habitat specificity can influence genetic variation in a pair of closely related sister species distributed in the same seascape.

The mutual history of Schlegel's Japanese gecko (Reptilia: Squamata: Gekkonidae) and humans inscribed in genes and ancient literature.

Knowing how the present distribution of organisms was formed is an essential issue in evolutionary ecology. Recently, the distribution of organisms on Earth has been significantly changed by human-mediated dispersal due to globalization. Therefore, significant attention has been paid to such processes. However, although humankind has taken considerable time to achieve modernization, the impact of ancient human activity on ecosystems has not yet been thoroughly studied. We hypothesized that ancient urban development and transitions had a non-negligible effect on species distribution. Inferring the impact of past human activity on ecosystems from ancient literature and verifying that impact by genetic analysis and human history is an effective means of tackling this problem. As geckos, a popular neighbor of human dwellings, are good material for this model, we performed this combination approach using Schlegel's Japanese gecko, Gekko japonicus. We show that G. japonicus migrated from China to the western Japanese archipelago before Christ. The gecko species dispersed itself from western to eastern the archipelago on a time scale of thousands of years. There are many synchronizations between the dispersal history of G. japonicus and the historical development of human society. It is suggested by such synchronizations that humans have influenced the distribution of G. japonicus many times throughout its dispersal history.

Secondary contact of two cryptic Hokou gecko groups in the Izu Islands, Japan.

We analyzed the mitochondrial DNA of Gekko hokouensis collected from the Izu Islands (maybe an introduced population) and the Nansei Islands (native population), both in Japan. A molecular phylogenetic analysis suggested that G. hokouensis of Japan belongs to a cryptic monophyletic group different from that of the currently discovered sample of China. Furthermore, the Japanese clade of G. hokouensis is differentiated into two subclades (Clade 1 and Clade 2 in this article). In the Nansei Islands, these two subclades form a complicated nested-distribution pattern and do not coexist on any of the islands, whereas both clades appear to coexist in the Izu Islands. The two clades exhibit high genetic diversity in the Nansei islands, which are the source population. Surprisingly, it has been revealed that high genetic diversity has also been maintained in the Izu Islands, which are the introduced population, in each clade. AMOVA has also revealed that the genetic differentiation between the populations in the Izu Islands and the Nansei Islands was not significant in each clade. These results suggest that the population of the Izu Islands is now in secondary contact between two clades by multiple migrations from various regions of the Nansei Islands.

A complete dietary review of Japanese birds with special focus on molluscs.

Birds often hold important positions in the food webs of ecosystems. As a result, interactions between birds and their prey have attracted attention not only in ecology, but also in fields like agriculture and conservation. Avian food resources are well researched in Japan, however there is no database critically reviewing molluscs as a food resource for birds. Here, we present a new database reviewing dietary information for all Japanese bird species. In addition to addressing general diet categories and specific food habits for each bird, we include detailed data on the molluscan prey observed for all species that consume them. The information within this database was collected through intense literary review to provide a complete look at bird species historically present around the country. We also include new information on snail species found in the upper digestive tract of harvested wild birds. This database is publicly available in the Zenodo repository. The information should aid research around the Japanese archipelago, especially projects involving birds or molluscs.

Citizen science via social media revealed conditions of symbiosis between a marine gastropod and an epibiotic alga.

Environmental factors promote symbiosis, but its mechanism is not yet well understood. The alga Pseudocladophora conchopheria grows only on the shell of an intertidal gastropod Lunella correensis, and these species have a close symbiotic relationship which the alga reduces heat stress of the gastropod. In collaboration with general public, we investigated how environmental conditions alter the symbiotic interaction between the alga and the gastropod. Information about the habitats of each gastropod and images of shells was obtained from the Japanese and Korean coasts via social media. We constructed the hierarchical Bayesian model using the data. The results indicated that the proportion of shell area covered by P. conchopheria increased as the substrate size utilized by the gastropod increased. Meanwhile, temperature did not affect the proportion of P. conchopheria on the shell. These suggested that the alga provides no benefits for the gastropod on small substrates because gastropod can reduce the heat stress by diving into the small sediment. Further, the gastropod's cost incurred by growing the alga on the shell seems to be low as the algae can grow even in cooler places where no benefits of heat resistance for gastropods. Different environments can yield variable conditions in symbiosis.

Phylogeography of freshwater planorbid snails reveals diversification patterns in Eurasian continental islands.

BACKGROUND: Islands have traditionally been the centre of evolutionary biological research, but the dynamics of immigration and differentiation at continental islands have not been well studied. Therefore, we focused on the Japanese archipelago, the continental islands located at the eastern end of the Eurasian continent. While the Japanese archipelago is characterised by high biodiversity and rich freshwater habitats, the origin and formation mechanisms of its freshwater organisms are not clear. In order to clarify the history of the planorbid gastropod fauna, we conducted phylogenetic analysis, divergence time estimation, ancestral state reconstruction, and lineage diversity estimations. RESULTS: Our analyses revealed the formation process of the planorbid fauna in the Japanese archipelago. Most lineages in the Japanese archipelago have closely related lineages on the continent, and the divergence within the Japanese lineages presumably occurred after the late Pliocene. In addition, each lineage is characterised by different phylogeographical patterns, suggesting that immigration routes from the continent to the Japanese archipelago differ among lineages. Furthermore, a regional lineage diversity plot showed that the present diversity in the Japanese archipelago potentially reflects the differentiation of lineages within the islands after the development of the Japanese archipelago. CONCLUSIONS: Although additional taxon sampling and genetic analysis focused on each lineage are needed, our results suggest that immigration from multiple routes just prior to the development of the Japanese archipelago and subsequent diversification within the islands are major causes of the present-day diversity of the Japanese planorbid fauna.