Cavernacmella dentata Fukuda, Sawada & Kameda, n. sp. is described from the inside of the Sekigahara Limestone Cave, Gifu Prefecture, central Honsh & umacr;, Japan. The new species is unique in the genus and the Assimineidae in having a strong fold on the parietal lip of the aperture, but according to its internal anatomy and a molecular phylogenetic analysis it is a member of Cavernacmella Habe, 1942. (sic)(sic)(sic)(sic) (JAPANESE ABSTRACT)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic) Cavernacmella dentata Fukuda, Sawada and Kameda, n. sp. (sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic).(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic), (sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic) Cavernacmella Habe, 1942 (sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic).ZooBank registration: https://www.zoobank.org/NomenclaturalActs/6ace1748-1075-419a-9f84-231a10896df8

Abstract Characterized by the earliest use of pottery, the Jomon culture was a unique Neolithic culture that spread throughout the Japanese Archipelago. Previous archaeological evidence suggests that Jomon hunter-gatherers colonized the southernmost islands, the Ryukyu Archipelago, by approximately 7,000 years before present (YBP). However, genetic characteristics of the Ryukyu Jomon population and its contribution to the modern population have not been elucidated yet. In this study, we newly sequenced 273 modern and 25 ancient (6,700–900 YBP) whole genomes collected across the Ryukyu Archipelago. Our analysis demonstrated a genetic differentiation between the Hondo (Japanese mainland) and Ryukyu Jomon, dating back to ∼6,900 YBP. After the divergence from the Hondo Jomon, the Ryukyu Jomon experienced severe bottlenecks, with an effective population size of ∼2,000. Admixture between the Ryukyu Jomon and migrants from the historic Hondo population occurred ∼1,000 YBP, which corresponds to the widespread adoption of iron tools and agriculture in the Central Ryukyus. Different demographic histories between modern Hondo and Ryukyu populations resulted in different rates of Jomon ancestry in these populations. By providing a new perspective on the peopling of the Ryukyu Archipelago, this study significantly enhances our understanding of cultural transitions in the region.

Aim When a specific lineage evolves to become significantly larger compared to other lineages in island environments, this can be considered autapomorphic giantism, which follows the island rule. For historical evolutionary events that cannot be reproduced in the laboratory, pattern-based validation is essential to infer the most likely evolutionary processes. This study aimed to test whether the reduction in area due to fragmentation is consistently associated with increased body size in isolated populations, potentially reflecting autapomorphic giantism.Location Amami Islands, Ryukyu Islands, Japan.Taxon Operculate land snail complex Cyclophorus hirasei hirasei/oshimanus (Caenogastropoda: Cyclophoridae).Methods To investigate phylogenetic relationships and population structure within the C. h. hirasei/oshimanus complex, we performed phylogenomic analyses and admixture analyses based on MIG-seq data. Past population dynamics were inferred via approximate Bayesian computation (ABC), and divergence times were estimated using calibrated time-correlated trees. Migration rates among populations were estimated. Shell width variation was analysed using Gaussian mixture models (GMM), and its relationship with environmental factors was tested using phylogenetic generalized linear mixed models (PGLMM).Results We detected a repeated association between body size and habitat across genetically distinct populations, even when accounting for phylogenetic relationships (autapomorphic giantism in C. h. hirasei). Parallel evolution in body size occurred among populations on different islands within a short period since the formation of the current islands 2 Ma ago. The migration rate results indicate that the population was extremely sedentary and may have adapted to its habitat independently in each region. Many of the islands were once part of the mainland, and body size may have increased following their isolation due to island formation.Main Conclusions Our results demonstrate a spatially replicated pattern of body size divergence that aligns with predictions of the island rule. To our knowledge, this is among the first studies to investigate autapomorphic giantism in terrestrial molluscs from a phylogenomic perspective. These findings highlight Cyclophorus of the Ryukyu Islands as a promising model for studying the predictability of trait evolution and ecological diversification in insular systems.

Recent studies have shown the significant role of introgression in evolution, yet the confident evidence of introgression has come from biased taxa. Although the Mollusca were representative taxa to show introgression-driven evolution, conclusive examination has been rare. Here, we investigated the genetic structure and evolutionary history of terrestrial snails in the Euhadra peliomphala species complex (Heterobranchia: Camaenidae). We collected 197 individuals from 196 sites in Japan, covering the entire distribution, and obtained genome-wide single nucleotide polymorphisms (SNPs) using double digest restriction site-associated DNA sequencing. The population genetic analysis showed that the study individuals are clustered into 12 lineages, which are distributed parapatrically. Our hybrid test detected hybrid zones between several pairs of the lineages. Additionally, we reconstructed a phylogenetic network of these lineages and detected three reticulate events, including introgression from an extinct lineage. The ages of the introgression events suggests that geological activity and climate change were involved. We also found human-induced introgression. Overall, the present research showed a highly reticulated evolutionary history of the E. peliomphala species complex, and provided confident evidence of introgression in terrestrial snails. This study should play a major role as a basis for further research on introgression-driven evolution within the E. peliomphala species complex.

Environmental DNA (eDNA) measurements have been widely applied in the biomonitoring of organisms. However, different DNA extraction methods may cause changes in yield and consistency, resulting in an inaccurate estimation of eDNA, especially when quantitative measurements are performed. This study focused on evaluating the effects of DNA extraction methods on the magnitude and consistency of fish DNA yield from water samples. Samples were collected periodically over a year from river and lake water systems, and reference eDNA was spiked into them. The spiked samples were extracted using three methods: using lysis buffer for blood and cells (AL method) with the DNeasy Blood and Tissue Kit (Qiagen), using lysis buffer for tissue (ATL method), and the microfluidic-channel method (BC method). DNA yields were compared by determining the copy number using qPCR. The ATL method showed better stability and a higher yield than the AL method. In addition, despite being comparatively simple, the BC method performed extractions stably and at relatively high yields. Based on the long-term evaluation of eDNA measurements using various methods, we showed that differences in DNA extraction methods may alter DNA yield and stability.

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.

The all raw data of quantitative PCR for environmental DNA in Fukuzawa, T et al. "Environmental DNA extraction method for a high and stable DNA yield".

Environmental DNA measurement has been widely applied in organism biomonitoring. Different DNA extraction methods may cause changes in yield and stability, resulting in an inaccurate estimation of eDNA, especially when quantitative measurements are performed. This study focused on the DNA extraction method and compared its yield and stability for stream fish and spiked DNA samples. Samples were collected periodically over a year from river and lake water systems and eDNA was spiked into them. The samples were extracted and compared using three methods: using Buffer-AL for initial lysis with the DNeasy Blood and Tissue Kit (Qiagen); using Buffer-ATL for initial lysis and the microfluidic-channel method (BC method). The method using Buffer-ATL in the DNeasy Blood and Tissue Kit showed better stability and a higher yield than the Buffer-AL method. In addition, the BC method, despite being comparatively simple, performed the extraction stably and with relatively high yields. We showed that differences in DNA extraction methods based on the long-term evaluation of eDNA measurements with various methods may cause alterations in DNA yield and stability.

We demonstrated the genetic variation and population structure of a direct developing marine gastropod, Littorina horikawai which is endemic species distributed in the western coast of Kyushu, Japan and is found mainly on the adjacent islands. We conducted the field survey and sampling covering most of the distribution area of L. horikawai. In total, we collected 173 individuals from 13 localities (between Tsushima Island and Yakushima Island) and determined the sequences of mitochondrial DNA, the cytochrome b (CYTB) gene. We detected a significant genetic population differentiation in L. horikawai despite the limited distribution area of the western coast of Kyushu. Also, its haplotype distribution is mainly consistent with the geographical structure: the northern part of Kyushu, the central islands, and the southern islands. However, some haplotypes obtained from the Goto Islands which is located in the border between the northern part of Kyushu and the central islands do not correspond to the above geographic distribution. The high level of genetic differentiation of L. horikawai compared to other low dispersal Littorina species despite its narrow distribution range may be due to its predominantly fragmented island habitats. Although it is necessary for understanding the detailed evolutionary history of L. horikawai to perform the high-resolution genetic analysis, our present findings indicated that a high level of genetic differentiation of L. horikawai occurred even in a narrow distribution range because of not only the ecological traits linking to dispersal ability but also its discontinuous pattern such as the island inhabitation.

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.

The environmental DNA (eDNA) method, which is widely applied for biomonitoring, is limited to laboratory analysis and processing. In this study, we developed a filtration/extraction component using a microfluidic channel, Biryu-Chip (BC), and a filtration/extraction method, BC method, to minimize the volume of the sample necessary for DNA extraction and subsequent PCR amplification. We tested the performance of the BC method and compared it with the Sterivex filtration/extraction method using aquarium and river water samples. We observed that using the BC method, the same concentration of the extracted DNA was obtained with 1/20–1/40 of the filtration volume of the Sterivex method, suggesting that the BC method can be widely used for eDNA measurement. In addition, we could perform on-site measurements of eDNA within 30 min using a mobile PCR device. Using the BC method, filtration and extraction could be performed easily and quickly. The PCR results obtained by the BC method were similar to those obtained by the Sterivex method. The BC method required fewer steps and therefore, the risk of DNA contamination could be reduced. When combined with a mobile PCR, the BC method can be applied to easily detect eDNA within 30 min from a few 10 mL of the water sample, even on-site.

The metacercarial infections of door snails (Gastropoda: Clausiliidae) with unknown species of the genus Brachylaima (Trematoda: Brachylaimidae) have recently been reported in eastern Honshu and Kyushu, Japan. A large scale snail survey was carried out to clarify their taxonomic status. From the period of 2015 to 2020, a total of 1239 land snails (768 door snails and 471 others) were collected from 32 localities in Honshu, Shikoku, and Kyushu. The resulting trematode isolates were identified as Brachylaima sp. by mitochondrial DNA barcoding. The sporocysts were found only a few from Megalophaedusa sublunellata (Clausiliidae), Tauphaedusa subaculus (Clausiliidae), and Aegista trochula (Camaenidae), while the metacercariae were frequently detected from 14 species of Clausiliidae and 2 species of other families. Although Brachylaima sp. showed a broad range of intermediate hosts, door snails seem to be very important to drive the life cycle. The gravid adults of Brachylaima sp. was experimentally raised from metacercariae using immunosuppressed mice. Morphological, phylogenetical, and ecological considerations prompted us to propose Brachylaima phaedusae n. sp. for this unknown species. The definitive hosts of the new species are completely unknown. The wide geographic distribution and high genetic diversity of the new species suggest a possibility that the definitive host is ground-foraging birds, which prefer door snails.

Abstract To clarify the effect of niche conservatism on evolutionary history, we focused on freshwater snails, which have different ecological and phylogenetic properties from previously tested taxa. We conducted a phylogenetic analysis using 750 lymnaeid individuals from 357 sites of eleven Radix species. Then, we estimated the ancestral distribution using the geographic coordinates and colonization routes. In addition, a statistical test of the colonization distances in the latitudinal and longitudinal directions was performed. We also conducted ecological niche modeling for two widely distributed species using climatic data. Ancestral geographic reconstruction estimated the origin of the genus to be around the Indian subcontinental region and showed that latitudinal immigration distances were shorter than longitudinal immigration distances in the diversification process. Ecological niche models suggested that the current distribution was restricted by climate, with annual mean temperature and precipitation of the driest month as particularly strong factors. Niche conservatism to the climate can affect the diversification of freshwater snails.

Molecular methods, including those for environmental DNA (eDNA), provide essential information for biological and conservation sciences. Molecular measurements are often performed in the laboratory, which limits their scope, especially for rapid on‐site analysis. eDNA methods for species detection provide essential information for the management and conservation of species and communities in various environments. We have developed an innovative novel method for on‐site eDNA measurements using an ultrarapid mobile PCR platform. We tested the ability of our method to detect the distribution of silver carp, Hypophthalmichthys molitrix , in rivers and lakes. Our method reduced the measurement time to 30 min and provided high detectability of aquatic organisms compared to national observation surveys that use multiple fishing nets and laboratory extraction/detection using a benchtop qPCR platform. Our on‐site eDNA method can be immediately applied to various taxa and environments.

Molecular methods, including environmental DNA (eDNA) methods, provide essential information for biological and conservation sciences. Molecular measurements are often performed in the laboratory, which limits their scope, especially for rapid on-site analysis. eDNA methods for species detection provide essential information for the management and conservation of species and communities in various environments. We developed an innovative novel method for on-site eDNA measurements using an ultra-rapid mobile PCR platform. We tested the ability of our method to detect the distribution of silver carp, Hypophthalmichthys molitrix, an invasive fish in Japanese rivers and lakes. Our method reduced the measurement time to 30 min and provided high detectability of aquatic organisms compared to the national observation surveys using multiple fishing nets and laboratory extraction/detection using a benchtop qPCR platform. Our on-site eDNA method can be immediately applied to various taxa and environments.

Granulilimax fuscicornis Minato, 1989 is an endemic land slug of Japan . This slug was described as a member of the stylommatophoran family Philomycidae Gray, 1847, and subsequently transferred into the systellommatophoran family Rathouisiidae Heude, 1885. It is still unclear whether the taxonomic revision to Rathouisiidae is supported by genetic data. In this study, we investigated the phylogenetic position of the genus Gran ulilimax using G. fuscicornis and an undescribed species Gran ulilimax sp. Although the phylogenetic relationships between G. fuscicornis and rathouisiid slugs remain to be solved, our preliminary molecular analysis using mitochondrial COI and nuclear 18S genes strengthen the idea that the Japanese Granulilimax slugs belong to Systellommatophora.

Vicariance events have been proposed as a major source of lineage divergence on continental islands, whereas dispersal events followed by isolation have been proposed as the major cause on oceanic islands. However, organisms on continental islands may include taxa with characteristics similar to those on oceanic islands. Lineage divergence unassociated with the geological events that separated islands may also have occurred. This study addresses these possibilities through morphological and molecular phylogeographic analyses of land snails (Bradybaena) on continental islands.

We identified newly discovered pamphiliid larvae feeding on Cornus in Tochigi and Ibaraki Prefectures, Honshu, Japan, with Pamphilius japonicus Shinohara, 1985, by molecular methods using mitochondrial cytochrome oxidase subunit 1 (COI) gene sequences. The host plant of P. japonicus was unknown. This is the first record of the Cornaceae as a host plant of the Pamphiliidae in the Old World. The larvae were solitary leaf-rollers on Cornus and discovered mainly on small young trees in shadowy forests.

Taxonomic status of the widely spread xiphydriid woodwasp Xiphydria camelus (Linné, 1758) was revised by examining morphology of 964 specimens and by molecular analysis using COI barcode sequences. Both morphological and molecular approaches indicated existence of three separate species masquerading in X. camelus of authors in East Asia. The three species were finally determined as X. camelus, X. eborata Konow, 1899, and a new species, X. albopicta. Xiphydria eborata is revived from synonymy with X. camelus and X. albopicta is described as a new species from Russia (Primorskij Kraj), China (Heilongjiang) and Japan (Hokkaido). Collection records are given for all the specimens examined.

Sample information of Planorbidae in this study. See also Fig. 1. TUMC samples were deposited in the Tohoku University Museum Collection, Tohoku University; OKCABM samples were deposited in the Laboratory of Conservation of Aquatic Biodiversity, Faculity of Agriculture, Okayama University. (XLSX 26Â kb)