Phylogenomic analyses reveal incongruences between divergence times and fossil records of freshwater snails in East Asia.

Fossils provide important insight into our understanding of phylogenetic history by serving as calibration points for divergence time estimation. However, uncertainties in the fossil record due to parallel evolution and convergent evolution can critically affect estimates of node ages. Here, we compare and contrast estimates of phylogenetic divergence with geologic and fossil history for two freshwater snail genera of the family Viviparidae in East Asia (Cipangopaludina and Margarya). Cipangopaludina species are commonly widely distributed species in East Asia, but extant Margarya species are endemic to the ancient lakes in Yunnan, China. According to some previous studies, parallel evolution or convergent evolution of shell morphology has occurred in the family several times which may affect divergence time estimation using fossil records. In this study, we used SNP data derived from ddRAD-seq loci to investigate population demographic history of both genera. Our results show a common pattern of lake endemic lineages diversifying from widely distributed lineages in the Miocene, and multiple colonization to a single ancient lake occurred in the Pleistocene. Our results indicate substantial incongruence among estimated phylogenomic divergence times, some fossil records, and formation ages of ancient lakes. These findings suggest some fossil records may be misidentified in these groups and highlight the need to carefully evaluate geological evidence and fossil records when using these for divergence time estimation.

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.

A global agenda for advancing freshwater biodiversity research.

Global freshwater biodiversity is declining dramatically, and meeting the challenges of this crisis requires bold goals and the mobilisation of substantial resources. While the reasons are varied, investments in both research and conservation of freshwater biodiversity lag far behind those in the terrestrial and marine realms. Inspired by a global consultation, we identify 15 pressing priority needs, grouped into five research areas, in an effort to support informed stewardship of freshwater biodiversity. The proposed agenda aims to advance freshwater biodiversity research globally as a critical step in improving coordinated actions towards its sustainable management and conservation.

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.

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.

Role of ancient lakes in genetic and phenotypic diversification of freshwater snails.

Endemic organisms of ancient lakes have been studied as models to understand processes of speciation and adaptive radiation. However, it remains unclear how ancient lakes play roles in genetic and phenotypic diversity of freshwater mollusks. In the present study, we focus on viviparid freshwater snails in the ancient lakes of East and Southeast Asia (Japan and China) to address this question. Using molecular phylogenetic analyses based on mitochondrial (COI, 16S) and nuclear genes (18S, 28S, H3), we show that patterns of species diversification in viviparid lineages. Colonization to ancient lakes occurred independently in China and Japan at least four times, with subsequent diversification into more than two species within each lake group. Morphological analyses of fossil related viviparids suggest parallel phenotypic evolution occurred in the different lakes and ages. Each lake contained a single lineage which was phenotypically diversified relative to those from other sites. Using genome-wide SNPs obtained by MIG-seq, we also examined the genetic structure of three Japanese viviparids, including two endemic species of ancient Lake Biwa. The results suggest that these two species diversified from the population of the third species living in wetlands surrounding the lake. These findings suggest that rapid diversification of lineages and phenotypic divergence can occur in ancient lakes compared to other habitats. Formation of large lakes probably promotes speciation and phenotypic divergence as a result of adaptation into different microhabitats. High numbers of ancient lakes could be a driver of species diversity in Asian viviparid snails.

Expansion and systematics redefinition of the most threatened freshwater mussel family, the Margaritiferidae.

Two Unionida (freshwater mussel) families are present in the Northern Hemisphere; the Margaritiferidae, representing the most threatened of unionid families, and the Unionidae, which include several genera of unresolved taxonomic placement. The recent reassignment of the poorly studied Lamprotula rochechouartii from the Unionidae to the Margaritiferidae motivated a new search for other potential species of margaritiferids from members of Gibbosula and Lamprotula. Based on molecular and morphological analyses conducted on newly collected specimens from Vietnam, we here assign Gibbosula crassa to the Margaritiferidae. Additionally, we reanalyzed all diagnostic characteristics of the Margaritiferidae and examined museum specimens of Lamprotula and Gibbosula. As a result, two additional species are also moved to the Margaritiferidae, i.e. Gibbosula confragosa and Gibbosula polysticta. We performed a robust five marker phylogeny with all available margaritiferid species and discuss the taxonomy within the family. The present phylogeny reveals the division of Margaritiferidae into four ancient clades with distinct morphological, biogeographical and ecological characteristics that justify the division of the Margaritiferidae into two subfamilies (Gibbosulinae and Margaritiferinae) and four genera (Gibbosula, Cumberlandia, Margaritifera, and Pseudunio). The systematics of the Margaritiferidae family is re-defined as well as their distribution, potential origin and main biogeographic patterns.

Phylogeny of the most species-rich freshwater bivalve family (Bivalvia: Unionida: Unionidae): Defining modern subfamilies and tribes.

Freshwater mussels of the order Unionida are key elements of freshwater habitats and are responsible for important ecological functions and services. Unfortunately, these bivalves are among the most threatened freshwater taxa in the world. However, conservation planning and management are hindered by taxonomic problems and a lack of detailed ecological data. This highlights the urgent need for advances in the areas of systematics and evolutionary relationships within the Unionida. This study presents the most comprehensive phylogeny to date of the larger Unionida family, i.e., the Unionidae. The phylogeny is based on a combined dataset of 1032bp (COI+28S) of 70 species in 46 genera, with 7 of this genera being sequenced for the first time. The resulting phylogeny divided the Unionidae into 6 supported subfamilies and 18 tribes, three of which are here named for the first time (i.e., Chamberlainiini nomen novum, Cristariini nomen novum and Lanceolariini nomen novum). Molecular analyses were complemented by investigations of selected morphological, anatomical and behavioral characters used in traditional phylogenetic studies. No single morphological, anatomical or behavioral character was diagnostic at the subfamily level and few were useful at the tribe level. However, within subfamilies, many tribes can be recognized based on a subset of these characters. The geographical distribution of each of the subfamilies and tribes is also presented. The present study provides important advances in the systematics of these extraordinary taxa with implications for future ecological and conservation studies.

A new species of Tiwaripotamon Bott, 1970, from northern Vietnam, with notes on T. vietnamicum (Dang & Ho, 2002) and T. edostilus Ng & Yeo, 2001 (Crustacea, Brachyura, Potamidae).

A new species of potamid freshwater crab, Tiwaripotamon vixuyenense sp. nov., is described from caves in Ha Giang, northern Vietnam. The new species can be distinguished from congeners by a suite of characters, including the relatively small epibranchial tooth, more slender ambulatory legs and a proportionately stout male first gonopod. Molecular data using the mitochondrial cytochrome oxidase subunit I (COI) supports its placement as a new species. The molecular data also supports the inclusion of another species, Geothelphusa vietnamica Dang & Ho, 2002, from Ninh Binh, south of Hanoi, Vietnam in Tiwaripotamon Bott, 1970. The live coloration of T. vietnamicum and T. edostilus Ng & Yeo, 2001 is provided to help field identification.

A novel assembly pipeline and functional annotations for targeted sequencing: A case study on the globally threatened Margaritiferidae (Bivalvia: Unionida).

The proliferation of genomic sequencing approaches has significantly impacted the field of phylogenetics. Target capture approaches provide a cost-effective, fast and easily applied strategy for phylogenetic inference of non-model organisms. However, several existing target capture processing pipelines are incapable of incorporating whole genome sequencing (WGS). Here, we develop a new pipeline for capture and de novo assembly of the targeted regions using whole genome re-sequencing reads. This new pipeline captured targeted loci accurately, and given its unbiased nature, can be used with any target capture probe set. Moreover, due to its low computational demand, this new pipeline may be ideal for users with limited resources and when high-coverage sequencing outputs are required. We demonstrate the utility of our approach by incorporating WGS data into the first comprehensive phylogenomic reconstruction of the freshwater mussel family Margaritiferidae. We also provide a catalogue of well-curated functional annotations of these previously uncharacterized freshwater mussel-specific target regions, representing a complementary tool for scrutinizing phylogenetic inferences while expanding future applications of the probe set.

Diversity and Distribution of Fiddler Crabs (Crustacea: Brachyura: Ocypodidae) in Vietnam.

Based on recently collected material and records in the literature, 14 species of fiddler crabs (Crustacea: Ocypodidae: Gelasiminae) are reported from Vietnam. DNA barcoding analyses using the mitochondrial gene COI (cytochrome c oxidase subunit I) was performed to identify examined materials and their precise distributional range. Thirteen species-level taxa are identified and, with the exception of Galsimus borealis and G. vocans, have minimum interspcific divergences of at least 7.27%. The identified species include seven species of Tubuca Bott, 1973, three of Austruca Bott, 1973 and three of Gelasimus Latreille, 1817, and one Paraleptuca Bott, 1973. Two new records of Vietnam are herein reported: Tubuca rhizophorae and T. dussumieri. The southernmost distribution limits of East Asian G. borealis, T. acuta and T. arcuata are in northern Vietnam, A. lactea in central Vietnam, whereas northernmost limit of Southeast Asian T. rhizophorae and T. forcipata in southern Vietnam. A dichotomous key to identify the 14 Vietnamese species is provided.

Human-geographic effects on variations in the population genetics of Sinotaia quadrata (Gastropoda: Viviparidae) that historically migrated from continental East Asia to Japan.

BACKGROUND: Anthropogenic factors potentially affect observed biogeographical patterns in population genetics, but the effects of ancient human activities on the original patterns created by natural processes are unknown. Sinotaia quadrata, a widely distributed freshwater snail species in East Asia, was used to investigate this issue. It is unclear whether S. quadrata in Japan was introduced from China and how different human uses and varying geographic patterns affect the contemporary population genetics between the two regions. Thus, we investigated the demography of S. quadrata and detected its genetic structure in Japan and continental East Asia. RESULTS: Sinotaia quadrata populations first naturally migrated from continental East Asia to Japan, which is associated with the ancient period in Japanese geohistory (about 70,000 years ago). They were then artificially introduced in association with agriculture expansion by human movements in two recent periods (about 8,000 and 1,200 years ago). Populations in different parts of Japan have their own sources. Natural migration in the ancient period and artificial introduction in the recent period suggest that the population distribution is affected by both the geohistory of East Asia and the history of human expansion. In the background of the historical migration and introduction, contemporary populations in the two regions show different genetic patterns. Population divergence levels were significantly correlated with geographical patterns in Japan and significantly correlated with human interventions variables in continental East Asia, suggesting that long-term geographical isolation is likely the major factor that shaped the contemporary population genetics in Japan, while modern human uses are likely the major factor in continental East Asia. CONCLUSIONS: Our preliminary results show a complex demography and unusual genetic patterns in the contemporary populations for a common freshwater snail and are of significance to determine the historical formation and contemporary patterns of biogeography in Japan and continental East Asia.

Enigmatic incongruence between mtDNA and nDNA revealed by multi-locus phylogenomic analyses in freshwater snails.

Phylogenetic incongruence has frequently been encountered among different molecular markers. Recent progress in molecular phylogenomics has provided detailed and important information for evolutionary biology and taxonomy. Here we focused on the freshwater viviparid snails (Cipangopaludina chinensis chinensis and C. c. laeta) of East Asia. We conducted phylogenetic analyses and divergence time estimation using two mitochondrial markers. We also performed population genetic analyses using genome-wide SNPs. We investigated how and which phylogenetic patterns reflect shell morphology. The results showed these two species could be separated into four major mitochondrial clades, whereas the nuclear clusters supported two groups. The phylogenetic patterns of both mtDNA and nDNA largely reflected the geographical distribution. Shell morphology reflected the phylogenetic clusters based on nDNA. The findings also showed these two species diversified in the Pliocene to early Pleistocene era, and occurred introgressive hybridisation. The results also raise the taxonomic issue of the two species.

Binhthuanomon vinhtan, a new genus and new species of semi-terrestrial freshwater crab (Crustacea: Decapoda: Brachyura: Potamidae) from south central Vietnam.

A new genus and new species of semi-terrestrial crab of the family Potamidae, Binhthuanomon vinhtan n. sp., is described from south central Vietnam. The new genus is morphologically closest to Villopotamon Dang & Ho, 2003, and Balssipotamon Dang & Ho, 2008, in the shapes of the carapace and first gonopod but is easily distinguished by distinct carapace characteristics and gonopod 1 structures, and the habitat occupied.