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.

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.

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.

Substantial incongruence among the morphology, taxonomy, and molecular phylogeny of the land snails Aegista, Landouria, Trishoplita, and Pseudobuliminus (Pulmonata: Bradybaenidae) occurring in East Asia.

Analyses of molecular phylogeny have revealed that phenotypically identified taxa do not reflect phylogenetic relationships. Such taxa often are not evolutionary significant and are misleading in describing and evaluating biological diversity. Herein, we investigated the molecular phylogeny of the East Asian bradybaenid land snail genera Aegista, Landouria, Trishoplita, and Pseudobuliminus to test whether morphology and current taxonomy reflect phylogenetic relationships. Our results document extensive parallel evolution and substantial incongruence between taxonomy and molecular phylogeny. Species with an elongated turret shell evolved independently five times, and hair-like ornamentation of the shell was independently gained or lost in a number of lineages. Although genital anatomy reveals phylogenetic conservatism to some extent, love dart and dart-related organs were lost independently at least three times in these genera. Accordingly, classification of these genera based on morphological traits did not reflect phylogenetic relationships, and, overall, these genera except for a few species should be tentatively assigned to a single genus as Aegista. The present findings suggest that radical revision is required for the taxonomy of bradybaenid land snails.

Long-term stasis and short-term divergence in the phenotypes of microsnails on oceanic islands.

Phenotypic divergence is often unrelated to genotypic divergence. An extreme example is rapid phenotypic differentiation despite genetic similarity. Another extreme is morphological stasis despite substantial genetic divergence. These opposite patterns have been viewed as reflecting opposite properties of the lineages. In this study, phenotypic radiation accompanied by both rapid divergence and long-term conservatism is documented in the inferred molecular phylogeny of the micro land snails Cavernacmella (Assimineidae) on the Ogasawara Islands. The populations of Cavernacmella on the Sekimon limestone outcrop of Hahajima Island showed marked divergence in shell morphology. Within this area, one lineage diversified into types with elongated turret shells, conical shells and flat disc-like shells without substantial genetic differentiation. Additionally, a co-occurring species with these types developed a much larger shell size. Moreover, a lineage adapted to live inside caves in this area. In contrast, populations in the other areas exhibited no morphological differences despite high genetic divergence among populations. Accordingly, the phenotypic evolution of Cavernacmella in Ogasawara is characterized by a pattern of long-term stasis and periodic bursts of change. This pattern suggests that even lineages with phenotypic conservatism could shift to an alternative state allowing rapid phenotypic divergence.

Filtration extraction method using a microfluidic channel for measuring environmental DNA.

The environmental DNA (eDNA) method, which is widely applied in biomonitoring, is limited to laboratory analysis and processing. In this study, we developed a filtration/extraction component using a microfluidic channel, the Biryu-Chip (BC), and a filtration/extraction method, the 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 that of the Sterivex filtration/extraction method using aquarium and river water samples. We observed that using the BC method, the same concentration of 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 performed on-site measurements of eDNA within 30 min using a mobile PCR device, demonstrating that filtration and extraction can be performed easily and quickly using the BC method. The PCR results obtained using the BC method were similar to those obtained using the Sterivex method. The BC method requires fewer steps; therefore, the risk of DNA contamination can be reduced. When combined with mobile PCR, the BC method can be applied to easily detect eDNA within 30 min from the collection of water sample, even on-site.

Effects of mouthpiece shape and expiratory threshold loading on contraction of the lateral abdominal muscles: A cross-sectional study.

BACKGROUND: Expiratory tasks may facilitate transversus abdominis (TrA) activity for spinal stabilization. The purpose of this study was to verify whether a combination of pursed-lip breathing (PLB) and use of an expiratory threshold loading (ETL) device to increase expiratory resistance would promote TrA contraction comparable to that for a stabilization exercise. METHODS: Twenty healthy men performed expiratory tasks or an abdominal drawing-in maneuver (ADIM). Expiratory tasks comprised combinations of ETL settings with 0%, 5%, or 15% of maximum expiratory pressure, and mouthpieces with a normal shape or pursed-lip shape. B-mode ultrasound imaging of the TrA, internal oblique, and external oblique muscles was performed to determine percentage changes in muscle thickness. Percentage changes among tasks were statistically compared for each muscle. FINDINGS: TrA thickness increased with normal lips at 15% ETL, with PLB with 5% ETL, and with ADIM (p < 0.01 each). Internal oblique thickness increased under all PLB conditions (p < 0.01 each). No significant differences in external oblique thickness were seen for any tasks. The total thickness of the lateral abdominal muscles was significantly increased not only for 15% ETL tasks and ADIM, but also for PLB with 5% ETL (p < 0.02 each). INTERPRETATION: These results indicated that PLB with 5% ETL could facilitate the same level of TrA activity as the ADIM. PLB with 5% ETL was the only task that simultaneously increased overall lateral abdominal muscles, including the internal obliques, and might be readily applicable in clinical situations.

Brachylaima phaedusae n. sp. (Trematoda: Brachylaimidae) from door snails in Japan.

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.

Terrestrial invasion of pomatiopsid gastropods in the heavy-snow region of the Japanese Archipelago.

BACKGROUND: Gastropod mollusks are one of the most successful animals that have diversified in the fully terrestrial habitat. They have evolved terrestrial taxa in more than nine lineages, most of which originated during the Paleozoic or Mesozoic. The rissooidean gastropod family Pomatiopsidae is one of the few groups that have evolved fully terrestrial taxa during the late Cenozoic. The pomatiopsine diversity is particularly high in the Japanese Archipelago and the terrestrial taxa occur only in this region. In this study, we conducted thorough samplings of Japanese pomatiopsid species and performed molecular phylogenetic analyses to explore the patterns of diversification and terrestrial invasion. RESULTS: Molecular phylogenetic analyses revealed that Japanese Pomatiopsinae derived from multiple colonization of the Eurasian Continent and that subsequent habitat shifts from aquatic to terrestrial life occurred at least twice within two Japanese endemic lineages. Each lineage comprises amphibious and terrestrial species, both of which are confined to the mountains in heavy-snow regions facing the Japan Sea. The estimated divergence time suggested that diversification of these terrestrial lineages started in the Late Miocene, when active orogenesis of the Japanese landmass and establishment of snowy conditions began. CONCLUSIONS: The terrestrial invasion of Japanese Pomatiopsinae occurred at least twice beside the mountain streamlets of heavy-snow regions, which is considered the first case of this event in the area. Because snow coverage maintains stable temperatures and high humidity on the ground surface, heavy-snow conditions may have paved the way for these organisms from freshwater to land via mountain streamlets by preventing winter desiccation in mountain valleys. The fact that the terrestrialization of Pomatiopsidae occurred only in year-round wet environments, but not in seasonally dried regions, provides new insight into ancient molluscan terrestrialization.

Pattern and process of diversification in an ecologically diverse epifaunal bivalve group Pterioidea (Pteriomorphia, Bivalvia).

The pterioid bivalves (superfamily Pterioidea) are epifaunal filter feeders that attach to various substrata, including rocks, corals, gorgonians, and sponges. An intriguing question is how different substratum types have affected the diversification of pterioid bivalves. To elucidate the evolutionary pathway of Pterioidea, we conducted a phylogenetic analysis of 49 individuals belonging to 18 pterioid and 5 outgroup species using 18S and 28S ribosomal RNA genes. The results supported the monophyly of superfamily Pterioidea and recovered three major clades within Pterioidea: Malleus, Pteria and Electroma (Pterelectroma) zebra and the rest. This result contradicts the current circumscription of the pterioid families, Pteriidae, Malleidae, and Isognomonidae, and suggests that hinge morphology, which has long been used as a diagnostic character, does not reflect phylogenetic relationships. The monophyly of most genera, however, was confirmed. Mapping substrata types on the phylogenetic tree indicated that the ancestors of pterioid bivalves were epifaunal on rocks, and that epizoic life on sessile cnidarians had a single origin. Although we could not ascertain whether endozoic life in sponges evolved once or twice, our results suggest that colonization of biotic substrata resulted in diversification and morphological and ecological adaptation to epi-/endozoic life. We estimated that the genus Pteria has diversified since the late Cretaceous in response to gorgonian diversification. These results emphasize the importance of substratum shifts in speciation and diversification of pterioid bivalves.

On-site environmental DNA detection of species using ultrarapid mobile PCR.

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.

A comprehensive phylogeography of the widespread pond snail genus Radix revealed restricted colonization due to niche conservatism.

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.

Reproductive character displacement in genital morphology in Satsuma land snails.

When species diverge in allopatry and come into secondary contact, natural selection might favor character divergence to facilitate partner discrimination and avoid mating interference. This process is called reinforcement, which typically results in greater differentiation in mating-associated traits in areas of sympatry (reproductive character displacement). Such patterns are usually manifest in premating signals, but little is known about how interspecific mating affects the morphological evolution of genitalia. Here, we present evidence of reproductive character displacement in genital morphology in two land snail species of the genus Satsuma. Morphometric analyses clearly revealed greater sympatric divergences in penis and vagina lengths, regardless of the environmental, genetic, and geographic effects. This is the first demonstration of reproductive character displacement in genital characters in an organism other than insects and highlights the importance of analyzing previously overlooked genital morphologies in understanding the general mechanisms of prezygotic isolation in land snails.

DNA barcodes and morphology revealed three species masquerading in Xiphydria camelus of authors (Hymenoptera, Xiphydriidae) in Northeast Asia: X. eborata sp. rev. and X. albopicta sp. nov.

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.

Pamphilius japonicus feeds on Cornus: the first Old World record of Pamphiliidae (Hymenoptera) associated with Cornaceae.

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.

Redundant species, cryptic host-associated divergence, and secondary shift in Sennertia mites (Acari: Chaetodactylidae) associated with four large carpenter bees (Hymenoptera: Apidae: Xylocopa) in the Japanese island arc.

Sennertia mites live as inquilines in the nests of carpenter bees and disperse as deutonymphs on newly emerged adult bees. Because their life cycle is tightly linked to that of the host bees, Sennertia may diverge in response to speciation in the hosts. However, the majority of Sennertia species are associated with several closely related carpenter bees, suggesting that host speciation may not be reflected in mite genetic structure. Here we investigate the extent of host-associated genetic differentiation in two Sennertia mites (S. alfkeni and S. japonica) that share four closely related, strictly allopatric large carpenter bees (Xylocopa). Analysis of the mitochondrial cytochrome oxidase subunit I (COI) gene in Sennertia unexpectedly indicates that the two species represent morphological variants of a single species, and they collectively group into four distinct, allopatric clades that are uniquely associated with a single Xylocopa host. An exception is the mites associated with X. amamensis of the northernmost populations, which have genotypes typical of those associated with neighboring X. appendiculatacircumvolans. Additional analysis using amplified fragment length polymorphism (AFLP) further corroborates the presence of four mite clades but contrary to the COI data, suggests that the mites of the southernmost population of X. appendiculatacircumvolans have genetic profiles typical of those associated with X. amamensis. These results indicate that some mites have undergone secondary host switch after the formation of the four mite lineages and further experienced mitochondrial introgression during period of lineage coexistence. Overall, our results strongly urge reappraisal of deutonymph-based mite taxonomy and illuminate the importance of host-associated divergence during incipient stage of speciation in chaetodactylid mites. Furthermore, the occurrence of host switch and introgression between genetically differentiated mites entails that two host species have co-occurred in the past, thus providing a unique source of evidence for migration and competitive exclusion between the presently allopatric Xylocopa hosts.

DNA Barcodes Identify the Larvae and Unassociated Male of Three Onycholyda Sawflies (Hymenoptera, Pamphiliidae) from China.

A molecular analysis based on mitochondrial cytochrome oxidase subunit 1 (COI) gene sequences has indicated that larvae collected in Sichuan and Zhejiang Provinces, China, belong to Onycholyda xanthogaster Shinohara, 1999, and O. fulvicornis Shinohara, in Shinohara Wei, 2016 (Hymenoptera, Pamphiliidae), and that a male Onycholyda specimen from Mt. Tianmushan, Zhejiang Province is the hitherto unknown male of O. tianmushana Shinohara Xiao, 2006. The first host plant records are Rubus inopertus (Focke) Focke (Rosaceae) for O. xanthogaster and Rubus hirsutus Thunb. for O. fulvicornis. The larvae of O. xanthogaster and O. fulvicornis are briefly described and O. xanthogaster is newly recorded from Sichuan Province. The male of O. tianmushana is described for the first time.

Bird droppings on chestnut leaves or sawfly larvae: DNA barcodes verify the occurrence of the archaic Megaxyela togashii (Hymenoptera, Xyelidae) in Hokkaido, Japan.

We made a molecular phylogenetic analysis using mitochondrial cytochrome oxidase subunit 1 (COI) gene sequences for ten unidentified Megaxyela larvae from Hokkaido, Honshu and Shikoku, Japan, and 15 identified adults of four Megaxyela, one Macroxyela and three Xyela species. It revealed that all larvae belonged to M. togashii Shinohara, 1992, which showed rather large intraspecific genetic variability even among the individuals from the same population. This is the first distribution record of M. togashii from Hokkaido. Megaxyela togashii is a univoltine species with a very short larval feeding period, only nine days in one rearing experiment from egg to larval maturation. The larva is a solitary, external leaf-feeder on Juglans ailanthifolia, resting curled around the central leaf vein at the apex of a leaflet, and may resemble the excrement of birds. The prepupa overwinters in an earthen cell whose wall is made only of soil, neither parchment-like nor containing fiber. The mature larva is described and several life traits are discussed.

Pamphilius ishikawai feeds on Astilbe: the first record of Pamphiliidae (Hymenoptera) associated with Saxifragaceae.

Based on field observations and rearing experiments in Nagano and Tochigi prefectures in central Honshu, Japan, host plants and life history of a leaf-rolling sawfly, Pamphilius ishikawai Shinohara, 1979, are recorded for the first time. The larva is a solitary leaf-roller on Astilbe. This is the first record of the Saxifragaceae as a host plant of the Pamphiliidae. We made molecular phylogenetic analysis using mitochondrial cytochrome oxidase subunit 1 (COI) gene sequences for nine individuals of P. ishikawai feeding on three different species of Astilbe in two localities with different climates and for six outgroup pamphiliine species. It revealed very small genetic variability within P. ishikawai and suggested probable monophyly of the P. sulphureipes group.

A speciation gene for left-right reversal in snails results in anti-predator adaptation.

How speciation genes can spread in a population is poorly understood. In land snails, a single gene for left-right reversal could be responsible for instant speciation, because dextral and sinistral snails have difficulty in mating. However, the traditional two-locus speciation model predicts that a mating disadvantage for the reversal should counteract this speciation. In this study, we show that specialized snake predation of the dextral majority drives prey speciation by reversal. Our experiments demonstrate that sinistral Satsuma snails (Stylommatophora: Camaenidae) survive predation by Pareas iwasakii (Colubroidea: Pareatidae). Worldwide biogeography reveals that stylommatophoran snail speciation by reversal has been accelerated in the range of pareatid snakes, especially in snails that gain stronger anti-snake defense and reproductive isolation from dextrals by sinistrality. Molecular phylogeny of Satsuma snails further provides intriguing evidence of repetitive speciation under snake predation. Our study demonstrates that a speciation gene can be fixed in populations by positive pleiotropic effects on survival.