X-ray phase contrast reveals soft tissue and shell growth lines in mollusks.

High-resolution 3D imaging of species with exoskeletons such as shell-bearing mollusks typically involves destructive steps. Nondestructive alternatives are desirable since samples can be rare and valuable, and destructive steps are time-consuming and may distort the tissue. Here, we show for the first time that propagation-based phase-contrast X-ray imaging can significantly increase contrast in mollusks with intact shells. By using the recently upgraded monochromator at the SPring-8 BL20B2 synchrotron beamline, we imaged six species of mollusks, showing that X-ray phase contrast enhances soft-tissue contrast. Features that are almost invisible in conventional attenuation-based micro-computed tomography (micro-CT) are clearly reproduced with phase-contrast imaging under the same scan conditions. Furthermore, this method can reveal features such as growth rings in the shell and differentiate between calcite and aragonite crystal forms. Phase-contrast imaging can thus serve as a compelling alternative when destructive methods are not an option.

Phylogenomic resolution of Imparidentia (Mollusca: Bivalvia) diversification through mitochondrial genomes.

Despite significant advances in the phylogenomics of bivalves over the past decade, the higher-level phylogeny of Imparidentia (a superorder of Heterodonta) remains elusive. Here, a total of five new mitochondrial sequences (Chama asperella, Chama limbula, Chama dunkeri, Barnea manilensis and Ctena divergens) was added to provide resolution in nodes that required additional study. Although the monophyly of Lucinida remains less clear, the results revealed the overall backbone of the Imparidentia tree and the monophyly of Imparidentia. Likewise, most relationships among the five major Imparidentia lineages-Lucinida, Cardiida, Adapedonta, Myida and Venerida-were addressed with a well-supported topology. Basal relationships of Imparidentia recovered Lucinidae as the sister group to all remaining imparidentian taxa. Thyasiridae is a sister group to other imparidentian bivalves (except Lucinidae species) which is split into Cardiida, Adapedonta and the divergent clade of Neoheterodontei. Neoheterodontei was comprised of Venerida and Myida, the former of which now also contains Chamidae as the sister group to all the remaining venerid taxa. Moreover, molecular divergence times were inferred by calibrating nine nodes in the Imparidentia tree of life by extinct taxa. The origin of these major clades ranged from Ordovician to Permian with the diversification through the Palaeozoic to Mesozoic. Overall, the results obtained in this study demonstrate a better-resolved Imparidentia phylogeny based on mitochondrial genomes. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00178-x.

Introgressive hybridization in the west Pacific pen shells (genus Atrina): Restricted interspecies gene flow within the genome.

A compelling interest in marine biology is to elucidate how species boundaries between sympatric free-spawning marine invertebrates such as bivalve molluscs are maintained in the face of potential hybridization. Hybrid zones provide the natural resources for us to study the underlying genetic mechanisms of reproductive isolation between hybridizing species. Against this backdrop, we examined the occurrence of introgressive hybridization (introgression) between two bivalves distributed in the western Pacific margin, Atrina japonica and Atrina lischkeana, based on single-nucleotide polymorphisms (SNPs) derived from restriction site-associated DNA sequencing. Using 1066 ancestry-informative SNP sites, we also investigated the extent of introgression within the genome to search for SNP sites with reduced interspecies gene flow. A series of our individual-level clustering analyses including the principal component analysis, Bayesian model-based clustering, and triangle plotting based on ancestry-heterozygosity relationships for an admixed population sample from the Seto Inland Sea (Japan) consistently suggested the presence of specimens with varying degrees of genomic admixture, thereby implying that the two species are not completely isolated. The Bayesian genomic cline analysis identified 10 SNP sites with reduced introgression, each of which was located within a genic region or an intergenic region physically close to a functional gene. No, or very few, heterozygotes were observed at these sites in the hybrid zone, suggesting that selection acts against heterozygotes. Accordingly, we raised the possibility that the SNP sites are within genomic regions that are incompatible between the two species. Our finding of restricted interspecies gene flow at certain genomic regions gives new insight into the maintenance of species boundaries in hybridizing broadcast-spawning molluscs.

Gene Recruitments and Dismissals in the Argonaut Genome Provide Insights into Pelagic Lifestyle Adaptation and Shell-like Eggcase Reacquisition.

The paper nautilus or greater argonaut, Argonauta argo, is a species of octopods which is characterized by its pelagic lifestyle and by the presence of a protective spiral-shaped shell-like eggcase in females. To reveal the genomic background of how the species adapted to the pelagic lifestyle and acquired its shell-like eggcase, we sequenced the draft genome of the species. The genome size was 1.1 Gb, which is the smallest among the cephalopods known to date, with the top 215 scaffolds (average length 5,064,479 bp) covering 81% (1.09 Gb) of the total assembly. A total of 26,433 protein-coding genes were predicted from 16,802 assembled scaffolds. From these, we identified nearly intact HOX, Parahox, Wnt clusters, and some gene clusters that could probably be related to the pelagic lifestyle, such as reflectin, tyrosinase, and opsin. The gene models also revealed several homologous genes related to calcified shell formation in Conchiferan mollusks, such as Pif-like, SOD, and TRX. Interestingly, comparative genomics analysis revealed that the homologous genes for such genes were also found in the genome of the shell-less octopus, as well as Nautilus, which has a true outer shell. Therefore, the draft genome sequence of Arg. argo presented here has helped us to gain further insights into the genetic background of the dynamic recruitment and dismissal of genes to form an important, converging extended phenotypic structure such as the shell and the shell-like eggcase. Additionally, it allows us to explore the evolution of from benthic to pelagic lifestyles in cephalopods and octopods.

Molecular phylogeny of Nipponacmea (Patellogastropoda, Lottiidae) from Japan: a re-evaluation of species taxonomy and morphological diagnosis.

The patellogastropod limpet genus Nipponacmea is widely distributed in Japan and adjacent East Asia. Species identification within Nipponacmea is challenging due to the high variation in shell morphology. In this study, we examined the taxonomy of this genus represented by nine nominal species from 43 localities (including type localities). Results of the molecular phylogenetic analysis revealed that: (1) N.gloriosa, the sole species in this genus inhabiting the subtidal zone, represents the most basal independent branch; (2) the remaining species are divided into two large clades with lower- and higher-apex shell profiles; and (3) the high-apex morphology was derived from the low-apex type. The terminal clades defined using the molecular data were consistent with nine morphospecies and had 100% bootstrap values, strongly supporting the conventional taxonomy of Nipponacmea. Although morphological similarities do not always reflect phylogeny, the set of morphological characters used in the current taxonomy were proven to be adequate for diagnosis. In conclusion, this study provided solid evidence to uphold the monophyly of known species of Nipponacmea in Japan and demonstrated the usefulness of morphological characters for species diagnosis.

Sinking Down or Floating Up? Current State of Taxonomic Studies on Marine Invertebrates in Japan Inferred from the Number of New Species Published between the Years 2003 and 2020.

Taxonomic studies on marine invertebrates have been prominent in Japan since the 19th century. Globally, taxonomy was reported to have been in recession since the early 21st century, but it is not clear if it is still in hardship or is making a recovery in recent years in Japan. In order to comprehend the status quo of taxonomic studies on marine invertebrates in Japan, we compiled a list of marine invertebrate species newly described from the exclusive economic zone of this country during the period between 2003 and 2020 and investigated trends by making comparisons of higher taxa and academic journals in terms of the numbers of new species and taxonomic authors. We noticed that recruitment of new researchers into taxonomic studies is evident in some taxa. We also found that certain articles with descriptions of new species are now being published in journals aimed at general biology/zoology, not oriented principally to taxonomy. We consider from our analyses that taxonomic studies on marine invertebrates in Japan show signs of resurgence, but development of new taxonomists is awaited in certain taxa.

Hydrophilic Shell Matrix Proteins of Nautilus pompilius and the Identification of a Core Set of Conchiferan Domains.

Despite being a member of the shelled mollusks (Conchiferans), most members of extant cephalopods have lost their external biomineralized shells, except for the basally diverging Nautilids. Here, we report the result of our study to identify major Shell Matrix Proteins and their domains in the Nautilid Nautilus pompilius, in order to gain a general insight into the evolution of Conchiferan Shell Matrix Proteins. In order to do so, we performed a multiomics study on the shell of N. pompilius, by conducting transcriptomics of its mantle tissue and proteomics of its shell matrix. Analyses of obtained data identified 61 distinct shell-specific sequences. Of the successfully annotated 27 sequences, protein domains were predicted in 19. Comparative analysis of Nautilus sequences with four Conchiferans for which Shell Matrix Protein data were available (the pacific oyster, the pearl oyster, the limpet and the Euhadra snail) revealed that three proteins and six protein domains were conserved in all Conchiferans. Interestingly, when the terrestrial Euhadra snail was excluded, another five proteins and six protein domains were found to be shared among the four marine Conchiferans. Phylogenetic analyses indicated that most of these proteins and domains were probably present in the ancestral Conchiferan, but employed in shell formation later and independently in most clades. Even though further studies utilizing deeper sequencing techniques to obtain genome and full-length sequences, and functional analyses, must be carried out in the future, our results here provide important pieces of information for the elucidation of the evolution of Conchiferan shells at the molecular level.

Complete mitochondrial genome of the Pacific limpet Cellana nigrolineata (Gastropoda: Patellogastropoda) determined by shotgun sequencing using the Illumina NGS platform.

The Pacific limpet Cellana nigrolineata is one of the most commonly found limpets in the intertidal shores of Japan. Here, we report the full mitogenome sequence of an individual specimen of the species, which was collected from the intertidal rocky beach in the Nada beach of Gobo City, Wakayama, Japan (33.8316 N, 135.1751 E), in 2018. The sequence was determined by the shotgun sequencing method using the NGS Illumina MiSeq platform. The genomic structure of C. nigrolineata is the same as the previously reported congener, C. radiata, which shows a representative Nacellidae and metazoan mitogenomic structures. The mitogenome has all of its 37 genes included in its 16,153 bp, with one control region located between the tRNA-Cys and tRNA-Gly genes. In order to clarify the phylogenetic position of C. nigrolineata in Gastropoda, a data set including the mitogenomes of 10 patellogastropods, 10 non-patellogastropod gastropods, and four outgroups were used in maximum likelihood inferences. Although with some exceptions, the resulting phylogeny supported the monophylies of traditionally accepted gastropod subclasses, and thus confirms the position of C. nigrolineata in Patellogastropoda.

A New Species of the Intertidal Limpet Eoacmaea (Patellogastropoda: Eoacmaeidae) from Yonaguni Island, Japan and Taiwan.

A new species of intertidal limpet was identified from Yonaguni Island, Japan and southern Taiwan in the present study and described as Eoacmaea nivea n. sp. It was previously reported by different authors as Cellana mauritiana, or as Acmaeid sp. in Taiwan, and as Patelloida sp. in Yonaguni Island, Japan. Based on detailed morphological observations and molecular analyses (COI), E. nivea n. sp. is distinguished from other Eoacmaea species. Eoacmaea nivea n. sp. is presently known from southern Taiwan, and a population on Yonaguni Island, Japan that is very limited. The present study reports a total 11 species of patellogastropod limpet species in Taiwan. The seven species-Cellana grata, Cellana toreuma, Nipponacmea nigrans, Nipponacmea fuscoviridis, Lottia dorsuosa, Lottia luchuana and Lottia tenuisculpta-were found in the intertidal on rocky shores along the north to northeastern shores of Taiwan. The five species-Cellana radiata, Lottia luchuana, Scutellastra flexuosa, Patelloida saccharina and E. nivea n. sp.-can be found mostly within depressions in limestone substrate in southeastern Taiwan. Of these, only Lottia luchuana is found throughout Taiwan, and overall has a tropical, not warm-temperate, distribution.

Molecular Species Delimitation of the Genus Reishia (Mollusca: Gastropoda) along the Coasts of China and Korea.

Species of the predatory gastropod genus Reishia Kuroda and Habe, 1971 (Muricidae) inhabit intertidal rocky shores in East Asia. Due to their highly variable external shell morphology, the taxonomy of this genus at species-level is still in need of re-evaluation. Using DNA-based delimitation methods, we aimed to ascertain the number of species of Reishia along the coasts of China and adjacent Asian areas. Also, we looked for diagnostic traits using morphology-based statistical approaches. Our genetic data suggest that the studied individuals comprised two separate species of a Reishia complex in this region, in contrast to the previously proposed four or more taxa. This conclusion is further supported by statistical analyses of shell morphological characteristics. The morphospecies R. bronni (Dunker, 1860), R. jubilaea (Tan and Sigurdsson, 1990), and R. luteostoma (Holten, 1803) were assigned to a single taxon, indicating that they might be synonyms of the same species. The morphospecies R. clavigera (Küster, 1860) singly formed one group, suggesting that it is likely a valid name. The estimated divergence time of the two identified taxa indicates that speciation might have been associated with the sea level and temperature fluctuations during the Plio-Pleistocene period. Our study on Reishia species provides crucial information for further research on the ecology, evolutionary biology, and conservation of this genus.

Morphological characterization of domatium development in Callicarpa saccata.

BACKGROUND AND AIMS: Domatia are plant structures within which organisms reside. Callicarpa saccata (Lamiaceae) is the sole myrmecophyte, or 'ant plant', that develops foliar (leaf-borne) myrmeco-domatia in this genus. In this work we examined domatium development in C. saccata to understand the developmental processes behind pouch-like domatia. METHODS: Scanning electron microscopy, sectioning and microcomputed tomography were carried out to compare the leaves of C. saccata with those of the closely related but domatia-less myrmecophyte Callicarpa subaequalis, both under cultivation without ants. KEY RESULTS: Callicarpa saccata domatia are formed as a result of excess cell proliferation at the blade/petiole junctions of leaf primordia. Blade/petiole junctions are important meristematic sites in simple leaf organogenesis. We also found that the mesophyll tissue of domatia does not clearly differentiate into palisade and spongy layers. CONCLUSIONS: Rather than curling of the leaf margins, a perturbation of the normal functioning of the blade/petiole junction results in the formation of domatium tissue. Excess cell proliferation warps the shape of the blade and disturbs the development of the proximal-distal axis. This process leads to the generation of distinct structures that facilitate interaction between C. saccata and ants.

Complex factors shape phenotypic variation in deep-sea limpets.

Pectinodontid limpets are important members of deep-sea hot vents and cold seeps as can be seen by their conspicuous presence in both extant and extinct systems. They have traditionally been classified into different genera and species based on shell and radula characteristics; the reliability of these characters has been questioned but not tested thoroughly. Here, for the first time in taxa endemic to deep-sea chemosynthetic ecosystems, we combine substrate translocation with molecular data to assess the plasticity and variability of key phenotypic characters. Molecular data revealed that several 'species' of extant vent/seep pectinodontids actually represent intergrading morphotypes of a single, highly plastic, evolutionary lineage, with each morphological trait being possibly influenced differently by environmental and genetic factors. Our results challenge previous interpretations of paleoecology at fossil chemosynthetic ecosystems and highlight the importance of modern analogues in understanding fossil systems.

Four new deep-sea provannid snails (Gastropoda: Abyssochrysoidea) discovered from hydrocarbon seep and hydrothermal vents in Japan.

Recently, the species richness of provannid gastropods inhabiting chemosynthetic ecosystems in the northwestern Pacific has been reassessed, revealing a much higher diversity than previously realized. Here, we describe four further new species, two in the genus Desbruyeresia and two in the genus Provanna. Their generic placement was confirmed by both shell and radula morphology, as well as phylogenetic reconstruction using the mitochondrial cytochrome c oxidase subunit I gene. Desbruyeresia armata n. sp. from vent fields in the Izu-Ogasawara Arc is characterized by a stout shell with numerous tubercles or short spines and marginal teeth coarsely serrated into only four denticles. Desbruyeresia costata n. sp. from Okinawa Trough vents is distinguished from other congeners by an elongate shell with strong axial ribs and obsolete spiral ribs. These represent the first Desbruyeresia species from Japanese waters. Provanna fenestrata n. sp. discovered from two vent fields in the Okinawa Trough is instantly recognizable from its uniquely regular rectangular lattice sculpture; Provanna stephanos n. sp. is a surprising new discovery from the supposedly well-explored 'Off Hatsushima' hydrocarbon seep site in Sagami Bay, and is highly distinctive with two characteristic rows of strongly spinous spiral ribs. The discovery of these new species in relatively well-explored chemosynthetic ecosystems in Japan indicates that the biodiversity of such systems remains poorly documented.

Sample checklist of Gastropoda and Bivalvia in Cham Islands, Vietnam.

BACKGROUND: Cham Islands (Cu Lao Cham) is a group of 8 small islands in the Quang Nam province, Central Vietnam. There is only one study that mentioned the diversity of marine molluscs in this area. However, the data on species composition have not been digitised and not stored or shared for other purposes. Our paper aims to share the checklist of marine mollusc species (Bivalvia and Gastropoda) species that we collected from the littoral zone of Cham Islands in May 2017. This is the first digitised and online data of marine molluscs in Vietnam. It is very important for researchers in various fields such as the structure and function of ecology and biodiversity monitoring. NEW INFORMATION: This study provides a checklist of the marine bivalves and snails in the Cham Islands of Vietnam. Moreover, this first widely shared data of biodiversity in Vietnam can trigger biodiversity data papers in this data-poor country.The data of this study will be important inputs for better understanding biodiversity on the Cham Islands and Vietnam as well as for forming the basis for monitoring, exploitation and conservation of biodiversity in this area.In total, 145 taxa were recorded, 46 bivalve taxa and 99 snail taxa, from which 128 were identified to the species level and 17 were identified to the genus level. There are 116 new species records for the Cham Islands.The specimens are currently deposited in the Institute of Ecology and Biological Resources (IEBR), Vietnam Academy of Science and Technology (VAST).

Population history of deep-sea vent and seep Provanna snails (Mollusca: Abyssochrysoidea) in the northwestern Pacific.

BACKGROUND: Gastropods of the genus Provanna are abundant and widely distributed in deep-sea chemosynthetic environments with seven extant species described in the northwestern Pacific. METHODS: We investigated the population history and connectivity of five Provanna species in the northwestern Pacific through population genetic analyses using partial sequences of the cytochrome c oxidase subunit I gene. RESULTS: We found that P. subglabra, the most abundant and genetically diverse species, is genetically segregated by depth. Among the five species, the three comparatively shallower species (P. lucida, P. kuroshimensis, P. glabra) had a more constant demographic history compared to the deeper species (P. subglabra, P.  clathrata). DISCUSSION: Environmental differences, especially depth, appears to have a role in the segregation of Provanna snails. The population of P. clathrata in the Irabu Knoll appears to have expanded after P. subglabra population. The remaining three species, P. lucida, P. kuroshimensis, and P. glabra, are only known from a single site each, all of which were shallower than 1,000 m. These data indicate that Provanna gastropods are vertically segregated, and that their population characteristics likely depend on hydrothermal activities.

Prenatal cranial bone development of Thomas's horseshoe bat (Rhinolophus thomasi): with special reference to petrosal morphology.

Cochlear morphology has been regarded as one of the key traits to understand the origin and evolution of echolocation in bats, given its functionality and performance for receiving echolocation sonar. While numerous researchers have compared adult-stage morphology, few have studied the prenatal development of the cochlea. Here, we provide the first detailed three-dimensional description of the prenatal cranial development in bats, using Rhinolophus thomasi as a model, with particular interest to the petrosal which houses the cochlea. Results revealed that among all cranial bones the onset of the ossification of the petrosal is earlier in R. thomasi when compared to other reported mammals. Generally, the cochlea reaches adult size and shape before or around birth in placental mammals including bats, but we found that its shape and size growths continue until maturity in Rhinolophus species. The relationship of cochlear size and skull size is maintained constant throughout the postnatal ontogeny to adulthood in Rhinolophus, a pattern previously reported neither in any other bats nor other mammals. The peculiar developmental pattern in Rhinolophus possibly allows them to form their characteristically large cochlea and facilitate their distinctive echolocation behavior. A recent study reported that non-echolocating Pteropodidae shares a similar prenatal cochlear size to laryngeal echolocating bats. The apparent resemblance of fetal cochlear size was proposed to be a vestigial signal of large cochlear size in the last common ancestor of bats and thus as supporting evidence for the single origin of laryngeal echolocation. However, results from the present observations suggest that limited aspects of the cochlear development were captured in this previous investigation and that the resulting interpretations may be questionable. We point out that diversity and patterns of cochlear development among bats are still not resolved, and the controversy on the origins of laryngeal echolocation is still open to discussion.

Non-destructive morphological observations of the fleshy brittle star, Asteronyx loveni using micro-computed tomography (Echinodermata, Ophiuroidea, Euryalida).

The first morphological observation of a euryalid brittle star, Asteronyx loveni, using non-destructive X-ray micro-computed tomography (µCT) was performed. The body of euryalids is covered by thick skin, and it is very difficult to observe the ossicles without dissolving the skin. Computed tomography with micrometer resolution (approximately 4.5-15.4 µm) was used to construct 3D images of skeletal ossicles and soft tissues in the ophiuroid's body. Shape and positional arrangement of taxonomically important ossicles were clearly observed without any damage to the body. Detailed pathways inside the vertebral ossicles, lateral arm plates, and arm spines for passage of nerves and water vascular structures were observed. Inter-vertebral muscles were also observed. Forms and 3D arrangements of many important taxonomical characters of the euryalids were scrutinized by µCT in high enough resolution for taxonomic description of ophiuroids.

Molecular analysis reveals a new cryptic species in a limpet Lottia kogamogai (Patellogastropoda: Lottiidae) from Japan.

A group of small lottiid species in Japan, the Lottia kogamogai complex, was phylogenetically analyzed based on the mitochondrial COI gene. As a result, L. kogamogai is subdivided into two geographic clades. Lottia kogamogai s.s. is limited to southern temperate Japan. The northern population formerly considered an intraspecific variation of the species is shown to be a distinct new species, here described as Lottia goshimai n. sp. The new species is distinguishable from L. kogamogai not only genetically, but in radular morphology and shell sculpture.

Geometric analysis and estimation of the growth rate gradient on gastropod shells.

The morphology of gastropod shells provides a record of the growth rate at the aperture of the shell, and molecular biological studies have shown that the growth rate gradient along the aperture of a gastropod shell can be closely related to gene expression at the aperture. Here, we develop a novel method for deriving microscopic growth rates from the macroscopic shapes of gastropod shells. The growth vector map of a shell provides information on the growth rate gradient as a vector field along the aperture, over the growth history. However, it is difficult to estimate the growth vector map directly from the macroscopic shape of a specimen, because the degree of freedom of the growth vector map is very high. In order to overcome this difficulty, we develop a method of estimating the growth vector map based on a growing tube model, where the latter includes fewer parameters to be estimated. In addition, we calculate an aperture map specifying the magnitude of the growth vector at each location, which can be compared with the expression levels of several genes or proteins that are important in morphogenesis. Finally, we show a concrete example of how macroscopic shell shapes evolve in a morphospace when microscopic growth rate gradient changes.

[Case of urgent surgical cricothyrotomy in CICV situation during induction of anesthesia for hemostasis after tonsillectomy].

A 38-year-old man (BMI 31) underwent bilateral tonsillectomy for sleep apnea syndrome under general anesthesia without any airway difficulty. On the fifth post-operative day excessive bleeding occurred suddenly. Emergency tracheostomy was planned, not under local anesthesia but general anesthesia in the presence of otolaryngologists for urgent tracheostomy, since the patient could not lie in the supine position. Tracheal intubation was tried using rapid sequence technique. However, excessive bleeding in the oral cavity did not allow successful direct laryngoscopy, resulting in CICV situation. When CICV situation was con- firmed associated with SpO2 91%, surgical cricothyrotomy was started by otolaryngologists. SpO2 decreased to 13% associated with heart rate of 38 beats · min-1 immediately before restoration of ventilation and oxygenation. After hemostasis, he showed uneventful post-operative course. On the occasion of airway management for excessive laryngopharyngeal bleeding, emergency surgical crycothyrotomy should be performed immediately before the fall of oxygen tension, if rapid sequence tracheal intubation had failed.