Redescription and first Japanese seamount record of Stethopristes eos (Zeiformes; Parazenidae).

During a biodiversity survey conducted in 2020, focusing on seamounts located in the southern region of Japan, specifically designated as marine protected areas, a single specimen of Stethopristes eos Gilbert, 1905 measuring 124.2 mm in standard length was obtained via the use of a remotely operated vehicle (ROV). The collection occurred at a depth of 519 meters on the Ritto Seamount, located along the western Mariana Ridge. However, this species is poorly known, with only a limited dataset available concerning its morphology. In this study, we present a comprehensive redescription of the species, utilizing information obtained from the type specimens and a newly discovered specimen from Japanese waters. The Japanese specimen constitutes the first recorded occurrence of this species within the western Pacific Ocean. As part of this redescription, we suggest new standard Japanese names for both the genus and species.

Collection of environmental DNA from stemflow for monitoring arboreal biodiversity: Preliminary validation using lichens.

The forest canopy harbors a diverse array of organisms. However, monitoring their biodiversity poses challenges due to limited accessibility and the vast taxonomic diversity. To address these challenges, we present a novel method for capturing arboreal biodiversity by harnessing stemflow as a source of DNA from organisms inhabiting trees. Our method involves encircling the tree trunk with gauze, directing the stemflow along the gauze into a funnel, and collecting it in a plastic bag. We employed dual collection systems to retrieve environmental DNA (eDNA) from the stemflow: the gauze trap, designed to capture macroscopic biological fragments, and the plastic bag trap, which collected the stemflow itself. The trapped fragments and stemflow were separately filtered, and eDNA was subsequently extracted from the filter membranes. To validate our method, we focused on foliose lichens, which are easily observable on tree surfaces. We performed eDNA metabarcoding and successfully detected a majority of the observed foliose lichen species, including those not identified through visual observation alone.•We have developed a non-invasive and straightforward method for monitoring arboreal biodiversity by collecting eDNA from stemflow, which has been validated using lichens for its efficacy.•This cost-effective approach minimizes disruptions to tree ecosystems and is expected to provide an efficient means of sampling and monitoring arboreal organisms.

MitoFish, MitoAnnotator, and MiFish Pipeline: Updates in 10 Years.

MitoFish, MitoAnnotator, and MiFish Pipeline are comprehensive databases of fish mitochondrial genomes (mitogenomes), accurate annotation software of fish mitogenomes, and a web platform for metabarcoding analysis of fish mitochondrial environmental DNA (eDNA), respectively. The MitoFish Suite currently receives over 48,000 visits worldwide every year; however, the performance and usefulness of the online platforms can still be improved. Here, we present essential updates on these platforms, including an enrichment of the reference data sets, an enhanced searching function, substantially faster genome annotation and eDNA analysis with the denoising of sequencing errors, and a multisample comparative analysis function. These updates have made our platform more intuitive, effective, and reliable. These updated platforms are freely available at http://mitofish.aori.u-tokyo.ac.jp/.

Gravity filtration of environmental DNA: A simple, fast, and power-free method.

Filtration is required during the collection of trace amounts of environmental DNA (eDNA) from water samples to achieve a concentration sufficient for downstream molecular experiments. To date, collected water samples have been filtered by humans or electric power using various instruments. We developed a simple gravity filtration system that does not need for an external force. The system comprises a plastic bag filled with a water sample (1 L), a filter cartridge, and a long plastic tube (e.g., 2 m). When hung at a height equal to the tube length, this filtration unit can enable power-free collection and concentration of eDNA at any remote location within a reasonable time (10-60 min).•A simple, rapid, power-free, practical filtration system for environmental DNA analysis is reported.•If there is a place to hang the filtration system, filtration can be performed anywhere.•The filtration speed increased when the system was hung higher.

Discovery of a colossal slickhead (Alepocephaliformes: Alepocephalidae): an active-swimming top predator in the deep waters of Suruga Bay, Japan.

A novel species of the family Alepocephalidae (slickheads), Narcetes shonanmaruae, is described based on four specimens collected at depths greater than 2171 m in Suruga Bay, Japan. Compared to other alepocephalids, this species is colossal (reaching ca. 140 cm in total length and 25 kg in body weight) and possesses a unique combination of morphological characters comprising anal fin entirely behind the dorsal fin, multiserial teeth on jaws, more scale rows than congeners, precaudal vertebrae less than 30, seven branchiostegal rays, two epurals, and head smaller than those of relatives. Mitogenomic analyses also support the novelty of this large deep-sea slickhead. Although most slickheads are benthopelagic or mesopelagic feeders of gelatinous zooplankton, behavioural observations and dietary analyses indicate that the new species is piscivorous. In addition, a stable nitrogen isotope analysis of specific amino acids showed that N. shonanmaruae occupies one of the highest trophic positions reported from marine environments to date. Video footage recorded using a baited camera deployed at a depth of 2572 m in Suruga Bay revealed the active swimming behaviour of this slickhead. The scavenging ability and broad gape of N. shonanmaruae might be correlated with its colossal body size and relatively high trophic position.

New PCR primers for metabarcoding environmental DNA from freshwater eels, genus Anguilla.

Freshwater eels of the genus Anguilla comprise 16 species that include three subspecies and are characterized by their unique catadromous life cycles. Their life histories and nocturnal life styles make it difficult to observe them in freshwater and marine habitats. To investigate their distribution and ecology in aquatic environments, we developed new PCR primers for metabarcoding environmental DNA (eDNA) from Anguilla. The new primers (MiEel) were designed for two conserved regions of the mitochondrial ATP6 gene, which amplify a variable region with sufficient interspecific variations ranging from five to 22 nucleotide differences (one to three nucleotide differences between three subspecies pairs). We confirmed the versatility of the MiEel primers for all freshwater eels using tissue DNA extracts when analyzed separately. The metabarcoding combined with the MiEel primers using mock communities enabled simultaneous detection of Anguilla at the species level. Analysis of eDNA samples from aquarium tanks, a controlled pond and natural rivers demonstrated that the MiEel metabarcoding could successfully detect the correct Anguilla species from water samples. These results suggested that eDNA metabarcoding with MiEel primers would be useful for non-invasively monitoring the presence of the endangered anguillid eels in aquatic environments where sampling surveys are difficult.

Resolving deep-sea pelagic saccopharyngiform eel mysteries: Identification of Neocyema and Monognathidae leptocephali and establishment of a new fish family "Neocyematidae" based on larvae, adults and mitogenomic gene orders.

Deep-sea midwater "saccopharyngiform" eels of the families Cyematidae, Monognathidae, Eurypharyngidae and Saccopharyngidae (order Anguilliformes) are extraordinary fishes having major skeletal reductions and modifications compared to the general anguilliform body structure. Little is known about most aspects of the systematics, phylogeny, and ecology of these families, and few of the approximately 30 species described from adult specimens have been matched with their leptotocephalus larvae. Based on mitogenomic sequence data from rare new specimens, we show that the long-speculated-about larval form referred to as "Leptocephalus holti", which was thought to possibly be the larva of the rare orange-colored eels of Neocyema (5 known specimens; speculated to belong to the Cyematidae) are actually the larvae of the one-jaw eels of the family Monognathidae. One of the 5 types of L. holti larvae that were collected in the Pacific is genetically matched with Monognathus jesperseni, but multiple species exist based on larval sequence data and the morphology of adult specimens. A rare leptocephalus from the Sargasso Sea, with unique morphological characteristics including many small orange spots on the gut, was found to be the larva of Neocyema, which is presently only known from the Atlantic Ocean. We demonstrate that Neocyema constitutes a separate family being most closely related to Eurypharyngidae and Saccopharyngidae based on mitogenomic DNA sequences and unique mitochondrial gene orders.

MitoFish and MiFish Pipeline: A Mitochondrial Genome Database of Fish with an Analysis Pipeline for Environmental DNA Metabarcoding.

Fish mitochondrial genome (mitogenome) data form a fundamental basis for revealing vertebrate evolution and hydrosphere ecology. Here, we report recent functional updates of MitoFish, which is a database of fish mitogenomes with a precise annotation pipeline MitoAnnotator. Most importantly, we describe implementation of MiFish pipeline for metabarcoding analysis of fish mitochondrial environmental DNA, which is a fast-emerging and powerful technology in fish studies. MitoFish, MitoAnnotator, and MiFish pipeline constitute a key platform for studies of fish evolution, ecology, and conservation, and are freely available at http://mitofish.aori.u-tokyo.ac.jp/ (last accessed April 7th, 2018).

Demonstration of the potential of environmental DNA as a tool for the detection of avian species.

Birds play unique functional roles in the maintenance of ecosystems, such as pollination and seed dispersal, and thus monitoring bird species diversity is a first step towards avoiding undesirable consequences of anthropogenic impacts on bird communities. In the present study, we hypothesized that birds, regardless of their main habitats, must have frequent contact with water and that tissues that contain their DNA that persists in the environment (environmental DNA; eDNA) could be used to detect the presence of avian species. To this end, we applied a set of universal PCR primers (MiBird, a modified version of fish/mammal universal primers) for metabarcoding avian eDNA. We confirmed the versatility of MiBird primers by performing in silico analyses and by amplifying DNAs extracted from bird tissues. Analyses of water samples from zoo cages of birds with known species composition suggested that the use of MiBird primers combined with Illumina MiSeq could successfully detect avian species from water samples. Additionally, analysis of water samples collected from a natural pond detected five avian species common to the sampling areas. The present findings suggest that avian eDNA metabarcoding would be a complementary detection/identification tool in cases where visual census of bird species is difficult.

Multilocus phylogenetic analysis of the first molecular data from the rare and monotypic Amarsipidae places the family within the Pelagia and highlights limitations of existing data sets in resolving pelagian interrelationships.

The Pelagia is a recently delineated group of fishes, comprising fifteen families formerly placed in six perciform suborders. The Pelagia was lately recognized as it encompasses huge morphological diversity and only in the last few years have large-scale molecular phylogenetic studies been undertaken that could unite such morphologically disparate lineages. Due to the recent erection of Pelagia, the composition of the taxon is not entirely certain. Five families of the former perciform suborder Stromateoidei have been identified as pelagians. However, the sixth stromateoid subfamily Amarsipidae is a rare monotypic family that has distinctive meristic and morphological characteristics from that of other stromateoids such as the lack of a pharyngeal sac. We examine molecular data generated from the sole species in Amarsipidae, Amarsipus carlsbergi, and demonstrate that it is clearly nested within Pelagia. As with two previous studies that have the breadth of sampling to evaluate pelagian intra-relationships, we find high support for monophyly of most family-level taxonomic units but statistical support for early-branching nodes in the pelagian tree is very low. We conduct the first analyses of Pelagia incorporating the multispecies coalescent and are limited by a high degree of missing loci, or, incomplete taxon sampling. The high degree of missing data across a complete sampling of pelagian lineages along with the deep time scale and rapid radiation of the lineage contribute to poor resolution of early-branching relationships within Pelagia that cannot be resolved with current data sets. Currently available data are either mitochondrial genomes or a super matrix of relatively few loci with a high degree of missing data. A new and independent dataset of numerous phylogenetic loci derived from high-throughput sequencing technology may reduce uncertainty within the Pelagia and provide insights into this adaptive radiation.

Molecular systematics of the anchovy genus Encrasicholina in the Northwest Pacific.

The anchovy genus Encrasicholina is an important coastal marine resource of the tropical Indo-West Pacific (IWP) region for which insufficient comparative data are available to evaluate the effects of current exploitation levels on the sustainability of its species and populations. Encrasicholina currently comprises nine valid species that are morphologically very similar. Only three, Encrasicholina punctifer, E. heteroloba, and E. pseudoheteroloba, occur in the Northwest Pacific subregion of the northeastern part of the IWP region. These species are otherwise broadly distributed and abundant in the IWP region, making them the most important anchovy species for local fisheries. In this study, we reconstructed the phylogeny of these three species of Encrasicholina within the Engraulidae. We sequenced 10 complete mitochondrial genomes (using high-throughput and Sanger DNA sequencing technologies) and compared those sequences to 21 previously published mitochondrial genomes from various engraulid taxa. The phylogenetic results showed that the genus Encrasicholina is monophyletic, and it is the sister group to the more-diverse "New World anchovy" clade. The mitogenome-based dating results indicated that the crown group Encrasicholina originated about 33.7 million years ago (nearby the limit Eocene/Oligocene), and each species of Encrasicholina has been reproductively isolated from the others for more than 20 million years, despite their morphological similarities. In contrast, preliminary population genetic analyses across the Northwest Pacific region using four mitogenomic sequences revealed very low levels of genetic differentiation within Encrasicholina punctifer. These molecular results combined with recent taxonomic revisions are important for designing further studies on the population structure and phylogeography of these anchovies.

Environmental DNA enables detection of terrestrial mammals from forest pond water.

Terrestrial animals must have frequent contact with water to survive, implying that environmental DNA (eDNA) originating from those animals should be detectable from places containing water in terrestrial ecosystems. Aiming to detect the presence of terrestrial mammals using forest water samples, we applied a set of universal PCR primers (MiMammal, a modified version of fish universal primers) for metabarcoding mammalian eDNA. The versatility of MiMammal primers was tested in silico and by amplifying DNAs extracted from tissues. The results suggested that MiMammal primers are capable of amplifying and distinguishing a diverse group of mammalian species. In addition, analyses of water samples from zoo cages of mammals with known species composition suggested that MiMammal primers could successfully detect mammalian species from water samples in the field. Then, we performed an experiment to detect mammals from natural ecosystems by collecting five 500-ml water samples from ponds in two cool-temperate forests in Hokkaido, northern Japan. MiMammal amplicon libraries were constructed using eDNA extracted from water samples, and sequences generated by Illumina MiSeq were subjected to data processing and taxonomic assignment. We thereby detected multiple species of mammals common to the sampling areas, including deer (Cervus nippon), mouse (Mus musculus), vole (Myodes rufocanus), raccoon (Procyon lotor), rat (Rattus norvegicus) and shrew (Sorex unguiculatus). Many previous applications of the eDNA metabarcoding approach have been limited to aquatic/semiaquatic systems, but the results presented here show that the approach is also promising even for forest mammal biodiversity surveys.

Phylogenetic position of the rainbow sardine Dussumieria (Dussumieriidae) and its bearing on the early evolution of the Clupeoidei.

The fish family Dussumieriidae (suborder Clupeoidei), commonly called round herrings, is traditionally considered to be a key taxon for understanding the evolution of the Clupeoidei because some of its morphological characteristics have been interpreted as being either derived or primitive, such as the nearly complete absence of abdominal scutes. Recent molecule-based studies showed that the Dussumieriidae is likely not a monophyletic group. None of those studies, however, included the genus Dussumieria (rainbow sardines) which is the type genus of the family Dussumieriidae. Herein, we investigated the phylogenetic position of Dussumieria within the Clupeoidei, using a dataset of complete mitogenomic sequences, including five newly determined using high-throughput sequencing technology. In the inferred phylogenetic reconstructions, the Dussumieriidae was never recovered as monophyletic, and Dussumieria was not exclusively related to any other of the three dussumieriid genera. Although the position of Dussumieria is not fully resolved, this genus represents a major evolutionary lineage within the Clupeoidei, along with the Engraulidae, Pristigasteridae, Clupeinae, Etrumeus, and two unnamed clades, one containing the Chirocentridae and Spratelloidinae and the other containing the Ehiravinae, Dorosomatinae, and Alosinae. Our results allow some comments regarding the early evolution of the Clupeoidei. In particular, they strongly support the hypothesis that the W-shaped pelvic scute does not represent a good phylogenetic character within the Clupeoidei as either it is primitive or, alternatively, it has independently evolved several times.

Evolution of bidirectional sex change and gonochorism in fishes of the gobiid genera Trimma, Priolepis, and Trimmatom.

Size-advantage and low-density models have been used to explain how mating systems favor hermaphroditism or gonochorism. However, these models do not indicate historical transitions in sexuality. Here, we investigate the evolution of bidirectional sex change and gonochorism by phylogenetic analysis using the mitochondrial gene of the gobiids Trimma (31 species), Priolepis (eight species), and Trimmatom (two species). Trimma and Priolepis formed a clade within the sister group Trimmatom. Gonadal histology and rearing experiments revealed that Trimma marinae, Trimma nasa, and Trimmatom spp. were gonochoric, whereas all other Trimma and Priolepis spp. were bidirectional sex changers or inferred ones. A maximum-likelihood reconstruction analysis demonstrated that the common ancestor of the three genera was gonochoristic. Bidirectional sex change probably evolved from gonochorism in a common ancestor of Trimma and Priolepis. As the gonads of bidirectional sex changers simultaneously contain mature ovarian and immature testicular components or vice versa, individuals are always potentially capable of functioning as females or males, respectively. Monogamy under low-density conditions may have been the ecological condition for the evolution of bidirectional sex change in a common ancestor. As T. marinae and T. nasa are a monophyletic group, gonochorism should have evolved from bidirectional sex change in a common ancestor.

Evolutionary affinities of the unfathomable Parabrotulidae: Molecular data indicate placement of Parabrotula within the family Bythitidae, Ophidiiformes.

Fishes are widely diverse in shape and body size and can quite rapidly undergo these changes. Consequently, some relationships are not clearly resolved with morphological analyses. In the case of fishes of small body size, informative characteristics can be absent due to simplification of body structures. The Parabrotulidae, a small family of diminutive body size consisting of two genera and three species has most recently been classified as either a perciform within the suborder Zoarcoidei or an ophidiiform. Classification of parabrotulids as ophidiiforms has become predominant; however the Parabrotulidae has not yet been investigated in a molecular phylogenetic framework. We examine molecular data from ten genetic loci to more specifically place the Parabrotulidae within the fish tree of life. In a hypothesis testing framework, the Parabrotulidae as a zoarcoid taxon is rejected. Previous identity with zoarcoids due to the one fin ray for each vertebra being present, a characteristic for the Zoarcidae, appears to be an example of convergence. Our results indicate that parabrotulids are viviparous ophidiiforms within the family Bythitidae.

Environmental DNA metabarcoding reveals local fish communities in a species-rich coastal sea.

Environmental DNA (eDNA) metabarcoding has emerged as a potentially powerful tool to assess aquatic community structures. However, the method has hitherto lacked field tests that evaluate its effectiveness and practical properties as a biodiversity monitoring tool. Here, we evaluated the ability of eDNA metabarcoding to reveal fish community structures in species-rich coastal waters. High-performance fish-universal primers and systematic spatial water sampling at 47 stations covering ~11 km2 revealed the fish community structure at a species resolution. The eDNA metabarcoding based on a 6-h collection of water samples detected 128 fish species, of which 62.5% (40 species) were also observed by underwater visual censuses conducted over a 14-year period. This method also detected other local fishes (≥23 species) that were not observed by the visual censuses. These eDNA metabarcoding features will enhance marine ecosystem-related research, and the method will potentially become a standard tool for surveying fish communities.

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA.

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes ≤4 L or >4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m3) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

Preservation Obscures Pelagic Deep-Sea Fish Diversity: Doubling the Number of Sole-Bearing Opisthoproctids and Resurrection of the Genus Monacoa (Opisthoproctidae, Argentiniformes).

The family Opisthoproctidae (barreleyes) constitutes one of the most peculiar looking and unknown deep-sea fish groups in terms of taxonomy and specialized adaptations. All the species in the family are united by the possession of tubular eyes, with one distinct lineage exhibiting also drastic shortening of the body. Two new species of the mesopelagic opisthoproctid mirrorbelly genus Monacoa are described based on pigmentation patterns of the "sole"-a unique vertebrate structure used in the reflection and control of bioluminescence in most short-bodied forms. Different pigmentation patterns of the soles, previously noted as intraspecific variations based on preserved specimens, are here shown species-specific and likely used for communication in addition to counter-illumination of down-welling sunlight. The genus Monacoa is resurrected from Opisthoproctus based on extensive morphological synaphomorphies pertaining to the anal fin and snout. Doubling the species diversity within sole-bearing opisthoproctids, including recognition of two genera, is unambiguously supported by mitogenomic DNA sequence data. Regular fixation with formalin and alcohol preservation is shown problematic concerning the retention of species-specific pigmentation patterns. Examination or photos of fresh material before formalin fixation is shown paramount for correct species recognition of sole-bearing opisthoproctids-a relatively unknown issue concerning species diversity in the deep-sea pelagic realm.

Phylogeny and polyploidy: resolving the classification of cyprinine fishes (Teleostei: Cypriniformes).

Cyprininae is the largest subfamily (>1300 species) of the family Cyprinidae and contains more polyploid species (∼400) than any other group of fishes. We examined the phylogenetic relationships of the Cyprininae based on extensive taxon, geographical, and genomic sampling of the taxa, using both mitochondrial and nuclear genes to address the phylogenetic challenges posed by polyploidy. Four datasets were analyzed in this study: two mitochondrial gene datasets (465 and 791 taxa, 5604bp), a mitogenome dataset (85 taxa, 14,771bp), and a cloned nuclear RAG1 dataset (97 taxa, 1497bp). Based on resulting trees, the subfamily Cyprininae was subdivided into 11 tribes: Probarbini (new; Probarbus+Catlocarpio), Labeonini Bleeker, 1859 (Labeo & allies), Torini Karaman, 1971 (Tor, Labeobarbus & allies), Smiliogastrini Bleeker, 1863 (Puntius, Enteromius & allies), Poropuntiini (Poropuntius & allies), Cyprinini Rafinesque, 1815 (Cyprinus & allies), Acrossocheilini (new; Acrossocheilus & allies), Spinibarbini (new; Spinibarbus), Schizothoracini McClelland, 1842 (Schizothorax & allies), Schizopygopsini Mirza, 1991 (Schizopygopsis & allies), and Barbini Bleeker, 1859 (Barbus & allies). Phylogenetic relationships within each tribe were discussed. Two or three distinct RAG1 lineages were identified for each of the following tribes Torini, Cyprinini, Spinibarbini, and Barbini, indicating their hybrid origin. The hexaploid African Labeobarbus & allies and Western Asian Capoeta are likely derived from two independent hybridization events between their respective maternal tetraploid ancestors and Cyprinion.

Phylogenetic relationships of Acheilognathidae (Cypriniformes: Cyprinoidea) as revealed from evidence of both nuclear and mitochondrial gene sequence variation: evidence for necessary taxonomic revision in the family and the identification of cryptic species.

Bitterlings are relatively small cypriniform species and extremely interesting evolutionarily due to their unusual reproductive behaviors and their coevolutionary relationships with freshwater mussels. As a group, they have attracted a great deal of attention in biological studies. Understanding the origin and evolution of their mating system demands a well-corroborated hypothesis of their evolutionary relationships. In this study, we provide the most comprehensive phylogenetic reconstruction of species relationships of the group based on partitioned maximum likelihood and Bayesian methods using DNA sequence variation of nuclear and mitochondrial genes on 41 species, several subspecies and three undescribed species. Our findings support the monophyly of the Acheilognathidae. Two of the three currently recognized genera are not monophyletic and the family can be subdivided into six clades. These clades are further regarded as genera based on both their phylogenetic relationships and a reappraisal of morphological characters. We present a revised classification for the Acheilognathidae with five genera/lineages: Rhodeus, Acheilognathus (new constitution), Tanakia (new constitution), Paratanakia gen. nov., and Pseudorhodeus gen. nov. and an unnamed clade containing five species currently referred to as "Acheilognathus". Gene trees of several bitterling species indicate that the taxa are not monophyletic. This result highlights a potentially dramatic underestimation of species diversity in this family. Using our new phylogenetic framework, we discuss the evolution of the Acheilognathidae relative to classification, taxonomy and biogeography.