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/.

Pilot study of a comprehensive resource estimation method from environmental DNA using universal D-loop amplification primers.

Many studies have investigated the ability of environmental DNA (eDNA) to identify the species. However, when individual species are to be identified, accurate estimation of their abundance using traditional eDNA analyses is still difficult. We previously developed a novel analytical method called HaCeD-Seq (haplotype count from eDNA by sequencing), which focuses on the mitochondrial D-loop sequence for eels and tuna. In this study, universal D-loop primers were designed to enable the comprehensive detection of multiple fish species by a single sequence. To sequence the full-length D-loop with high accuracy, we performed nanopore sequencing with unique molecular identifiers (UMI). In addition, to determine the D-loop reference sequence, whole genome sequencing was performed with thin coverage, and complete mitochondrial genomes were determined. We developed a UMI-based Nanopore D-loop sequencing analysis pipeline and released it as open-source software. We detected 5 out of 15 species (33%) and 10 haplotypes out of 35 individuals (29%) among the detected species. This study demonstrates the possibility of comprehensively obtaining information related to population size from eDNA. In the future, this method can be used to improve the accuracy of fish resource estimation, which is currently highly dependent on fishing catches.

Testing the Utility of Alternative Metrics of Branch Support to Address the Ancient Evolutionary Radiation of Tunas, Stromateoids, and Allies (Teleostei: Pelagiaria).

The use of high-throughput sequencing technologies to produce genome-scale data sets was expected to settle some long-standing controversies across the Tree of Life, particularly in areas where short branches occur at deep timescales. Instead, these data sets have often yielded many well-supported but conflicting topologies, and highly variable gene-tree distributions. A variety of branch-support metrics beyond the nonparametric bootstrap are now available to assess how robust a phylogenetic hypothesis may be, as well as new methods to quantify gene-tree discordance. We applied multiple branch-support metrics to a study of an ancient group of marine fishes (Teleostei: Pelagiaria) whose interfamilial relationships have proven difficult to resolve due to a rapid accumulation of lineages very early in its history. We analyzed hundreds of loci including published ultraconserved elements and newly generated exonic data along with their flanking regions to represent all 16 extant families for more than 150 out of 284 valid species in the group. Branch support was typically lower at inter- than intra-familial relationships regardless of the type of marker used. Several nodes that were highly supported with bootstrap had a very low site and gene-tree concordance, revealing underlying conflict. Despite this conflict, we were able to identify four consistent interfamilial clades, each comprised of two or three families. Combining exons with their flanking regions also produced increased branch lengths at the deep branches of the pelagiarian tree. Our results demonstrate the limitations of employing current metrics of branch support and species-tree estimation when assessing the confidence of ancient evolutionary radiations and emphasize the necessity to embrace alternative measurements to explore phylogenetic uncertainty and discordance in phylogenomic data sets.[Concatenation; exons; introns; phylogenomics; species-tree methods; target capture.].

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).

A phylogenomic framework for pelagiarian fishes (Acanthomorpha: Percomorpha) highlights mosaic radiation in the open ocean.

The fish clade Pelagiaria, which includes tunas as its most famous members, evolved remarkable morphological and ecological variety in a setting not generally considered conducive to diversification: the open ocean. Relationships within Pelagiaria have proven elusive due to short internodes subtending major lineages suggestive of rapid early divergences. Using a novel sequence dataset of over 1000 ultraconserved DNA elements (UCEs) for 94 of the 286 species of Pelagiaria (more than 70% of genera), we provide a time-calibrated phylogeny for this widely distributed clade. Some inferred relationships have clear precedents (e.g. the monophyly of 'core' Stromateoidei, and a clade comprising 'Gempylidae' and Trichiuridae), but others are unexpected despite strong support (e.g. Chiasmodontidae + Tetragonurus). Relaxed molecular clock analysis using node-based fossil calibrations estimates a latest Cretaceous origin for Pelagiaria, with crown-group families restricted to the Cenozoic. Estimated mean speciation rates decline from the origin of the group in the latest Cretaceous, although credible intervals for root and tip rates are broad and overlap in most cases, and there is higher-than-expected partitioning of body shape diversity (measured as fineness ratio) between clades concentrated during the Palaeocene-Eocene. By contrast, more direct measures of ecology show either no substantial deviation from a null model of diversification (diet) or patterns consistent with evolutionary constraint or high rates of recent change (depth habitat). Collectively, these results indicate a mosaic model of diversification. Pelagiarians show high morphological disparity and modest species richness compared to better-studied fish radiations in contrasting environments. However, this pattern is also apparent in other clades in open-ocean or deep-sea habitats, and suggests that comparative study of such groups might provide a more inclusive model of the evolution of diversity in fishes.

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.

Phylogenetic classification of bony fishes.

BACKGROUND: Fish classifications, as those of most other taxonomic groups, are being transformed drastically as new molecular phylogenies provide support for natural groups that were unanticipated by previous studies. A brief review of the main criteria used by ichthyologists to define their classifications during the last 50 years, however, reveals slow progress towards using an explicit phylogenetic framework. Instead, the trend has been to rely, in varying degrees, on deep-rooted anatomical concepts and authority, often mixing taxa with explicit phylogenetic support with arbitrary groupings. Two leading sources in ichthyology frequently used for fish classifications (JS Nelson's volumes of Fishes of the World and W. Eschmeyer's Catalog of Fishes) fail to adopt a global phylogenetic framework despite much recent progress made towards the resolution of the fish Tree of Life. The first explicit phylogenetic classification of bony fishes was published in 2013, based on a comprehensive molecular phylogeny ( www.deepfin.org ). We here update the first version of that classification by incorporating the most recent phylogenetic results. RESULTS: The updated classification presented here is based on phylogenies inferred using molecular and genomic data for nearly 2000 fishes. A total of 72 orders (and 79 suborders) are recognized in this version, compared with 66 orders in version 1. The phylogeny resolves placement of 410 families, or ~80% of the total of 514 families of bony fishes currently recognized. The ordinal status of 30 percomorph families included in this study, however, remains uncertain (incertae sedis in the series Carangaria, Ovalentaria, or Eupercaria). Comments to support taxonomic decisions and comparisons with conflicting taxonomic groups proposed by others are presented. We also highlight cases were morphological support exist for the groups being classified. CONCLUSIONS: This version of the phylogenetic classification of bony fishes is substantially improved, providing resolution for more taxa than previous versions, based on more densely sampled phylogenetic trees. The classification presented in this study represents, unlike any other, the most up-to-date hypothesis of the Tree of Life of fishes.

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.

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.

Structure and variation of the mitochondrial genome of fishes.

BACKGROUND: The mitochondrial (mt) genome has been used as an effective tool for phylogenetic and population genetic analyses in vertebrates. However, the structure and variability of the vertebrate mt genome are not well understood. A potential strategy for improving our understanding is to conduct a comprehensive comparative study of large mt genome data. The aim of this study was to characterize the structure and variability of the fish mt genome through comparative analysis of large datasets. RESULTS: An analysis of the secondary structure of proteins for 250 fish species (248 ray-finned and 2 cartilaginous fishes) illustrated that cytochrome c oxidase subunits (COI, COII, and COIII) and a cytochrome bc1 complex subunit (Cyt b) had substantial amino acid conservation. Among the four proteins, COI was the most conserved, as more than half of all amino acid sites were invariable among the 250 species. Our models identified 43 and 58 stems within 12S rRNA and 16S rRNA, respectively, with larger numbers than proposed previously for vertebrates. The models also identified 149 and 319 invariable sites in 12S rRNA and 16S rRNA, respectively, in all fishes. In particular, the present result verified that a region corresponding to the peptidyl transferase center in prokaryotic 23S rRNA, which is homologous to mt 16S rRNA, is also conserved in fish mt 16S rRNA. Concerning the gene order, we found 35 variations (in 32 families) that deviated from the common gene order in vertebrates. These gene rearrangements were mostly observed in the area spanning the ND5 gene to the control region as well as two tRNA gene cluster regions (IQM and WANCY regions). Although many of such gene rearrangements were unique to a specific taxon, some were shared polyphyletically between distantly related species. CONCLUSIONS: Through a large-scale comparative analysis of 250 fish species mt genomes, we elucidated various structural aspects of the fish mt genome and the encoded genes. The present results will be important for understanding functions of the mt genome and developing programs for nucleotide sequence analysis. This study demonstrated the significance of extensive comparisons for understanding the structure of the mt genome.

Wide distribution range of rhizobial symbionts associated with pantropical sea-dispersed legumes.

To understand the geographic distributions of rhizobia that associated with widely distributed wild legumes, 66 nodules obtained from 41 individuals including three sea-dispersed legumes (Vigna marina, Vigna luteola, and Canavalia rosea) distributed across the tropical and subtropical coastal regions of the world were studied. Partial sequences of 16S rRNA and nodC genes extracted from the nodules showed that only Bradyrhizobium and Sinorhizobium were associated with the pantropical legumes, and some of the symbiont strains were widely distributed over the Pacific. Horizontal gene transfer of nodulation genes were observed within the Bradyrhizobium and Sinorhizobium lineages. BLAST searches in GenBank also identified records of these strains from various legumes across the world, including crop species. However, one of the rhizobial strains was not found in GenBank, which implies the strain may have adapted to the littoral environment. Our results suggested that some rhizobia, which associate with the widespread sea-dispersed legume, distribute across a broad geographic range. By establishing symbiotic relationships with widely distributed rhizobia, the pantropical legumes may also be able to extend their range much further than other legume species.

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.

MitoFish and MitoAnnotator: a mitochondrial genome database of fish with an accurate and automatic annotation pipeline.

Mitofish is a database of fish mitochondrial genomes (mitogenomes) that includes powerful and precise de novo annotations for mitogenome sequences. Fish occupy an important position in the evolution of vertebrates and the ecology of the hydrosphere, and mitogenomic sequence data have served as a rich source of information for resolving fish phylogenies and identifying new fish species. The importance of a mitogenomic database continues to grow at a rapid pace as massive amounts of mitogenomic data are generated with the advent of new sequencing technologies. A severe bottleneck seems likely to occur with regard to mitogenome annotation because of the overwhelming pace of data accumulation and the intrinsic difficulties in annotating sequences with degenerating transfer RNA structures, divergent start/stop codons of the coding elements, and the overlapping of adjacent elements. To ease this data backlog, we developed an annotation pipeline named MitoAnnotator. MitoAnnotator automatically annotates a fish mitogenome with a high degree of accuracy in approximately 5 min; thus, it is readily applicable to data sets of dozens of sequences. MitoFish also contains re-annotations of previously sequenced fish mitogenomes, enabling researchers to refer to them when they find annotations that are likely to be erroneous or while conducting comparative mitogenomic analyses. For users who need more information on the taxonomy, habitats, phenotypes, or life cycles of fish, MitoFish provides links to related databases. MitoFish and MitoAnnotator are freely available at http://mitofish.aori.u-tokyo.ac.jp/ (last accessed August 28, 2013); all of the data can be batch downloaded, and the annotation pipeline can be used via a web interface.

Phylogeny of the Elopomorpha (Teleostei): evidence from six nuclear and mitochondrial markers.

The Elopomorpha (eels and relatives) is a morphologically diverse group of predominantly marine teleost fishes comprising about 1000 species placed in 25 families. It is one of the three major living teleost lineages along with the Osteoglossomorpha and Clupeocephala. Among a few morphological synapomorphies that have been offered as evidence for the monophyly of the Elopomorpha, the remarkable leptocephalus larvae stand out. Several studies aiming at reconstructing the elopomorph phylogeny using morphological or molecular characters led to inconsistent results. In this study, we have tested previous hypotheses regarding inter- and intra-relationships of the Elopomorpha using a multi-locus dataset composed of three nuclear and three mitochondrial genes. Our analyses were based on likelihood phylogenetic reconstruction methods using different character-weighting data matrices and gene partition schemes to assess reliability of our findings. Our results confirm the respective monophyly of the Elopomorpha, Osteoglossomorpha and Clupeocephala. The majority of our analyses identify the Elopomorpha as the sister group of a clade containing the rest of the Teleostei. Within the Elopomorpha, the Elopiformes is the sister group of the remaining taxa. The Albuliformes sensuForey et al. (1996) and the Notacanthiformes are never sister-taxa in our phylogenetic trees, in contradiction with the recent mitogenomic hypothesis and current classification. Our results place the Notacanthiformes as the sister group of the Anguilliformes, including Saccopharyngiformes. Among anguilliforms, the families Congridae and Muraenesocidae are not monophyletic. The recently discovered anguilliform "living fossil" family Protanguillidae is not the sister group of the remaining Anguilliformes, instead, the sister group of the Synaphobranchidae. Based on the results presented here, we propose a revised classification for the Elopomorpha, comprised of four orders, including a resurrected Notacanthiformes but surrendering the Saccopharyngiformes. Within Anguilliformes, we recognized four monophyletic suborders named Protanguilloidei, Muraenoidei, Anguilloidei, and Congroidei.

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.

Mitogenomic sequences and evidence from unique gene rearrangements corroborate evolutionary relationships of myctophiformes (Neoteleostei).

BACKGROUND: A skewed assemblage of two epi-, meso- and bathypelagic fish families makes up the order Myctophiformes - the blackchins Neoscopelidae and the lanternfishes Myctophidae. The six rare neoscopelids show few morphological specializations whereas the divergent myctophids have evolved into about 250 species, of which many show massive abundances and wide distributions. In fact, Myctophidae is by far the most abundant fish family in the world, with plausible estimates of more than half of the oceans combined fish biomass. Myctophids possess a unique communication system of species-specific photophore patterns and traditional intrafamilial classification has been established to reflect arrangements of photophores. Myctophids present the most diverse array of larval body forms found in fishes although this attribute has both corroborated and confounded phylogenetic hypotheses based on adult morphology. No molecular phylogeny is available for Myctophiformes, despite their importance within all ocean trophic cycles, open-ocean speciation and as an important part of neoteleost divergence. This study attempts to resolve major myctophiform phylogenies from both mitogenomic sequences and corroborating evidence in the form of unique mitochondrial gene order rearrangements. RESULTS: Mitogenomic evidence from DNA sequences and unique gene orders are highly congruent concerning phylogenetic resolution on several myctophiform classification levels, corroborating evidence from osteology, larval ontogeny and photophore patterns, although the lack of larval morphological characters within the subfamily Lampanyctinae stands out. Neoscopelidae is resolved as the sister family to myctophids with Solivomer arenidens positioned as a sister taxon to the remaining neoscopelids. The enigmatic Notolychnus valdiviae is placed as a sister taxon to all other myctophids and exhibits an unusual second copy of the tRNA-Met gene - a gene order rearrangement reminiscent of that found in the tribe Diaphini although our analyses show it to be independently derived. Most tribes are resolved in accordance with adult morphology although Gonichthyini is found within a subclade of the tribe Myctophini consisting of ctenoid scaled species. Mitogenomic sequence data from this study recognize 10 reciprocally monophyletic lineages within Myctophidae, with five of these clades delimited from additional rearranged gene orders or intergenic non-coding sequences. CONCLUSIONS: Mitogenomic results from DNA sequences and unique gene orders corroborate morphology in phylogeny reconstruction and provide a likely scenario for the phylogenetic history of Myctophiformes. The extent of gene order rearrangements found within the mitochondrial genomes of myctophids is unique for phylogenetic purposes.

Limits and phylogenetic relationships of East Asian fishes in the subfamily Oxygastrinae (Teleostei: Cypriniformes: Cyprinidae).

The cyprinid subfamily Oxygastrinae is composed of a diverse group of fishes that has been taxonomically and phylogenetically problematic. Their great variation in appearance, life histories, and trophic diversity resulted in uncertainty regarding their relationships, which led to their historical classification across many disparate subfamilies. The phylogenetic relationships of Oxygastrinae are resolved based on sequence data from four loci: cytochrome b, cytochrome c oxidase I, opsin, and recombination activating gene 1. A combined data matrix consisting of 4114 bp for 144 taxa was compiled and analyzed using maximum likelihood and parsimony optimality criteria. The subfamily Oxygastrinae is recovered as a monophyletic group that includes Ancherythroculter, Aphyocypris, Candidia, Chanodichthys, Ctenopharyngodon, Culter, Distoechodon, Elopichthys, Hainania, Hemiculter, Hemiculterella, Hemigrammocypris, Hypophthalmichthys, Ischikauia, Macrochirichthys, Megalobrama, Metzia, Mylopharyngodon, Nicholsicypris, Nipponocypris, Ochetobius, Opsariichthys, Oxygaster, Parabramis, Parachela, Paralaubuca, Pararasbora, Parazacco, Plagiognathops, Pseudobrama, Pseudohemiculter, Pseudolaubuca, Sinibrama, Squaliobarbus, Toxabramis, Xenocyprioides, Xenocypris, Yaoshanicus, and Zacco. Of these genera, the following were found to be monophyletic: Aphyocypris, Distoechodon, Hypophthalmichthys, Nipponocypris, Opsariichthys, Parachela, Paralaubuca, Plagiognathops, Xenocyprioides, and Xenocypris. The following genera were not monophyletic: Metzia, Hemiculter, Toxabramis, Ancherythroculter, Chanodichthys, Culter, Megalobrama. The remainder are either monotypic or were represented by only a single species. Four genera not examined in this study are provisionally classified in Oxygastrinae: Anabarilius, Longiculter, Pogobrama, and Rasborichthys.

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.

Temperature sensitivity of the interspecific interaction strength of coastal marine fish communities.

The effects of temperature on interaction strengths are important for understanding and forecasting how global climate change impacts marine ecosystems; however, tracking and quantifying interactions of marine fish species are practically difficult especially under field conditions, and thus, how temperature influences their interaction strengths under field conditions remains poorly understood. We herein performed quantitative fish environmental DNA (eDNA) metabarcoding on 550 seawater samples that were collected twice a month from 11 coastal sites for 2 years in the Boso Peninsula, Japan, and analyzed eDNA monitoring data using nonlinear time series analytical tools. We detected fish-fish interactions as information flow between eDNA time series, reconstructed interaction networks for the top 50 frequently detected species, and quantified pairwise, fluctuating interaction strengths. Although there was a large variation, water temperature influenced fish-fish interaction strengths. The impact of water temperature on interspecific interaction strengths varied among fish species, suggesting that fish species identity influences the temperature effects on interactions. For example, interaction strengths that Halichoeres tenuispinis and Microcanthus strigatus received strongly increased with water temperature, while those of Engraulis japonicus and Girella punctata decreased with water temperature. An increase in water temperature induced by global climate change may change fish interactions in a complex way, which consequently influences marine community dynamics and stability. Our research demonstrates a practical research framework to study the effects of environmental variables on interaction strengths of marine communities in nature, which would contribute to understanding and predicting natural marine ecosystem dynamics.

The world's oceans are home to tens of thousands of fish species, many of which live in nutrient-rich coastal waters. Different species living in a particular environment interact with each other in many ways. For example, a predatory fish may prey on some species of small fish but avoid feeding on others that help it by removing parasites from its skin. Rising ocean temperatures caused by global climate change could affect how different fish species interact with one another and, as a result, impact their communities. One of the first steps to understanding how fish interact with each other in nature typically requires researchers to count the number of different species present and observe how they behave, which is time-consuming and labor-intensive. An alternative is to use an emerging technique in which researchers extract DNA from water, soil or air ­ known as environmental DNA ­ and analyze it to identify the species present and estimate their numbers. Ushio et al. analyzed hundreds of samples of seawater that had been collected over a two-year period from the Boso Peninsula in Japan. Statistical methods were used to quantify how strongly fish species interact with each other and determine whether the temperature of the water influenced how different species of fish interacted over time. The findings showed that water temperature had a significant but complex effect on how strongly pairs of fish species interacted, with both positive and negative effects depending on the conditions. The impact of water temperature on the strength of the interactions varied between species, for example, Japanese anchovy and largescale blackfish interacted less strongly with other fish species in warmer water, whereas the Stripey and a species of wrasse interacted with other fish species more strongly. The findings provide new insights into how water temperature affects the communities of fish living in coastal areas. Alongside complementing existing knowledge in the field, refining the research framework used in this work will benefit those working in fishery science by providing valuable insights into how natural and commercially important fish species respond to climate change.

A 'living fossil' eel (Anguilliformes: Protanguillidae, fam. nov.) from an undersea cave in Palau.

We report the discovery of an enigmatic, small eel-like fish from a 35 m-deep fringing-reef cave in the western Pacific Ocean Republic of Palau that exhibits an unusual suite of morphological characters. Many of these uniquely characterize the Recent members of the 19 families comprising the elopomorph order Anguilliformes, the true eels. Others are found among anguilliforms only in the Cretaceous fossils, and still others are primitive with respect to both Recent and fossil eels. Thus, morphological evidence explicitly places it as the most basal lineage (i.e. the sister group of extant anguilliforms). Phylogenetic analysis and divergence time estimation based on whole mitogenome sequences from various actinopterygians, including representatives of all eel families, demonstrate that this fish represents one of the most basal, independent lineages of the true eels, with a long evolutionary history comparable to that of the entire Anguilliformes (approx. 200 Myr). Such a long, independent evolutionary history dating back to the early Mesozoic and a retention of primitive morphological features (e.g. the presence of a premaxilla, metapterygoid, free symplectic, gill rakers, pseudobranch and distinct caudal fin rays) warrant recognition of this species as a 'living fossil' of the true eels, herein described as Protanguilla palau genus et species nov. in the new family Protanguillidae.