Ryuji J. Machida’s research while affiliated with Academia Sinica and other places

We investigated the species diversity of the demersal copepod genus Pseudodiaptomus in the coastal waters of northern Taiwan, and recorded four species new to Taiwanese waters: P. echinatus n. sp., P. ishigakiensis Nishida, 1985, P. philippinensis Walter, 1986, and P. zhengi Wang & Guo, 2019. Pseudodiaptomus echinatus n. sp. is distinguished from the allied species P. serricaudatus (Scott T., 1894) by the female genital double-somite, which is laterally swollen and of subequal length and width, and the male fifth leg, the right endopod of which is oval with a row of spinules increasing in size toward the tip, and the first exopod segment of which has a distomedial digitiform process with rounded tip. In addition, out of the 11 nominal species previously recorded from Taiwanese waters, we found only three species ( P. annandalei Sewell, 1919, P. bispinosus Walter, 1984, P. trihamatus Wright, 1937). We were unable to confirm the presence of P. forbesi (Poppe & Richard, 1890), P. incisus Shen & Lee, 1963, P. inopinus Burckhardt, 1913, P. marinus Sato, 1913, P. pacificus Walter, 1986, P. penicillus Li & Huang, 1984, P. poplesia (Shen, 1955), and P. serricaudatus (T. Scott, 1894) by a critical review of available literature based on morphology, locality, habitat, etc. In this regard, it is necessary for a future study to try to verify the presence of these eight species in Taiwanese waters.

PCR‐based high‐throughput sequencing has permitted comprehensive resolution analyses of zooplankton diversity dynamics. However, significant methodological issues still surround analyses of complex bulk community samples, not least as in prevailing PCR‐based approaches. Marine drifting animals—zooplankton—play essential ecological roles in the pelagic ecosystem, transferring energy and elements to higher trophic levels, such as fishes, cetaceans and others. In the present study, we collected 48 size‐fractionated zooplankton samples in the vicinity of a coral reef island with environmental gradients. To investigate the spatiotemporal dynamics of zooplankton diversity patterns and the effect of PCR amplification biases across these complex communities, we first took metatranscriptomics approach. Comprehensive computational analyses revealed a clear pattern of higher/lower homogeneity in smaller/larger zooplankton compositions across samples respectively. Our study thus suggests changes in the role of dispersal across the sizes. Next, we applied in silico PCR to the metatranscriptomics datasets, in order to estimate the extent of PCR amplification bias. Irrespective of stringency criteria, we observed clear separations of size fraction sample clusters in both metatranscriptomics and in silico datasets. In contrast, the pattern—smaller‐fractioned communities had higher compositional homogeneity than larger ones—was observed in the metatranscriptomics data but not in the in silico datasets. To investigate this discrepancy further, we analysed the mismatches of widely used mitochondrial CO1 primers and identified priming site mismatches likely driving PCR‐based biases. Our results suggest the use of metatranscriptomics or, although less ideal, redesigning the CO1 primers is necessary to circumvent these issues.

Due to the paucity of morphological characters and extensive convergent evolution, bivalve classification is a challenging, two-centuries-old problem (Giribet & Wheeler, 2005; Bieler et al., 2014; Lemer et al., 2016; Giribet & Edgecombe, 2020). Consequently, it is not surprising that morphology-based phylogenies conflict with sequence-based phylogenies (Giribet & Wheeler, 2005; Tëmkin, 2010; Bieler et al., 2014; Lemer et al., 2016; Giribet & Edgecombe, 2020). Pearl oysters (superfamily Pterioidea) make critical contributions to shellfisheries and pearl harvesting. Despite extensive study, their systematic placement and relationships with other taxa within the subclass Pteriomorphia remain disputed (Steiner & Hammer, 2000; Giribet & Distel, 2003; Matsumoto, 2003; Tëmkin, 2006, 2010). Previously, a phylogeny of pearl oysters was estimated using four genetic markers (three nuclear and one mitochondrial) in combination with morphological characters (Tëmkin, 2010). Despite the high taxonomic coverage of Pterioidea species (c. >80%), these four genes may not accurately capture deep divergences, some of which are more than 470 Myr old (Tëmkin, 2010). Resolving deep relationships may require phylogenomic approaches, which are starting to be applied to bivalves and other molluscs (Kocot et al., 2011; González et al., 2015; Lemer et al., 2016; Lemer, Bieler & Giribet, 2019). Nevertheless, despite decreasing sequencing costs, obtaining transcriptome and genome data still requires considerable resources, and assembling high-quality mollusc genomes is hindered by the difficulty in isolating long DNA fragments (due to the presence of polysaccharides), high heterozygosity and abundant long repeats (Sigwart et al., 2021).

The enigmatic “y-larvae” (Pancrustacea: Facetotecta) still have an incompletely understood lifecycle, and their adult forms remain unknown despite their discovery more than 100 years ago and their documented global occurrence from shallow waters to the deep-sea. Only two of the 17 formally described species, all based on larval stages, have been investigated using an integrative taxonomic approach that, besides providing descriptions of the morphology of the naupliar and cyprid stages, also made use of exuvial voucher material and DNA barcodes. To improve our knowledge about the systematics and phylogenetics of y-larvae, we developed a novel protocol that maximizes the amount of morphological, ecological, and molecular data that can be harvested from single individuals of these tiny larvae. This revolves around single larva barcoding, and includes daily imaging of y-nauplii reared in culture dishes, mounting of their last naupliar exuviae on a slide as a reference voucher, live imaging of the y-cyprid instar that follows, and fixation, DNA extraction, amplification, and sequencing of the y-cyprid specimen. By developing and testing a suite of new primers for both nuclear and mitochondrial protein-coding and ribosomal genes, we estimated the most comprehensive phylogeny of Facetotecta to date. We expect that our novel procedure will help to unravel the complex systematics of y-larvae and show how these fascinating larval forms have evolved. Moreover, we posit that our protocols should work on larval specimens of a diverse array of molting marine invertebrate taxa.

Marine drifting animals — zooplankton — play essential ecological roles in the pelagic ecosystem, transferring energy and elements to higher trophic levels, such as fishes, cetaceans, and others. Zooplankton are generally considered passive drifting organisms homogeneously distributed throughout waters, where high dispersal is expected. Although empirical observations have demonstrated that many species possess active swimming mechanisms that generate metacommunities with high beta diversity, the role of animal sizes in the process of marine zooplankton community dynamics remains unexplored. Here, we collected a total of 48 size-fractionated zooplankton samples in the vicinity of a coral reef island with environmental gradients and performed metatranscriptome analyses. The samples were collected in two transects (from nearshore to offshore) twice a day (morning and night). Sample size fraction was the only variable that rendered apparent differences in species composition between the samples. Our results demonstrate differential dispersal through the size fractions — smaller size fraction communities had higher compositional homogeneity than larger ones. Contrary to expectation, distance to shore had no significant influence on the composition or diversity of zooplankton communities. This study offers novel insights on the use of metatranscriptomics for analyzing community structures and the role size plays for the marine zooplankton community assembly processes.

Metatranscriptomics allows profiling of community messenger RNA (mRNA) and ribosomal RNA (rRNA) transcript abundance under certain environmental conditions. However, variations in the proportion of RNA transcripts across different community size structures remain less explained, thus limiting the possible applications of metatranscriptomics in community studies. Here, we extended the assumptions of the growth‐rate hypothesis (GRH) and the metabolic theory of ecology (MTE) to validate the allometric scaling of interspecific RNA transcript (mRNA and rRNA) abundance through metatranscriptomic analysis of mock communities consisting of model organisms. Results suggest that body size imposes significant constraints on RNA transcript abundance. Interestingly, the relationship between the total mitochondrial transcript abundance (mRNA and rRNA slopes were –0.30 and –0.28, respectively) and body size aligned with the MTE assumptions with slopes close to –¼, while the nuclear transcripts displayed much steeper slopes (mRNA and rRNA slopes were –0.33 and –0.40, respectively). The assumed temperature dependence was not observed in this study. At the gene level, the allometric slopes range from 0 to –1. Overall, the above results showed that larger individuals have lesser RNA transcript abundance per tissue mass than smaller ones regardless of temperature. Analyses of field‐collected microcrustacean zooplankton samples demonstrated that the correction of size effect, using the allometric exponents derived from the model organism mock community, explains better the patterns of interspecific RNA transcripts abundance within the metatranscriptome. Integrating allometry with metatranscriptomics can extend the use of RNA transcript reads in estimating ecological processes within complex communities.

High-throughput sequencing has enabled genome skimming approaches to produce complete mitochondrial genomes (mitogenomes) for species identification and phylogenomics purposes. In particular, the portable sequencing device from Oxford Nanopore Technologies (ONT) has the potential to facilitate hands-on training from sampling to sequencing and interpretation of mitogenomes. In this study, we present the results from sampling and sequencing of six gastropod mitogenomes (Aplysia argus, Cellana orientalis, Cellana toreuma, Conus ebraeus, Conus miles and Tylothais aculeata) from a graduate level biodiversity course. The students were able to produce mitogenomes from sampling to annotation using existing protocols and programs. Approximately 4 Gb of sequence was produced from 16 Flongle and one MinION flow cells, averaging 235 Mb and N50 = 4.4 kb per flow cell. Five of the six 14.1–18 kb mitogenomes were circlised containing all 13 core protein coding genes. Additional Illumina sequencing revealed that the ONT assemblies spanned over highly AT rich sequences in the control region that were otherwise missing in Illumina-assembled mitogenomes, but still contained a base error of one every 70.8–346.7 bp under the fast mode basecalling with the majority occurring at homopolymer regions. Our findings suggest that the portable MinION device can be used to rapidly produce low-cost mitogenomes onsite and tailored to genomics-based training in biodiversity research.

Growth rate estimation is important to understand the flow of energy and nutrient elements in an ecosystem, but it has remained challenging, especially on microscopic organisms. In this study, we propose four growth rate indices that use mRNA abundance ratios between nuclear and mitochondrial genes: (1) total nuclear and mitochondrial mRNA ratio (Nuc:Mito‐TmRNA), (2) nuclear and mitochondrial ribosomal protein mRNA ratio (Nuc:Mito‐RPmRNA), (3) gene ontology (GO) terms and total mitochondrial mRNA ratios and (4) nuclear and mitochondrial specific gene mRNA ratio. We examine these proposed ratios using RNA‐Seq datasets of Daphnia magna, and Saccharomyces cerevisiae retrieved from the NCBI Short Read Archive. The results showed that both Nuc:Mito‐TmRNA and Nuc:Mito‐RPmRNA ratio indices showed significant correlations with the growth rate for both species. A large number of GO terms mRNA ratios showed significant correlations with the growth rate of S. cerevisiae. Lastly, we identified mRNA ratios of several specific nuclear and mitochondrial gene pairs that showed significant correlations. We foresee future implications for the proposed mRNA ratios used in metatranscriptome analyses to estimate the growth rate of communities and species.

Analysis of environmental DNA is increasingly used to characterize ecological communities, but the effectiveness of this approach depends on the accuracy of taxonomic reference databases. The MIDORI databases, first released in 2017, were built to improve accuracy for mitochondrial metazoan (animal) sequences. MIDORI has now been significantly improved and renamed MIDORI2 (available at http://www.reference‐midori.info). Like MIDORI, MIDORI2 is built from GenBank and contains curated sequences of thirteen protein‐coding and two ribosomal RNA mitochondrial genes. Coverage has been substantially expanded to cover all eukaryotes, including fungi, green algae and land plants, other multicellular algal groups, and diverse protist lineages. MIDORI2 also now includes not only species with full binomials, but also taxa referred to by genus with species left unspecified (“sp.”). Another new feature is the updating of the databases approximately every two months with version numbers corresponding to each new GenBank release. Additional potentially erroneously annotated sequences have also been removed. Finally, the ability to export data files to BLAST+ has been added to the original ability to export preformatted data to five taxonomic assignment programs, and databases of amino acid sequences are also made available for protein‐coding genes. As a technical validation, we conducted a preliminary comparison of the performance of MIDORI2 with five taxonomic assignment programs. Results suggest that BLAST+ top hits performed better for assigning CO1 sequences than alignment‐free methods based on compositional features. Comparing MIDORI2 with two other commonly used curated databases of mitochondrial sequences, CO‐ARBitrator and BOLD, we show that MIDORI2 includes sequences from a broader range of metazoan and non‐metazoan taxa. Overall, in many contexts, MIDORI2 offers clear advantages: a higher diversity of taxa than other databases, a variety of user‐friendly features, and regular updates. MIDORI2 is particularly well‐suited for environmental DNA studies that target mitochondrial genes with broad primers. In MIDORI2, we extended the taxonomic scope from metazoans to all eukaryote groups represented in GenBank. Databases now include both sequences with binomial identification and sequences without a species name (“sp.”). We built a pipeline to update the databases approximately every two months following the release of each GenBank database. We also increased the quality of the databases by removing sequences that we identified as potentially mislabeled in a previous study. Databases are formatted for six taxonomic assignment software programs. Databases of amino acid sequences are also available for protein‐coding genes

High-throughput sequencing has enabled genome skimming approaches to produce complete mitochondrial genomes (mitogenomes) for species identification and phylogenomics purposes. In particular, the portable sequencing device from Oxford Nanopore Technologies (ONT) has the potential to facilitate hands-on training from sampling to sequencing and interpretation of mitogenomes. In this study, we present the results from sampling and sequencing six gastropod mitogenomes ( Aplysia argus, Cellana orientalis, Cellana toreuma, Conus ebraeus, Conus miles and Tylothais aculeata ) from a graduate level biodiversity course. The students were able to produce mitochondrial genomes from sampling to annotation using existing protocols and programs. Approximately 4Gb of sequence was produced from 15 Flongle and two R9.4 Flowcells, averaging 235Mb and N50=4.4kb per Flowcell. Five of the six 14.1-18kb mitogenomes were circlised containing all 13 core protein coding genes. Additional Illumina sequencing reveal that the ONT assemblies were able to span over highly AT rich sequence in the control region that was otherwise missing in Illumina-assembled mitogenomes, but still contained a base error of one every 70.8-346.7bp with the majority occurring at homopolymer regions. Our findings suggest that ONT are portable and can be used to rapidly produce mitogenomes at low cost and tailored to genomics-based training in biodiversity research.