GenBank is a reliable resource for 21st century biodiversity research.

Traditional methods of characterizing biodiversity are increasingly being supplemented and replaced by approaches based on DNA sequencing alone. These approaches commonly involve extraction and high-throughput sequencing of bulk samples from biologically complex communities or samples of environmental DNA (eDNA). In such cases, vouchers for individual organisms are rarely obtained, often unidentifiable, or unavailable. Thus, identifying these sequences typically relies on comparisons with sequences from genetic databases, particularly GenBank. While concerns have been raised about biases and inaccuracies in laboratory and analytical methods, comparatively little attention has been paid to the taxonomic reliability of GenBank itself. Here we analyze the metazoan mitochondrial sequences of GenBank using a combination of distance-based clustering and phylogenetic analysis. Because of their comparatively rapid evolutionary rates and consequent high taxonomic resolution, mitochondrial sequences represent an invaluable resource for the detection of the many small and often undescribed organisms that represent the bulk of animal diversity. We show that metazoan identifications in GenBank are surprisingly accurate, even at low taxonomic levels (likely <1% error rate at the genus level). This stands in contrast to previously voiced concerns based on limited analyses of particular groups and the fact that individual researchers currently submit annotated sequences to GenBank without significant external taxonomic validation. Our encouraging results suggest that the rapid uptake of DNA-based approaches is supported by a bioinformatic infrastructure capable of assessing both the losses to biodiversity caused by global change and the effectiveness of conservation efforts aimed at slowing or reversing these losses.

Seabed mining could come at a high price for a unique fauna.

The deep seafloor is teeming with life, most of which remains poorly known to science. It also constitutes an important reserve of natural resources, particularly minerals, that mining companies will start harvesting in the next few years (Nat Rev Earth Environ, 1, 2020, 158). In this context, broad biodiversity assessments of deep-sea ecosystems are urgently needed to establish a baseline prior to mining. However, significant gaps in our taxonomic knowledge and the high cost of sampling in the deep sea limit the effectiveness of conventional morphology-based surveys. In this issue of Molecular Ecology, Laroche et al. (Mol Ecol, 2020) capitalize on high throughput molecular methods to conduct one of the most detailed and rigorous surveys of the composition and biogeography of deep-seafloor metazoan communities to date. The authors show that deep seamounts in the Clarion Clipperton Zone are inhabited by rich metazoan communities that are distinct from those of the surrounding abyssal plains. These results have important conservation implications: if communities on deep seamounts were to persist after large-scale industrial mining operations on the surrounding plains, the seamounts would not serve as appropriate reservoirs to repopulate impacted areas.

MIDORI server: a webserver for taxonomic assignment of unknown metazoan mitochondrial-encoded sequences using a curated database.

Summary: We present MIDORI server, a user-friendly web platform that uses a curated reference dataset, MIDORI, for high throughput taxonomic classification of unknown metazoan mitochondrial-encoded gene sequences. Currently three methods of taxonomic assignments: RDP Classifier, SPINGO and SINTAX, are implemented. Availability and implementation: The web server is freely available at {http://reference-midori.info/server.php}.

Novel molecular resources for single-specimen barcoding of enigmatic crustacean y-larvae.

Despite discovery more than 100years ago and documented global occurrence from shallow waters to the deep sea, the life cycle of the enigmatic crustacean y-larvae isincompletely understood and adult forms remain unknown. To date, only 2 of the 17 formally described species, all based on larval stages, have been investigated using an integrative taxonomic approach. This approach provided descriptions of the morphology of the naupliar and cyprid stages, and made use of exuvial voucher material and DNA barcodes. To improve our knowledge about the evolutionary history and ecological importance of y-larvae, we developed a novel protocol that maximises the amount of morpho-ecological and molecular data that can be harvested from single larval specimens. This includes single-specimen DNA barcoding and daily imaging of y-nauplii reared in culture dishes, mounting of the 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. Through development and testing of a suite of new primers for both nuclear and mitochondrial protein-coding and ribosomal genes, we showcase how new sequence data can be used to estimate the phylogeny of Facetotecta. 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 from a diverse array of moulting marine invertebrate taxa.

Potential PCR amplification bias in identifying complex ecological patterns: Higher species compositional homogeneity revealed in smaller-size coral reef zooplankton by metatranscriptomics.

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.

A new versatile primer set targeting a short fragment of the mitochondrial COI region for metabarcoding metazoan diversity: application for characterizing coral reef fish gut contents.

INTRODUCTION: The PCR-based analysis of homologous genes has become one of the most powerful approaches for species detection and identification, particularly with the recent availability of Next Generation Sequencing platforms (NGS) making it possible to identify species composition from a broad range of environmental samples. Identifying species from these samples relies on the ability to match sequences with reference barcodes for taxonomic identification. Unfortunately, most studies of environmental samples have targeted ribosomal markers, despite the fact that the mitochondrial Cytochrome c Oxidase subunit I gene (COI) is by far the most widely available sequence region in public reference libraries. This is largely because the available versatile ("universal") COI primers target the 658 barcoding region, whose size is considered too large for many NGS applications. Moreover, traditional barcoding primers are known to be poorly conserved across some taxonomic groups. RESULTS: We first design a new PCR primer within the highly variable mitochondrial COI region, the "mlCOIintF" primer. We then show that this newly designed forward primer combined with the "jgHCO2198" reverse primer to target a 313 bp fragment performs well across metazoan diversity, with higher success rates than versatile primer sets traditionally used for DNA barcoding (i.e. LCO1490/HCO2198). Finally, we demonstrate how the shorter COI fragment coupled with an efficient bioinformatics pipeline can be used to characterize species diversity from environmental samples by pyrosequencing. We examine the gut contents of three species of planktivorous and benthivorous coral reef fish (family: Apogonidae and Holocentridae). After the removal of dubious COI sequences, we obtained a total of 334 prey Operational Taxonomic Units (OTUs) belonging to 14 phyla from 16 fish guts. Of these, 52.5% matched a reference barcode (>98% sequence similarity) and an additional 32% could be assigned to a higher taxonomic level using Bayesian assignment. CONCLUSIONS: The molecular analysis of gut contents targeting the 313 COI fragment using the newly designed mlCOIintF primer in combination with the jgHCO2198 primer offers enormous promise for metazoan metabarcoding studies. We believe that this primer set will be a valuable asset for a range of applications from large-scale biodiversity assessments to food web studies.

Allometric scaling of interspecific RNA transcript abundance to extend the use of metatranscriptomics in characterizing complex communities.

Metatranscriptomics allows profiling of community mRNA and 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. The 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 -», 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.

Development of transcriptomics-based growth rate indices in two model eukaryotes and relevance to metatranscriptomics datasets.

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.

Using metatranscriptomics to estimate the diversity and composition of zooplankton communities.

DNA metabarcoding is a rapid, high-resolution tool used for biomonitoring complex zooplankton communities. However, diversity estimates derived with this approach can be biased by the co-detection of sequences from environmental DNA (eDNA), nuclear-encoded mitochondrial (NUMT) pseudogene contamination, and taxon-specific PCR primer affinity differences. To avoid these methodological uncertainties, we tested the use of metatranscriptomics as an alternative approach for characterizing zooplankton communities. Specifically, we compared metatranscriptomics with PCR-based methods using genomic (gDNA) and complementary DNA (cDNA) amplicons, and morphology-based data for estimating species diversity and composition for both mock communities and field-collected samples. Mock community analyses showed that the use of gDNA mitochondrial cytochrome c oxidase I (mtCO1) amplicons inflates species richness due to the co-detection of extra-organismal eDNA. Significantly more amplicon sequence variants, nucleotide diversity, and indels were observed with gDNA amplicons than with cDNA, indicating the presence of putative NUMT pseudogenes. Moreover, PCR-based methods failed to detect the most abundant species in mock communities due to priming site mismatch. Overall, metatranscriptomics provided estimates of species richness and composition that closely resembled those derived from morphological data. The use of metatranscriptomics was further tested using field-collected samples, with the results showing consistent species diversity estimates among biological and technical replicates. Additionally, temporal zooplankton species composition changes could be monitored using different mitochondrial markers. These findings demonstrate the advantages of metatranscriptomics as an effective tool for monitoring diversity in zooplankton research.

Utilisation of Oxford Nanopore sequencing to generate six complete gastropod mitochondrial genomes as part of a biodiversity curriculum.

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.

Zooplankton diversity analysis through single-gene sequencing of a community sample.

BACKGROUND: Oceans cover more than 70% of the earth's surface and are critical for the homeostasis of the environment. Among the components of the ocean ecosystem, zooplankton play vital roles in energy and matter transfer through the system. Despite their importance, understanding of zooplankton biodiversity is limited because of their fragile nature, small body size, and the large number of species from various taxonomic phyla. Here we present the results of single-gene zooplankton community analysis using a method that determines a large number of mitochondrial COI gene sequences from a bulk zooplankton sample. This approach will enable us to estimate the species richness of almost the entire zooplankton community. RESULTS: A sample was collected from a depth of 721 m to the surface in the western equatorial Pacific off Pohnpei Island, Micronesia, with a plankton net equipped with a 2-m2 mouth opening. A total of 1,336 mitochondrial COI gene sequences were determined from the cDNA library made from the sample. From the determined sequences, the occurrence of 189 species of zooplankton was estimated. BLASTN search results showed high degrees of similarity (>98%) between the query and database for 10 species, including holozooplankton and merozooplankton. CONCLUSION: In conjunction with the Census of Marine Zooplankton and Barcode of Life projects, single-gene zooplankton community analysis will be a powerful tool for estimating the species richness of zooplankton communities.

Taiwanese Leucothoidae (Crustacea: Amphipoda), Including Three New Species from Dongsha Atoll.

Kristine N. White and Ryuji J. Machida (2018) Examination of leucothoid amphipods from Penghu, Green Island, and Dongsha Atoll revealed 16 species not previously reported from Taiwan. Leucothoe batillum sp. nov., Leucothoe cracentis sp. nov., and Paranamixis lunata sp. nov. are described from Dongsha Atoll. The ranges of 13 Leucothoe species are expanded to include the South China and Philippine Seas, suggesting genetic or geographic-driven connectivity between the South and East China Seas and the Philippine Sea. Leucothoe furina (Savigny, 1816), originally described from the Mediterranean or Red Sea, is reported from Taiwan, which suggests the occurrence of another species complex in the Leucothoidae.

Occurrence of mitochondrial CO1 pseudogenes in Neocalanus plumchrus (Crustacea: Copepoda): Hybridization indicated by recombined nuclear mitochondrial pseudogenes.

A portion of the mitochondrial cytochrome c oxidase I gene was sequenced using both genomic DNA and complement DNA from three planktonic copepod Neocalanus species (N. cristatus, N. plumchrus, and N. flemingeri). Small but critical sequence differences in CO1 were observed between gDNA and cDNA from N. plumchrus. Furthermore, careful observation revealed the presence of recombination between sequences in gDNA from N. plumchrus. Moreover, a chimera of the N. cristatus and N. plumchrus sequences was obtained from N. plumchrus gDNA. The observed phenomena can be best explained by the preferential amplification of the nuclear mitochondrial pseudogenes from gDNA of N. plumchrus. Two conclusions can be drawn from the observations. First, nuclear mitochondrial pseudogenes are pervasive in N. plumchrus. Second, a mating between a female N. cristatus and a male N. plumchrus produced viable offspring, which further backcrossed to a N. plumchrus individual. These observations not only demonstrate intriguing mating behavior in these species, but also emphasize the importance of careful interpretation of species marker sequences amplified from gDNA.

Metazoan mitochondrial gene sequence reference datasets for taxonomic assignment of environmental samples.

Mitochondrial-encoded genes are increasingly targeted in studies using high-throughput sequencing approaches for characterizing metazoan communities from environmental samples (e.g., plankton, meiofauna, filtered water). Yet, unlike nuclear ribosomal RNA markers, there is to date no high-quality reference dataset available for taxonomic assignments. Here, we retrieved all metazoan mitochondrial gene sequences from GenBank, and then quality filtered and formatted the datasets for taxonomic assignments using taxonomic assignment tools. The reference datasets-'Midori references'-are available for download at www.reference-midori.info. Two versions are provided: (I) Midori-UNIQUE that contains all unique haplotypes associated with each species and (II) Midori-LONGEST that contains a single sequence, the longest, for each species. Overall, the mitochondrial Cytochrome oxidase subunit I gene was the most sequence-rich gene. However, sequences of the mitochondrial large ribosomal subunit RNA and Cytochrome b apoenzyme genes were observed for a large number of species in some phyla. The Midori reference is compatible with some taxonomic assignment software. Therefore, automated high-throughput sequence taxonomic assignments can be particularly effective using these datasets.

Large-scale gene rearrangements in the mitochondrial genomes of two calanoid copepods Eucalanus bungii and Neocalanus cristatus (Crustacea), with notes on new versatile primers for the srRNA and COI genes.

Partial sequences of the mitochondrial genomes were determined for two calanoid copepods, Eucalanus bungii [9530 bp (base pairs)] and Neocalanus cristatus (7965 bp), using an approach that employs a long polymerase chain reaction (PCR) technique and primer walking. In contrast to Tigriopus japonicus (Harpacticoida), in which the complete mitochondrial genome has been determined, genes are encoded on both strands of the genome. In spite of the close relationship between E. bungii and N. cristatus, the gene orders of these calanoid copepods differed greatly from each other. From the sequence we obtained, new copepod-specific versatile primers were designed for the cytochrome c oxidase I (COI) gene (1200 bp). These primer pairs, as well as the previously designed primer pairs for the small ribosomal RNA (srRNA) gene (500 bp), were applied for 20 species representing four orders of both calanoid and non-calanoid copepods and amplification of sequences from at least one species from each order was successful.

Complete mitochondrial DNA sequence of Tigriopus japonicus (Crustacea: Copepoda).

The complete nucleotide sequence of the mitochondrial genome was determined for a harpacticoid copepod, Tigriopus japonicus (Crustacea), using an approach that employs a long polymerase chain reaction technique and primer walking. Although the genome (14,628 bp) contained the same set of 37 genes (2 ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes) as found in other metazoan animals, none of the previously reported gene orders were comparable to that of T. japonicus. Furthermore, all genes were encoded on one strand, unlike the mitochondrial genomes of most metazoan animals. Size reductions were notable for tRNA and rRNA genes, resulting in one of the smallest mitochondrial genomes in the arthropod lineage. Although it appears that such large-scale gene rearrangements have occurred in the ancestral species of T. japonicus, none of the proposed mechanisms parsimoniously account for this eccentric gene arrangement.

PCR-based approach for sequencing mitochondrial genomes of decapod crustaceans, with a practical example from kuruma prawn (Marsupenaeus japonicus).

An approach for sequencing the entire mitochondrial genomes (mitogenomes) of decapod crustaceans using 79 newly designed and 7 published polymerase chain reaction (PCR) primers is described. The approach comprises the following steps: (1) the entire mitogenome is amplified in 2 or 3 long PCRs; (2) the 86 primers are used in different combinations to amplify contiguous, overlapping short segments of the entire mitogenome with the diluted long PCR products as templates; (3) direct cycle sequencing is conducted using the short PCR products. This strategy allows a more rapid determination of decapod mitogenomic sequences than a traditional method using cloned mitochondrial DNA and primer walking strategy. As a practical example, the mitogenomic sequence for a kuruma prawn Marsupenaeus japonicus (Crustacea: Decapoda), was determined using the PCR-based approach.

Organization of the mitochondrial genome of Antarctic krill Euphausia superba (Crustacea: Malacostraca).

We determined the nearly complete DNA sequence of the mitochondrial genome of Antarctic krill Euphausia superba (Crustacea: Malacostraca), one of the most ecologically and commercially important zooplankters in Antarctic waters. All of the genome sequences were purified by gene amplification using long polymerase chain reaction (PCR), and the products were subsequently used as templates for either direct sequencing using a primer-walking strategy or nested PCR with crustacea-versatile primers. Although we were unable to determine a portion of the genome owing to technical difficulties, the sequenced position, 14,606 bp long, contained all of the 13 protein-coding genes, 19 of the 22 transfer RNA genes, and the large subunit as well as a portion of the small subunit ribosomal RNA genes. Gene rearrangement was observed for 3 transfer RNA genes (tRNACys, tRNATyr, and tRNATrp) and the 2 leucine tRNA genes.

Four methods of preparing mRNA 5' end libraries using the Illumina sequencing platform.

BACKGROUND: The 5' untranslated regions of mRNA play an important role in their translation. RESULTS: Here, we describe the development of four methods of profiling mRNA 5' ends using the Illumina sequencing platform; the first method utilizes SMART (Switching Mechanism At 5' end of RNA Transcript) technology, while the second involves replacing the 5' cap structure with RNA oligomers via ligation. The third and fourth methods are modifications of SMART, and involve enriching mRNA molecules with (nuclear transcripts) and without (mitochondrial transcripts) 5' end cap structures, respectively. Libraries prepared using SMART technology gave more reproducible results, but the ligation method was advantageous in that it only sequenced mRNAs with a cap structure at the 5' end. CONCLUSIONS: These methods are suitable for global mapping of mRNA 5' ends, both with and without cap structures, at a single molecule resolution. In addition, comparison of the present results obtained using different methods revealed the presence of abundant messenger RNAs without a cap structure.