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Dr.Weibo Song received his Ph.D.degree in the Zoological Institute,Bonn University,Germany,in January 1989.He then joined the College of Fisheries,Ocean University of China（OUC）,and was qualified as a lecturer,associate professor and full professor in the following 4 years.During the past two decades,Dr.Song established the Laboratory

Freshwater ecosystems are being heavily exploited and degraded by human activities all over the world, including in North America, where fishes and fisheries are strongly affected. Despite centuries of taxonomic inquiry, problems inherent to species identification continue to hamper the conservation of North American freshwater fishes. Indeed, nearly 10% of species diversity is thought to remain undescribed. To provide an independent calibration of taxonomic uncertainty and to establish a more accessible molecular identification key for its application, we generated a standard reference library of mtDNA sequences (DNA barcodes) derived from expert-identified museum specimens for 752 North American freshwater fish species. This study demonstrates that 90% of known species can be delineated using barcodes. Moreover, it reveals numerous genetic discontinuities indicative of independently evolving lineages within described species, which points to the presence of morphologically cryptic diversity. From the 752 species analyzed, our survey flagged 138 named species that represent as many as 347 candidate species, which suggests a 28% increase in species diversity. In contrast, several species of parasitic and nonparasitic lampreys lack such discontinuity and may represent alternative life history strategies within single species. Therefore, it appears that the current North American freshwater fish taxonomy at the species level significantly conceals diversity in some groups, although artificially creating diversity in others. In addition to providing an easily accessible digital identification system, this study identifies 151 fish species for which taxonomic revision is required.

Anthropogenically forced changes in global freshwater biodiversity demand more efficient monitoring approaches. Consequently, environmental DNA (eDNA) analysis is enabling ecosystem-scale biodiversity assessment, yet the appropriate spatio-temporal resolution of robust biodiversity assessment remains ambiguous. Here, using intensive, spatio-temporal eDNA sampling across space (five rivers in Europe and North America, with an upper range of 20–35 km between samples), time (19 timepoints between 2017 and 2018) and environmental conditions (river flow, pH, conductivity, temperature and rainfall), we characterise the resolution at which information on diversity across the animal kingdom can be gathered from rivers using eDNA. In space, beta diversity was mainly dictated by turnover, on a scale of tens of kilometres, highlighting that diversity measures are not confounded by eDNA from upstream. Fish communities showed nested assemblages along some rivers, coinciding with habitat use. Across time, seasonal life history events, including salmon and eel migration, were detected. Finally, effects of environmental conditions were taxon-specific, reflecting habitat filtering of communities rather than effects on DNA molecules. We conclude that riverine eDNA metabarcoding can measure biodiversity at spatio-temporal scales relevant to species and community ecology, demonstrating its utility in delivering insights into river community ecology during a time of environmental change. Using intensive eDNA sampling in space and time across five rivers in Europe and North America, this study shows that eDNA gives relevant information on freshwater diversity and ecology across broad taxonomic groups, and with limited downstream transport. The findings demonstrate that eDNA is vital for freshwater biodiversity monitoring in a time of anthropogenic change.

DNA barcoding is critical to conservation and biodiversity research, yet public reference databases are incomplete. Existing barcode databases are biased toward cytochrome oxidase subunit I (COI) and frequently lack associated voucher specimens or geospatial metadata, which can hinder reliable species assignments. The emergence of metabarcoding approaches such as environmental DNA (eDNA) has necessitated multiple marker techniques combined with barcode reference databases backed by voucher specimens. Reference barcodes have traditionally been generated by Sanger sequencing, however sequencing multiple markers is costly for large numbers of specimens, requires multiple separate PCR reactions, and limits resulting sequences to targeted regions. High-throughput sequencing techniques such as genome skimming enable assembly of complete mitogenomes, which contain the most commonly used barcoding loci ( e.g., COI, 12S, 16S), as well as nuclear ribosomal repeat regions ( e.g., ITS1&2, 18S). We evaluated the feasibility of genome skimming to generate barcode references databases for marine fishes by assembling complete mitogenomes and nuclear ribosomal repeats. We tested genome skimming across a taxonomically diverse selection of 12 marine fish species from the collections of the National Museum of Natural History, Smithsonian Institution. We generated two sequencing libraries per species to test the impact of shearing method (enzymatic or mechanical), extraction method (kit-based or automated), and input DNA concentration. We produced complete mitogenomes for all non-chondrichthyans (11/12 species) and assembled nuclear ribosomal repeats (18S-ITS1-5.8S-ITS2-28S) for all taxa. The quality and completeness of mitogenome assemblies was not impacted by shearing method, extraction method or input DNA concentration. Our results reaffirm that genome skimming is an efficient and (at scale) cost-effective method to generate all mitochondrial and common nuclear DNA barcoding loci for multiple species simultaneously, which has great potential to scale for future projects and facilitate completing barcode reference databases for marine fishes.

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.

The challenge of sequencing ancient DNA has led to the development of specialized laboratory protocols that have focused on reducing contamination and maximizing the number of molecules that are extracted from ancient remains. Despite the fact that success in ancient DNA studies is typically obtained by screening many samples to identify a promising subset, ancient DNA protocols have not, in general, focused on reducing the time required to screen samples. We present an adaptation of a popular ancient library preparation method that makes screening more efficient. First, the DNA extract is treated using a protocol that causes characteristic ancient DNA damage to be restricted to the terminal nucleotides, while nearly eliminating it in the interior of the DNA molecules, allowing a single library to be used both to test for ancient DNA authenticity and to carry out population genetic analysis. Second, the DNA molecules are ligated to a unique pair of barcodes, which eliminates undetected cross-contamination from this step onwards. Third, the barcoded library molecules include incomplete adapters of short length that can increase the specificity of hybridization-based genomic target enrichment. The adapters are completed just before sequencing, so the same DNA library can be used in multiple experiments, and the sequences distinguished. We demonstrate this protocol on 60 ancient human samples.

Increasing fishing effort, including bycatch and discard practices, are impacting marine biodiversity, particularly among slow-to-reproduce taxa such as elasmobranchs, and specifically sharks. While some fisheries involving sharks are sustainably managed, collateral mortalities continue, contributing towards > 35% of species being threatened with extinction. To effectively manage shark stocks, life-history information, including resource use and feeding ecologies is pivotal, especially among those species with wide-ranging distributions. Two cosmopolitan sharks bycaught off eastern Australia are the common blacktip shark ( Carcharhinus limbatus ; globally classified as Near Threatened) and great hammerhead ( Sphyrna mokarran ; Critically Endangered). We opportunistically sampled the digestive tracts of these two species (and also any whole prey; termed the ‘Russian-doll’ approach), caught in bather-protection gillnets off northern New South Wales, to investigate the capacity for DNA metabarcoding to simultaneously determine predator and prey regional feeding ecologies. While sample sizes were small, S. mokkaran fed predominantly on stingrays and skates (Myliobatiformes and Rajiformes), but also teleosts, while C. limbatus mostly consumed teleosts. Metabarcoding assays showed extensive intermixing of taxa from the digestive tracts of predators and their whole prey, likely via the predator’s stomach chyme, negating the opportunity to distinguish between primary and secondary predation. This Russian-doll effect requires further investigation in DNA metabarcoding studies focussing on dietary preferences and implies that any outcomes will need to be interpreted concomitant with traditional visual approaches.

Biodiversity is an important parameter for the evaluation of the extant environmental conditions. Here, we used environmental DNA (eDNA) metabarcoding to investigate fish biodiversity in five different estuaries in Japan. Water samples for eDNA were collected from river mouths and adjacent coastal areas of two estuaries with high degrees of development (the Tama and Miya Rivers) and three estuaries with relatively low degrees of development (the Aka, Takatsu, and Sendai Rivers). A total of 182 fish species across 67 families were detected. Among them, 11 species occurred in all the rivers studied. Rare fishes including endangered species were successfully detected in rich natural rivers. Biodiversity was the highest in the Sendai River and lowest in the Tama River, reflecting the degree of human development along each river. Even though nutrient concentration was low in both the Aka and Sendai Rivers, the latter exhibited greater diversity, including many tropical or subtropical species, owing to its more southern location. Species composition detected by eDNA varied among rivers, reflecting the distribution and migration of fishes. Our results are in accordance with the ecology of each fish species and environmental conditions of each river.

Interception of potential invasive species at ports-of-entry is essential for effective biosecurity and biosurveillance programs. However, taxonomic assessment of the immature stages of most arthropods is challenging; characters for identification are often dependent on adult morphology and reproductive structures. This study aims to strengthen the identification of such specimens through DNA barcoding, with a focus on microlepidoptera. A sample of 241 primarily immature microlepidoptera specimens intercepted at U.S. ports-of-entry from 2007 to 2011 were selected for analysis. From this sample, 201 COI-5P sequences were generated and analyzed for concordance between morphology-based and DNA-based identifications. The retrospective analysis of the data over 10 years (2009 to 2019) using the Barcode of Life Data (BOLD) system demonstrates the importance of establishing and growing DNA barcode reference libraries for use in specimen identification. Additionally, analysis of specimen identification using public data (43.3% specimens identified) vs. non-public data (78.6% specimens identified) highlights the need to encourage researchers to make data publicly accessible. DNA barcoding surpassed morphological identification with 42.3% (public) and 66.7% (non-public) of the sampled specimens achieving a species-level identification, compared to 38.3% species-level identification by morphology. Whilst DNA barcoding was not able to identify all specimens in our dataset, its incorporation into border security programs as an adjunct to morphological identification can provide secondary lines of evidence and lower taxonomic resolution in many cases. Furthermore, with increased globalization, database records need to be clearly annotated for suspected specimen origin versus interception location.

Traditional methods of forage identification are impractical with non-leafcutting fungus gardening ants, making diet-related ecological and life history questions difficult to study. To address this limitation, we utilized dietary DNA metabarcoding on excavated ant fungus gardens to generate forage diversity metrics for the two co-occurring species Trachymyrmex septentrionalis and Mycetomoellerius turrifex. Ten fungus garden samples from each species were collected from a 60x70 m plot in East Texas. Each of the colonies we sampled was paired with a colony from the other species within 3 m of it. Plant forage diversity was assessed with chloroplast trnL primers, and insect frass forage diversity was assessed with mitochondria COI primers. DNA metabarcoding identified a total of 44 plant taxa across all samples, but performed poorly when characterizing foraged insect frass. Plant beta diversity was significantly different between the gardens of T. septentrionalis and M. turrifex colonies, as well as paired colonies. Colony pairs also had significantly different plant alpha diversity. This indicates that diet preference is likely driven both by ant species-specific plant preference, and colony location-specific plant resource availability. Overall, our results show that dietary DNA techniques are a promising tool for the identification of plant forage in ant fungus gardens, enabling the study of future diet-based ecological and natural history questions.