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DNA metabarcoding is used to generate species composition data for entire communities. However, sequencing errors in high-throughput sequencing instruments are fairly common, usually requiring reads to be clustered into operational taxonomic units (OTUs), losing information on intraspecific diversity in the process. While Cytochrome c oxidase subunit I (COI) haplotype information is limited in resolving intraspecific diversity it is nevertheless often useful e.g. in a phylogeographic context, helping to formulate hypotheses on taxon distribution and dispersal. This study combines sequence denoising strategies, normally applied in microbial research, with additional abundance-based filtering to extract haplotype information from freshwater macroinvertebrate metabarcoding datasets. This novel approach was added to the R package \"JAMP\" and can be applied to COI amplicon datasets. We tested our haplotyping method by sequencing (i) a single-species mock community composed of 31 individuals with 15 different haplotypes spanning three orders of magnitude in biomass and (ii) 18 monitoring samples each amplified with four different primer sets and two PCR replicates. We detected all 15 haplotypes of the single specimens in the mock community with relaxed filtering and denoising settings. However, up to 480 additional unexpected haplotypes remained in both replicates. Rigorous filtering removes most unexpected haplotypes, but also can discard expected haplotypes mainly from the small specimens. In the monitoring samples, the different primer sets detected 177-200 OTUs, each containing an average of 2.40-3.30 haplotypes per OTU. The derived intraspecific diversity data showed population structures that were consistent between replicates and similar between primer pairs but resolution depended on the primer length. A closer look at abundant taxa in the dataset revealed various population genetic patterns, e.g. the stonefly and the caddisfly showed a distinct north-south cline with respect to haplotype distribution, while the beetle and the isopod displayed no clear population pattern but differed in genetic diversity. We developed a strategy to infer intraspecific genetic diversity from bulk invertebrate metabarcoding data. It needs to be stressed that at this point this metabarcoding-informed haplotyping is not capable of capturing the full diversity present in such samples, due to variation in specimen size, primer bias and loss of sequence variants with low abundance. Nevertheless, for a high number of species intraspecific diversity was recovered, identifying potentially isolated populations and taxa for further more detailed phylogeographic investigation. While we are currently lacking large-scale metabarcoding datasets to fully take advantage of our new approach, metabarcoding-informed haplotyping holds great promise for biomonitoring efforts that not only seek information about species diversity but also underlying genetic diversity.

Metabarcoding can rapidly determine the species composition of bulk samples and thus aids biodiversity and ecosystem assessment. However, it is essential to use primer sets that minimize amplification bias among taxa to maximize species recovery. Despite this fact, the performance of primer sets employed for metabarcoding terrestrial arthropods has not been sufficiently evaluated. This study tests the performance of 36 primer sets on a mock community containing 374 insect species. Amplification success was assessed with gradient PCRs and the 21 most promising primer sets selected for metabarcoding. These 21 primer sets were also tested by metabarcoding a Malaise trap sample. We identified eight primer sets, mainly those including inosine and/or high degeneracy, that recovered more than 95% of the species in the mock community. Results from the Malaise trap sample were congruent with the mock community, but primer sets generating short amplicons produced potential false positives. Taxon recovery from both mock community and Malaise trap sample metabarcoding were used to select four primer sets for additional evaluation at different annealing temperatures (40–60 °C) using the mock community. The effect of temperature varied by primer pair but overall it only had a minor effect on taxon recovery. This study reveals the weak performance of some primer sets employed in past studies. It also demonstrates that certain primer sets can recover most taxa in a diverse species assemblage. Thus, based our experimental set up, there is no need to employ several primer sets targeting the same gene region. We identify several suitable primer sets for arthropod metabarcoding, and specifically recommend BF3 + BR2, as it is not affected by primer slippage and provides maximal taxonomic resolution. The fwhF2 + fwhR2n primer set amplifies a shorter fragment and is therefore ideal when targeting degraded DNA (e.g., from gut contents).

During the last years DNA barcoding has become a popular method of choice for molecular specimen identification. Here we present a comprehensive DNA barcode library of various crustacean taxa found in the North Sea, one of the most extensively studied marine regions of the world. Our data set includes 1,332 barcodes covering 205 species, including taxa of the Amphipoda, Copepoda, Decapoda, Isopoda, Thecostraca, and others. This dataset represents the most extensive DNA barcode library of the Crustacea in terms of species number to date. By using the Barcode of Life Data Systems (BOLD), unique BINs were identified for 198 (96.6%) of the analyzed species. Six species were characterized by two BINs (2.9%), and three BINs were found for the amphipod species Gammarus salinus Spooner, 1947 (0.4%). Intraspecific distances with values higher than 2.2% were revealed for 13 species (6.3%). Exceptionally high distances of up to 14.87% between two distinct but monophyletic clusters were found for the parasitic copepod Caligus elongatus Nordmann, 1832, supporting the results of previous studies that indicated the existence of an overlooked sea louse species. In contrast to these high distances, haplotype-sharing was observed for two decapod spider crab species, Macropodia parva Van Noort & Adema, 1985 and Macropodia rostrata (Linnaeus, 1761), underlining the need for a taxonomic revision of both species. Summarizing the results, our study confirms the application of DNA barcodes as highly effective identification system for the analyzed marine crustaceans of the North Sea and represents an important milestone for modern biodiversity assessment studies using barcode sequences.

Sustainably feeding the next generation is often described as one of the most pressing \"grand challenges\" facing the 21st century. Generally, scholars propose addressing this problem by increasing agricultural production, investing in technology to boost yields, changing diets, or reducing food waste. In this paper, we explore whether global food production is nutritionally balanced by comparing the diet that nutritionists recommend versus global agricultural production statistics. Results show that the global agricultural system currently overproduces grains, fats, and sugars while production of fruits and vegetables and protein is not sufficient to meet the nutritional needs of the current population. Correcting this imbalance could reduce the amount of arable land used by agriculture by 51 million ha globally but would increase total land used for agriculture by 407 million ha and increase greenhouse gas emissions. For a growing population, our calculations suggest that the only way to eat a nutritionally balanced diet, save land and reduce greenhouse gas emissions is to consume and produce more fruits and vegetables as well as transition to diets higher in plant-based protein. Such a move will help protect habitats and help meet the Sustainable Development Goals.

Background Advancing our knowledge of vector species genomes is a key step in our battle against the spread of diseases. Biting midges of the genus Culicoides are vectors of arboviruses that significantly affect livestock worldwide. Culicoides stellifer is a suspected vector with a wide range distribution in North America, for which cryptic diversity has been described. Results With just one specimen of C. stellifer , we assembled and annotated the nuclear and mitochondrial genome using the ultra-low input DNA PacBio protocol. The genome assembly is 119 Mb in length with a contig N50 value of 479.3 kb, contains 11% repeat sequences and 18,895 annotated protein-coding genes. To further elucidate the role of this species as a vector, we provide genomic evidence of a non-retroviral endogenous viral element integrated into the genome that corresponds to rhabdovirus nucleocapsid proteins, the same family as the vesicular stomatitis virus. Conclusions This genomic information will pave the way for future investigations into this species’s putative vector role. We also demonstrate the practicability of completing genomic studies in small dipterans using single specimens preserved in ethanol as well as introduce a workflow for data analysis that considers the challenges of insect genome assembly.

Continuously increasing demand for plant and animal products causes unsustainable depletion of biological resources. It is estimated that one-quarter of sharks and rays are threatened worldwide and although the global fin trade is widely recognized as a major driver, demand for meat, liver oil, and gill plates also represents a significant threat. This study used DNA barcoding and 16 S rRNA sequencing as a method to identify shark and ray species from dried fins and gill plates, obtained in Canada, China, and Sri Lanka. 129 fins and gill plates were analysed and searches on BOLD produced matches to 20 species of sharks and five species of rays or – in two cases – to a species pair. Twelve of the species found are listed or have been approved for listing in 2017 in the appendices of the Convention on International Trade in Endangered Species of Fauna and Flora (CITES), including the whale shark ( Rhincodon typus ), which was surprisingly found among both shark fin and gill plate samples. More than half of identified species fall under the IUCN Red List categories ‘Endangered’ and ‘Vulnerable’, raising further concerns about the impacts of this trade on the sustainability of these low productivity species.

The Coral Triangle (CT), a region spanning across Indonesia and Philippines, is home to about 4,350 marine fish species and is among the world's most emblematic regions in terms of conservation. Threatened by overfishing and oceans warming, the CT fisheries have faced drastic declines over the last decades. Usually monitored through a biomass‐based approach, fisheries trends have rarely been characterized at the species level due to the high number of taxa involved and the difficulty to accurately and routinely identify individuals to the species level. Biomass, however, is a poor proxy of species richness, and automated methods of species identification are required to move beyond biomass‐based approaches. Recent meta‐analyses have demonstrated that species richness peaks at intermediary levels of biomass. Consequently, preserving biomass is not equal to preserving biodiversity. We present the results of a survey to estimate the shore fish diversity retailed at the harbor of Ambon Island, an island located at the center of the CT that display exceptionally high biomass despite high levels of threat, while building a DNA barcode reference library of CT shore fishes targeted by artisanal fisheries. We sampled 1,187 specimens and successfully barcoded 696 of the 760 selected specimens that represent 202 species. Our results show that DNA barcodes were effective in capturing species boundaries for 96% of the species examined, which opens new perspectives for the routine monitoring of the CT fisheries. The Coral Triangle is home of the highest marine biodiversity. Artisanal fisheries are extremely important in the Coral Triangle. Fish species are poorly known and hardly identifiable. We propose a DNA barcode reference library for the fish harvested by the artisanal fisheries in Ambon Island.

Background: Trade in ornamental fishes represents, by far, the largest route for the importation of exotic vertebrates. There is growing pressure to regulate this trade with the goal of ensuring that species are sustainably harvested and that their point of origin is accurately reported. One important element of such regulation involves easy access to specimen identifications, a task that is currently difficult for all but specialists because of the large number of species involved. The present study represents an important first step in making identifications more accessible by assembling a DNA barcode reference sequence library for nearly half of the ornamental fish species imported into North America. Methodology/Principal Findings: Analysis of the cytochrome c oxidase subunit I (COI) gene from 391 species from 8 coral reef locations revealed that 98% of these species exhibit distinct barcode clusters, allowing their unambiguous identification. Most species showed little intra-specific variation (adjusted mean = 0.21%), but nine species included two or three lineages showing much more divergence (2.19–6.52%) and likely represent overlooked species complexes. By contrast, three genera contained a species pair or triad that lacked barcode divergence, cases that may reflect hybridization, young taxa or taxonomic over-splitting. Conclusions/Significance: Although incomplete, this barcode library already provides a new species identification tool for the ornamental fish industry, opening a realm of applications linked to collection practices, regulatory control and conservation.

Taxonomic and distributional information on each fish species found in arctic marine waters is reviewed, and a list of families and species with commentary on distributional records is presented. The list incorporates results from examination of museum collections of arctic marine fishes dating back to the 1830s. It also incorporates results from DNA barcoding, used to complement morphological characters in evaluating problematic taxa and to assist in identification of specimens collected in recent expeditions. Barcoding results are depicted in a neighbor-joining tree of 880 CO1 (cytochrome c oxidase 1 gene) sequences distributed among 165 species from the arctic region and adjacent waters, and discussed in the family reviews. Using our definition of the arctic region, we count 242 species with documented presence, if 12 species that likely are synonyms are excluded. The 242 species are distributed among 45 families. Six families in Cottoidei with 72 species and five in Zoarcoidei with 55 species account for more than half (52.5%) the species. This study produced CO1 sequences for 106 of the 242 species. Sequence variability in the barcode region permits discrimination of all species. The average sequence variation within species was 0.3% (range 0–3.5%), while the average genetic distance between congeners was 4.7% (range 3.7–13.3%). The CO1 sequences support taxonomic separation of some species, such as Osmerus dentex and O. mordax and Liparis bathyarcticus and L. gibbus; and synonymy of others, like Myoxocephalus verrucosus in M. scorpius and Gymnelus knipowitschi in G. hemifasciatus . They sometimes revealed the presence of additional species that were not entirely expected, such as an unidentified species of Ammodytes in the western Gulf of Alaska, most likely A. personatus; and an unidentified Icelus species of the I. spatula complex with populations in the western Gulf of Alaska and the northern Bering and Chukchi Seas which could be a new species or a species in synonymy. Reviewing distribution, we found that for 24 species the patterns assigned by authors understated historical presence in the arctic region, and for 12 species they overstated presence. For instance, Hippoglossoides robustus is counted as an arctic–boreal species rather than predominantly boreal, and Artediellus uncinatus as predominantly arctic rather than predominantly boreal. Species with arctic, predominantly arctic, or arctic–boreal distributions composed 41% of the 242 species in the region, and predominantly boreal, boreal, and widely distributed species composed 59%. For some continental shelf species, such as the primarily amphiboreal Eumesogrammus praecisus and Leptoclinus maculatus, distributions appear to reflect changes, including reentry into Arctic seas and reestablishment of continuous ranges, that zoogeographers believe have been going on since the end of land bridge and glacial times.

Environmental DNA (eDNA) metabarcoding is widely used to detect animals from environmental samples and on the brink of being implemented into routine species monitoring. Surprisingly, birds are among the taxonomic groups which have received comparably little attention in eDNA research regarding primer optimization—particularly reducing the amplification of non‐target taxa, the availability of appropriate reference sequence databases for the targeted fragments, and the evaluation of different filter types for their capability to detect avian eDNA from water samples. Here, we present a novel primer pair (BirT) for metabarcoding avian eDNA. We optimized specificity and fragment length with regard to taxonomic resolution and available sequencing platforms. Additionally, we evaluated the availability of 12S rRNA gene reference sequences for birds and filled database gaps by generating novel barcodes. Finally, we tested the applicability of the BirT primer pair using field‐collected eDNA samples obtained with three different filter types and compared the eDNA metabarcoding results to visual observations uploaded to eBird ( www.eBird.org ). Our results confirm the suitability of the BirT primer pair for avian eDNA metabarcoding with no amplification of key non‐target groups and improved taxonomic resolution. Albeit there are still substantial gaps in the 12S reference sequence database, the analysis of bird eDNA from water samples resulted in species‐level taxonomic resolution for 91% of the detected taxa. All tested filters delivered similar results for total read numbers per sample (mean: 623,990 ± 331,236 SD) and species detected per sample (mean: 5.0 ± 2.0 SD). Ninety‐five percent of the bird detections were highly plausible based on eBird data collected at the time of eDNA sampling and 64% were directly confirmed by visual observations. Most detected species were closely associated with aquatic habitats confirming the suitability of water samples for the detection of waterfowl and species inhabiting similar ecological niches via eDNA.