Ground-truthing Phylotype Assignments for Antarctic Invertebrates

Biodiversity information from Antarctic terrestrial habitats helps conservation efforts, but the distribution and diversity particularly of microinvertebrates remains poorly understood. Springtails, mites, tardigrades, nematodes and rotifers are difficult to identify using morphological features, hence DNA-based metabarcoding methods are well suited for their study. We compared taxonomy assignments of a high throughput sequencing metabarcoding approach using one ribosomal DNA (18S rDNA) and one mitochondrial DNA (cytochrome c oxidase subunit I - COI) marker with morphological reference data. Specifically, we compared metabarcoding or morphological taxonomic assignments on multiple taxonomic levels in an artificial DNA blend containing Australian invertebrates, and in seven extracts of Antarctic soils containing known micro-faunal taxa. Avoiding arbitrary application of metabarcoding analysis parameters, we calibrated those parameters with metabarcoding data from non-Antarctic soils. Metabarcoding approaches employing 18S rDNA and COI markers enabled detection of small and cryptic Antarctic invertebrates, and on low taxonomic ranks 18S data outperformed COI data in this respect. Morphological taxonomy determination did not outperform metabarcoding approaches. Our study demonstrates how barcoding markers can be tested prior to their application to specific taxonomic groups, and that taxonomy fidelity of markers needs to be validated in relation to environment, taxa, and available reference information.

Sequencing of COI gene in four rodent pests for species identification

Among mammals, rodents are the most important group since they influence the crop production and protection. Species identification of rodents on morphometric parameters is cumbersome and often misleading. However, DNA based identification would enable accurate species identification. For this purpose, we examined 650 bp of Cytochrome c oxidase subunit1 (COI) in four rodent pest species namely

Barcoding, phylogeography and species boundaries in clownfishes of the Indian Ocean

In this study, barcoding of 13 clownfish species of the Indian Ocean was carried out to infer the phylogenetic relationships among them by analyzing cytochrome oxidase 1 (CO1) and cytochrome

Estimating the effectiveness of species identification by sequencing of two chloroplast DNA loci (matK and rbcL) in selected groups of Polish flora

This paper focuses on assessing the effectiveness of species identification using barcoding standard loci (matK and rbcL) for a selected group of Polish flora. 55 samples of 17 species from six taxonomic groups representing various parts of the system, including Monocots and Dicots as well as Pteridophytes, were selected. PCR amplification success was 100% (of all samples) for the rbcL primer and 64% for matK primers giving in sum 87 fragments for sequencing. The aligned sequences show that in the

Extending DNA barcoding coverage for Lake Whitefish (Coregonus clupeaformis) across the three major basins of Lake Huron

DNA barcoding is a useful tool for both species identification and discovery, but the latter requires denser sampling than typically used in barcode studies. Lake Whitefish (Coregonus clupeaformis) is a valuable species, fished traditionally, commercially, and recreationally in Lake Huron. Based on the natural geographic and bathymetric separation of the three major basins in Lake Huron, the potential separation of Lake Whitefish within these basins, and the variation among life history (early and late spawning), we predicted that Lake Huron might harbour cryptic lineages of Lake Whitefish at the basin level. To test this prediction, DNA barcodes of the mitochondrial 5’ cytochrome c oxidase subunit I (COI) gene sequences were recovered from spawning phase Lake Whitefish (n = 5 per site), which were collected from sites (n = 28) around Lake Huron during Fall 2012. These sequences, combined with other publically available DNA barcodes from the Barcode of Life Data System (BOLD), revealed twelve unique haplotypes across North America, with seven unique to Lake Huron. The dominant haplotype was found throughout Lake Huron and east to the St. Lawrence River. No deep divergences were revealed. This comprehensive lake-wide sampling effort offers a new perspective on C. clupeaformis, and can provide insight for environmental assessments and fisheries management.

DNA barcoding increases resolution and changes structure in Canadian boreal shield lake food webs

Food webs are important in understanding the structure, function, and behaviour of ecosystems, but, due to methodological limitations, are often poorly resolved in ways that impact food-web properties. Although DNA barcoding has proven useful in determining the diet of consumers, few studies have used this technique to determine food-web structure. These studies report mixed impacts on various food-web properties, but are limited by their taxonomic focus and their failure to evaluate DNA barcoding for both diet analysis and food-web structure. In this study, we show that, when compared to a morphological approach, DNA barcoding increases foodweb resolution by increasing the number and frequency of prey species identified in the stomach contents of eight species of Canadian boreal shield predatory fishes. In addition, we observed differences in food-web structure, such as increased generalism, habitat coupling, and omnivory, that have strong implications for food-web stability and dynamics. We conclude that DNA barcoding is a powerful tool to evaluate how resolution impacts foodweb properties and can help further our understanding of how food webs are structured by identifying feeding interactions in an unprecedented and highly detailed manner.

Standards for Conducting a DNA Barcoding Market Survey: Minimum Information and Best Practices

DNA barcoding has been applied as a method to test seafood authenticity in numerous market surveys. This trend is continuing to gain momentum as DNA barcoding is employed as a regulatory tool, by the media, and by students to test seafood products, in addition to its use by scientific researchers to monitor seafood substitution. However, as market surveys documenting mislabeling continue to be published by both the press and scientific journals, there is a need for standardization in practices to aid in comparing and verifying results. This communication provides an overview of best practices for conducting and reporting DNA barcoding market studies for seafood. These standards can also be used as a guideline for other methods for conducting market surveys, or for market surveys employing DNA barcoding of other groups of organisms.

Fishing for barcodes in the Torrent: from COI to complete mitogenomes on NGS platforms

The adoption of Next-Generation Sequencing (NGS) by the field of DNA barcoding of Metazoa has been hindered by the fit between the classical COI barcode and the Sanger-based sequencing method. Here we describe a framework for the sequencing and multiplexing of mitogenomes on NGS platforms that implements (I) a universal long-range PCR-based amplification technique, (II) a two-level multiplexing approach (i.e. divergence-based and specific tag indexing), and (III) a dedicated demultiplexing and assembling script from an Ion Torrent sequencing platform. We provide a case study of mitogenomes obtained for two vouchered individuals of daces Leuciscus burdigalensis and L. oxyrrhis and show that this workflow enables to recover over 100 mitogenomes per sequencing chip on a PGM sequencer, bringing the individual cost down below 7,50€ per mitogenome (as of current 2015 sequencing costs). The use of several kilobases for identification purposes, as involved in the improved DNA-barcode we propose, stress the need for data reliability, especially through metadata. Based on both scientific and economic considerations, this framework presents a relevant approach for multiplexing samples, adaptable on any desktop NGS platform. It enables to extend from the prevalent barcoding approach by shifting from the single COI to complete mitogenome sequencing

Cods for sale Do we know what we are buying?

The Atlantic Cod Gadus morhua is fished in Northern Atlantic and Arctic waters. In Mexico cod is imported from Norway and the United States, and is traditionally eaten at Christmas and Easter. In Mexico City several stores sell dry-salted cod, but due to the high price of the imported Atlantic Cod, other fish species are sold. In this project we examined six samples of dried-salted fish from different stores and used DNA barcoding of the COI gene to corroborate the identity of the product sold as Atlantic Cod. Barcoding revealed that only two of the six samples (33%) were Atlantic cod, with two being Ling, one Alaska Pollock, and one Blue Shark. The high rate of mislabeling (67%) is a major concern. It is especially worrying that Blue Shark is being sold as Atlantic cod, since the Blue Shark is listed as a near-threatened species by the IUCN. We must be aware of the damages that overexploitation and uninformed consumption cause to cod and shark populations. There needs to be stricter policing of seafood product labeling in México.

Adding nuclear rhodopsin data where mitochondrial COI indicates discrepancies – can this marker help to explain conflicts in cyprinids?

DNA barcoding is a fast and reliable tool for species identification, and has been successfully applied to a wide range of freshwater fishes. The limitations reported were mainly attributed to effects of geographic scale, taxon-sampling, incomplete lineage sorting, or mitochondrial introgression. However, the metrics for the success of assigning unknown samples to species or genera also depend on a suited taxonomic framework. A simultaneous use of the mitochondrial COI and the nuclear RHO gene turned out to be advantageous for the barcode efficiency in a few previous studies. Here, we examine 14 cyprinid fish genera, with a total of 74 species, where standard DNA barcoding failed to identify closely related species unambiguously. Eight of the genera (Acanthobrama, Alburnus, Chondrostoma, Gobio, Mirogrex, Phoxinus, Scardinius, and Squalius) contain species that exhibit very low interspecific divergence, or haplotype sharing (12 species pairs) with presumed introgression based on mtCOI data. We aimed to test the utility of the nuclear rhodopsin marker to uncover reasons for the high similarity and haplotype sharing in these different groups. The included labeonine species belonging to Crossocheilus, Hemigrammocapoeta, Tylognathus and Typhlogarra were found to be nested within the genus Garra based on mtCOI. This specific taxonomic uncertainty was also addressed by the use of the additional nuclear marker. As a measure of the delineation success we computed barcode gaps, which were present in 75% of the species based on mtCOI, but in only 39% based on nuclear rhodopsin sequences. Most cases where standard barcodes failed to offer unambiguous species identifications could not be resolved by adding the nuclear marker. However, in the labeonine cyprinids included, nuclear rhodopsin data generally supported the lineages as defined by the mitochondrial marker. This suggests that mitochondrial patterns were not mislead by introgression, but are caused by an inadequate taxonomy. Our findings support the transfer of the studied species of Crossocheilus, Hemigrammocapoeta, Tylognathus and Typhlogarra to Garra.