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.
Background: Barcode coverage is difficult to assess for large regions due to incomplete species lists, inaccurate identifications, and cryptic diversity. However, as coverage approaches completion, it becomes possible to critically evaluate identifications and validate barcode lineages. We collate the results of the FISH-BOL barcode project and assess coverage for each family of bony shorefishes and reef fishes from the tropical western Atlantic Ocean. Methodology: We identify to species the public and private barcode lineages from the region on BOLD, confirming identifications by vouchers, phylogeographic deduction, and the process of elimination. The lineages and BINs are assigned to species from a comprehensive species list for the region. Results: We estimate 1029 of 1311 total bony shorefish species in the region are barcoded (78.5%). For reef-associated fishes, 902 of 1083 species are barcoded (83.3%). About 70 of the 181 species not yet barcoded are endemic species from Florida/ Gulf of Mexico or Venezuela, leaving about 90% of the central Caribbean reef fish species barcoded to date. Most species are represented by one barcode lineage, but among the gobioids and blennioids there are many more lineages (BINs) than species, indicating substantial cryptic diversity. Conclusions: As barcode coverage for a region approaches completion, a robust assessment of coverage can be made. The reef fish fauna of the tropical western Atlantic now has the highest coverage for a large marine area, from about 80 to 90% depending on definitions and geographic limits.
AbstractAnostomidae is a widely distributed group that includes the species rich Leporinus. This genus is widespread in South America, including its south portion where they are mainly distributed in the São Francisco and La Plata basins. Here we obtained sequences of mitochondrial and nuclear genes of 19 species of Leporinus with the objective of testing our methods of molecular species identification and to test the similarities between species of Leporinus of the São Francisco and La Plata basins. Results show that two species pair have a genetic distance below 2% (L. paranensis x L. obtusidens and L. paranensis x L. aff. paranensis). Our results also show that a high genetic distance occurs in L. obtusidens and L. striatus suggesting a possible local differentiation of these species. An interesting finding of our study is that the genetic similarities observed among species does not corroborate the division of species of Leporinus in groups according to color patterns. Additionally, our results show that the species of Leporinus from the La Plata and São Francisco are not more similar among themselves than to species of other drainages which could be explained by the occurrence of recent fauna exchanges between these two basins and adjacent basins.
AbstractDNA 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.
Species identification is a difficult task, ranging from the definition of the species concept itself to the definition of the threshold for speciation. DNA Barcode technology uses a fragment of the Cytochrome Oxidase I (COI) gene as a molecular tool that many studies have already validated as a tool for species identification. DNA barcode sequences for COI were generated and analyzed from 805 specimens. The General Mixed Yule Coalescent (GMYC) analysis recognized 99 independent evolution units, and the Barcode Index Numbers (BIN) approach pointed to the existence of 104 BINs (interpreted as distinct species). By cross-tabulating the results of all approaches, we identified 109 Molecular Operational Taxonomic Units (MOTU) by at least one methodology. In most cases (89 MOTUs), the genetic approaches are in agreement with morphological identification, and the discrepant results of MOTUs are in the complex groups, which have many morphological similarities but may represent species complexes.
AbstractSeafood authenticity is a global concern. As seafood consumption increases, so does public awareness of the associated nutritional and environmental issues related to seafood mislabeling. Cases of substitution continue to be observed, even after the adoption of DNA barcoding as a regulatory tool by the Food and Drug Administration in the United States in 2011. Although media coverage of these cases has highlighted the incidence of fraud in Canada, more in-depth engagement of the public is lacking. By partnering with community members to conduct research, knowledge about the incidence and impact of seafood mislabeling can be directly communicated to consumers. In this study high school students and educators participated in a market survey using DNA barcoding to identify seafood. The Canadian Food Inspection Agency Fish List was used to determine if mislabeling had occurred. Twenty-three percent of samples surveyed were mislabeled, suggesting that the incidence of retail seafood mislabeling continues to be significant in Canada. Continued involvement of the public in market surveys will help to monitor trends in seafood mislabeling, and may help to increase awareness of potential seafood fraud.
A record of a juvenile Porbeagle, Lamna nasus, in the coastal waters of Argentina is confirmed by DNA barcoding, since the specimen lacked some external diagnostic features. We discuss the occurrence of this species in the coastal waters of Argentina, its molecular affinity with conspecifics from a broad range of the species, and relevant conservation issues.
AbstractThe 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
AbstractOne of the problems that arose from the meetings of the Barcode in Mexico project was the urgency of having a method in which Mexican authorities could trust for detecting shark finning. This study examined DNA barcoding as a method to identify 14 dried shark fins confiscated by the Mexican Government in two exportation shipments in Mazatlán and Manzanillo ports. Fins were DNA barcoded using the COI mitochondrial gene and provided matching sequences of six species: Prionace glauca, Carcharhinus falciformis, Carcharhinus limbatus, Alopias pelagicus, Mustelus henlei and Rhizoprionodon longurio. There is no information on DNA barcoding sharkfin trade in the Mexican Pacific and this is the first group effort with Mexican Government Agencies for the conservation of sharks.
AbstractDNA 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.
AbstractEstimating appropriate sample sizes to measure species abundance and richness is a fundamental problem for most biodiversity research. In this study, we explore a method to measure sampling sufficiency based on haplotype diversity in the ray-finned fishes (Animalia: Chordata: Actinopterygii). To do this, we use linear regression and hypothesis testing methods on haplotype accumulation curves from DNA barcodes for 18 species of fishes, in the statistics platform R. We use a simple mathematical model to estimate sampling sufficiency from a sample-number based prediction of intraspecific haplotype diversity, given an assumption of equal haplotype frequencies. Our model finds that haplotype diversity for most of the 18 fish species remains largely unsampled, and this appears to be a result of small sample sizes. Lastly, we discuss how our overly simple model may be a useful starting point to develop future estimators for intraspecific sampling sufficiency in studies using DNA barcodes.
AbstractIn Japan, overgrazing by sika deer (Cervus nippon) has been suggested to cause a decline in forest understory vegetation. DNA barcoding has become an accepted method for analyzing the diets of animals and may be useful for evaluating the impact of sika deer on vegetation. However, the applicability of DNA barcoding in the dietary analysis of sika deer, particularly whether all of the food plants can be detected with sufficient taxonomic resolution and whether the results can be evaluated quantitatively, has not been investigated. We conducted a feeding trial by feeding five plant species to a captive sika deer and sequenced the chloroplast trnL P6 loop region from the sika deer’s fecal DNA using the Ion PGM sequencer. We detected the sequences of all of the food plants at the species level using the local (selfproduced) database and at the genus or family level with the global database. Although the sequences of some major food plants were detected with high frequency, the proportion of consumed food plants did not match the proportion of sequences obtained from fecal DNA. With further technical advances and the further completeness of the sequence database for vegetation, DNA barcoding will be a useful tool for the dietary study of sika deer.
AbstractWe used the DNA barcoding technique (COI and CytB markers) combined with GMYC analysis to characterize the genetics of the widely distributed Neotropical fish species Knodus moenkhausii from three different isolated hydrographic basins. Despite the fact that most of the Neotropical hydrographic basins have been isolated for millions of years, species could be shared between basins due to natural events (stream capture) or anthropogenic activities. Recent surveys, however, have shown that many widely distributed species are actually species complexes divided into previously unrecognized cryptic species. In this study, we tested the hypothesis that K. moenkhausii from three hydrographic basins represent a single panmictic species and discuss the most likely explanation of its present geographical distribution. The GMYC analysis revealed that all specimens of K. moenkhausii represent a single species: the intra- and intergroup minimum K2P genetic distances for both genes were zero and haplotypes were shared among the three hydrographic basins. This suggests there has been recent interchange of K. moenkhausii throughout the three hydrographic basins. It is likely that this is due to recent human activities, either the transposition of natural barriers or intentional introduction or accidental escape due to the ornamental fish trade.
AbstractDNA 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.
With 1172 native species, the Indonesian ichthyofauna is among the world's most speciose. Despite that the inventory of the Indonesian ichthyofauna started during the eighteen century, the numerous species descriptions during the last decades highlight that the taxonomic knowledge is still fragmentary. Meanwhile, the fast increase of anthropogenic perturbations during
: DNA barcoding is a system designed to provide species identification by using standardized gene regions as internal species tag. Foreseen since its earlier development as a solution to speed up the pace of species discovery, DNA barcoding has established as a mature field of biodiversity sciences filing the conceptual gap between traditional taxonomy and different fields of molecular systematics. Initially proposed as a tool for species identification, DNA barcoding has also been applied in taxonomy routines for automated species delineation. Species identification and species delineation, however, should be considered as distinct activities relying on different theoretical and methodological backgrounds. The aim of the present review is to provide an overview of the use of DNA sequences in taxonomy, since the earliest development of molecular taxonomy until the development of DNA barcoding. We further present the differences between procedures of species identification and species delineation and highlight how DNA barcoding proposed a new paradigm that helps promote more sustainable practices in taxonomy.
AbstractThe 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.
AbstractThis 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
AbstractIn the present study, we compare the results of alpha taxonomy (based on morphology), DNA Barcoding method with a 2% genetic divergence threshold, and the GMYC (General Model Yule Coalescent) technique to identify species clusters in Neoplecostomus. We used partial sequences of mitochondrial COI (Cytochrome oxidase subunit I) for 59 specimens representing 13 valid species (Neoplecostomus bandeirante, N. jaguari, N. langeanii, N. paranensis, N. yapo, N. botucatu, N. selenae, N. doceensis, N. corumba, N. ribeirensis, N. microps, N. espiritosantensis and N. franciscoensis) of Neoplecostomus collected in all its distribution area. For the analysis we used Bayesian inference of phylogeny with relaxed clock methods on an arbitrary timescale, using BEAST. The ultrametrics genes trees obtained for each tree prior evaluated (Yule, Birth-death and coalescent Population Size) were used in the GMYC analysis to identify a time in the tree when the branching rate shifts (in forward time) from Yule and Birth-Death (species) to a coalescent (population) process. We found that the GMYC model using the Yule prior identified 11 groups, closer to the current taxonomy (13 species). GMYC analyses using other ultrametric gene trees estimated under alternative prior such as Birth-Death and Coalescent Population Size, identified 9 groups, results observed in the traditional 2% genetic distance threshold, resulting in a low number of species recognized compared to the number of species identified with current taxonomy. Based on these results we conclude that the molecular approaches are helpful to distinguish species of Neoplecostomus, nevertheless it is important to combine molecular methodologies with current taxonomy in order to identify correctly species that recently originated.
AbstractAmong 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
