Molecular identification of three Ompok species using mitochondrial COI gene

A DNA-based barcode identification system that is applicable to all animal species will provide a simple, universal tool for the identification of fish species. The barcode system is based on sequence diversity in subunit 1 cytochrome c oxidase (COI) gene. Identification and characterization of fish species based on morphological characters are sometimes found to be erroneous and environmentally affected. There are no studies on the genus Ompok in India at molecular level and species identification of the Ompok is usually carried out through morphological features. A total of 106 samples from three species Ompok pabda, O. pabo and O. bimaculatus were collected from eight sampling sites of seven Indian rivers. One hundred and six sequences were generated from COI region of three Ompok species and 21 haplotypes were observed. The sequence analysis of COI gene revealed three genetically distinct Ompok species and exhibited identical phylogenetic resolution among them. The partial COI gene sequence can be used as a diagnostic molecular marker for identification and resolution of taxonomic ambiguity of Ompok species.     

Discrimination of Cricotopus species (Diptera: Chironomidae) by DNA barcoding

Chironomids (Diptera) typically comprise the most abundant group of macroinvertebrates collected in water quality surveys. Species in the genus Cricotopus display a wide range of tolerance for manmade pollutants, making them excellent bioindicators. Unfortunately, the usefulness of Cricotopus is overshadowed by the difficulty of accurately identifying larvae using current morphological keys. Molecular approaches are now being used for identification and taxonomic resolution in many animal taxa. In this study, a sequence-based approach for the mitochondrial gene, cytochrome oxidase I (COI), was developed to facilitate identification of Cricotopus species collected from Baltimore area streams. Using unique COI sequence variations, we developed profiles for seven described Cricotopus sp., four described Orthocladius sp., one described Paratrichocladius sp. and one putative species of Cricotopus. In addition to providing an accurate method for identification of Cricotopus, this method will make a useful contribution to the development of keys for Nearctic Cricotopus.     

MOTU analysis of ichthyoplankton biodiversity in the upper Yangtze River,China

Identification of ichthyoplankton is difficult because fish during early life stages often lack stable morphological characteristics; such difficulty in species identification can be a major hindrance in conducting ichthyoplankton surveys for fish biodiversity investigations. Here, we evaluated the feasibility of a molecular operational taxonomic unit (MOTU) approach for ichthyoplankton investigations, and describe fish biodiversity in the Jinshajiang section of the upper Yangtze River, China. The MOTUs were established by grouping specimens diverging less than 1.00% Kimura two‐parameter (K2P) distance units from their nearest neighbor within the same MOTU, based on previous work on between‐species divergences of the mitochondrial cytochrome C oxidase subunit I (COI) gene. Taxonomic assignment of the MOTUs was performed by comparing the MOTU sequences with the COI sequences of taxonomic species. Sixty‐eight MOTUs were inferred from 818 COI sequences of ichthyoplankton in the Jinshajiang river section. Among those, one MOTU was composed of two identified taxonomic species, and each of the other MOTUs was linked to a single, identified taxonomic species. Only 26 MOTUs were successfully identified to taxonomic species due to the limited reference database. Our results demonstrate that the MOTU approach can be applied successfully for analyzing biodiversity and identifying species of freshwater ichthyoplankton. Compared with previous ichthyoplankton investigations the richness of ichthyoplankton was very high. High diversity of ichthyoplankton noted in our study suggests that the Jinshajiang section should be an important target for fish biodiversity conservation in the Yangtze River.     

A new approach to an old conundrum--DNA barcoding sheds new light on phenotypic plasticity and morphological stasis in microsnails (Gastropoda, Pulmonata, Carychiidae)

The identification of microsnail taxa based on morphological characters is often a time-consuming and inconclusive process. Aspects such as morphological stasis and phenotypic plasticity further complicate their taxonomic designation. In this study, we demonstrate that the application of DNA barcoding can alleviate these problems within the Carychiidae (Gastropoda, Pulmonata). These microsnails are a taxon of the pulmonate lineage and most likely migrated onto land independently of the Stylommatophora clade. Their taxonomical classification is currently based on conchological and anatomical characters only. Despite much confusion about historic species assignments, the Carychiidae can be unambiguously subdivided into two taxa: (i) Zospeum species, which are restricted to karst caves, and (ii) Carychium species, which occur in a broad range of environmental conditions. The implementation of discrete molecular data (COI marker) enabled us to correctly designate 90% of the carychiid microsnails. The remaining cases were probably cryptic Zospeum and Carychium taxa and incipient species, which require further investigation into their species status. Because conventional reliance upon mostly continuous (i.e. nondiscrete) conchological characters is subject to fallibility for many gastropod species assignments, we highly recommend the use of DNA barcoding as a taxonomic, cutting-edge method for delimiting microsnail taxa.     

Identifying chironomids (Diptera: Chironomidae) for biological monitoring with PCR-RFLP

Chironomids are excellent biological indicators for the health of aquatic ecosystems, but their use at finer taxonomic levels is hindered by morphological similarity of species at each life stage. Molecular markers have the potential to overcome these problems by facilitating species identification particularly in large-scale surveys. In this study, the potential of the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) approach was tested to rapidly distinguish among chironomids within a geographic area, by considering chironomid species from Melbourne, Australia. By comparing molecular markers with diagnostic morphological traits, RFLP profiles of the cytochrome oxidase I (COI) region were identified that were specific to genera and some common species. These profiles were used to develop an RFLP-based key, which was validated by testing the markers on samples from several wetlands and streams. As well as allowing for rapid identification of species that are difficult to separate on morphological grounds, this approach also has the potential to resolve current taxonomic ambiguities.     

Environmental metabarcodes for insects: in silico PCR reveals potential for taxonomic bias

Studies of insect assemblages are suited to the simultaneous DNA‐based identification of multiple taxa known as metabarcoding. To obtain accurate estimates of diversity, metabarcoding markers ideally possess appropriate taxonomic coverage to avoid PCR‐amplification bias, as well as sufficient sequence divergence to resolve species. We used in silico PCR to compare the taxonomic coverage and resolution of newly designed insect metabarcodes (targeting 16S) with that of existing markers [16S and cytochrome oxidase c subunit I (COI)] and then compared their efficiency in vitro. Existing metabarcoding primers amplified in silico <75% of insect species with complete mitochondrial genomes available, whereas new primers targeting 16S provided >90% coverage. Furthermore, metabarcodes targeting COI appeared to introduce taxonomic PCR‐amplification bias, typically amplifying a greater percentage of Lepidoptera and Diptera species, while failing to amplify certain orders in silico. To test whether bias predicted in silico was observed in vitro, we created an artificial DNA blend containing equal amounts of DNA from 14 species, representing 11 insect orders and one arachnid. We PCR‐amplified the blend using five primer sets, targeting either COI or 16S, with high‐throughput amplicon sequencing yielding more than 6 million reads. In vitro results typically corresponded to in silico PCR predictions, with newly designed 16S primers detecting 11 insect taxa present, thus providing equivalent or better taxonomic coverage than COI metabarcodes. Our results demonstrate that in silico PCR is a useful tool for predicting taxonomic bias in mixed template PCR and that researchers should be wary of potential bias when selecting metabarcoding markers.     

Effect of marker choice and thermal cycling protocol on zooplankton DNA metabarcoding studies

DNA metabarcoding is a promising approach for rapidly surveying biodiversity and is likely to become an important tool for measuring ecosystem responses to environmental change. Metabarcoding markers need sufficient taxonomic coverage to detect groups of interest, sufficient sequence divergence to resolve species, and will ideally indicate relative abundance of taxa present. We characterized zooplankton assemblages with three different metabarcoding markers (nuclear 18S rDNA, mitochondrial COI, and mitochondrial 16S rDNA) to compare their performance in terms of taxonomic coverage, taxonomic resolution, and correspondence between morphology‐ and DNA‐based identification. COI amplicons sequenced on separate runs showed that operational taxonomic units representing >0.1% of reads per sample were highly reproducible, although slightly more taxa were detected using a lower annealing temperature. Mitochondrial COI and nuclear 18S showed similar taxonomic coverage across zooplankton phyla. However, mitochondrial COI resolved up to threefold more taxa to species compared to 18S. All markers revealed similar patterns of beta‐diversity, although different taxa were identified as the greatest contributors to these patterns for 18S. For calanoid copepod families, all markers displayed a positive relationship between biomass and sequence reads, although the relationship was typically strongest for 18S. The use of COI for metabarcoding has been questioned due to lack of conserved primer‐binding sites. However, our results show the taxonomic coverage and resolution provided by degenerate COI primers, combined with a comparatively well‐developed reference sequence database, make them valuable metabarcoding markers for biodiversity assessment.     

DNA barcode assessment of Mediterranean mayflies (Ephemeroptera), benchmark data for a regional reference library for rapid biomonitoring of freshwaters

Accurate identification of aquatic species is fundamental to freshwater research. In this paper, we targeted Ephemeroptera, a key taxonomic insect group for biomonitoring of water bodies and present an overview on the efficacy of the DNA barcoding approach to document species identity in the Mediterranean region. We sequenced the mitochondrial cytochrome c oxidase (COI) in 39 nominal species. Sample discrimination and species identification were investigated by evaluating haplotype identity and similarity, intra-/interspecific genetic distances, optimal identification of barcoding gap thresholds, estimates of species monophyly and comparative species matches on available reference libraries. The resolving power of the obtained data was discussed in the light of statistical tools such as Spider R-package and Poisson Tree Processes. High levels of species identification were achieved with all the used methodologies, and the occurrence of cryptic species was suggested. We conclude that DNA barcoding is a powerful tool for taxonomic research in Mediterranean mayflies, with great promise to ameliorate biodiversity inventories of freshwater ecosystems and to provide the necessary accuracy for water quality assessment programs. Our results further indicated we need to upgrade the current regional mayfly diversity knowledge. The development of a Mediterranean reference library could integrate this new information system.     

Efficacy of using DNA barcoding to identify parasitoid wasps of the melon-cotton aphid (<Emphasis Type="Italic">Aphis gossypii</Emphasis>) in watermelon cropping system

Parasitoid wasps have received a great deal of attention in the biological control of melon-cotton aphid (Aphis gossypii Glover). The species of parasitoids are often difficult to identify because of their small body size and profound diversity. DNA barcoding offers scientists who are not expert taxonomists a powerful tool to render their field studies more accurate. Using DNA barcodes to identify aphid parasitoid wasps in specific cropping systems may provide valuable information for biological control. Here, we report the use of DNA barcoding to confirm the morphological identification of 14 species (belonging to 13 genera of 7 families) of parasitoid wasps from two-year field samples in a watermelon cropping system. We generated DNA sequences from the mitochondrial COI gene and the nuclear D2 region of 28S rDNA to assess the genetic variation within and between parasitoid species. Automatic Barcode Gap Discovery (ABGD) supported the presence of 14 genetically distinct groups in the dataset. Among the COI sequences, we found no overlap between the maximum K2P distance within species (0.49%) and minimum distance between species (6.85%). The 28S sequences also showed greater interspecific distance than intraspecific distance. DNA barcoding confirmed the morphological identification. However, inconsistency and ambiguity of taxonomic information available in the online databases has limited the successful use of DNA barcoding. Only five species matched those in the BOLD and GenBank. Four species did not match the entries in GenBank and five species showed ambiguous results in BOLD due to confusing nomenclature. We suggested that species identification based on DNA barcodes should be performed using both COI and other genes. Nonetheless, we demonstrate the potential of the DNA barcoding approach to confirm field identifications and to provide a foundation for studies aimed at improving the understanding of the biocontrol services provided by parasitoids in the melon ecosystem.