DNA barcoding of endangered Indian Paphiopedilum species

The indiscriminate collections of Paphiopedilum species from the wild for their exotic ornamental flowers have rendered these plants endangered. Although the trade of these endangered species from the wild is strictly forbidden, it continues unabated in one or other forms that elude the current identification methods. DNA barcoding that offers identification of a species even if only a small fragment of the organism at any stage of development is available could be of great utility in scrutinizing the illegal trade of both endangered plant and animal species. Therefore, this study was undertaken to develop DNA barcodes of Indian species of Paphiopedilum along with their three natural hybrids using loci from both the chloroplast and nuclear genomes. The five loci tested for their potential as effective barcodes were RNA polymerase-β subunit (rpoB), RNA polymerase-β' subunit (rpoC1), Rubisco large subunit (rbcL) and maturase K (matK) from the chloroplast genome and nuclear ribosomal internal transcribed spacer (nrITS) from the nuclear genome. The intra- and inter-specific divergence values and species discrimination rates were calculated by Kimura 2 parameter (K2P) method using mega 4.0. The matK with 0.9% average inter-specific divergence value yielded 100% species resolution, thus could distinguish all the eight species of Paphiopedilum unequivocally. The species identification capability of these sequences was further confirmed as each of the matK sequences was found to be unique for the species when a blast analysis of these sequences was carried out on NCBI. nrITS, although had 4.4% average inter-specific divergence value, afforded only 50% species resolution. DNA barcodes of the three hybrids also reflected their parentage.     

DNA barcoding gap: reliable species identification over morphological and geographical scales

The philosophical basis and utility of DNA barcoding have been a subject of numerous debates. While most literature embraces it, some studies continue to question its use in dipterans, butterflies and marine gastropods. Here, we explore the utility of DNA barcoding in identifying spider species that vary in taxonomic affiliation, morphological diagnosibility and geographic distribution. Our first test searched for a ‘barcoding gap’ by comparing intra‐ and interspecific means, medians and overlap in more than 75 000 computed Kimura 2‐parameter (K2P) genetic distances in three families. Our second test compared K2P distances of congeneric species with high vs. low morphological distinctness in 20 genera of 11 families. Our third test explored the effect of enlarging geographical sampling area at a continental scale on genetic variability in DNA barcodes within 20 species of nine families. Our results generally point towards a high utility of DNA barcodes in identifying spider species. However, the size of the barcoding gap strongly depends on taxonomic groups and practices. It is becoming critical to define the barcoding gap statistically more consistently and to document its variation over taxonomic scales. Our results support models of independent patterns of morphological and molecular evolution by showing that DNA barcodes are effective in species identification regardless of their morphological diagnosibility. We also show that DNA barcodes represent an effective tool for identifying spider species over geographic scales, yet their variation contains useful biogeographic information.     

Collecting in collections: a PCR strategy and primer set for DNA barcoding of decades‐old dried museum specimens

Natural history museums are vastly underutilized as a source of material for DNA analysis because of perceptions about the limitations of DNA degradation in older specimens. Despite very few exceptions, most DNA barcoding projects, which aim to obtain sequence data from all species, generally use specimens collected specifically for that purpose, instead of the wealth of identified material in museums, constrained by the lack of suitable PCR methods. Any techniques that extend the utility of museum specimens for DNA analysis therefore are highly valuable. This study first tested the effects of specimen age and PCR amplicon size on PCR success rates in pinned insect specimens, then developed a PCR primer set and amplification strategy allowing greatly increased utilization of older museum specimens for DNA barcoding. PCR success rates compare favourably with the few published studies utilizing similar aged specimens, and this new strategy has the advantage of being easily automated for high‐throughput laboratory workflows. The strategy uses hemi‐nested, degenerate, M13‐tailed PCR primers to amplify two overlapping amplicons, using two PCRs per amplicon (i.e. four PCRs per DNA sample). Initial PCR products are reamplified using an internal primer and a M13 primer. Together the two PCR amplicons yield 559 bp of the COI gene from Coleoptera, Lepidoptera, Diptera, Hemiptera, Odonata and presumably also other insects. BARCODE standard‐compliant data were recovered from 67% (56 of 84) of specimens up to 25 years old, and 51% (102 of 197) of specimens up to 55 years old. Given the time, cost and specialist expertise required for fieldwork and identification, ‘collecting in collections’ is a viable alternative allowing researchers to capitalize on the knowledge captured by curation work in decades past.     

A DNA barcoding approach for identification of hidden diversity in Parmeliaceae (Ascomycota): Parmelia sensu stricto as a case study

Accurate specimen identification is challenging in groups with subtle or scarce taxonomically diagnostic characters, and the use of DNA barcodes can provide an effective means for consistent identification. Here, we investigate the utility of DNA barcode identification of species in a cosmopolitan genus of lichen‐forming fungi, Parmelia (Parmeliaceae). Two hundred and two internal transcribed spacer (ITS) sequences generated from specimens collected from all continents, including Antarctica, were analysed, and DNA barcodes of 14 species of Parmelia s.s. are reported. Almost all species show a barcode gap. Overall, intraspecific divergence values were lower than the threshold previously established for Parmeliaceae. However, the mean and range were elevated by deep barcode divergences in three species, indicating the likely occurrence of overlooked species‐level lineages. Here, we provide a DNA barcode reference library with well‐identified specimens sampled worldwide and sequences from most of the type material to enable easy and fast accurate sample identification and to assist in uncovering overlooked species in Parmelia s.s. Further, our results confirm the efficiency of the ITS region in the identification of species of Parmelia s.s. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180 , 21–29.     

Tissue segregation of mitochondrial haplotypes in heteroplasmic Hawaiian bees: implications for DNA barcoding

The issue of mitochondrial heteroplasmy has been cited as a theoretical problem for DNA barcoding but is only beginning to be examined in natural systems. We sequenced multiple DNA extractions from 20 individuals of four Hawaiian Hylaeus bee species known to be heteroplasmic. All species showed strong differences at polymorphic sites between abdominal and muscle tissue in most individuals, and only two individuals had no obvious segregation. Two specimens produced completely clean sequences from abdominal DNA. The fact that these differences are clearly visible by direct sequencing indicates that substantial intra-individual mtDNA diversity may be overlooked when DNA is taken from small tissue fragments. At the same time, differences in haplotype distribution among individuals may result in incorrect recognition of cryptic species. Because DNA barcoding studies typically use only a small fragment of an organism, they are particularly vulnerable to sequencing bias where heteroplasmy and haplotype segregation are present. It is important to anticipate this possibility prior to undertaking large-scale barcoding projects to reduce the likelihood of haplotype segregation confounding the results.     

COI barcoding of true bugs (Insecta, Heteroptera)

Several recent studies have proposed that partial DNA sequences of the cytochrome c oxidase I (COI) mitochondrial gene might serve as DNA barcodes for identifying and differentiating between animal species, such as birds, fish and insects. In this study, we tested the effectiveness of a COI barcode to identify true bugs from 139 species collected from Korea and adjacent regions (Japan, Northeastern China and Fareast Russia). All the species had a unique COI barcode sequence except for the genus Apolygus (Miridae), and the average interspecific genetic distance between closely related species was about 16 times higher than the average intraspecific genetic distance. DNA barcoding identified one probable new species of true bug and revealed identical or very recently divergent species that were clearly distinguished by morphological characteristics. Therefore, our results suggest that COI barcodes can reveal new cryptic true bug species and are able to contribute for the exact identification of the true bugs.     

DNA barcoding meets molecular scatology: short mtDNA sequences for standardized species assignment of carnivore noninvasive samples

Although species assignment of scats is important to study carnivore biology, there is still no standardized assay for the identification of carnivores worldwide, which would allow large-scale routine assessments and reliable cross-comparison of results. Here, we evaluate the potential of two short mtDNA fragments [ATP6 (126 bp) and cytochrome oxidase I gene (COI) (187 bp)] to serve as standard markers for the Carnivora. Samples of 66 species were sequenced for one or both of these segments. Alignments were complemented with archival sequences and analysed with three approaches (tree-based, distance-based and character-based). Intraspecific genetic distances were generally lower than between-species distances, resulting in diagnosable clusters for 86% (ATP6) and 85% (COI) of the species. Notable exceptions were recently diverged species, most of which could still be identified using diagnostic characters and uniqueness of haplotypes or by reducing the geographic scope of the comparison. In silico analyses were also performed for a 110-bp cytochrome b (cytb) segment, whose identification success was lower (70%), possibly due to the smaller number of informative sites and/or the influence of misidentified sequences obtained from GenBank. Finally, we performed case studies with faecal samples, which supported the suitability of our two focal markers for poor-quality DNA and allowed an assessment of prey DNA co-amplification. No evidence of prey DNA contamination was found for ATP6, while some cases were observed for COI and subsequently eliminated by the design of more specific primers. Overall, our results indicate that these segments hold good potential as standard markers for accurate species-level identification in the Carnivora.     

Unraveling the plant diversity of the Amazonian canga through DNA barcoding

The canga of the Serra dos Carajás, in Eastern Amazon, is home to a unique open plant community, harboring several endemic and rare species. Although a complete flora survey has been recently published, scarce to no genetic information is available for most plant species of the ironstone outcrops of the Serra dos Carajás. In this scenario, DNA barcoding appears as a fast and effective approach to assess the genetic diversity of the Serra dos Carajás flora, considering the growing need for robust biodiversity conservation planning in such an area with industrial mining activities. Thus, after testing eight different DNA barcode markers (matK, rbcL, rpoB, rpoC1, atpF‐atpH, psbK‐psbI, trnH‐psbA, and ITS2), we chose rbcL and ITS2 as the most suitable markers for a broad application in the regional flora. Here we describe DNA barcodes for 1,130 specimens of 538 species, 323 genera, and 115 families of vascular plants from a highly diverse flora in the Amazon basin, with a total of 344 species being barcoded for the first time. In addition, we assessed the potential of using DNA metabarcoding of bulk samples for surveying plant diversity in the canga. Upon achieving the first comprehensive DNA barcoding effort directed to a complete flora in the Brazilian Amazon, we discuss the relevance of our results to guide future conservation measures in the Serra dos Carajás.     

DNA barcoding reveals the temporal community composition of drifting fish eggs in the lower Hongshui River,China

Determining the temporal community composition of fish eggs in particular regions and understanding the reproductive times of regional fish taxa are key aspects of the management and regulation of regional fish stocks. However, it is extremely difficult to accurately identify fish eggs due to the absence of diagnostic morphological characters. We sampled fish eggs in the lower Hongshuihe River (an upper mainstem of the Pearl River) between May and September 2020. We then used DNA barcoding to determine the species composition of the egg pool and to predict the spawning periods of the identified species. A total of 641 eggs and 17 larvae were chosen for molecular identification; 397 eggs and 17 larvae yielded high‐quality barcoding sequences. The high failure rate (~38%) was most likely due to long‐term storage in low concentrations of ethanol prior to molecular analysis. We successfully classified 392 eggs into 10 species and 13 larvae into four species using public databases. Most of the species identified in the egg pool were small and/or benthic, and migratory species were rare. This may partially reflect the adverse effects of hydropower cascade development in this river section. We also found that spawning periods tended to be species‐specific. Our study provides a reference for the conservation and management of regional fishery stocks.     

DNA barcoding the ichthyofauna of the Yangtze River: Insights from the molecular inventory of a mega‐diverse temperate fauna

Intensification of inland fisheries and aquatic landscape conversion led to a drastic decline of fish populations in the Yangtze River (YR) during the last decades. This situation urges for the development of a large‐scale molecular assessment of YR ichthyofauna to further develop standardized methods of molecular identification for conservation and fisheries management purposes. We present here the results of a large‐scale campaign to DNA barcode YR freshwater fishes that succeeded in producing 1,424 new DNA barcodes for 123 species. Together with 1,406 sequences mined from BOLD and GenBank, a reference library including 2,830 DNA barcodes for 238 species was compiled. By using four DNA‐based species delimitation methods, RESL, ABGD, mPTP and mGMYC, 230 operational taxonomic units (OTUs) were identified and 195 species displayed OTUs that tightly match species boundaries. No barcoding gap was observed; however, and conflicting cases of species and OTU delimitation were identified. A total of 23 species with maximum intraspecific distances above 2% were detected and null genetic distances to the nearest phylogenetic relatives were detected in 11 species. Among those 23 species, 16 were represented by multiple OTUs amounting to 40 OTUs delineated. Several cases of multiple OTUs confined to species boundaries were detected suggesting the presence of overlooked species. A total of 18 OTUs, however, were shared by several species and particularly so for the Qinghai‐Tibet plateau endemic species. These results are discussed with reference to previous large‐scale DNA barcoding campaign and compared to previous phylogeographic studies in the YR.     

DNA barcoding of Canada's skates

DNA-based identifications have been employed across broad taxonomic ranges and provide an especially useful tool in cases where external identification may be problematic. This study explored the utility of DNA barcoding in resolving skate species found in Atlantic Canadian waters. Most species were clearly resolved, expanding the utility for such identification on a taxonomically problematic group. Notably, one genus (Amblyraja) contained three of four species whose distributions do not overlap that could not be readily identified with this method. On the other hand, two common and partially sympatric species (Little and Winter skates) were readily identifiable. There were several instances of inconsistency between the voucher identification and the DNA sequence data. In some cases, these were at the intrageneric level among species acknowledged to be prone to misidentification. However, several instances of intergeneric discrepancies were also identified, suggesting either evidence of past introgressive hybridization or misidentification of vouchered specimens across broader taxonomic ranges. Such occurrences highlight the importance of retaining vouchered specimens for subsequent re-examination in the light of conflicting DNA evidence.     

Single-locus DNA barcoding and species delimitation of the sandfly subgenus Evandromyia (Aldamyia)

Sandfly specimens from the subgenus Evandromyia (Aldamyia) Galati, 2003 (Diptera: Psychodidae: Phlebotominae) were collected between 2012 and 2019 from nine localities in seven Brazilian states, morphologically-identified, and then DNA barcoded by sequencing the mitochondrial cytochrome c oxidase subunit I (coi) gene. Forty-four new barcode sequences generated from 10 morphospecies were combined with 49 previously published sequences from the same subgenus and analysed using sequence-similarity methods (best-match criteria) to assess their ability at specimen identification, while four different species delimitation methods (ABGD, GMYC, PTP and TCS) were used to infer molecular operational taxonomic units (MOTUs). Overall, seven of the 11 morphospecies analysed were congruent with both the well-supported clades identified by phylogenetic analysis and the MOTUs inferred by species delimitation, while the remaining four morphospecies – E. carmelinoi, E. evandroi, E. lenti and E. piperiformis – were merged into a single well-supported clade/MOTU. Although E. carmelinoi, E. evandroi and E. lenti were indistinguishable using coi DNA barcodes, E. piperiformis did form a distinct phylogenetic cluster and could be correctly identified using best-match criteria. Despite their apparent morphological differences, we propose on the basis of the molecular similarity of their DNA barcodes that these latter four morphospecies should be considered members of a recently-diverged species complex.     

DNA barcoding of stylommatophoran land snails: a test of existing sequences

DNA barcoding has attracted attention because it is a potentially simple and universal method for taxonomic assignment. One anticipated problem in applying the method to stylommatophoran land snails is that they frequently exhibit extreme divergence of mitochondrial DNA sequences, sometimes reaching 30% within species. We therefore trialled the utility of barcodes in identifying land snails, by analysing the stylommatophoran cytochrome oxidase subunit I sequences from GenBank. Two alignments of 381 and 228 base pairs were used to determine potential error rates among a test data set of 97 or 127 species, respectively. Identification success rates using neighbour‐joining phylogenies were 92% for the longer sequence and 82% for the shorter sequence, indicating that a high degree of mitochondrial variation may actually be an advantage when using phylogeny‐based methods for barcoding. There was, however, a large overlap between intra‐ and interspecific variation, with assignment failure (per cent of samples not placed with correct species) particularly associated with a low degree of mitochondrial variation (Kimura 2‐parameter distance < 0.05) and a small GenBank sample size (< 25 per species). Thus, while the optimum intra/interspecific threshold value was 4%, this was associated with an overall error of 32% for the longer sequences and 44% for the shorter sequences. The high error rate necessitates that barcoding of land snails is a potentially useful method to discriminate species of land snail, but only when a baseline has first been established using conventional taxonomy and sample DNA sequences. There is no evidence for a barcoding gap, ruling out species discovery based on a threshold value alone.     

‘Direct PCR’ optimization yields a rapid,cost‐effective,nondestructive and efficient method for obtaining DNA barcodes without DNA extraction

Macroinvertebrates that are collected in large numbers pose major problems in basic and applied biodiversity research: identification to species via morphology is often difficult, slow and/or expensive. DNA barcodes are an attractive alternative or complementary source of information. Unfortunately, obtaining DNA barcodes from specimens requires many steps and thus time and money. Here, we promote a short cut to DNA barcoding, that is, a nondestructive PCR method that skips DNA extraction (‘direct PCR’) and that can be used for a broad range of invertebrate taxa. We demonstrate how direct PCR can be optimized for the larvae and adults of nonbiting midges (Diptera: Chironomidae), a typical invertebrate group that is abundant, contains important bioindicator species, but is difficult to identify based on morphological features. After optimization, direct PCR yields high PCR success rates (>90%), preserves delicate morphological features (e.g. details of genitalia, and larval head capsules) while allowing for the recovery of genomic DNA. We also document that direct PCR can be successfully optimized for a wide range of other invertebrate taxa that need routine barcoding (flies: Culicidae, Drosophilidae, Dolichopodidae, Sepsidae; sea stars: Oreasteridae). Key for obtaining high PCR success rates is optimizing (i) tissue quantity, (ii) body part, (iii) primer pair and (iv) type of Taq polymerase. Unfortunately, not all invertebrates appear suitable because direct PCR has low success rates for other taxa that were tested (e.g. Coleoptera: Dytiscidae, Copepoda, Hymenoptera: Formicidae and Odonata). It appears that the technique is less successful for heavily sclerotized insects and/or those with many exocrine glands.     

Utility of DNA barcoding to identify rare endemic vascular plant species in Trinidad

The islands of the Caribbean are considered to be a “biodiversity hotspot.” Collectively, a high level of endemism for several plant groups has been reported for this region. Biodiversity conservation should, in part, be informed by taxonomy, population status, and distribution of flora. One taxonomic impediment to species inventory and management is correct identification as conventional morphology‐based assessment is subject to several caveats. DNA barcoding can be a useful tool to quickly and accurately identify species and has the potential to prompt the discovery of new species. In this study, the ability of DNA barcoding to confirm the identities of 14 endangered endemic vascular plant species in Trinidad was assessed using three DNA barcodes (matK, rbcL, and rpoC1). Herbarium identifications were previously made for all species under study. matK, rbcL, and rpoC1 markers were successful in amplifying target regions for seven of the 14 species. rpoC1 sequences required extensive editing and were unusable. rbcL primers resulted in cleanest reads, however, matK appeared to be superior to rbcL based on a number of parameters assessed including level of DNA polymorphism in the sequences, genetic distance, reference library coverage based on BLASTN statistics, direct sequence comparisons within “best match” and “best close match” criteria, and finally, degree of clustering with moderate to strong bootstrap support (>60%) in neighbor‐joining tree‐based comparisons. The performance of both markers seemed to be species‐specific based on the parameters examined. Overall, the Trinidad sequences were accurately identified to the genus level for all endemic plant species successfully amplified and sequenced using both matK and rbcL markers. DNA barcoding can contribute to taxonomic and biodiversity research and will complement efforts to select taxa for various molecular ecology and population genetics studies.     

Evaluating next‐generation sequencing (NGS) methods for routine monitoring of wild bees: Metabarcoding,mitogenomics or NGS barcoding

Implementing cost‐effective monitoring programs for wild bees remains challenging due to the high costs of sampling and specimen identification. To reduce costs, next‐generation sequencing (NGS)‐based methods have lately been suggested as alternatives to morphology‐based identifications. To provide a comprehensive presentation of the advantages and weaknesses of different NGS‐based identification methods, we assessed three of the most promising ones, namely metabarcoding, mitogenomics and NGS barcoding. Using a regular monitoring data set (723 specimens identified using morphology), we found that NGS barcoding performed best for both species presence/absence and abundance data, producing only few false positives (3.4%) and no false negatives. In contrast, the proportion of false positives and false negatives was higher using metabarcoding and mitogenomics. Although strong correlations were found between biomass and read numbers, abundance estimates significantly skewed the communities' composition in these two techniques. NGS barcoding recovered the same ecological patterns as morphology. Ecological conclusions based on metabarcoding and mitogenomics were similar to those based on morphology when using presence/absence data, but different when using abundance data. In terms of workload and cost, we show that metabarcoding and NGS barcoding can compete with morphology, but not mitogenomics which was consistently more expensive. Based on these results, we advocate that NGS barcoding is currently the seemliest NGS method for monitoring of wild bees. Furthermore, this method has the advantage of potentially linking DNA sequences with preserved voucher specimens, which enable morphological re‐examination and will thus produce verifiable records which can be fed into faunistic databases.     

DNA barcoding for the identification of eight species members of the Thai Hyrcanus Group and investigation of their stenogamous behavior

Eight species members of the Thai Hyrcanus Group were identified based on the intact morphology and molecular analysis (COI barcoding, 658 bp) of F₁-progenies. Five iso-female lines of each species were pooled in order to establish stock colonies. A stenogamous colony of each species was investigated by making 200 and 300 newly emerged adult females and males co-habit in a 30 cm cubic cage for one week. After ovipositon, the spermathecae of females were examined for sperms. The results revealed that Anopheles argyropus, Anopheles crawfordi, Anopheles nitidus, Anopheles pursati, Anopheles sinensis, Anopheles nigerrimus, Anopheles paraliae and Anopheles peditaeniatus yielded insemination rates of 0%, 0%, 0%, 31%, 33%, 42%, 50% and 77%, respectively. Continuous selection to establish stenogamous colonies indicated that An. sinensis, An. pursati, An. nigerrimus, An. paraliae and An. peditaeniatus provided insemination rates of 33–34%, 27–31%, 42–58%, 43–57% and 61–86% in 1, 2, 5, 6 and 20 generations of passages, respectively.