Redesign of PCR primers for mitochondrial cytochrome c oxidase subunit I for marine invertebrates and application in all‐taxa biotic surveys

DNA barcoding is a powerful tool for species detection, identification and discovery. Metazoan DNA barcoding is primarily based upon a specific region of the cytochrome c oxidase subunit I gene that is PCR amplified by primers HCO2198 and LCO1490 (‘Folmer primers’) designed by Folmer et al. (Molecular Marine Biology and Biotechnology, 3 , 1994, 294). Analysis of sequences published since 1994 has revealed mismatches in the Folmer primers to many metazoans. These sequences also show that an extremely high level of degeneracy would be necessary in updated Folmer primers to maintain broad taxonomic utility. In primers jgHCO2198 and jgLCO1490, we replaced most fully degenerated sites with inosine nucleotides that complement all four natural nucleotides and modified other sites to better match major marine invertebrate groups. The modified primers were used to amplify and sequence cytochrome c oxidase subunit I from 9105 specimens from Moorea, French Polynesia and San Francisco Bay, California, USA representing 23 phyla, 42 classes and 121 orders. The new primers, jgHCO2198 and jgLCO1490, are well suited for routine DNA barcoding, all‐taxon surveys and metazoan metagenomics.     

Streamlining DNA Barcoding Protocols: Automated DNA Extraction and a New cox1 Primer in Arachnid Systematics

Background

DNA barcoding is a popular tool in taxonomic and phylogenetic studies, but for most animal lineages protocols for obtaining the barcoding sequences—mitochondrial cytochrome C oxidase subunit I (cox1 AKA CO1)—are not standardized. Our aim was to explore an optimal strategy for arachnids, focusing on the species-richest lineage, spiders by (1) improving an automated DNA extraction protocol, (2) testing the performance of commonly used primer combinations, and (3) developing a new cox1 primer suitable for more efficient alignment and phylogenetic analyses.

Methodology

We used exemplars of 15 species from all major spider clades, processed a range of spider tissues of varying size and quality, optimized genomic DNA extraction using the MagMAX Express magnetic particle processor—an automated high throughput DNA extraction system—and tested cox1 amplification protocols emphasizing the standard barcoding region using ten routinely employed primer pairs.

Results

The best results were obtained with the commonly used Folmer primers (LCO1490/HCO2198) that capture the standard barcode region, and with the C1-J-2183/C1-N-2776 primer pair that amplifies its extension. However, C1-J-2183 is designed too close to HCO2198 for well-interpreted, continuous sequence data, and in practice the resulting sequences from the two primer pairs rarely overlap. We therefore designed a new forward primer C1-J-2123 60 base pairs upstream of the C1-J-2183 binding site. The success rate of this new primer (93%) matched that of C1-J-2183.

Conclusions

The use of C1-J-2123 allows full, indel-free overlap of sequences obtained with the standard Folmer primers and with C1-J-2123 primer pair. Our preliminary tests suggest that in addition to spiders, C1-J-2123 will also perform in other arachnids and several other invertebrates. We provide optimal PCR protocols for these primer sets, and recommend using them for systematic efforts beyond DNA barcoding.     

Assessing the effects of primer specificity on eliminating numt coamplification in DNA barcoding: a case study from Orthoptera (Arthropoda: Insecta)

DNA barcoding is a diagnostic method of species identification based on sequencing a short mitochondrial DNA fragment of cytochrome oxidase I (COI), but its ability to correctly diagnose species is limited by the presence of nuclear mitochondrial pseudogenes (numts). Numts can be coamplified with the mitochondrial orthologue when using universal primers, which can lead to incorrect species identification and an overestimation of the number of species. Some researchers have proposed that using more specific primers may help eliminate numt coamplification, but the efficacy of this method has not been thoroughly tested. In this study, we investigate the taxonomic distribution of numts in 11 lineages within the insect order Orthoptera, by analysing cloned COI sequences and further test the effects of primer specificity on eliminating numt coamplification in four lineages. We find that numts are coamplified in all 11 taxa using universal (barcoding) primers, which suggests that numts may be widespread in other taxonomic groups as well. Increased primer specificity is only effective at reducing numt coamplification in some species tested, and only eliminates it in one species tested. Furthermore, we find that a number of numts do not have stop codons or indels, making it difficult to distinguish them from mitochondrial orthologues, thus putting the efficacy of barcoding quality control measures under question. Our findings suggest that numt coamplification is a serious problem for DNA barcoding and more quality control measures should be implemented to identify and eliminate numts prior to using mitochondrial barcodes for species diagnoses.     

Estimating diversity of crabs (Decapoda: Brachyura) in a no-take marine protected area of the SW Atlantic coast through DNA barcoding of larvae

DNA barcoding was used to identify crab larvae from the Marine Biological Reserve of Arvoredo, encompassing a coastal archipelago off the SW Atlantic coast (27°S, 48°W). Partial mitochondrial COI or 16S rRNA gene sequences were obtained for 488 larvae, leading to the identification of 20 species. The COI sequences generated 13 barcode index numbers (BINs) within Barcode of Life Data Systems (BOLD), among which 11 were concordant with single species. DNA from ～ 6% of the larvae did not amplify using the primers tested; based on external morphological characteristics, these larvae represented four possible additional operational taxonomic units (OTUs) at the family level. Intraspecific variation for the COI and 16S rRNA genes was found to be < 2.6% and < 2.1% respectively (Kimura 2-parameter distance), whereas interspecific divergence ranged from 7.9% to 21.5% and 6.4% to 14.5%, respectively. These results imply that both genes are suitable for use in species identification of brachyuran crabs of this area. Molecular identification of this group successfully enabled the diagnosis of larvae of closely related species, including congeners in Mithrax, Achelous and Callinectes. In addition, eight out of 20 species recognized represent new records for the reserve suggesting that the brachyuran fauna in the area has been underestimated based on traditional biodiversity measures. The availability of primers suited to the targeted species, and the development of a taxonomically comprehensive DNA barcoding database are the major recommendations to improve the accuracy and feasibility of using DNA barcoding for species identification of SW Atlantic brachyuran crabs.     

Species discrimination in Sisyrinchium (Iridaceae): assessment of DNA barcodes in a taxonomically challenging genus

DNA barcoding aims to develop an efficient tool for species identification based on short and standardized DNA sequences. In this study, the DNA barcode paradigm was tested among the genera of the tribe Sisyrinchieae (Iridoideae). Sisyrinchium, with more than 77% of the species richness in the tribe, is a taxonomically complex genus. A total of 185 samples belonging to 98 species of Sisyrinchium, Olsynium, Orthrosanthus and Solenomelus were tested using matK, trnH‐psbA and internal transcribed spacer (ITS). Candidate DNA barcodes were analysed either as single markers or in combination. Detection of a barcoding gap, similarity‐based methods and tree‐based analyses were used to assess the discrimination efficiency of DNA barcodes. The levels of species identification obtained from plastid barcodes were low and ranged from 17.35% to 20.41% for matK and 5.11% to 7.14% for trnH‐psbA. The ITS provided better results with 30.61–38.78% of species identified. The analyses of the combined data sets did not result in a significant improvement in the discrimination rate. Among the tree‐based methods, the best taxonomic resolution was obtained with Bayesian inference, particularly when the three data sets were combined. The study illustrates the difficulties for DNA barcoding to identify species in evolutionary complex lineages. Plastid markers are not recommended for barcoding Sisyrinchium due to the low discrimination power observed. ITS gave better results and may be used as a starting point for species identification.     

Cold Code: the global initiative to DNA barcode amphibians and nonavian reptiles

DNA barcoding facilitates the identification of species and the estimation of biodiversity by using nucleotide sequences, usually from the mitochondrial genome. Most studies accomplish this task by using the gene encoding cytochrome oxidase subunit I (COI; Entrez COX1). Within this barcoding framework, many taxonomic initiatives exist, such as those specializing in fishes, birds, mammals, and fungi. Other efforts center on regions, such as the Arctic, or on other topics, such as health. DNA barcoding initiatives exist for all groups of vertebrates except for amphibians and nonavian reptiles. We announce the formation of Cold Code, the international initiative to DNA barcode all species of these ‘cold‐blooded’ vertebrates. The project has a Steering Committee, Coordinators, and a home page. To facilitate Cold Code, the Kunming Institute of Zoology, Chinese Academy of Sciences will sequence COI for the first 10 specimens of a species at no cost to the steward of the tissues.     

Cryptic diversity in flathead fishes (Scorpaeniformes: Platycephalidae) across the Indo‐West Pacific uncovered by DNA barcoding

Identification of taxonomical units underpins most biological endeavours ranging from accurate biodiversity estimates to the effective management of sustainably harvested, protected or endangered species. Successful species identification is now frequently based on a combination of approaches including morphometrics and DNA markers. Sequencing of the mitochondrial COI gene is an established methodology with an international campaign directed at barcoding all fishes. We employed COI sequencing alongside traditional taxonomic identification methods and uncovered instances of deep intraspecific genetic divergences among flathead species. Sixty‐five operational taxonomic units (OTUs) were observed across the Indo‐West Pacific from just 48 currently recognized species. The most comprehensively sampled taxon, Platycephalus indicus, exhibited the highest levels of genetic diversity with eight lineages separated by up to 16.37% genetic distance. Our results clearly indicate a thorough reappraisal of the current taxonomy of P. indicus (and its three junior synonyms) is warranted in conjunction with detailed taxonomic work on the other additional Platycephalidae OTUs detected by DNA barcoding.     

Development of internal COI primers to improve and extend barcoding of fruit flies (Diptera: Tephritidae: Dacini)

Accurate species-level identifications underpin many aspects of basic and applied biology;however,identifications can be hampered by a lack of discriminating morphological characters,taxonomic expertise or time.Molecular approaches,such as DNA"barcoding"of the cytochrome c oxidase(COI)gene,are argued to overcome these issues.However,nuclear encoding of mitochondrial genes(numts)and poor amplification success of suboptimally preserved specimens can lead to erroneous identifications.One insect group for which these molecular and morphological problems are significant are the dacine fruit flies(Diptera:Tephritidae:Dacini).We addressed these issues associated with COI barcoding in the dacines by first assessing several"universal"COI primers against public mitochondrial genome and numt sequences for dacine taxa.We then modified a set of four primers that more closely matched true dacine COI sequence and amplified two overlapping portions of the COI barcode region.Our new primers were tested alongside universal primers on a selection of dacine species,including both fresh preserved and decades-old dry specimens.Additionally,Bactrocera tiyoni mitochondrial and nuclear genomes were compared to identify putative numts.Four numt clades were identified,three of which were amplified using existing universal primers.In contrast,our new primers preferentially amplified the"true"mitochondrial COI barcode in all dacine species tested.The new primers also successfully amplified partial barcodes from dry specimens for which full length barcodes were unobtainable.Thus we recommend these new primers be incorporated into the suites of primers used by diagnosticians and quarantine labs for the accurate identification of dacine species.     

Molecular identification of Bactrocera passiflorae (Diptera: Tephritidae): Challenge and solution for DNA barcoding

Fruit flies cause significant damage to crop and fruit production worldwide. Therefore, it is essential to identify these organisms to species level; however, immature stages are often impossible to be identified morphologically; thus, the application of DNA barcoding has greatly assisted in species identification. Nuclear, mitochondrial pseudo-COI (NUMT) can be co-amplified with mitochondrial DNA when using generic primers and therefore impair the efficacy of DNA barcoding. This study detected two types of NUMTs from Bactrocera passiflorae, one of them is novel. Therefore, the new finding will assist future species identification by avoiding misidentification using ambiguous NUMT sequences. In addition, this study has developed primers to target the COI gene of B. passiflorae, not the NUMT copies. The newly designed primers have demonstrated its efficiency in amplifying the Mt-COI of B. passiflorae and can be used in routine diagnostics.     

DNA barcoding commercially important fish species of Turkey

DNA barcoding was used in the identification of 89 commercially important freshwater and marine fish species found in Turkish ichthyofauna. A total of 1765 DNA barcodes using a 654‐bp‐long fragment of the mitochondrial cytochrome c oxidase subunit I gene were generated for 89 commercially important freshwater and marine fish species found in Turkish ichthyofauna. These species belong to 70 genera, 40 families and 19 orders from class Actinopterygii, and all were associated with a distinct DNA barcode. Nine and 12 of the COI barcode clusters represent the first species records submitted to the BOLD and GenBank databases, respectively. All COI barcodes (except sequences of first species records) were matched with reference sequences of expected species, according to morphological identification. Average nucleotide frequencies of the data set were calculated as T = 29.7%, C = 28.2%, A = 23.6% and G = 18.6%. Average pairwise genetic distance among individuals were estimated as 0.32%, 9.62%, 17,90% and 22.40% for conspecific, congeneric, confamilial and within order, respectively. Kimura 2‐parameter genetic distance values were found to increase with taxonomic level. For most of the species analysed in our data set, there is a barcoding gap, and an overlap in the barcoding gap exists for only two genera. Neighbour‐joining trees were drawn based on DNA barcodes and all the specimens clustered in agreement with their taxonomic classification at species level. Results of this study supported DNA barcoding as an efficient molecular tool for a better monitoring, conservation and management of fisheries.     

DNA barcoding of Murinae (Rodentia: Muridae) and Arvicolinae (Rodentia: Cricetidae) distributed in China

Identification of rodents is very difficult mainly due to high similarities in morphology and controversial taxonomy. In this study, mitochondrial cytochrome oxidase subunit I (COI) was used as DNA barcode to identify the Murinae and Arvicolinae species distributed in China and to facilitate the systematics studies of Rodentia. In total, 242 sequences (31 species, 11 genera) from Murinae and 130 sequences (23 species, 6 genera) from Arvicolinae were investigated, of which 90 individuals were novel. Genetic distance, threshold method, tree‐based method, online BLAST and BLOG were employed to analyse the data sets. There was no obvious barcode gap. The average K2P distance within species and genera was 2.10% and 12.61% in Murinae, and 2.86% and 11.80% in Arvicolinae, respectively. The optimal threshold was 5.62% for Murinae and 3.34% for Arvicolinae. All phylogenetic trees exhibited similar topology and could distinguish 90.32% of surveyed species in Murinae and 82.60% in Arvicolinae with high support values. BLAST analyses yielded similar results with identification success rates of 92.15% and 93.85% for Murinae and Arvicolinae, respectively. BLOG successfully authenticated 100% of detected species except Leopoldamys edwardsi based on the latest taxonomic revision. Our results support the species status of recently recognized Micromys erythrotis, Eothenomys tarquinius and E. hintoni and confirm the important roles of comprehensive taxonomy and accurate morphological identification in DNA barcoding studies. We believe that, when proper analytic methods are applied or combined, DNA barcoding could serve as an accurate and effective species identification approach for Murinae and Arvicolinae based on a proper taxonomic framework.     

High mitochondrial diversity in geographically widespread butterflies of Madagascar: A test of the DNA barcoding approach

The standardized use of mitochondrial cytochrome c oxidase subunit I (COI) gene sequences as DNA barcodes has been widely promoted as a high-throughput method for species identification and discovery. Species delimitation has been based on the following criteria: (1) monophyletic association and less frequently (2) a minimum 10× greater divergence between than within species. Divergence estimates, however, can be inflated if sister species pairs are not included and the geographic extent of variation within any given taxon is not sampled comprehensively. This paper addresses both potential biases in DNA divergence estimation by sampling range-wide variation in several morphologically distinct, endemic butterfly species in the genus Heteropsis, some of which are sister taxa. We also explored the extent to which mitochondrial DNA from the barcode region can be used to assess the effects of historical rainforest fragmentation by comparing genetic variation across Heteropsis populations with an unrelated forest-associated taxon Saribia tepahi. Unexpectedly, generalized primers led to the inadvertent amplification of the endosymbiont Wolbachia, undermining the use of universal primers and necessitating the design of genus-specific COI primers alongside a Wolbachia-specific PCR assay. Regardless of the high intra-specific genetic variation observed, most species satisfy DNA barcoding criteria and can be differentiated in the nuclear phylogeny. Nevertheless, two morphologically distinguishable candidate species fail to satisfy the barcoding 10× genetic distance criterion, underlining the difficulties of applying a standard distance threshold to species delimitation. Phylogeographic analysis of COI data suggests that forest fragmentation may have played an important role in the recent evolutionary diversification of these butterflies. Further work on other Malagasy taxa using both mitochondrial and nuclear data will provide better insight into the role of historical habitat fragmentation in species diversification and may potentially contribute to the identification of priority areas for conservation.     

Daphnia diversity on the Tibetan Plateau measured by DNA taxonomy

Daphnia on the Tibetan Plateau has been little studied, and information on species diversity and biogeography is lacking. Here, we conducted a 4‐year survey using the barcoding fragment of the mitochondrial COI gene to determine the distribution and diversity of Daphnia species found across the Plateau. Our results show that species richness is higher than previously thought, with total described and provisional species number doubling from 5 to 10. Six of the taxonomic units recovered by DNA taxonomy agreed well with morphology, but DNA barcoding distinguished three clades each for the D. longispina (D. galeata, D. dentifera, and D. longispina) and D. pulex (D. pulex, D. cf. tenebrosa, and D. pulicaria) complexes. The sequence divergence between congeneric species varied within a large range, from 9.25% to 30.71%. The endemic D. tibetana was the most common and widespread species, occurring in 12 hyposaline to mesosaline lakes. The lineage of D. longispina is the first confirmed occurrence in west Tibet.     

线粒体COⅠ基因在昆虫DNA条形码中的研究与应用

自2003年DNA条形码(DNA barcodes)概念出现以来,DNA条形码技术(DNA barcoding)受到生物分类学领域普遍关注,线粒体细胞色素氧化酶亚基I(mtDNACOⅠ)被用作动物类群的主要条形码序列,基于该基因片段的昆虫条形码研究在国内外广泛开展。本文在概述DNA条形码、条形码技术及已开展的昆虫条形码研究计划的基础上,总结了昆虫mtDNACOⅠ条形码及其技术在发现和描述隐种、种类分子鉴定以及系统发育等方面的研究进展,分析了细胞核线粒体假基因(Numts)对mtDNACOⅠ条形码扩增的影响,提出检测和避免Numts的方法,并对DNA条形码技术的进一步研究和应用进行了讨论和展望。     

Is DNA barcoding child's play? Science education and the utility of DNA barcoding for the discrimination of UK tree species

We present the findings of a DNA barcoding study of the UK tree flora, implemented as part of an innovative, research‐based science education programme called ‘Tree School’. The UK tree flora comprises native and introduced species, and is a taxonomically diverse study group for the exploration of the potential and limitations of DNA barcoding. The children participating in the project collected voucher specimens and generated DNA barcode sequences from trees and shrubs found in the grounds and surrounding woodlands of a residential field centre in Dorset, UK. We assessed the potential of rbcL and matK markers for amplification and DNA sequencing success and for species discrimination among the 67 tree and shrub species included in this study. Although we achieved 100% PCR amplification and sequencing success for rbcL and matK, mononucleotide repeats affected sequence quality in matK for some taxonomic groups (e.g. Rosaceae). Species discrimination success ranged from 65% to 71% using tree‐based methods to 86% using BLASTN. The occurrence of known hybrids (diploid and polyploid) and their progenitors on the study site reduced the overall species discrimination success for both loci. This study demonstrates that, even in a floristic context, rbcL and matK alone are insufficient for the discrimination of UK tree species, especially where taxonomically complex groups are present. From a science education perspective, DNA barcoding represents a compelling and accessible platform for the engagement of non‐experts in ongoing research, providing an opportunity for them to contribute authentic scientific data to an international research campaign.     

Deep sympatric mtDNA divergence in the autumnal moth (Epirrita autumnata)

Deep sympatric intraspecific divergence in mtDNA may reflect cryptic species or formerly distinct lineages in the process of remerging. Preliminary results from DNA barcoding of Scandinavian butterflies and moths showed high intraspecific sequence variation in the autumnal moth, Epirrita autumnata. In this study, specimens from different localities in Norway and some samples from Finland and Scotland, with two congeneric species as outgroups, were sequenced with mitochondrial and nuclear markers to resolve the discrepancy found between mtDNA divergence and present species‐level taxonomy. We found five COI sub‐clades within the E. autumnata complex, most of which were sympatric and with little geographic structure. Nuclear markers (ITS2 and Wingless) showed little variation and gave no indications that E. autumnata comprises more than one species. The samples were screened with primers for Wolbachia outer surface gene (wsp) and 12% of the samples tested positive. Two Wolbachia strains were associated with different mtDNA sub‐clades within E. autumnata, which may indicate indirect selection/selective sweeps on haplotypes. Our results demonstrate that deep mtDNA divergences are not synonymous with cryptic speciation and this has important implications for the use of mtDNA in species delimitation, like in DNA barcoding.     

DNA barcoding of bark and ambrosia beetles reveals excessive NUMTs and consistent east‐west divergence across Palearctic forests

A comprehensive DNA barcoding library is very useful for rapid identification and detection of invasive pest species. We tested the performance of species identification in the economically most damaging group of wood‐boring insects – the bark and ambrosia beetles – with particular focus on broad geographical sampling across the boreal Palearctic forests. Neighbour‐joining and Bayesian analyses of cytochrome oxidase I (COI) sequences from 151 species in 40 genera revealed high congruence between morphology‐based identification and sequence clusters. Inconsistencies with morphological identifications included the discovery of a likely cryptic Nearctic species of Dryocoetes autographus, the possible hybrid origin of shared mitochondrial haplotypes in Pityophthorus micrographus and P. pityographus, and a possible paraphyletic Xyleborinus saxeseni. The first record of Orthotomicus suturalis in North America was confirmed by DNA barcoding. The mitochondrial data also revealed consistent divergence across the Palearctic or Holarctic, confirmed in part by data from the large ribosomal subunit (28S). Some populations had considerable variation in the mitochondrial barcoding marker, but were invariant in the nuclear ribosomal marker. These findings must be viewed in light of the high number of nuclear insertions of mitochondrial DNA (NUMTs) detected in eight bark beetle species, suggesting the possible presence of additional cryptic NUMTs. The occurrence of paralogous COI copies, hybridization or cryptic speciation demands a stronger focus on data quality assessment in the construction of DNA barcoding databases.     

Next‐generation DNA barcoding: using next‐generation sequencing to enhance and accelerate DNA barcode capture from single specimens

DNA barcoding is an efficient method to identify specimens and to detect undescribed/cryptic species. Sanger sequencing of individual specimens is the standard approach in generating large‐scale DNA barcode libraries and identifying unknowns. However, the Sanger sequencing technology is, in some respects, inferior to next‐generation sequencers, which are capable of producing millions of sequence reads simultaneously. Additionally, direct Sanger sequencing of DNA barcode amplicons, as practiced in most DNA barcoding procedures, is hampered by the need for relatively high‐target amplicon yield, coamplification of nuclear mitochondrial pseudogenes, confusion with sequences from intracellular endosymbiotic bacteria (e.g. Wolbachia) and instances of intraindividual variability (i.e. heteroplasmy). Any of these situations can lead to failed Sanger sequencing attempts or ambiguity of the generated DNA barcodes. Here, we demonstrate the potential application of next‐generation sequencing platforms for parallel acquisition of DNA barcode sequences from hundreds of specimens simultaneously. To facilitate retrieval of sequences obtained from individual specimens, we tag individual specimens during PCR amplification using unique 10‐mer oligonucleotides attached to DNA barcoding PCR primers. We employ 454 pyrosequencing to recover full‐length DNA barcodes of 190 specimens using 12.5% capacity of a 454 sequencing run (i.e. two lanes of a 16 lane run). We obtained an average of 143 sequence reads for each individual specimen. The sequences produced are full‐length DNA barcodes for all but one of the included specimens. In a subset of samples, we also detected Wolbachia, nontarget species, and heteroplasmic sequences. Next‐generation sequencing is of great value because of its protocol simplicity, greatly reduced cost per barcode read, faster throughout and added information content.     

DNA barcoding of crickets,katydids and grasshoppers (Orthoptera) from Central Europe with focus on Austria,Germany and Switzerland

We present a DNA barcoding study on the insect order Orthoptera that was generated in collaboration between four barcoding projects in three countries, viz. Barcoding Fauna Bavarica (Germany), German Barcode of Life, Austrian Barcode of Life and Swiss Barcode of Life. Our data set includes 748 COI sequences from 127 of the 162 taxa (78.4%) recorded in the three countries involved. Ninety‐three of these 122 species (76.2%, including all Ensifera) can be reliably identified using DNA barcodes. The remaining 26 caeliferan species (families Acrididae and Tetrigidae) form ten clusters that share barcodes among up to five species, in three cases even across different genera, and in six cases even sharing individual barcodes. We discuss incomplete lineage sorting and hybridization as most likely causes of this phenomenon, as the species concerned are phylogenetically young and hybridization has been previously observed. We also highlight the problem of nuclear mitochondrial pseudogenes (numts), a known problem in the barcoding of orthopteran species, and the possibility of Wolbachia infections. Finally, we discuss the possible taxonomic implications of our barcoding results and point out future research directions.     

DNA Barcodes indicate members of the Anopheles fluviatilis (Diptera: Culicidae) species complex to be conspecific in India

Anopheles fluviatilis, a major vector of malaria in India has been described as a complex of three sibling species members, named as S, T and U, based on variations in chromosomal inversions. Also, ribosomal DNA markers (repetitive Internal Transcribed Spacer 2 (ITS2) and 28S D3 region) were described to differentiate these three sibling species members. However, controversies prevail on the genetic isolation status of these cryptic species. Hence, we evaluated this taxonomic incongruence employing DNA barcoding, the well established methodology for species identification, using 60 An. fluviatilis sensu lato specimens, collected from two malaria endemic eastern states of India. These specimens were also subjected to sibling species characterization by ITS2 and D3 DNA markers. The former marker identified 31 specimens among these as An. fluviatilis S and 21 as An. fluviatilis T. Eight specimens amplified DNA fragments specific for both S and T. The D3 marker characterized 39 specimens belonging to species S and 21 to species T. Neither marker identified species U. Neighbor Joining analysis of mitochondrial cytochrome c oxidase gene 1 sequences (the DNA barcode) categorized all the 60 specimens into a single operational taxonomic unit, their Kimura 2 parameter (K2P) genetic variability being only 0.8%. The genetic differentiation (F_ST) and gene flow (N_m) estimates were 0.00799 and 62.07, respectively, indicating these two ‘species’ (S & T) as genetically con‐specific intermixing populations with negligible genetic differentiation. Earlier investigations have refuted the existence of species U. Also, this study demonstrated that An. fluviatilis and the closely related An. minimus could be taxonomically differentiated by the DNA Barcode approach (K2P = 5.0%).