Species identification of aphids (Insecta: Hemiptera: Aphididae) through DNA barcodes

A 658-bp fragment of mitochondrial DNA from the 5' region of the mitochondrial cytochrome c oxidase 1 (COI) gene has been adopted as the standard DNA barcode region for animal life. In this study, we test its effectiveness in the discrimination of over 300 species of aphids from more than 130 genera. Most (96%) species were well differentiated, and sequence variation within species was low, averaging just 0.2%. Despite the complex life cycles and parthenogenetic reproduction of aphids, DNA barcodes are an effective tool for identification.     

DNA barcoding of genus Toxoptera Koch (Hemiptera: Aphididae): Identification and molecular phylogeny inferred from mitochondrial COI sequences

Abstract Identification of aphid species is always difficult due to the shortage of easily distinguishable morphological characters. Aphid genus Toxoptera consists of species with similar morphology and similar to Aphis in most morphological characters except the stridulatory apparatus. DNA barcodes with 1 145 bp sequences of partial mitochondrial cytochrome‐coxidase I (COI) genes were used for accurate identification of Toxoptera. Results indicated mean intraspecific sequence divergences were 1.33%, whereas mean interspecific divergences were greater at 8.29% (0.13% and 7.79% if T. aurantii 3 and T. aurantii 4 are cryptic species). Sixteen samples were distinguished to four species correctly by COI barcodes, which implied that DNA barcoding was successful in discrimination of aphid species with similar morphology. Phylogenetic relationships among species of this genus were tested based on this portion of COI sequences. Four species of Toxoptera assembled a clade with low support in maximum‐parsimony (MP) analysis, maximum‐likelihood (ML) analysis and Bayesian phylogenetic trees, the genus Toxoptera was not monophyletic, and there were two sister groups, such as T. citricidus and T. victoriae, and two clades of T. aurantii which probably presented cryptic species in the genus.     

Assessing the diversity of New Zealand freshwater harpacticoid copepods (Crustacea: Copepoda) using mitochondrial DNA (COI) barcodes

Taxonomic and ecological studies of freshwater harpacticoid copepods are limited globally by the ability to easily and accurately identify specimens. Here, we test the use of the mitochondrial cytochrome c oxidase subunit I (COI) gene locus as a tool for assessing the diversity of freshwater Harpacticoida. We obtained sequences from New Zealand harpacticoid copepods, representing two families, five genera and nine species, including the non-indigenous Elaphoidella sewelli. All species were delineated by the COI gene. However, high intraspecific diversity was evident among populations of Elaphoidella bidens (>12%), and between North and South Island populations of Bryocamptus pygmaeus (>18%), potentially indicating the presence of morphologically cryptic taxa. We suggest that mitochondrial DNA (COI) sequences can provide a useful tool for the routine identification of freshwater harpacticoid copepods. Applications of these data will include assessing species diversity and biogeography as well as assisting with the detection of non-indigenous species.     

DNA barcoding of Caenogastropoda along coast of China based on the COI gene

DNA barcoding provides an efficient method for species-level identifications. In this study, we have amplified partial sequences of mitochondrial cytochrome c oxidase I (COI) gene from 110 specimens of 45 species of Caenogastropoda collected from the coast along China to evaluate whether DNA barcodes can distinguish these species accurately. The average Kimura 2-parameter (K2P) distances within species, genera and families were 0.44%, 13.96% and 22.27%, respectively. Both the neighbour-joining tree and the Bayesian tree showed a clear discrimination of all the species in our study with highly supported clades. These results proved that the species of Caenogastropoda can be efficiently and accurately identified by DNA barcoding based on the COI gene.     

Nondestructive DNA extraction from blackflies (Diptera: Simuliidae): retaining voucher specimens for DNA barcoding projects

A nondestructive, chemical-free method is presented for the extraction of DNA from small insects. Blackflies were submerged in sterile, distilled water and sonicated for varying lengths of time to provide DNA which was assessed in terms of quantity, purity and amplification efficiency. A verified DNA barcode was produced from DNA extracted from blackfly larvae, pupae and adult specimens. A 60-second sonication period was found to release the highest quality and quantity of DNA although the amplification efficiency was found to be similar regardless of sonication time. Overall, a 66% amplification efficiency was observed. Examination of post-sonicated material confirmed retention of morphological characters. Sonication was found to be a reliable DNA extraction approach for barcoding, providing sufficient quality template for polymerase chain reaction amplification as well as retaining the voucher specimen for post-barcoding morphological evaluation.     

Conflicting patterns of DNA barcoding and taxonomy in the cicada genus Tettigettalna from southern Europe (Hemiptera: Cicadidae)

DNA barcodes have great potential to assist in species identification, especially when high taxonomical expertise is required. We investigated the utility of the 5′ mitochondrial cytochrome c oxidase I (COI) region to discriminate between 13 European cicada species. These included all nine species currently recognized under the genus Tettigettalna, from which seven are endemic to the southern Iberian Peninsula. These cicadas have species‐specific male calling songs but are morphologically very similar. Mean COI divergence between congeners ranged from 0.4% to 10.6%, but this gene was proven insufficient to determine species limits within genus Tettigettalna because a barcoding gap was absent for several of its species, that is, the highest intraspecific distance exceeded the lowest interspecific distance. The genetic data conflicted with current taxonomic classification for T. argentata and T. mariae. Neighbour‐joining and Bayesian analyses revealed that T. argentata is geographically structured (clades North and South) and might constitute a species complex together with T. aneabi and T. mariae. The latter diverges very little from the southern clade of T. argentata and shares with it its most common haplotype. T. mariae is often in sympatry with T. argentata but it remains unclear whether introgression or incomplete lineage sorting may be responsible for the sharing of haplotypes. T. helianthemi and T. defauti also show high intraspecific variation that might signal hidden cryptic diversity. These taxonomic conflicts must be re‐evaluated with further studies using additional genes and extensive morphological and acoustic analyses.     

Identifying the true oysters (Bivalvia: Ostreidae) with mitochondrial phylogeny and distance-based DNA barcoding

Oysters (family Ostreidae), with high levels of phenotypic plasticity and wide geographic distribution, are a challenging group for taxonomists and phylogenetics. As a useful tool for molecular species identification, DNA barcoding might offer significant potential for oyster identification and taxonomy. This study used two mitochondrial fragments, cytochrome c oxidase I (COI) and the large ribosomal subunit (16S rDNA), to assess whether oyster species could be identified by phylogeny and distance-based DNA barcoding techniques. Relationships among species were estimated by the phylogenetic analyses of both genes, and then pairwise inter- and intraspecific genetic divergences were assessed. Species forming well-differentiated clades in the molecular phylogenies were identical for both genes even when the closely related species were included. Intraspecific variability of 16S rDNA overlapped with interspecific divergence. However, average intra- and interspecific genetic divergences for COI were 0-1.4% (maximum 2.2%) and 2.6-32.2% (minimum 2.2%), respectively, indicating the existence of a barcoding gap. These results confirm the efficacy of species identification in oysters via DNA barcodes and phylogenetic analysis.     

Rapid diagnostic method for discriminating two types of COI sequences in the gall‐forming aphid Tetraneura nigriabdominalis (Hemiptera: Aphididae) by multiplex polymerase chain reaction

Tetraneura nigriabdominalis (Sasaki, 1899) is a galling aphid that is distributed widely in Eurasia. In the present study, a diagnostic multiplex polymerase chain reaction (PCR) protocol was developed to discriminate two types of the cytochrome oxidase subunit I (COI) sequences in this species, and its usefulness was examined from 25 samples of T. nigriabdominalis. Multiplex PCR and electrophoresis revealed that 10 and 15 samples had one band of 133 bp (type A) and 426 bp (type B), respectively. This discrimination was identical to the division of the 25 samples based on the alignments of COI sequences (511 bp), indicating high accuracy of this protocol in distinguishing the two types. This protocol is useful for studying microgeographic distributions of the two types and genetic diversities in T. nigriabdominalis.     

The origin of the Tibetan Mastiff and species identification of Canis based on mitochondrial cytochrome c oxidase subunit I (COI) gene and COI barcoding

DNA barcoding is an effective technique to identify species and analyze phylogenesis and evolution. However, research on and application of DNA barcoding in Canis have not been carried out. In this study, we analyzed two species of Canis, Canis lupus (n = 115) and Canis latrans (n = 4), using the cytochrome c oxidase subunit I (COI) gene (1545 bp) and COI barcoding (648 bp DNA sequence of the COI gene). The results showed that the COI gene, as the moderate variant sequence, applied to the analysis of the phylogenesis of Canis members, and COI barcoding applied to species identification of Canis members. Phylogenetic trees and networks showed that domestic dogs had four maternal origins (A to D) and that the Tibetan Mastiff originated from Clade A; this result supports the theory of an East Asian origin of domestic dogs. Clustering analysis and networking revealed the presence of a closer relative between the Tibetan Mastiff and the Old English sheepdog, Newfoundland, Rottweiler and Saint Bernard, which confirms that many well-known large breed dogs in the world, such as the Old English sheepdog, may have the same blood lineage as that of the Tibetan Mastiff.     

Mitochondrial DNA sequences support allozyme evidence for cryptic radiation of New Zealand Peripatoides (Onychophora)

A combination of single-strand conformation polymorphism analysis (SSCP) and sequencing were used to survey cytochrome oxidase I (COI) mitochondrial DNA (mtDNA) diversity among New Zealand ovoviviparous Onychophora. Most of the sites and individuals had previously been analysed using allozyme electrophoresis. A total of 157 peripatus collected at 54 sites throughout New Zealand were screened yielding 62 different haplotypes. Comparison of 540-bp COI sequences from Peripatoides revealed mean among-clade genetic distances of up to 11. 4% using Kimura 2-parameter (K2P) analysis or 17.5% using general time-reversible (GTR + I + Gamma) analysis. Phylogenetic analysis revealed eight well-supported clades that were consistent with the allozyme analysis. Five of the six cryptic peripatus species distinguished by allozymes were confirmed by mtDNA analysis. The sixth taxon appeared to be paraphyletic, but genetic and geographical evidence suggested recent speciation. Two additional taxa were evident from the mtDNA data but neither occurred within the areas surveyed using allozymes. Among the peripatus surveyed with both mtDNA and allozymes, only one clear instance of recent introgression was evident, even though several taxa occurred in sympatry. This suggests well-developed mate recognition despite minimal morphological variation and low overall genetic diversity.     

Hidden diversity of Ctenophora revealed by new mitochondrial COI primers and sequences

The mitochondrial gene cytochrome-c-oxidase subunit 1 (COI) is useful in many taxa for phylogenetics, population genetics, metabarcoding, and rapid species identifications. However, the phylum Ctenophora (comb jellies) has historically been difficult to study due to divergent mitochondrial sequences and the corresponding inability to amplify COI with degenerate and standard COI “barcoding” primers. As a result, there are very few COI sequences available for ctenophores, despite over 200 described species in the phylum. Here, we designed new primers and amplified the COI fragment from members of all major groups of ctenophores, including many undescribed species. Phylogenetic analyses of the resulting COI sequences revealed high diversity within many groups that was not evident from more conserved 18S rDNA sequences, in particular among the Lobata (Ctenophora; Tentaculata; Lobata). The COI phylogenetic results also revealed unexpected community structure within the genus Bolinopsis, suggested new species within the genus Bathocyroe, and supported the ecological and morphological differences of some species such as Lampocteis cruentiventer and similar undescribed lobates (Lampocteis sp. “V” stratified by depth, and “A” differentiated by colour). The newly designed primers reported herein provide important tools to enable researchers to illuminate the diversity of ctenophores worldwide via quick molecular identifications, improve the ability to analyse environmental DNA by improving reference libraries and amplifications, and enable a new breadth of population genetic studies.     

DNA barcoding analysis of Coleoidea (Mollusca: Cephalopoda) from Chinese waters

Coleoids are part of the Cephalopoda class, which occupy an important position in most oceans both at an ecological level and at a commercial level. Nevertheless, some coleoid species are difficult to distinguish with traditional morphological identification in cases when specimens are heavily damaged during collection or when closely related taxa are existent. As a useful tool for rapid species assignment, DNA barcoding may offer significant potential for coleoid identification. Here, we used two mitochondrial fragments, cytochrome c oxidase I and the large ribosomal subunit (16S rRNA), to assess whether 34 coleoids accounting for about one-third of the Chinese coleoid fauna could be identified by DNA barcoding technique. The pairwise intra- and interspecific distances were assessed, and relationships among species were estimated by NJ and bayesian analyses. High levels of genetic differentiation within Loliolus beka led to an overlap between intra- and interspecific distances. All remaining species forming well-differentiated clades in the NJ and bayesian trees were identical for both fragments. Loliolus beka possessed two mitochondrial lineages with high levels of intraspecific distances, suggesting the occurrence of cryptic species. This study confirms the efficacy of DNA barcoding for identifying species as well as discovering cryptic diversity of Chinese coleoids. It also lays a foundation for other ecological and biological studies of Coleoidea.     

Incomplete estimates of genetic diversity within species: Implications for DNA barcoding

DNA barcoding has greatly accelerated the pace of specimen identification to the species level, as well as species delineation. Whereas the application of DNA barcoding to the matching of unknown specimens to known species is straightforward, its use for species delimitation is more controversial, as species discovery hinges critically on present levels of haplotype diversity, as well as patterning of standing genetic variation that exists within and between species. Typical sample sizes for molecular biodiversity assessment using DNA barcodes range from 5 to 10 individuals per species. However, required levels that are necessary to fully gauge haplotype variation at the species level are presumed to be strongly taxon‐specific. Importantly, little attention has been paid to determining appropriate specimen sample sizes that are necessary to reveal the majority of intraspecific haplotype variation within any one species. In this paper, we present a brief outline of the current literature and methods on intraspecific sample size estimation for the assessment of COI DNA barcode haplotype sampling completeness. The importance of adequate sample sizes for studies of molecular biodiversity is stressed, with application to a variety of metazoan taxa, through reviewing foundational statistical and population genetic models, with specific application to ray‐finned fishes (Chordata: Actinopterygii). Finally, promising avenues for further research in this area are highlighted.     

DNA barcoding and genetic diversity of phyllostomid bats from the Yucatan Peninsula with comparisons to Central America

The mitochondrial cytochrome c oxidase subunit I gene is the standard DNA barcoding region used for species identification and discovery. We examined the variation of COI (454 bp) to discriminate 20 species of bats in the family Phyllostomidae that are found in the Yucatan Peninsula of southeastern Mexico and northern Guatemala and compared them genetically to other samples from Central America. The majority of these species had low intraspecific variation (mean = 0.75%), but some taxa had intraspecific variation ranging to 8.8%, suggesting the possibility of cryptic species (i.e. Desmodus rotundus and Artibeus jamaicensis). There was a recurring biogeographic pattern in eight species with a separation of northern and southern Middle American localities. The Yucatan Peninsula was a discrete area identified in four species, whereas Panama was recovered in five species of phyllostomid bats. Our study establishes a foundation for further molecular work incorporating broader taxonomic and geographic coverage to better understand the phylogeography and genetic diversity that have resulted from the ecological constraints in this region and the remarkable differentiation of bats in the Neotropics.     

Genetic diversity of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) populations and presence of the B biotype and a non-B biotype that can induce silverleaf symptoms in squash, in Uganda

The extent of genetic variability and host‐plant distribution of Bemisia tabaci (Gennadius) genotypes colonising cultivated and uncultivated plant species occurring adjacent to cassava fields in selected cassava‐producing areas of Uganda in 2003/04 were investigated using the mitochondrial cytochrome oxidase I (mtCOI) gene as the molecular marker. Eight genotype clusters, Ug1–Ug8, which are supported by high bootstrap values (≥80), at 3–18% nt divergence, were revealed among the collective Ugandan B. tabaci populations. Ug1 and Ug2 (both cassava‐associated) and Ug8 (sweetpotato‐associated) have been reported previously in Uganda. Ug3 was genetically dissimilar to B. tabaci described elsewhere and colonised a single species, Ocimum gratissimum. Ug4–Ug7 formed four closely related subclusters (93–97% nt identity) and diverged by 15–18% from Ug1, Ug2, Ug3 and Ug8, respectively. Ug4 had as its closest relatives (at 97–99% nt identity) the Ivory Coast okra biotype, whereas genotypes Ug5 and Ug6 had as their closest relatives (at 95–99% and 99% nt identity, respectively) the Mediterranean–North Africa–Middle East (MED‐NAFR‐ME) biotypes, which also include the well‐studied B and Q biotypes. Ug7 was closely related (at 98–99% nt identity) to biotype Ms from the Reunion Island in the Indian Ocean. Ug4 colonised Cucurbita pepo, Cucurbita sativus, Leonotis nepetifolia and Pavonia urens, while Ug7 colonised Commelina benghalensis, Gossypium hirsutum and Phaseolus vulgaris. Ug6, the B‐biotype‐like genotype colonised Abelmoschus esculentus and C. benghalensis only. None of Ug4–Ug7 genotypes was found associated with, or colonising, cassava or sweetpotato plants. In addition to colonising sweetpotato, the Ug8 genotypes colonised Lycopersicon esculentum and L. nepetifolia. Ug6 and Ug7, both members of the B biotype/B‐like cluster, induced silverleaf symptoms on Cucurbita sp. The discovery of five previously identified B. tabaci genotype clusters, Ug3–Ug7, in Uganda, among which are some of the world's most economically important biotypes, namely B and Q, is particularly significant in the spread of geminiviruses with devastating effects to crop production in Africa.     

Environmental calcium and mitochondrial DNA polymorphism among local populations of Gammarus fossarum (Crustacea, Amphipoda)

Mitochondrial DNA sequence analysis was used to investigate genetic diversity and phylogeographic population structure among geographically isolated alpine populations of the amphipod Gammarus fossarum. This study was performed across a region with an important glacial history and substantial geographical variations in calcium levels, in order to evaluate the relative impact on the genetic diversity of the population history and this environmental factor known to be associated with local adaptation and population growth. Analysis of the nucleotide sequences for a 376-bp segment of the cytochrome c oxidase I gene in 84 specimens from seven different populations revealed 20 different haplotypes distributed into five major lineages that are not geographically structured. Analysis of molecular variance indicates that the populations from the crystalline massif are less diverse than those living on limestone, suggesting a global correlation between genetic diversity and calcium concentration of the water. However, the role of this environmental factor appears to be indirect, with smaller population size leading to lower genetic diversity.     

Identification of elmid larvae (Coleoptera: Elmidae) from Sanin District of Honshu,Japan, based on mitochondrial DNA sequences

Larvae of Elmidae from the Sanin District, Honshu, Japan, were classified into 14 types based on morphological features, of which 11 types were unidentified for species. Species or genus of the unidentified types were determined by comparing their mitochondrial cytochrome oxidase subunit I gene sequences with those of identified adult specimens. A new key to species/genera of elmid larvae was proposed.     

DNA barcoding of the genus Lepidion (Gadiformes: Moridae) with recognition of Lepidion eques as a junior synonym of Lepidion lepidion

DNA sequences of cytochrome c oxidase I gene (COI) from Lepidion spp. were employed to test the efficiency of species identification. A sample of 32 individuals from five Lepidion species was sequenced and combined with 26 sequences from other BOLD projects. As a result, 58 Lepidion DNA sequences of the COI gene belonging to eight of the nine recognized Lepidion species were analysed. Sequences were aligned and formed seven clades in a Bayesian phylogenetic tree, where Lepidion lepidion and Lepidion eques grouped jointly. The Kimura 2‐parameter genetic distances, among congeners were, on average, 4.28%, 16 times greater than among conspecifics (0.27%). The main diagnostic meristic data of Lepidion spp. were compiled and a detailed morphological revision of the congeneric species L. eques and L. lepidion was made. The eye diameter was significantly different between L. eques and L. lepidion (P < 0.001). The number of anal fin rays ranged from 45 to 51 in L. lepidion and from 47 to 54 in L. eques, but no significant differences were obtained in the mean values of this variable (P = 0.07). According to the morphological and genetic analyses, the results strongly suggest that the Mediterranean codling L. lepidion and the North Atlantic codling L. eques are conspecific, making L. eques a junior synonym of L. lepidion.     

Rapid and accurate taxonomic classification of insect (class Insecta) cytochrome c oxidase subunit 1 (COI) DNA barcode sequences using a naïve Bayesian classifier

Current methods to identify unknown insect (class Insecta) cytochrome c oxidase (COI barcode) sequences often rely on thresholds of distances that can be difficult to define, sequence similarity cut‐offs, or monophyly. Some of the most commonly used metagenomic classification methods do not provide a measure of confidence for the taxonomic assignments they provide. The aim of this study was to use a naïve Bayesian classifier (Wang et al. Applied and Environmental Microbiology, 2007; 73: 5261) to automate taxonomic assignments for large batches of insect COI sequences such as data obtained from high‐throughput environmental sequencing. This method provides rank‐flexible taxonomic assignments with an associated bootstrap support value, and it is faster than the blast ‐based methods commonly used in environmental sequence surveys. We have developed and rigorously tested the performance of three different training sets using leave‐one‐out cross‐validation, two field data sets, and targeted testing of Lepidoptera, Diptera and Mantodea sequences obtained from the Barcode of Life Data system. We found that type I error rates, incorrect taxonomic assignments with a high bootstrap support, were already relatively low but could be lowered further by ensuring that all query taxa are actually present in the reference database. Choosing bootstrap support cut‐offs according to query length and summarizing taxonomic assignments to more inclusive ranks can also help to reduce error while retaining the maximum number of assignments. Additionally, we highlight gaps in the taxonomic and geographic representation of insects in public sequence databases that will require further work by taxonomists to improve the quality of assignments generated using any method.     

High genetic diversity in the harvestman Geraeocormobius sylvarum (Arachnida,Opiliones, Gonyleptidae) from subtropical forests in north‐eastern Argentina revealed by mitochondrial DNA sequences

This research was aimed to analyse the genetic diversity of Geraeocormobius sylvarum, a forest‐dwelling Neotropical harvestman with a disjunct distribution, separated by approximately 630 km of semi‐arid environments. The usefulness of a fragment of the cytochrome c oxidase subunit I (COI) mitochondrial gene as molecular marker was tested in 109 individuals. Results showed high levels of both haplotype and nucleotide diversity in populations corresponding to north‐eastern Argentina, the core area of the species range. A strong genetic structuring was detected, supported by both the phylogenetic trees and the haplotype network, with six identifiable haplogroups. Populations of the Yungas ecoregion did not show significant diversity levels, suggesting a putative recent introduction of the species into that region. The overall results suggest that the present genetic diversity of the species is consistent with past fragmentation events of the species range (in refuges?), probably during the Last Glacial Maximum. The COI gene was concluded to be a well‐suited marker to associate past environmental events with the high genetic diversity observed in this species.