Assessing the diversity and distribution of Cephalothrix species (Nemertea: Palaeonemertea) in European waters by comparing different species delimitation methods

Soft‐bodied marine taxa, like ribbon worms (Nemertea), often lack clear diagnostic morphological characters impeding traditional species delimitation. Therefore, recent studies concentrated on molecular genetic methods to solve taxonomic issues. Different delimitation methods were employed to explore species boundaries and the presence of cryptic species. However, the performance of the different delimitation methods needs to be tested. A particularly promising nemertean genus in this regard is the palaeonemertean genus Cephalothrix that is commonly found in European waters. In order to gain information on the number and distribution of European cephalotrichids and to test different tree‐based and non‐tree‐based delimitation methods, we analyzed a dataset comprising the barcoding region of the mitochondrial cytochrome c oxidase subunit I (COI) of 215 European Cephalothrix specimens, of which 78 were collected for this study. Our results show the presence of 12–13 European lineages of which several can be assigned to known European species. Analyzing a second dataset comprising 74 additional sequences from the Pacific and the Atlantic Oceans helped identify some of the unassigned European specimens. One resulting clade seems to represent a non‐native introduced Cephalothrix species, while another has never been recorded from Europe before. In our analysis, especially the tree‐based methods and the phylogenetic analysis proved to be a useful tool when delimiting species. It remains unclear whether the different identified clades result from cryptic speciation or from a high genetic variability of the COI gene.     

DNA barcoding supports reclassification of Japanese Chironomus species (Diptera: Chironomidae)

Non‐biting midges (Diptera: Chironomidae) adapt to species‐specific environmental conditions and hence are promising bioindicators for aquatic and ecotoxicological monitoring. Although their utility for these purposes was historically limited by difficulties in their morphological identification, DNA barcoding offers a possible solution. Here, eight Japanese species of the genus Chironomus, which is characterized by its worldwide distribution and abundance among Chironomidae, were subjected to DNA barcoding using cytochromec oxidase subunit I (COI). To examine whether this DNA barcode is a useful indicator for Japanese species of Chironomus, we calculated genetic distances within and between the COI sequences of Chironomus species both from this study and worldwide and constructed phylogenetic trees. Based on 415 bp COI sequences and the Kimura two‐parameter model, the average genetic distances within 37 species and between 72 species were 2.6% and 17.2%, respectively. Although the ranges of genetic distances within and between species overlapped from 0.8% to 17.3%, 99.7% of average genetic distances between species were >3.0%. Some of this overlap is attributable to distances within species that were “too large” as well as those between species that were “too small”. Of eight Japanese species examined, two showed genetic distances between species that were below a 3.0% threshold, and four had distances within species that were greater than 3.0%. These results suggest a possible reclassification of these species and the need for further sampling to unveil biogeographic variations among different countries and regions.     

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.     

Population structure,population history and DNA barcoding of fruit fly Bactrocera latifrons (Hendel) (Diptera: Tephritidae)

The fruit fly Bactrocera latifrons (Hendel) is an important pest of commercially significant plants such as chili, tomato and eggplant. The species is native to South and Southeast Asia, but has now invaded Japan, Hawaii and Africa. In this study, mitochondrial DNA sequences were used to infer genetic structure and demographic history of B. latifrons. The efficiency of DNA barcodes for identification of B. latifrons was also tested. Ninety‐three specimens infesting four host‐plant species were obtained from 11 sampling locations in Thailand. The mitochondrial haplotype network revealed no major divergent lineage, which was consistent with a phylogenetic analysis that found strong support for the monophyly of B. latifrons. Population pairwise F_ST revealed that most (65%) comparisons were not significantly different, suggesting a high rate of gene flow. Analysis of molecular variance (amova ) found no significant genetic differentiation among populations from different host‐plant species. Sharing of several haplotypes among flies from different host‐plants indicates that the flies were moved freely across the plant species. Demographic history analysis revealed that the population has undergone recent expansion dating back to the end of the last glaciation. Thus, the results indicate that both ongoing and historical factors have played important roles in determining the genetic structure and diversity of B. latifrons. DNA barcoding analysis revealed that B. latifrons specimens were clearly differentiated from other species with 100% correct identification. Therefore, cytochrome oxidase I (COI) barcoding sequences could be effectively used to identify this important pest species, which could encourage monitoring and control efforts for this species.     

Analysis of mitochondrial COI sequences of the Diachasmimorpha longicaudata (Hymenoptera: Braconidae) species complex in Thailand

Diachasmimorpha longicaudata is an Opiinae parasitoid used to control tephritid fruit flies, which cause tremendous economic losses of fruits worldwide. In Thailand, D. longicaudata is classified as three sibling species, DLA, DLB and DLBB, based on the morphological and biological species concepts but their genetic variation has not been studied. Therefore, we investigated the genetic differentiation of the mitochondrial COI gene to clarify the ambiguous taxonomy of this species complex. The 603‐bp COI region was sequenced from laboratory‐bred colonies and field‐collected specimens from seven locations representing five geographical regions in Thailand. DLA was associated with the host Bactrocera correcta while DLB and DLBB were associated with Bactrocera dorsalis. The interspecific nucleotide differences of COI sequences among the three groups ranged from 6.70% to 7.62% (Kimura 2‐parameter distance), which adequately separates species complexes within the order Hymenoptera and supports the current sibling species classification. The neighbor joining, maximum likelihood and consensus Bayesian phylogenetic trees constructed from COI sequences revealed that the three sibling species of laboratory and field‐collected D. longicaudata are monophyletic with 100% support. The high genetic variation and molecular phylogeny of the COI sequences were shown to discriminate between the D. longicaudata species examined in this study.     

The gnd gene of Buchnera as a new,effective DNA barcode for aphid identification

DNA barcoding uses a standard DNA sequence to facilitate species identification. Although the COI gene has been adopted as the standard, COI alone is imperfect due to several shortcomings. The primary endosymbiont of aphids, Buchnera, has higher evolutionary rates and interspecies divergence than its co‐diverging aphid hosts, making it a potential tool for resolving the ambiguities in aphid taxonomy. We compared the effectiveness of employing two different DNA regions, gnd and COI, for the discrimination of over 100 species of aphids. The mean interspecific divergence of the gnd region was significantly higher than the mean intraspecific variation; there were nearly nonoverlapping distributions between the intra‐ and interspecific samples. In contrast, COI showed a lower interspecific divergence, which led to difficulties in identifying closely related species. Our results show that gnd can identify species in the Aphididae, which suggests that the gnd region of Buchnera is a potentially effective barcode for aphid species identification. We also recommend the 2‐locus combination of gnd + COI as the aphid barcode. This will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of aphids.     

Revised status of Chloridea Duncan and (Westwood), 1841, for the Heliothis virescens species group (Lepidoptera: Noctuidae: Heliothinae) based on morphology and three genes

The Heliothinae comprise some of the world's most injurious agricultural pests. This study reanalyses a subsample of the Heliothis group to determine the monophyly of Chloridea (Heliothis virescens and H. subflexa). Two nuclear gene regions, elongation factor‐1α (EF‐1α; 1240 bp) and dopa decarboylase (DDC; 687 bp), and the barcoding region of mitochondrial cytochrome oxidase I (COI; 708 bp) were used in this analysis for a total of 2635 bp and a morphological dataset of 20 characters and 62 character states. Sixteen species representing five genera plus two outgroup species were used in the analysis. Analyses used were Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian Inference (BI). The revised status for the monophyletic genus Chloridea Duncan and (Westwood) was supported by a very strong bootstrap support (BP = 98–100). Larval host‐plant usage is discussed within the Heliothis clade. Polyphagy is most likely the ancestral condition with a host shift to monophagy and oligophagy. Based on known larval hosts, Heliocheilus is oligophagous on Poaceae. Traits of host plant use in Helicoverpa and Chloridea where both polyphagy and oligophagy occur in closely related species are discussed.     

Protein–protein interactions of the cytochrome c oxidase DNA barcoding region

Enzymatic amplification of homologous regions of DNA using ‘universal’ polymerase chain reaction primers has provided insight into insect systematics, phylogeography, molecular evolution and species identification. One of the more commonly amplified and sequenced regions is a short region of the cytochrome c oxidase subunit I gene (COI), commonly called the barcoding region. COI is one of three mitochondrial‐encoded subunits of complex IV (Cox) of the electron transport chain. In addition to the mitochondrial subunits there are nine nuclear‐encoded subunits of the complex in Drosophila. Whereas a number of phylogenetic biases associated with this region have been examined and the quaternary structure of Cox has been modelled, the influence of protein–protein interactions on the observed patterns of evolution in this barcoding region of insects has never been examined critically. Using a well‐resolved independently derived phylogeny of 38 Diptera species, we examined the homogeneity of the substitution processes within the barcoding region. We show that, within Diptera, amino acid residues interacting with nuclear‐encoded subunits of Cox are evolving at elevated rates across the phylogeny. Furthermore, we show that codon position two is biased by protein–protein interactions. In contrast, third codon positions provide a less biased estimate of genetic variation in the region. This study highlights the need to examine the potential for systematic bias in DNA barcoding regions as part of the critical assessment of evidence in systematics and in biodiversity assessments.     

The first confirmed cases of full albinism in rajid species

Three albino skate specimens (Rajidae) were captured from the North Sea and English Channel between 2008 and 2011. Using DNA barcoding (COI gene) and morphometric analyses, species were identified as a spotted ray Raja montagui, a blonde ray Raja brachyura and a thornback ray Raja clavata. This finding represents the first record of full albinism (a lack of skin and retinal pigmentation) in rajid species.     

Genetic diversity and population structure of Culex modestus across Europe: does recent appearance in the United Kingdom reveal a tendency for geographical spread?

In mainland Europe, the mosquito species Culex modestus Ficalbi (1890) is a bridge vector for West Nile virus (WNV) from its natural bird-mosquito cycle to mammals. The present study assessed the genetic diversity of Cx. modestus, as well as related Culex species, using the mitochondrial COI DNA barcoding region and compared this with the population structure across Europe. A haplotype network was mapped to determine genealogical relationships among specimens. The intraspecific genetic diversity within individual Culex species was below 2%, whereas the interspecific genetic divergence varied from 2.99% to 13.74%. In total, 76 haplotypes were identified among 198 sequences. A median-joining network determined from 198 COI sequences identified two major lineages that were separated by at least four mutation steps. A high level of intraspecific genetic diversity was not detected in Cx. modestus in samples submitted from different European populations, which indicates that morphologically identified specimens represent a single species and not a species complex. Therefore, it is deduced that different populations of Cx. modestus will show a similar potential to transmit WNV, lending support to concerns that the population present in southeast England represents a risk of transmission to humans.     

DNA barcoding of economically important freshwater fish species from north‐central Nigeria uncovers cryptic diversity

This study examines the utility of morphology and DNA barcoding in species identification of freshwater fishes from north‐central Nigeria. We compared molecular data (mitochondrial cytochrome c oxidase subunit I (COI) sequences) of 136 de novo samples from 53 morphologically identified species alongside others in GenBank and BOLD databases. Using DNA sequence similarity‐based (≥97% cutoff) identification technique, 50 (94.30%) and 24 (45.30%) species were identified to species level using GenBank and BOLD databases, respectively. Furthermore, we identified cases of taxonomic problems in 26 (49.00%) morphologically identified species. There were also four (7.10%) cases of mismatch in DNA barcoding in which our query sequence in GenBank and BOLD showed a sequence match with different species names. Using DNA barcode reference data, we also identified four unknown fish samples collected from fishermen to species level. Our Neighbor‐joining (NJ) tree analysis recovers several intraspecific species clusters with strong bootstrap support (≥95%). Analysis uncovers two well‐supported lineages within Schilbe intermedius. The Bayesian phylogenetic analyses of Nigerian S. intermedius with others from GenBank recover four lineages. Evidence of genetic structuring is consistent with geographic regions of sub‐Saharan Africa. Thus, cryptic lineage diversity may illustrate species’ adaptive responses to local environmental conditions. Finally, our study underscores the importance of incorporating morphology and DNA barcoding in species identification. Although developing a complete DNA barcode reference library for Nigerian ichthyofauna will facilitate species identification and diversity studies, taxonomic revisions of DNA sequences submitted in databases alongside voucher specimens are necessary for a reliable taxonomic and diversity inventory.     

Complete mitochondrial genome and assembled DNA barcoding analysis of Lutjanus fulgens (Valenciennes, 1830) and its comparison with other Lutjanus species

Lutjanus fulgens (Valenciennes, 1830) is a teleost species classified under the family Lutjanidae which is a native of the Eastern Atlantic Ocean. Though highly commercialized due to its abundance and good taste, the production output has declined in recent years. This is an indication of the need for effective management and conservation measures. However, accurate species identification will ensure strategic management and conservation measure. DNA‐based species identification has proven its reliability in this regard via precise species identification. Several researchers have confirmed the accuracy of DNAbarcode as a species identification tool as well as species phylogeny analysis based on both the complete mitogenome and COI gene. Currently, nine specimens of L. fulgens were sampled from Ghana and subjected to DNA‐based analysis, namely, complete mitochondrial DNAand COI gene (DNA barcoding) analyses. The mitogenomic result revealed that L. fulgens is made up of a 16,500 base pairs (bp) mtDNA which consists of 22 transfer RNAs, 13 protein‐coding genes, and two ribosomal RNAs (GenBank Accession Number: MN398650). Furthermore, a sequence polymorphism analysis of the COIgene (MN986442–MN986450) detected two haplotypes. These haplotypes were both collected from the same fish landing site which suggests a possible cryptic linage diversity in the L. fulgens population at Vodza. According to the phylogeny examination, a close taxonomic relationship exists between L. fulgens and Lutjanus buccanella caused by a recent evolution termed as sympatric speciation. This study serves as a novel study for this species, building the foundation for future molecular‐based study for this species and as a DNA barcode reference data.     

Barcoding,molecular taxonomy,and exploration of the diversity of shrews (Soricomorpha: Soricidae) on Mount Nimba (Guinea)

We tested the efficiency of cytochrome oxidase I (COI)‐barcoding as a taxonomic tool to discriminate and identify sympatric shrew species on Mount Nimba (Guinea). We identified 148 specimens at the species level using morphological characters and comparison with type specimens, including several taxa from Mount Nimba. We identified ten morphospecies and tested aspects of genetic diversity and monophyly using genetic data from three mitochondrial (16S, cytochrome b, and COI) and one nuclear marker (the breast cancer gene, BRCA). Nine morphospecies were validated under the phylogenetic and genetic species concepts, including the recently diverged species Crocidura buettikoferi, Crocidura theresae, and Crocidura grandiceps. Under the same concepts, our analyses revealed the presence of two cryptic species amongst animals identified as Crocidura muricauda. We then tested the efficiency of barcoding thanks to commonly used phenetic methods, with the 148 specimens representing 11 potentially valid species based on morphological and molecular data. We show that COI‐barcoding is a powerful tool for shrew identification and can be used for taxonomic surveys. The comparison of genetic divergence values shows the presence of a barcoding gap (i.e. difference between the highest intraspecific and the lowest interspecific genetic divergence values). Given that only a few COI sequences are available for Afrotropical shrews, our work is an important step forward toward their enrichment. We also tested the efficiency of the three other sequenced markers and found that cytochrome b is as efficient as COI for barcoding shrews. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166 , 672–687.     

DNA barcoding of microgastrine parasitoid wasps (Hymenoptera: Braconidae) using high‐throughput methods more than doubles the number of species known for Australia

The Microgastrinae are a hugely diverse subfamily of endoparasitoid wasps of lepidopteran caterpillars. They are important in agriculture as biological control agents and play a significant ecological role in the regulation of caterpillar populations. Whilst the group has been the focus of intensive rearing and DNA barcoding studies in the Northern Hemisphere, the Australian fauna has received little attention. In total, 99 species have been described from or have been introduced into Australia, but the real species diversity for the region is clearly much larger than this. In this study, museum ethanol samples and recent field collections were mined for hundreds of specimens of microgastrine wasps, which were then barcoded for the COI region, ITS2 ribosomal spacer and the wingless nuclear genes, using a pooled sequencing approach on an Illumina Miseq system. Full COI sequences were obtained for 525 individuals which, when combined with 162 publicly available sequences, represented 417 haplotypes, and a total of 236 species were delimited using a consensus approach. By more than doubling the number of known microgastrine wasp species in Australia, our study highlights the value of DNA barcoding in the context of employing high‐throughput sequencing methods of bulk ethanol museum collections for biodiversity assessment.     

The cold‐adapted population of Folsomia manolachei (Hexapoda,Collembola) from a glaciated karst doline of Central Europe: evidence for a cryptic species?

Folsomia manolachei Bagnall, 1939 (Collembola), is a widespread and common European species. However, it may represent a complex of species also associated with the climatically more extreme environments of the karst landforms. Three species of the genus Folsomia, distributed in the Slovak Karst region (Central Europe), namely three different populations of F. manolachei, and one population of F. penicula and F. candida, were analysed using a partial sequence of the mitochondrial cytochrome c oxidase subunit I (COI barcoding section). The DNA barcoding suggested the existence of cryptic diversity in populations of the eurytopic species Folsomia manolachei. The cold‐adapted population of ‘F. manolachei’ was abundant in primary soil on stony debris near the permanent floor ice (yearly air temperature ~0°C) in the collapsed karst doline of the Silická ?adnica Ice Cave. It showed a genetic differentiation supported by intra‐ and interspecific distances that ranged from 0.0% to 1.4% and from 19.2% to 24.0%, respectively. Analysis using Taxon DNA showed a large barcoding gap between intra‐ and interspecific COI sequences. Genetic differentiation suggests a scenario of cryptic speciation in the population of ‘F. manolachei’ occupying harsh soils near the floor ice. A survival test showed the different responses of ‘F. manolachei’ and other populations to low temperature. Within a temperature range from ?3 to ?10°C, the ‘F. manolachei’ population from Silická ?adnica was the most cold‐resistant, showing a lethal dose LD₅₀ of ?7.8°C. The two forest populations of F. manolachei had LD₅₀ ?6.1°C and ?6.0°C, respectively; the most cold‐sensitive F. penicula showed an LD₅₀ of ?5.4°C. The survival of the tested springtails significantly decreased with temperature (p < 0.0001). The lethal temperature and the shape of the survival–temperature curves were different in different populations. The impact of population was significant at p < 0.0001, and the interaction between population and temperature at p < 0.039 was significant as well. Crypticity vs. phenotypic plasticity in Folsomia manolachei populations is discussed in terms of DNA barcoding and the cold tolerance data provided by this study.     

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.     

Species diversity can be overestimated by a fixed empirical threshold: insights from DNA barcoding of the genus Cletus (Hemiptera: Coreidae) and the meta‐analysis of COI data from previous phylogeographical studies

The use of genetic distances to identify species within the framework of DNA barcoding has to some extent improved the development of biodiversity studies. However, using a fixed empirical threshold to delimit species may lead to overestimating species diversity. In this study, we use a new data set of COI sequences for 366 specimens within the genus of Cletus as well as conduct an analysis on the same genetic data for collected morphologically defined species from previous phylogeographical studies, to test whether high intraspecific genetic divergences are common with the premises of comprehensive sampling. The results indicate C. graminis Hsiao & Cheng 1964 , is the same species with C. punctiger (Dallas, 1852) and should be synonymized and that the distributional record of C. pugnator (Fabricius, 1787) in China is correct. High intraspecific genetic differentiations (0%–4.35%) were found in C. punctiger. Furthermore, as to the mined data, the maximum intraspecific K2P distances of 186 species (48.44% of 384) exceed 3%, and 101 species (26.30%) can be divided into two or more clusters with a threshold of 3% in cluster analysis. If genetic distance is used to delimit species boundaries, the minimum interspecific K2P distance of the congeneric species should be considered rather than only using the fixed empirical value; otherwise, the species richness may be overestimated in some cases.     

Phylogenetic congruence between Mollitrichosiphum (Aphididae: Greenideinae) and Buchnera indicates insect–bacteria parallel evolution

We wanted to test whether Mollitrichosiphum, an aphid genus with life cycles on subtropical woody host plants, and Buchnera, the primary endosymbiont of aphids, evolve in parallel. We used three aphid genes (mitochondrial COI, cytochrome oxidase subunit I and Cytb, cytochrome b; nuclear EF1α, translation elongation factor 1 alpha) and two Buchnera genes (16S rDNA; gnd, gluconate‐6‐phosphate dehydrogenase) to reconstruct phylogenies. The congruence between the phylogenetic trees of aphids and Buchnera was then measured. The results present phylogenetic evidence for the parallel evolution of Mollitrichosiphum and Buchnera at the intraspecific as well as the interspecific levels. Our results support the possibility of using endosymbiont genes to study host evolutionary history and biogeographical patterns. We also investigated the usability of the Buchnera gnd gene as a barcoding marker for aphid identification.     

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.