Cytochrome C Oxidase Subunit I Markers Research Articles

Genetic diversity, demography and connectivity in the marine snail Thaisella chocolata (Duclos, 1832): implications for sustainable fisheries management along the Peruvian coast

ABSTRACT Thaisella chocolata is a marine gastropod (family Muricidae) occurring from northern Peru to central Chile. It is of economic importance in these countries, where hundreds of tons are caught every year. In combination with El Niño–Southern Oscillation events, fishing activity has led to the collapse of Chilean populations, raising concerns about the state of Peruvian populations. The aim of this study is to assess the demography, genetic diversity and connectivity of T. chocolata along the Peruvian coast. This would provide important insights into the resilience of this species to anthropogenic disturbance. In this study, four locations along the coast of Peru were investigated for the first time. Mitochondrial cytochrome c oxidase subunit I (COI) sequences were obtained from 116 individuals and four microsatellites from 95 individuals. Both COI (combined with 156 publicly available sequences) and microsatellite data revealed very low genetic differentiation, despite a few significant φST and FST values between population pairs. The most likely gene flow model (MIGRATE-N) is panmixia between all locations, with gene flow occurring equally northwards and southwards. This pattern may be related to the surface currents along the Peruvian coastline combined with the high pelagic larval duration of T. chocolata. Hyperdiversity was found for the COI marker (πsyn > 5%). Microsatellite loci were also polymorphic, with a mean diversity of 12.69 alleles per locus per population. No recent population bottleneck was observed (microsatellite data), while at least one past demographic expansion was suggested (COI data). Given the high genetic diversity and connectivity, T. chocolata is expected to show high evolutionary resilience and can be considered as one management unit in terms of fisheries management. However, further studies, on, for example, the characterization of effective population size (using numerous nuclear loci), are required to confirm this hypothesis. In addition, precautionary fisheries measures should be maintained, given the recent collapse of T. chocolata stocks in Chile.

Integrated taxonomy of black flies (Diptera: Simuliidae) reveals unexpected diversity in the most arid ecosystem of Europe.

The family Simuliidae includes more than 2000 species of black flies worldwide. Their morphological uniformity creates difficulty for species identification, which limits our knowledge of their ecology and vectorial role. We investigated the systematics of black flies in a semi-arid area of the Iberian Peninsula, an ecologically harsh environment for these organisms. Sampling adult black flies in three different habitats (by means of CDC traps) and in avian nest boxes and collecting immature stages in high-salinity rills provided a representative sample of the component species. A combination of approaches, including morphological, chromosomal, and molecular (based on the mitochondrial cytochrome C oxidase subunit I (COI) and internal transcribed spacer 2 (ITS2) genes) revealed five species: four common species (Simulium intermedium, S. petricolum, S. pseudequinum, and S. rubzovianum) and the first European record for S. mellah. Barcoding gap and phylogenetic analyses revealed that ITS2 is a key marker to identify the species, whereas the COI marker does not provide enough resolution to identify some species or infer their phylogenetic relationships. Morphological and chromosomal features are also provided to identify S. mellah unequivocally. Our study highlights the need for integrated studies of black flies in ecologically extreme habitats to increase our knowledge of their distribution, ecology, and potential risks for public health.

New Morphological and Molecular Data Reveal an Underestimation of Species Diversity of Mites of the Genus Geckobia (Acariformes: Pterygosomatidae) in India

Mites of the genus Geckobia (Acariformes: Pterygosomatidae) are permanent and highly specialised ectoparasites of geckos (Gekkota). We conducted a local study on Geckobia mites associated with the geckos of the family Gekkonidae found mainly in the territory of the Indian Institute of Science’s campus (Bangalore, India). In total, we examined 208 lizards belonging to two genera: Hemidactylus and Cnemaspis. We assessed the prevalence of the mites and identified the preferred site for their infestation. We extended the standard morphological identification of the mite species by using DNA barcode markers, partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene and nuclear ribosomal gene sequences: 18S rRNA and hypervariable region D2 of nuclear 28S rRNA. We checked the suitability of COI and nuclear (D2 of 28S rRNA) markers for species delimitations and identification purposes of the genus. The distance- and phylogeny-based approaches were applied: (i) to test the presence of a barcoding gap, we used the automated barcoding gap discovery tool (ABGD) and investigated intra- and interspecific genetic distances, and (ii) to reconstruct evolutionary relationships within the species, we performed maximum likelihood (ML) and Bayesian inference with Markov-Chain Monte Carlo (BI) analyses. As a result, we described five new species—Geckobia gigantea sp. n., G. treutleri sp. n., G. unica sp. n. and G. brevicephala sp. n.—from four Hemidactylus species: H. giganteus, H. treutleri, H. parvimaculatus and H. frenatus, respectively, and G. mysoriensis sp. n. from Cnemaspis mysoriensis. Additionally, we found three already described species: Geckobia indica Hirst, 1917 on H. treutleri (new host), Geckobia bataviensis Vitzhum, 1926 on H. parvimaculatus (new host) and H. frenatus (new locality) and Geckobia phillipinensis Lawrence, 1953 on H. frenatus (new locality). The diagnoses of G. indica and G. phillipinensis were improved and supplemented by descriptions of the males and juveniles. Both topologies of the BI and ML phylogenetic trees, as well as genetic distances, supported the species boundaries in the mite population shown by the morphological data. Hemidactylus frenatus was the most infected gecko species (61% prevalence), with the highest number of mite species (three spp.). The scale-mite richness was higher than expected; therefore, further research is required to evaluate the true diversity of Geckobia mites.

Assessing population genetic structure of three New Zealand stream insects using mitochondrial and nuclear DNA markers.

Assessing genetic differentiation among natural populations can aid understanding of dispersal patterns and connectivity among habitats. Several molecular markers have become increasingly popular in determining population genetic structure for this purpose. Here, we compared the resolution of mitochondrial cytochrome c oxidase subunit I (COI) and nuclear single nucleotide polymorphism (SNP) markers for detecting population structure among stream insects at small spatial scales. Individuals of three endemic taxa-Coloburiscus humeralis (Ephemeroptera), Zelandobius confusus (Plecoptera), and Hydropsyche fimbriata (Trichoptera)-were collected from forested streams that flow across open pasture in the North Island of New Zealand. Both COI and SNP data indicated limited population structure across the study area, and small differences observed among these species were likely related to their putative dispersal abilities. For example, fine-scale genetic differentiation between and among neighbouring stream populations for H. fimbriata suggests that gene flow, and hence dispersal, may be more limited for this species relative to the others. Based on the generally similar results provided by both types of markers, we suggest that either COI or SNP markers can provide suitable initial estimates of fine-scale population genetic differentiation in stream insects.

Molecular characterization of spatially heterogeneous populations of the malaria vector (Anopheles gambiae s.l) in Niger State, Nigeria

In this study, Anopheles mosquitoes were characterized through an investigation of adult female mosquitoes for sibling species. For the first time, researchers combined efforts on morphology and DNA barcoding utilizing the markers cytochrome c oxidase subunit I (COI) gene and internal transcribed spacer 2 (ITS2) region was made. Cytochrome c oxidase subunit I and ITS2 regions of morphologically differentiated, Anopheline mosquitoes were sequenced. According to the results of molecular identification (PCR) of the distinctive sibling species of the Anopheline mosquitoes, two sibling species from An. gambiae complex and An. rufipes group were identified viz; An. gambiae ss (Giles 1902) and An. rufipes (Gough 1910), these sibling species were discovered in Bosso LGA's Large water and Gutter. An. gambiae s.s was collected in all the breeding habitats sampled in Katcha, Lapai and Shiroro LGAs. In this report, the habitat breeding density of the An. gambiae s.s. was found to be higher compared to An. rufipes, which was restricted to large water and gutter breeding habitats. This study highlights the usefulness and feasibility of COI and ITS2 genetic markers in recognizing Anophelines, their sibling species, the importance of a unified, systematic approach in mosquito taxonomy and highlights the epidemiological role of An. gambiae s.s. For the first time in Niger State An. rufipes has appeared as a malaria vector, emphasising the need for proper monitoring of this species across the country to effectively set up vector management strategies.

Brazilian invasion and dispersion routes of the cotton boll weevil

AbstractThe cotton boll weevil, Anthonomus grandis (Coleoptera: Curculionidae), is the major insect pest of cotton in the Americas. Although the demographic and dispersion patterns of the cotton boll weevil in the USA and Mexico were well characterized, in Brazil the information is scarce and does not include the main cotton‐producing regions of the country. Thus, our objective was to apply a phylogeographic approach sequencing a gene fragment of the cytochrome c oxidase subunit I (COI) to estimate the genetic diversity, identify the genetic strains, and describe the dispersion routes of the cotton boll weevil in the Brazilian territory. The COI marker revealed a low genetic diversity of the cotton boll weevil with only two haplotypes in Brazil. The haplotypes are genetically related to individuals from the eastern region of the USA and Mexico, which suggests a recent invasion event in Brazil. The genetic structure of COI haplotypes indicates a low dispersion of boll weevils between north/northeast and midwest/southeast regions of Brazil. The genetic drift and low gene flow during the insect expansion explain the COI haplotype distribution. The genetic structure of the cotton boll weevil in Brazil is novel information and provides valuable insight for the design of suitable pest management practices against this key pest species in the country.

DNA metabarcoding marker choice skews perception of marine eukaryotic biodiversity

AbstractDNA metabarcoding is an increasingly popular technique to investigate biodiversity; however, many methodological unknowns remain, especially concerning the biases resulting from marker choice. Regions of the cytochrome c oxidase subunit I (COI) and 18S rDNA (18S) genes are commonly employed “universal” markers for eukaryotes, but the extent of taxonomic biases introduced by these markers and how such biases may impact metabarcoding performance is not well quantified. Here, focusing on macroeukaryotes, we use standardized sampling from autonomous reef monitoring structures (ARMS) deployed in the world's most biodiverse marine ecosystem, the Coral Triangle, to compare the performance of COI and 18S markers. We then compared metabarcoding data to image‐based annotations of ARMS plates. Although both markers provided similar estimates of taxonomic richness and total sequence reads, marker choice skewed estimates of eukaryotic diversity. The COI marker recovered relative abundances of the dominant sessile phyla consistent with image annotations. Both COI and the image annotations provided higher relative abundance estimates of Bryozoa and Porifera and lower estimates of Chordata as compared to 18S, but 18S recovered 25% more phyla than COI. Thus, while COI more reliably reflects the occurrence of dominant sessile phyla, 18S provides a more holistic representation of overall taxonomic diversity. Ideal marker choice is, therefore, contingent on study system and research question, especially in relation to desired taxonomic resolution, and a multimarker approach provides the greatest application across a broad range of research objectives. As metabarcoding becomes an essential tool to monitor biodiversity in our changing world, it is critical to evaluate biases associated with marker choice.

Morphological and molecular characterization of Eustrongylides excisus larvae (Nematoda: Dioctophymatidae) in Sander lucioperca (L.) from Northern Turkey.

The genus Eustrongylides Jägerskiöld, 1909 includes parasitic nematodes (Dioctophymatidae) affecting various fish species and piscivorous birds of freshwater ecosystems. Currently, there is little information on the molecular characterization of E. excisus based on nuclear ribosomal internal transcribed spacer (ITS) rDNA regions. However, before the present study, there had been no reports of characterizing the E. excisus using nuclear small subunit ribosomal RNA (SSU rRNA) and mitochondrial cytochrome c oxidase subunit I (COI) genes sequences. In the present study, Eustrongylides spp. larvae were collected from pike-perch Sander lucioperca (L.) in Northern Turkey, and characterized by sequencing of ITS regions, SSU rRNA, and COI markers. Larvae herein morphologically identified as the fourth stage of Eustrongylides spp. were genetically identified as E. excisus based on the ITS sequence analysis. This study is the first record of SSU rRNA and COI sequences for E. excisus in GenBank. This is also a molecular characterization of E. excisus for the first time in Turkey. The ITS, SSU rRNA, and COI sequences of E. excisus can be used to establish the phylogenetic relationships of Eustrongylides species from Turkey and worldwide for further studies.

Cardinium symbiosis as a potential confounder of mtDNA based phylogeographic inference in Culicoides imicola (Diptera: Ceratopogonidae), a vector of veterinary viruses

BackgroundCulicoides imicola (Diptera: Ceratopogonidae) is an important Afrotropical and Palearctic vector of disease, transmitting viruses of animal health and economic significance including African horse sickness and bluetongue viruses. Maternally inherited symbiotic bacteria (endosymbionts) of arthropods can alter the frequency of COI (cytochrome c oxidase subunit I) mitochondrial haplotypes (mitotypes) in a population, masking the true patterns of host movement and gene flow. Thus, this study aimed to assess the mtDNA structure of C. imicola in relation to infection with Candidatus Cardinum hertigii (Bacteroides), a common endosymbiont of Culicoides spp.MethodsUsing haplotype network analysis, COI Sanger sequences from Cardinium-infected and -uninfected C. imicola individuals were first compared in a population from South Africa. The network was then extended to include mitotypes from a geographic range where Cardinium infection has previously been investigated.ResultsThe mitotype network of the South African population demonstrated the presence of two broad mitotype groups. All Cardinium-infected specimens fell into one group (Fisher’s exact test, P = 0.00071) demonstrating a linkage disequilibrium between endosymbiont and mitochondria. Furthermore, by extending this haplotype network to include other C. imicola populations from the Mediterranean basin, we revealed mitotype variation between the Eastern and Western Mediterranean basins (EMB and WMB) mirrored Cardinium-infection heterogeneity.ConclusionsThese observations suggest that the linkage disequilibrium of Cardinium and mitochondria reflects endosymbiont gene flow within the Mediterranean basin but may not assist in elucidating host gene flow. Subsequently, we urge caution on the single usage of the COI marker to determine population structure and movement in C. imicola and instead suggest the complementary utilisation of additional molecular markers.Graphical

The efficacy of COI barcoding and ISSR markers in molecular identification of diverse bird Sternula saundersi populations along the Red Sea coast, Kingdom of Saudi Arabia

Aim: To demonstrate the first DNA barcode and molecular diversity of bird Sternula saundersi (Saunders’s tern) populations collected from the Red Sea coast at the Kingdom of Saudi Arabia. Methodology: The mitochondrial cytochrome c oxidase subunit I (COI) gene was isolated and sequenced from 26 different individuals of S. saundersi, whose blood samples were collected from two different areas at the Red Sea coast of the Kingdom of Saudi Arabia. Also, the genetic diversity of 26 individuals were investigated using highly polymorphic nuclear markers, i.e., inter simple sequence repeat [ISSR] technique. Results: The COI gene sequences of all 26 individuals were documented and submitted to the GenBank database. Five unique single nucleotide polymorphisms were found in all individuals of S. saundersi that were not observed in other species of the genus Sternula. Consequently, these five nucleotides can be used as a distinctive DNA fingerprint of S. saundersi. Additionally, it was possible to identify molecular diversity within different populations from two different areas using eight different ISSR markers. Interpretation: The present data demonstrate the power of COI marker gene in identifying S. saundersi, i.e., it can be utilized to discriminate between different species of the genus Sternula. In addition, highly polymorphic nuclear markers (ISSR) were demonstrated to have significant power to accurately distinguish between individuals collected from different widely spaced areas.

Exposing the Barcoding Void: An Integrative Approach to Study Snail-Borne Parasites in a One Health Context.

Trematodes are snail-borne parasites of major zoonotic importance that infect millions of people and animals worldwide and frequently hybridize with closely related species. Therefore, it is desirable to study trematodiases in a One Health framework, where human and animal trematodes are considered equally important. It is within this framework that we set out to study the snail and trematode communities in four artificial lakes and an abattoir in Zimbabwe. Trematode infections in snails were detected through multiplex PCR protocols. Subsequently, we identified snails by sequencing a partial mitochondrial cytochrome c oxidase subunit I (COI) fragment, and trematodes (adults from the abattoir and larval stages detected in snails) using COI and nuclear rDNA markers. Of the 1,674 collected snails, 699 were molecularly analyzed, in which we identified 12 snail and 19 trematode species. Additionally, three parasite species were sampled from the abattoir. Merely four trematode species were identified to species level through COI-based barcoding. Moreover, identification of members of the superfamilies Opisthorchioidea and Plagiorchioidea required a phylogenetic inference using the highly conserved 18S rDNA marker, as no related COI reference sequences were present in public databases. These barcoding challenges demonstrate a severe barcoding void in the available databases, which can be attributed to the neglected status of trematodiases. Adding to this, many available sequences cannot be used as different studies use different markers. To fill this gap, more studies on African trematodes, using a standardized COI barcoding region, are desperately needed.

The efficiency of universal mitochondrial DNA barcodes for species discrimination of Pomacea canaliculata and Pomacea maculata.

Invasive apple snails, Pomacea canaliculata and P. maculata, have a widespread distribution globally and are regarded as devastating pests of agricultural wetlands. The two species are morphologically similar, which hinders species identification via morphological approaches and species-specific management efforts. Advances in molecular genetics may contribute effective diagnostic tools to potentially resolve morphological ambiguity. DNA barcoding has revolutionized the field of taxonomy by providing an alternative, simple approach for species discrimination, where short sections of DNA, the cytochrome c oxidase subunit I (COI) gene in particular, are used as ‘barcodes’ to delineate species boundaries. In our study, we aimed to assess the effectiveness of two mitochondrial markers, the COI and 16S ribosomal deoxyribonucleic acid (16S rDNA) markers for DNA barcoding of P. canaliculata and P. maculata. The COI and 16S rDNA sequences of 40 Pomacea specimens collected from six localities in Peninsular Malaysia were analyzed to assess their barcoding performance using phylogenetic methods and distance-based assessments. The results confirmed both markers were suitable for barcoding P. canaliculata and P. maculata. The phylogenies of the COI and 16S rDNA markers demonstrated species-specific monophyly and were largely congruent with the exception of one individual. The COI marker exhibited a larger barcoding gap (6.06–6.58%) than the 16S rDNA marker (1.54%); however, the magnitude of barcoding gap generated within the barcoding region of the 16S rDNA marker (12-fold) was bigger than the COI counterpart (approximately 9-fold). Both markers were generally successful in identifying P. canaliculata and P. maculata in the similarity-based DNA identifications. The COI + 16S rDNA concatenated dataset successfully recovered monophylies of P. canaliculata and P. maculata but concatenation did not improve individual datasets in distance-based analyses. Overall, although both markers were successful for the identification of apple snails, the COI molecular marker is a better barcoding marker and could be utilized in various population genetic studies of P. canaliculata and P. maculata.

COI metabarcoding primer choice affects richness and recovery of indicator taxa in freshwater systems.

Mixed community or environmental DNA marker gene sequencing has become a commonly used technique for biodiversity analyses in freshwater systems. Many cytochrome c oxidase subunit I (COI) primer sets are now available for such work. The purpose of this study is to test whether COI primer choice affects the recovery of arthropod richness, beta diversity, and recovery of target assemblages in the benthos kick-net samples typically used in freshwater biomonitoring. We examine six commonly used COI primer sets on samples collected from six freshwater sites. Biodiversity analyses show that richness is sensitive to primer choice and the combined use of multiple COI amplicons recovers higher richness. Thus, to recover maximum richness, multiple primer sets should be used with COI metabarcoding. In ordination analyses based on community dissimilarity, samples consistently cluster by site regardless of amplicon choice or PCR replicate. Thus, for broadscale community analyses, overall beta diversity patterns are robust to COI marker choice. Recovery of traditional freshwater bioindicator assemblages such as Ephemeroptera, Trichoptera, Plectoptera, and Chironomidae as well as Arthropoda site indicators were differentially detected by each amplicon tested. This work will help future biodiversity and biomonitoring studies develop not just standardized, but optimized workflows that either maximize taxon-detection or the selection of amplicons for water quality or Arthropoda site indicators.

DNA barcoding as a tool to facilitate the taxonomy of hermit crabs (Decapoda: Anomura: Paguroidea)

DNA barcoding has become an important aspect in biodiversity research, but is ineffective without comprehensive databases that can relate molecular sequences to scientific names. Hermit crabs, or paguroids, are an important component of the marine benthos, and are vastly understudied in southern Africa, a globally important bioregion. The study provides 194 cytochrome c oxidase subunit I (COI) barcode sequences (> 550 bp) of 43 nominal and 12 putative species from 19 genera and four families of Paguroidea. Preliminary COI-based phylogenetic analysis suggested some overall pattern of monophyly in the Parapaguridae and PaguristesDana, 1851 and DardanusPaulˊson, 1875. Large groups in Diogenidae, however, such as CalcinusDana, 1851 and DiogenesDana, 1851, were recovered as poly- or paraphyletic. Testing the dataset for barcoding efficacy revealed that the technique can reach identification rates near 100% success. A barcoding gap was apparent for almost all but one problematic taxon. The optimum threshold range to discriminate between species for this set of data was calculated at 3.7–3.9%, although the overall mean intraspecific distance was well below 1%. Furthermore, glaucothoe larvae of two deep-water paguroids were matched to names in the new molecular library. The COI marker alone, however, did not provide clear species identifications in three problematic species, Areopaguristes cf. jousseaumei (Bouvier, 1892), Goreopagurus pooreiLemaitre & McLaughlin, 2003, and Pagurus cuanensisBell, 1846. Despite the dataset having considerable species coverage of 55 total taxa, it is still estimated to be only about 50% complete in terms of the South African fauna. Future work should therefore increase coverage to other species in South Africa and expand geographic coverage for widely distributed species.

Molecular characterization of Anopheline (Diptera: Culicidae) mosquitoes from eight geographical locations of Sri Lanka

BackgroundGenus Anopheles is a major mosquito group of interest in Sri Lanka as it includes vectors of malaria and its members exist as species complexes. Taxonomy of the group is mainly based on morphological features, which are not conclusive and can be easily erased while handling the specimens. A combined effort, using morphology and DNA barcoding (using the markers cytochrome c oxidase subunit I (COI) gene and internal transcribed spacer 2 (ITS2) region, was made during the present study to recognize anophelines collected from eight districts of Sri Lanka for the first time.MethodsCytochrome c oxidase subunit I and ITS2 regions of morphologically identified anopheline mosquitoes from Sri Lanka were sequenced. These sequences together with GenBank sequences were used in phylogenetic tree construction and molecular characterization of mosquitoes.ResultsAccording to morphological identification, the field-collected adult mosquitoes belonged to 15 species, i.e., Anopheles aconitus, Anopheles annularis, Anopheles barbirostris, Anopheles culicifacies, Anopheles jamesii, Anopheles karwari, Anopheles maculatus, Anopheles nigerrimus, Anopheles pallidus, Anopheles peditaeniatus, Anopheles pseudojamesi, Anopheles subpictus, Anopheles tessellatus, Anopheles vagus, and Anopheles varuna. However, analysis of 123 COI sequences (445 bp) (16 clades supported by strong bootstrap value in the neighbour joining tree and inter-specific distances of >3%) showed that there are 16 distinct species. Identity of the morphologically identified species, except An. subpictus, was comparable with the DNA barcoding results. COI sequence analysis showed that morphologically identified An. subpictus is composed of two genetic entities: An. subpictus species A and species B (inter-specific K2P distance 0.128). All the four haplotypes of An. culicifacies discovered during the present study belonged to a single species. ITS2 sequences (542 bp) were obtained for all the species except for An. barbirostris, An. subpictus species B, An. tessellatus, and An. varuna. Each of these sequences was represented by a single species-specific haplotype.ConclusionsThe present study reflects the importance and feasibility of COI and ITS2 genetic markers in identifying anophelines and their sibling species, and the significance of integrated systematic approach in mosquito taxonomy. Wide distribution of malaria vectors in the country perhaps indicates the potential for re-emergence of malaria in the country.

PrimerMiner: an r package for development and in silico validation of DNA metabarcoding primers

Summary DNA metabarcoding is a powerful tool to assess biodiversity by amplifying and sequencing a standardized gene marker region. Its success is often limited due to variable binding sites that introduce amplification biases. Thus, the development of optimized primers for communities or taxa under study in a certain geographic region and/or ecosystems is of critical importance. However, no tool for obtaining and processing of reference sequence data in bulk that can serve as a backbone for primer design is currently available. We developed the r package PrimerMiner, which batch downloads DNA barcode gene sequences from BOLD and NCBI data bases for specified target taxonomic groups and then applies sequence clustering into operational taxonomic units (OTUs) to reduce biases introduced by the different number of available sequences per species. Additionally, PrimerMiner offers functionalities to evaluate primers in silico, which are in our opinion more realistic than the strategy employed in another available software for that purpose, ecoPCR. We used PrimerMiner to download cytochrome c oxidase subunit I (COI) sequences for 15 important freshwater invertebrate groups, relevant for ecosystem assessment. By processing COI markers from both data bases, we were able to increase the amount of reference data 249‐fold on average, compared to using complete mitochondrial genomes alone. Furthermore, we visualized the generated OTU sequence alignments and describe how to evaluate primers in silico using PrimerMiner. With PrimerMiner, we provide a useful tool to obtain relevant sequence data for targeted primer development and evaluation. The OTU‐based reference alignments generated with PrimerMiner can be used for manual primer design or processed with bioinformatic tools for primer development.

Cytogenetics and DNA barcoding of the Round-eared bats, Tonatia (Chiroptera: Phyllostomidae): a new karyotype for Tonatia bidens

There are two species of Neotropical Round-eared bats, Tonatia bidens Spix, 1823 and Tonatia saurophila Koopman & Williams, 1951, which present highly similar morphological characteristics that can lead to errors of identification. Specimens originally identified as T. bidens have recently been reclassified as T. saurophila, and the only karyotype documented previously for these species was 2n = 16, FN = 20. In the present study, specimens of Tonatia collected in the municipality of Barra do Garcas, in the Brazilian state of Mato Grosso, were analyzed morphologically, using conventional cytogenetic techniques (C-banding, Ag-NOR, and CMA3), and through sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene. In the specimens morphologically identified as T. bidens, the diploid number (2n) was 26, and the fundamental number (FN), 38, while in T. saurophila, 2n = 16 and FN = 20, which is the karyotype also described previously for T. bidens. The dendograms obtained with sequences of the COI marker resulted in the formation of two distinct groups between T. bidens and T. saurophila, consistent with the two species, with a high sequence divergence value (14.22%). Distinct clades were also observed between T. bidens and the other phyllostomines analyzed in this study, with T. bidens also close to Phyllostomus hastatus (14.18% of sequence divergence).

Performance of cytochrome c oxidase subunit I (COI), ribosomal DNA Large Subunit (LSU) and Internal Transcribed Spacer 2 (ITS2) in DNA barcoding of Collembola

Studies in soil meiofauna with traditional methods are highly laborious and identification exclusively by means of morphological traits usually underestimates species richness. Although the mitochondrial DNA Cytochrome c Oxidase subunit I gene (COI, CO1, cox1, coxI) has been proposed as a standard DNA barcode for animals, nuclear ribosomal DNA markers such as Large Subunit (LSU, 28S) and Internal Transcribed Spacer (ITS) have proven efficient to distinguish among species of animals, eukaryotic microbes and plants. The COI and LSU markers discriminate among closely related species of Collembola, but the efficiency of ITS2 has never been tested. We evaluated the relative performance of these three markers for DNA barcoding of Collembola. All three markers proved highly efficient in discriminating species, but LSU exhibited very low differences among certain closely related species. We conclude that ITS2 may serve as an alternative barcode to identify Collembola and has a potential to be used for metabarcoding analyses.

DNA barcode assessment of Gracilaria salicornia (Gracilariaceae, Rhodophyta) from Southeast Asia.

BackgroundDNA barcoding is becoming a widely applied tool for the quick and accurate identification of species. The evolution of the mitochondrial cytochrome c oxidase subunit I (COI) gene is sufficiently rapid to allow discrimination between closely related species and biogeographic subgroups within species. Gracilaria salicornia was originally described as being from Manila, the Philippines, and is distributed throughout Asia and the Indian Ocean. To more accurately define this species and its genetic diversity owing to the confusion of identification historically, DNA barcoding using the 5’ end of the COI gene of the mitochondrial genome was applied to specimens collected from the Philippines, Malaysia, Thailand, China, and Japan, and they were compared to other gracilarian species.ResultsWithin species, the COI marker yielded two clusters with nucleotide divergences of 0.0–1.3%. This divergence is slightly higher than the typical intraspecific variation for red algae. A total of eight COI haplotypes were found for G. salicornia, comprising the following groups: H1–H3 from the Philippines; H4 from Okinawa in Japan; H5–H7 from Malaysia, Thailand, and China; and H8 from Thailand.ConclusionAlthough this work concentrated on a limited geographical region of a widespread taxon, the data shows intraspecific molecular divergences in G. salicornia and provides further evidence that DNA barcodes are useful tools for identifying species boundaries and examining biogeographical haplotypes for the genus Gracilaria.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-54-27) contains supplementary material, which is available to authorized users.

DNAbarcode reference data for the Korean herpetofauna and their applications

Recently, amphibians and reptiles have drawn attention because of declines in species and populations caused mainly by habitat loss, overexploitation and climate change. This study constructed a DNA barcode database for the Korean herpetofauna, including all the recorded amphibians and 68% of the recorded reptiles, to provide a useful, standardized tool for species identification in monitoring and management. A total of 103 individuals from 18 amphibian and 17 reptile species were used to generate barcode sequences using partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene and to compare it with other suggested barcode loci. Comparing 16S rRNA, cytochrome b (Cytb) and COI for amphibians and 12S rRNA, Cytb and COI for reptiles, our results revealed that COI is better than the other markers in terms of a high level of sequence variation without length variation and moderate amplification success. Although the COI marker had no clear barcoding gap because of the high level of intraspecific variation, all of the analysed individuals from the same species clustered together in a neighbour-joining tree. High intraspecific variation suggests the possibility of cryptic species. Finally, using this database, confiscated snakes were identified as Elaphe schrenckii, designated as endangered in Korea and a food contaminant was identified as the lizard Takydromus amurensis.