Optimizing the use of shed feathers for genetic analysis

Shed feathers obtained by noninvasive genetic sampling (NGS) are a valuable source of DNA for genetic studies of birds. They can be collected across a large geographical range and facilitate research on species that would otherwise be extremely difficult to study. A limitation of this approach is uncertainty concerning the quality of the extracted DNA. Here we investigate the relationship between feather type, feather condition and DNA quality (amplification success) in order to provide a simple, cost-effective method for screening samples prior to genetic analysis. We obtained 637 shed feathers of the powerful owl (Ninox strenua) from across its range in southeastern Australia. The extracted DNA was amplified using polymerase chain reaction for a range of markers including mitochondrial DNA, ND3 and nuclear DNA, a simple sequence repeat (Nst02) and a portion of the CHD-1 gene (P2/P8). We found that feather condition significantly influenced the amplification success of all three loci, with feathers characterized as 'good' having greater success. Feather type was found to be of lower importance, with good quality feathers of all types consistently producing high success for all three loci. We also found that the successful amplification of multilocus genotypes was dependant on the condition of the starting material and was highly correlated with successful amplification of the sex-linked CHD-1 locus. Samples with low DNA quality have a higher probability of amplification failure and are more likely to produce incorrect genotypes; therefore, identifying samples with high DNA quality can save substantial time and cost associated with the genetic analysis of NGS. As a result, we propose a method for screening shed feathers in order to provide a subset of samples which will have a greater probability of containing high quality DNA suitable for the amplification of multilocus genotypes.     

DNA barcoding of eight North American coregonine species

Coregonine fishes have a circumpolar distribution in the Arctic and sub-Arctic Northern Hemisphere. This subfamily of Salmonidae consists of three genera: Prosopium, Stenodus and Coregonus, including over 30 species. Many species overlap spatially and are difficult to distinguish based on morphological characteristics, especially as larvae or juveniles. Here we present a method for rapid and cost-effective species identification for representatives of the three genera based on sequence variation at the mitochondrial cytochrome c oxidase subunit I gene (COI). We examined eight species common to North America with distributional overlap in Alaska. Mean pairwise sequence divergence for all eight species was 7.04% and ranged from 0.46% to 14.23%. This sequence variation was used to develop a genetic assay based on restriction fragment length polymorphism. In a blind test, this assay provided correct species assignment for 48 of 49 individuals representing all eight species. The single incorrect assignment may reflect hybridization between two closely related species. This DNA barcode-based assay promises to aid fishery managers and researchers by providing a cost-effective alternative to large-scale sequence analysis for identification of North American coregonine fishes.     

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.     

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 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.     

DNA metabarcoding and the cytochrome c oxidase subunit I marker: not a perfect match

DNA metabarcoding enables efficient characterization of species composition in environmental DNA or bulk biodiversity samples, and this approach is making significant and unique contributions in the field of ecology. In metabarcoding of animals, the cytochrome c oxidase subunit I (COI) gene is frequently used as the marker of choice because no other genetic region can be found in taxonomically verified databases with sequences covering so many taxa. However, the accuracy of metabarcoding datasets is dependent on recovery of the targeted taxa using conserved amplification primers. We argue that COI does not contain suitably conserved regions for most amplicon-based metabarcoding applications. Marker selection deserves increased scrutiny and available marker choices should be broadened in order to maximize potential in this exciting field of research.     

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.     

Species identification of Tanzanian antelopes using DNA barcoding

Efficient tools for consistent species identification are important in wildlife conservation as it can provide information on the levels of species exploitation and assist in solving forensic-related problems. In this study, we evaluated the effectiveness of the mitochondrial cytochrome c oxidase subunit I (COI) barcode in species identification of Tanzanian antelope species. A 470 base-pair region of the COI gene was examined in 95 specimens representing 20 species of antelopes, buffalo and domestic Bovidae. All the Tanzanian species showed unique clades, and sequence divergence within species was <1%, whereas divergence between species ranged from 6.3% to 22%. Lowest interspecific divergence was noted within the Tragelaphus genus. Neighbour-joining phylogenetic analyses demonstrated that the examined COI region provided correct and highly supported species clustering using short fragments down to 100 base-pair lengths. This study demonstrates that even short COI fragments can efficiently identify antelope species, thus demonstrating its high potential for use in wildlife conservation activities.     

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.     

DNA barcoding and evaluation of genetic diversity in Cyprinidae fish in the midstream of the Yangtze River

The Yangtze River is the longest river in China and is divided into upstream and mid‐downstream regions by the Three Gorges (the natural barriers of the Yangtze River), resulting in a complex distribution of fish. Dramatic changes to habitat environments may ultimately threaten fish survival; thus, it is necessary to evaluate the genetic diversity and propose protective measures. Species identification is the most significant task in many fields of biological research and in conservation efforts. DNA barcoding, which constitutes the analysis of a short fragment of the mitochondrial cytochrome c oxidase subunit I (COI) sequence, has been widely used for species identification. In this study, we collected 561 COI barcode sequences from 35 fish from the midstream of the Yangtze River. The intraspecific distances of all species were below 2% (with the exception of Acheilognathus macropterus and Hemibarbus maculatus). Nevertheless, all species could be unambiguously identified from the trees, barcoding gaps and taxonomic resolution ratio values. Furthermore, the COI barcode diversity was found to be low (≤0.5%), with the exception of H. maculatus (0.87%), A. macropterus (2.02%) and Saurogobio dabryi (0.82%). No or few shared haplotypes were detected between the upstream and downstream populations for ten species with overall nucleotide diversities greater than 0.00%, which indicated the likelihood of significant population genetic structuring. Our analyses indicated that DNA barcoding is an effective tool for the identification of cyprinidae fish in the midstream of the Yangtze River. It is vital that some protective measures be taken immediately because of the low COI barcode diversity.     

DNA barcoding of the ichthyofauna of Taal Lake, Philippines

This study represents the first molecular survey of the ichthyofauna of Taal Lake and the first DNA barcoding attempt in Philippine fishes. Taal Lake, the third largest lake in the Philippines, is considered a very important fisheries resource and is home to the world's only freshwater sardine, Sardinella tawilis. However, overexploitation and introduction of exotic fishes have caused a massive decline in the diversity of native species as well as in overall productivity of the lake. In this study, 118 individuals of 23 native, endemic and introduced fishes of Taal Lake were barcoded using the partial DNA sequence of the mitochondrial cytochrome c oxidase subunit I (COI) gene. These species belong to 21 genera, 17 families and 9 orders. Divergence of sequences within and between species was determined using Kimura 2-parameter (K2P) distance model, and a neighbour-joining tree was generated with 1000 bootstrap replications using the K2P model. All COI sequences for each of the 23 species were clearly discriminated among genera. The average within species, within genus, within family and within order percent genetic divergence was 0.60%, 11.07%, 17.67% and 24.08%, respectively. Our results provide evidence that COI DNA barcodes are effective for the rapid and accurate identification of fishes and for identifying certain species that need further taxonomic investigation.     

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

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

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.     

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.     

DNA barcode and minibarcode identification of freshwater fishes from Cerrado headwater streams in Central Brazil

The extraordinary species diversity of the Neotropical freshwater fish fauna is world renown. Yet, despite rich species diversity, taxonomic and genetic resources for its Cerrado ichthyofauna remain poorly developed. We provide a reference library of 149 DNA barcodes for 39 species/lineages of Cerrado headwater stream fishes from the Brazilian Distrito Federal and nearby areas and test the utility of distance-based criteria, tree-based criteria and minibarcodes for specimen identification. Mean Kimura 2-parameter genetic distances within species to orders ranged 1·8–12·1%. However, mean intraspecific v. congeneric-interspecific distances (0·9–1·3%) overlapped extensively and distance-based barcoding failed to achieve correct identifications due to c. 4–12·1% error rates and 19·5% ambiguous identifications related to the presence of singletons. Overlap was reduced and best-match success rates improved drastically to 83·5% when Characidium barcodes representing potential misidentifications or undescribed species were removed. Tree-based monophyly criteria generally performed similarly to distance methods, correctly differentiating up to c. 85% of species/lineages despite neighbour-joining and Bayesian tree errors (random lineage-branching events, long-branch attraction). Five clusters (Ancistrus aguaboensis, Characidium spp., Eigenmannia trilineata, Hasemania hanseni and Hypostomus sp. 2) exhibited deep intraspecific divergences or para−/polyphyly and multiple Barcode Index Number assignments indicative of putative candidate species needing taxonomic re-examination. Sliding-window analyses also indicated that a 200 bp minibarcode region performed just as well at specimen identification as the entire barcode gene. Future DNA barcoding studies of Distrito Federal–Cerrado freshwater fishes will benefit from increased sampling coverage, as well as consideration of minibarcode targets for degraded samples and next-generation sequencing.     

DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer

Oomycete species occupy many different environments and many ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak. The use of DNA for oomycete species identification is well established, but DNA barcoding with cytochrome c oxidase subunit I (COI) is a relatively new approach that has yet to be assessed over a significant sample of oomycete genera. In this study we have sequenced COI, from 1205 isolates representing 23 genera. A comparison to internal transcribed spacer (ITS) sequences from the same isolates showed that COI identification is a practical option; complementary because it uses the mitochondrial genome instead of nuclear DNA. In some cases COI was more discriminative than ITS at the species level. This is in contrast to the large ribosomal subunit, which showed poor species resolution when sequenced from a subset of the isolates used in this study. The results described in this paper indicate that COI sequencing and the dataset generated are a valuable addition to the currently available oomycete taxonomy resources, and that both COI, the default DNA barcode supported by GenBank, and ITS, the de facto barcode accepted by the oomycete and mycology community, are acceptable and complementary DNA barcodes to be used for identification of oomycetes.     

基于线粒体细胞色素c氧化酶亚基I基因序列的帘蛤科贝类分子系统发育研究

对21种帘蛤科贝类线粒体细胞色素c氧化酶亚基Ⅰ(cytochrome c oxidase subunit I,COI)基因核苷酸序列进行了分析,以探讨这一序列在种质鉴定、分子系统发生研究中的应用价值。测序结果表明,所有物种扩增片段长度均为707 bp(含引物),序列A+T含量(62.4%—67.8%)明显高于G+C含量。物种间共有变异位点379个,其中简约信息位点334个;此区段共编码235个氨基酸,种间共有氨基酸变异位点100个。以COI基因片段序列为标记,用中国蛤蜊(Mactra chinensis)作外群,构建了35种帘蛤科贝类(其中14种贝类COI序列从GenBank下载)的系统发生树,结合拓扑结构分析和序列比对分析,结果表明:支持将短文蛤(Meretrix petechinalis)和丽文蛤(M.lusoria)订为文蛤(M.meretrix)的同物异名的观点,建议将丽文蛤和短文蛤订为文蛤的地理亚种;支持将薄片镜蛤(Dosinia corrugata)和D.angulosa订为2个独立种的观点;认为将波纹巴非蛤(Paphia undulata)和织锦巴非蛤(P.textile)订为2个独立种是合适的。COI基因序列含有丰富的遗传信息,适合作为帘蛤科贝类种群遗传结构和系统发生研究的分子标记。     

Study of COI sequences from endemic New Zealand aphids highlights high mitochondrial DNA diversity in Rhopalosiphina (Hemiptera: Aphididae)

Focussed searches were made across New Zealand between 2013 and 2016, for endemic aphids from the Schizaphis (Rhopalosiphina) genus, which is currently represented by two putative, undescribed species from the endemic host plants Aciphylla and Dracophyllum. Cytochrome c oxidase I (COI) gene sequences (48 in total) from the Schizaphis were analysed together with those from a broader collection of New Zealand endemic aphids that has been assembled since the year 2000. The bulk of the Schizaphis belonged to two clusters corresponding to the host plant genera. Two aphids from central North Island Dracophyllum represented a much diverged lineage without clear affiliations to other New Zealand Schizaphis. Inter-population variation in the New Zealand Schizaphis was high compared with that seen in international studies of Aphidinae and among populations of other endemic New Zealand Aphidina. Within Schizaphis from Dracophyllum, geography played an apparent role in genetic structuring, with populations from Taranaki (North Island) and especially Mt Lyford (South Island) being divergent from those on the South Island main divide. Two distinct lineages of Schizaphis, which co-occurred at some sites, were found on Aciphylla. Our sequence comparisons, including GMYC analyses, indicated up to five New Zealand Schizaphis lineages, and two newly discovered endemic Aphis species from the host plants Clematis and Hebe.     

Testing the potential of DNA barcoding in vertebrate radiations: the case of the littoral cichlids (Pisces,Perciformes, Cichlidae) from Lake Tanganyika

We obtained 398 cytochrome c oxidase subunit I barcodes of 96 morphospecies of Lake Tanganyika (LT) cichlids from the littoral zone. The potential of DNA barcoding in these fishes was tested using both species identification and species delineation methods. The best match (BM) and best close match (BCM) methods were used to evaluate the overall identification success. For this, three libraries were analysed in which the specimens were categorized into Operational Taxonomic Units (OTU) in three alternative ways: (A) morphologically distinct, including undescribed, species, (B) valid species and (C) complexes of morphologically similar or closely related species. For libraries A, B and C, 73, 73 and 96% (BM) and 72, 70 and 94% (BCM) of the specimens were correctly identified. Additionally, the potential of two species delineation methods was tested. The General Mixed Yule Coalescent (GMYC) analysis suggested 70 hypothetical species, while the Automatic Barcode Gap Discovery (ABGD) method revealed 115 putative species. Although the ABGD method had a tendency to oversplit, it outperformed the GMYC analysis in retrieving the species. In most cases where ABGD suggested oversplitting, this was due to intraspecific geographical variation. The failure of the GMYC method to retrieve many species could be attributed to discrepancies between mitochondrial gene trees and the evolutionary histories of LT cichlid species. Littoral LT cichlids have complex evolutionary histories that include instances of hybridization, introgression and rapid speciation. Nevertheless, although the utility of DNA barcoding in identification is restricted to the level of complexes, it has potential for species discovery in cichlid radiations.