DNA barcodes for marine fungal identification and discovery

We employed DNA barcodes for identification of fungal species in marine sediments. Sediments were collected seasonally along the Southeast coast of India from which a culturable fungal library was constructed. All cultured species were morphologically documented using microscopical analysis. A maximum population density of 19.3 × 10³ CFU/g was recorded in monsoon and minimum of 3 × 10³ CFU/g in premonsoon season. Two-way analysis of variance suggests that the fungal community varied significantly between the seasons (F = 9.543, P < 0.001) and at various depths sampled (F = 4.655, P < 0.05). In total, 54 fungal species belonging to 13 different families were documented and all species were sequenced for internal transcribed spacer genes. Each species was represented by at least two specimens constituting a total of 171 specimens for DNA barcoding. Twelve species of a marine fungi were sequenced for the first time. Branching patterns of phylogenetic tree strongly supported the sequence variations within and between all species barcoded. Based on the pairwise distance model we suggest barcode gaps of 15 %, 21 %, 30 %, 35 % and 51 % for genera, family, order, class and phyla respectively.     

DNA barcoding of Cuban freshwater fishes: evidence for cryptic species and taxonomic conflicts

Despite ongoing efforts to protect species and ecosystems in Cuba, habitat degradation, overuse and introduction of alien species have posed serious challenges to native freshwater fish species. In spite of the accumulated knowledge on the systematics of this freshwater ichthyofauna, recent results suggested that we are far from having a complete picture of the Cuban freshwater fish diversity. It is estimated that 40% of freshwater Cuban fish are endemic; however, this number may be even higher. Partial sequences (652 bp) of the mitochondrial gene COI (cytochrome c oxidase subunit I) were used to barcode 126 individuals, representing 27 taxonomically recognized species in 17 genera and 10 families. Analysis was based on Kimura 2-parameter genetic distances, and for four genera a character-based analysis (population aggregation analysis) was also used. The mean conspecific, congeneric and confamiliar genetic distances were 0.6%, 9.1% and 20.2% respectively. Molecular species identification was in concordance with current taxonomical classification in 96.4% of cases, and based on the neighbour-joining trees, in all but one instance, members of a given genera clustered within the same clade. Within the genus Gambusia, genetic divergence analysis suggests that there may be at least four cryptic species. In contrast, low genetic divergence and a lack of diagnostic sites suggest that Rivulus insulaepinorum may be conspecific with Rivulus cylindraceus. Distance and character-based analysis were completely concordant, suggesting that they complement species identification. Overall, the results evidenced the usefulness of the DNA barcodes for cataloguing Cuban freshwater fish species and for identifying those groups that deserve further taxonomic attention.     

Developing a DNA barcode library for perciform fishes in the South China Sea: Species identification,accuracy and cryptic diversity

DNA barcodes were studied for 1,353 specimens representing 272 morphological species belonging to 149 genera and 55 families of Perciformes from the South China Sea (SCS). The average Kimura 2‐parameter (K2P) distances within species, genera and families were 0.31%, 8.71% and 14.52%, respectively. A neighbour‐joining (NJ) tree, Bayesian inference (BI) and maximum‐likelihood (ML) trees and Automatic Barcode Gap Discovery (ABGD) revealed 260, 253 and 259 single‐species‐representing clusters, respectively. Barcoding gap analysis (BGA) demonstrated that barcode gaps were present for 178 of 187 species analysed with multiple specimens (95.2%), with the minimum interspecific distance to the nearest neighbour larger than the maximum intraspecific distance. A group of three Thunnus species (T. albacares, T. obesus and T. tonggol), a pair of Gerres species (G. oyena and G. japonicus), a pair of Istiblennius species (I. edentulous and I. lineatus) and a pair of Uranoscopus species (U. oligolepis and U. kaianus) were observed with low interspecific distances and overlaps between intra‐ and interspecific genetic distances. Three species (Apogon ellioti, Naucrates ductor and Psenopsis anomala) showed deep intraspecific divergences and generated two lineages each, suggesting the possibility of cryptic species. Our results demonstrated that DNA barcodes are highly reliable for delineating species of Perciformes in the SCS. The DNA barcode library established in this study will shed light on further research on the diversity of Perciformes in the SCS.     

Revisiting the ichthyodiversity of Java and Bali through DNA barcodes: taxonomic coverage,identification accuracy,cryptic diversity and identification of exotic species

Among the 899 species of freshwater fishes reported from Sundaland biodiversity hotspot, nearly 50% are endemics. The functional integrity of aquatic ecosystems is currently jeopardized by human activities, and landscape conversion led to the decline of fish populations in several part of Sundaland, particularly in Java. The inventory of the Javanese ichthyofauna has been discontinuous, and the taxonomic knowledge is scattered in the literature. This study provides a DNA barcode reference library for the inland fishes of Java and Bali with the aim to streamline the inventory of fishes in this part of Sundaland. Owing to the lack of available checklist for estimating the taxonomic coverage of this study, a checklist was compiled based on online catalogues. A total of 95 sites were visited, and a library including 1046 DNA barcodes for 159 species was assembled. Nearest neighbour distance was 28‐fold higher than maximum intraspecific distance on average, and a DNA barcoding gap was observed. The list of species with DNA barcodes displayed large discrepancies with the checklist compiled here as only 36% (i.e. 77 species) and 60% (i.e. 24 species) of the known species were sampled in Java and Bali, respectively. This result was contrasted by a high number of new occurrences and the ceiling of the accumulation curves for both species and genera. These results highlight the poor taxonomic knowledge of this ichthyofauna, and the apparent discrepancy between present and historical occurrence data is to be attributed to species extirpations, synonymy and misidentifications in previous studies.     

Genetic variation in a freshwater prawn species,Palaemon paucidens,in South Korea

The biogeography of freshwater biota in the Korean Peninsula has been affected by recent geological processes and anthropogenic activity. The freshwater prawn, Palaemon paucidens, can serve as a non-fish model organism suitable for assessing these factors, as it is found in all river systems in the Korean Peninsula and may have been introduced by humans in some regions. In this study, we investigated the geographical distribution of genetic variation and the genetic structure of P. paucidens populations using mitochondrial DNA sequences and genotypes identified from four microsatellite loci. Our results showed that populations from westward-flowing river systems that drain into the Yellow Sea have more genetic diversity than those from southward-flowing river systems, and that the highest genetic variance revealed by analysis of molecular variance (AMOVA) using both genetic markers was observed in river systems grouped as HAN + GEUM, NAKDONG + JEJU, YOUNGSAN, and SEOMJIN. These results suggest that HAN and GEUM, in which freshwater prawn populations have higher levels of genetic diversity, were the most recently isolated river systems from Asian continental systems. Therefore, populations from HAN and GEUM experienced less severe bottlenecks than those from YOUNGSAN and SEOMJIN.     

DNA barcoding for the discrimination of Eurasian yews (Taxus L., Taxaceae) and the discovery of cryptic species

There is currently international interest in the application of DNA barcoding as a tool for plant species discrimination and identification. In this study, we evaluated the utility of five candidate plant DNA barcoding regions [rbcL, matK, trnH-psbA, trnL-F and internal transcribed spacer (ITS)] in Eurasian yews. This group of species is taxonomically difficult because of a lack of clear-cut morphologically differences between species and hence represents a good test case for DNA barcoding. Forty-seven accessions were analysed, representing all taxa treated in current floristic works and covering most of the distribution range of Taxus in Eurasia. As single loci, trnL-F and ITS showed the highest species discriminatory power, each resolving 11 of 11 lineages (= barcode taxa). Species discrimination using matK, trnH-psbA and rbcL individually was lower, with matK resolving 8 of 10, trnH-psbA 7 of 11 and rbcL 5 of 11 successfully sequenced lineages. The proposed CBOL core barcode (rbcL + matK) resolved 8 of 11 lineages. Combining loci generally increased the robustness (measured by clade support) of the barcoding discrimination. Based on overall performance, trnL-F and ITS, separately or combined, are proposed as barcode for Eurasian Taxus. DNA barcoding discriminated recognized taxa of Eurasian Taxus, namely T. baccata, T. cuspidata, T. fuana and T. sumatrana, and identified seven lineages among the T. wallichiana group, some with distinct geographical distributions and morphologies, and potentially representing new species. Using the proposed DNA barcode, a technical system can be established to rapidly and reliably identify Taxus species in Eurasia for conservation protection and for monitoring illegal trade.     

Mayfly production in a Colorado mountain stream: an assessment of methods for synchronous and non-synchronous species

Year-round collections of mayflies (Ephemeroptera) from a Colorado mountain stream allowed critical examination of several methods of calculating production for species with different life cycles. Five of the six numerically dominant species exhibited slow seasonal, univoltine life cycles. Baetis tricaudatus was bivoltine. Two species demonstrated well synchronized development, three species were poorly synchronized and a sixth was intermediate. Mean density and biomass data from each sampling date were used to ascertain the goodness-of-fit of each species to the Allen curve. It is proposed that such information can provide quantitative criteria for identifying species with well synchronized development and thereby determine when it is appropriate to directly apply cohort methods while avoiding time intensive body size (e.g. head width) measurements necessary for size-frequency analyses. In addition, these data demonstrate that species specific production varies with gross changes in elevation.     

Developing DNA barcodes for species identification in Podophylloideae (Berberidaceae)

Species of Podophyllum, Dysosma, Sinopodophyllum, and Diphylleia, genera from Podophylloideae of Berberidaceae, have long been used in traditional herbal medicine in East Asia and/or North America. Accurate identification of the species of these four genera is crucial to their medicinal uses. In this study, we tested the utility of nine barcodes (matK, rbcL, atpH-atpI, rpl32-trnL^UAG, rps18-clpp, trnL-trnF, trnL-ndhJ, trnS-trnfM, and internal transcribed spacer (ITS)) to discriminate different species of Podophylloideae. Thirty-six individuals representing 12 species of Podophylloideae were collected from different locations in China, Japan, and North America. We assessed the feasibility of amplification and sequencing of all markers, examined the levels of the barcoding gap based on DNA sequence divergence between ranges of intra- and interspecific variation using pairwise distances, and further evaluated successful identifications using each barcode by similarity-based and tree-based methods. Results showed that nine barcodes, except rps18-clpp, have a high level of primer universality and sequencing success. As a single barcode, ITS has the most variable sites, greater intra- and interspecific divergences, and the highest species discrimination rate (83%), followed by matKwhich has moderate variation and also high species discrimination rates. However, these species can also be discriminated by ITS alone, except Dysosma versipellis (Hance) M. Cheng ex T. S. Ying and D. pleiantha (Hance) Woodson. The combination of ITS + matK did not improve species resolution over ITS alone. Thus, we propose that ITS may be used as a sole region for identification of most species in Podophylloideae. The failure of ITS to distinguish D. versipellis and D. pleiantha is likely attributed to incomplete lineage sorting due to recent divergence of the two species.     

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.     

Searching for evidence of selection in avian DNA barcodes

The barcode of life project has assembled a tremendous number of mitochondrial cytochrome c oxidase I (COI) sequences. Although these sequences were gathered to develop a DNA-based system for species identification, it has been suggested that further biological inferences may also be derived from this wealth of data. Recurrent selective sweeps have been invoked as an evolutionary mechanism to explain limited intraspecific COI diversity, particularly in birds, but this hypothesis has not been formally tested. In this study, I collated COI sequences from previous barcoding studies on birds and tested them for evidence of selection. Using this expanded data set, I re-examined the relationships between intraspecific diversity and interspecific divergence and sampling effort, respectively. I employed the McDonald-Kreitman test to test for neutrality in sequence evolution between closely related pairs of species. Because amino acid sequences were generally constrained between closely related pairs, I also included broader intra-order comparisons to quantify patterns of protein variation in avian COI sequences. Lastly, using 22 published whole mitochondrial genomes, I compared the evolutionary rate of COI against the other 12 protein-coding mitochondrial genes to assess intragenomic variability. I found no conclusive evidence of selective sweeps. Most evidence pointed to an overall trend of strong purifying selection and functional constraint. The COI protein did vary across the class Aves, but to a very limited extent. COI was the least variable gene in the mitochondrial genome, suggesting that other genes might be more informative for probing factors constraining mitochondrial variation within species.     

Review of Cotachena Moore, 1885 (Lepidoptera: Crambidae,Spilomelinae) from Korea based on morphology and DNA barcodes

The Korean species of Cotachena were reviewed, based on morphological characters and DNA barcodes. Our study recognized three species of Cotachena from Korea: C. alysoni Whalley, C. brunnealis Yamanaka and C. taiwanalis Yamanaka. The latter two were new to Korea. Dubious occurrence of C. pubescens (Warren) in Korea was discussed. The 23 individuals of COI barcodes were analyzed and assigned to the eleven species of Cotachena. The DNA barcodes of C. taiwanalis and C. brunnealis were provided for the first time and their taxonomic status was discussed. Host plants of C. taiwanalis were first reported. The Korean species of Cotachena were compared to the conspecific populations from neighboring countries. Photographs of habitus and genital features were provided for the Korean Cotachena, if available, to facilitate identification.     

Conservation of Hipparchia autonoe (Esper) (Lepidoptera: Nymphalidae), Natural Monument in South Korea

Hipparchia autonoe, designated Natural Monument no. 458, is a species inhabiting Mt. Halla on Jeju Island, which is the only habitat of H. autonoe in South Korea. Recently, this species is in danger of extinction as the population has been remarkably reduced because its host plants and nectar plants are gradually disappearing due to plant succession in its habitat. On the climbing road of Jeju Island Eorimok, H. autonoe used to be observed at and above 1300 m a.s.l. However, since Sasa quelpaertensis has recently expanded its habitat up to 1400 m, H. autonoe is now observed at and above 1500 m. In Mongolia, the population of H. autonoe seems quite stable as there are dense populations of a host plant and nectar plants. Accordingly it is judged that we can maintain a stable community for support of H. autonoe if we successfully manage the host plant and nectar plant communities by controlling the density of S. quelpaertensis using biological and physical control methods. Comparative analysis of DNA barcode region of COI (658 bp) was done to check the homogeneity and the genetic diversity of H. autonoe collected in Korea and Mongolia. The DNA sequence difference among individuals collected in South Korea was 0.0–0.2%, and in Mongolia 0.0–1.4%. This confirmed a 0.06–1.2% barcoding gap. We believe that this result will provide basic information useful to guide conservation of H. autonoe in South Korea.     

Development of high‐resolution DNA barcodes for Dioscorea species discrimination and phylogenetic analysis

The genus Dioscorea is widely distributed in tropical and subtropical regions, and is economically important in terms of food supply and pharmaceutical applications. However, DNA barcodes are relatively unsuccessful in discriminating between Dioscorea species, with the highest discrimination rate (23.26%) derived from matK sequences. In this study, we compared genic and intergenic regions of three Dioscorea chloroplast genomes and found that the density of SNPs and indels in intergenic sites was about twice and seven times higher than that of SNPs and indels in the genic regions, respectively. A total of 52 primer pairs covering highly variable regions were designed and seven pairs of primers had 80%–100% PCR success rate. PCR amplicons of 73 Dioscorea individuals and assembled sequences of 47 Dioscorea SRAs were used for estimating intraspecific and interspecific divergence for the seven loci: The rpoB‐trnC locus had the highest interspecific divergence. Automatic barcoding gap discovery (ABGD), Poisson tree processes (PTP), and generalized mixed Yule coalescence (GMYC) analysis were applied for species delimitation based on the seven loci and successfully identified the majority of species, except for species in the Enantiophyllum section. Phylogenetic analysis of 51 Dioscorea individuals (28 species) showed that most individuals belonging to the same species tended to cluster in the same group. Our results suggest that the variable loci derived from comparative analysis of plastid genome sequences could be good DNA barcode candidates for taxonomic analysis and species delimitation.     

Testing the potential of proposed DNA barcodes for species identification of Zingiberaceae

In 2009, the Consortium for the Barcode of Life (CBOL) recommended the combination of rbcL and matK as the plant barcode based on assessments of recoverability, sequencing quality, and levels of species discrimination. Subsequently, based on a study of more than 6600 samples belonging to 193 families from seven phyla, the internal transcribed spacer (ITS) 2 locus was proposed as a universal barcode sequence for all major plant taxa used in traditional herbal medicine. Neither of these two studies was based on a detailed analysis of a particular family. Here, Zingiberaceae plants, including many closely related species, were used to compare the genetic divergence and species identification efficiency of ITS2, rbcL, matK, psbK-psbI, trnH-psbA, and rpoB.The results indicate that ITS2 has the highest interspecific divergence and significant differences between inter- and intraspecific divergence, whereas matK and rbcL have much lower divergence values. Among 260 species belongingto 30 genera in Zingiberaceae, the discrimination ability of the ITS2 locus was 99.5% at the genus level and 73.1% at the species level. Thus, we propose that ITS2 is the preferred DNA barcode sequence for identifying Zingiberaceae plants.     

Efficiency of DNA barcodes for species delimitation: A case in Pterygiella Oliv.(Orobanchaceae)

DNA barcoding is becoming an increasingly popular means to identify species. The obscure discrimination in the genus Pterygiella calls into question the re-assessment of the criterion for species delimitation. We collected 20 individuals, representing all five described species of this genus in its distributional range. The aim was to use three proposed barcode DNA regions (rbcL, matK, and ITS) to diagnose Pterygiella species, and examine which barcode is more suitable for discerning the congeneric and related species. The results showed that the core barcodes matK and rbcL were comparatively less effective. However, the ITS region, especially ITS-1and ITS-2, successfully identified all species in the genus. Furthermore, the secondary structure of ITS-2 RNA, especially compensatory base changes, appears complementary to classical primary sequence analysis for DNA barcoding.     

Probing diversity in freshwater fishes from Mexico and Guatemala with DNA barcodes

The freshwater fish fauna of Mexico and Guatemala is exceptionally diverse with >600 species, many endemic. In this study, patterns of sequence divergence were analysed in representatives of this fauna using cytochrome c oxidase subunit 1 (COI) DNA barcodes for 61 species in 36 genera. The average divergence among conspecific individuals was 0·45%, while congeneric taxa showed 5·1% divergence. Three species of Poblana , each occupying a different crater lake in the arid regions of Central Mexico, have had a controversial taxonomic history but are usually regarded as endemics to a single lake. They possess identical COI barcodes, suggesting a very recent history of isolation. Representatives of the Cichlidae, a complex and poorly understood family, were well discriminated by barcodes. Many species of Characidae seem to be young, with low divergence values (<2%), but nevertheless, clear barcode clusters were apparent in the Bramocharax – Astyanax complex. The symbranchid, Opisthernon aenigmaticum , has been regarded as a single species ranging from Guatemala to Mexico, but it includes two deeply divergent barcode lineages, one a possible new endemic species. Aside from these special cases, the results confirm that DNA barcodes will be highly effective in discriminating freshwater fishes from Central America and that a comprehensive analysis will provide new important insights for understanding diversity of this fauna.     

Molecular identification of species in Prunus sect.Persica (Rosaceae), with emphasis on evaluation of candidate barcodes for plants

Species of Prunus L. sect. Persica are not only important fruit trees, but also popular ornamental and medicinal plants. Correct identification of seedlings, barks, or fruit kernels is sometimes required, but no reliable morphological characters are available. Nowadays, the technique of DNA barcoding has the potential to meet such requirements. In this study, we evaluated the suitability of 11 DNA loci (atpB-rbcL, trnH-psbA, trnL-F, trnS-G, atpF-H, rbcL, matK, rpoB, rpoC1, nad1, and internal transcribed spa...     

Bridging two scholarly islands enriches both: COI DNA barcodes for species identification versus human mitochondrial variation for the study of migrations and pathologies

DNA barcodes for species identification and the analysis of human mitochondrial variation have developed as independent fields even though both are based on sequences from animal mitochondria. This study finds questions within each field that can be addressed by reference to the other. DNA barcodes are based on a 648‐bp segment of the mitochondrially encoded cytochrome oxidase I. From most species, this segment is the only sequence available. It is impossible to know whether it fairly represents overall mitochondrial variation. For modern humans, the entire mitochondrial genome is available from thousands of healthy individuals. SNPs in the human mitochondrial genome are evenly distributed across all protein‐encoding regions arguing that COI DNA barcode is representative. Barcode variation among related species is largely based on synonymous codons. Data on human mitochondrial variation support the interpretation that most – possibly all – synonymous substitutions in mitochondria are selectively neutral. DNA barcodes confirm reports of a low variance in modern humans compared to nonhuman primates. In addition, DNA barcodes allow the comparison of modern human variance to many other extant animal species. Birds are a well‐curated group in which DNA barcodes are coupled with census and geographic data. Putting modern human variation in the context of intraspecies variation among birds shows humans to be a single breeding population of average variance.