DNA barcoding commercially important fish species of Turkey

DNA barcoding was used in the identification of 89 commercially important freshwater and marine fish species found in Turkish ichthyofauna. A total of 1765 DNA barcodes using a 654‐bp‐long fragment of the mitochondrial cytochrome c oxidase subunit I gene were generated for 89 commercially important freshwater and marine fish species found in Turkish ichthyofauna. These species belong to 70 genera, 40 families and 19 orders from class Actinopterygii, and all were associated with a distinct DNA barcode. Nine and 12 of the COI barcode clusters represent the first species records submitted to the BOLD and GenBank databases, respectively. All COI barcodes (except sequences of first species records) were matched with reference sequences of expected species, according to morphological identification. Average nucleotide frequencies of the data set were calculated as T = 29.7%, C = 28.2%, A = 23.6% and G = 18.6%. Average pairwise genetic distance among individuals were estimated as 0.32%, 9.62%, 17,90% and 22.40% for conspecific, congeneric, confamilial and within order, respectively. Kimura 2‐parameter genetic distance values were found to increase with taxonomic level. For most of the species analysed in our data set, there is a barcoding gap, and an overlap in the barcoding gap exists for only two genera. Neighbour‐joining trees were drawn based on DNA barcodes and all the specimens clustered in agreement with their taxonomic classification at species level. Results of this study supported DNA barcoding as an efficient molecular tool for a better monitoring, conservation and management of fisheries.     

DNA barcoding of stygofauna uncovers cryptic amphipod diversity in a calcrete aquifer in Western Australia's arid zone

The arid Yilgarn region of Western Australia contains numerous subterranean calcrete aquifers with unique assemblages of obligate groundwater invertebrates (stygofauna). We aimed to establish a DNA barcoding framework for the macro-invertebrates present in a single calcrete, as a basis for future assessment of biodiversity of the Yilgarn calcretes and for investigating food webs. Intense sampling of a bore field grid in the Sturt Meadows calcrete was undertaken to obtain representatives of the entire macro-invertebrate ecosystem. A 623-bp fragment of the mitochondrial cytochrome c oxidase 1 (COI) gene was used to provide DNA barcodes for stygobiont macro-invertebrates plus terrestrial organisms that are found in the calcrete. Phylogenetic analyses revealed the existence of 12 divergent monophyletic groups of haplotypes. Subterranean amphipods (Chiltoniidae) showed three groups of COI haplotypes with sequence divergences between them of >11%. Allozyme analyses found a large number of fixed allelic differences between these three amphipod groups, indicating that there are three morphologically cryptic species within the Sturt Meadows calcrete. Unlike the sister triplet of dytiscid beetles present, the amphipods are not sister clades and are more closely related to other Yilgarn and non-Yilgarn amphipods than to each other. Our results show that the aquifer contains at least 12 macro-invertebrate species and DNA barcoding provides a useful means for discriminating species in this system.     

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.     

中国东南沿海弹涂鱼科常见鱼类的遗传多样性和DNA条形码

为了探究中国东南沿海海岸线经济开发、海水污染等对该区域生物遗传多样性的潜在影响,本文使用COI基因作为DNA条形码,对从中国东南沿海广东汕头、福建东山、福建云霄、福建泉州、福建霞浦、上海崇明6个地点采集的大弹涂鱼(Boleophthalmus pectinirostris)、弹涂鱼(Periophthalmus modestus)和青弹涂鱼(Scartelaos histophorus)进行种间和种内的遗传学分析。遗传多样性分析显示,青弹涂鱼的核苷酸多样性(0.0018)在3个种中最低;青弹涂鱼广东汕头种群的核苷酸多样性(0.0018)比福建云霄种群低;大弹涂鱼的上海崇明种群核苷酸多样性(0.0021)低于其他5个地理种群。物种水平分析表明,3个物种间分化明显,种间与种内遗传距离有明显差异;不同种的单倍型在系统发育树上聚成3个高度支持的独立分支。种群水平分析显示,大弹涂鱼的6个不同地理种群之间有共享单倍型,地理种群间与地理种群内的遗传距离重叠;各地理种群之间的基因流较频繁(Nm>4);青弹涂鱼的分析情况与大弹涂鱼基本一致。大弹涂鱼和青弹涂鱼的单倍型在系统发育树上均呈现多系或并系分布格局,并未按地理位置分成不同的单系群。本研究表明,应及时加强3种鱼类及其野生种群遗传多样性的保护,且COI基因对种一级的识别明确可靠,而在地理种群水平的识别则比较困难,有待进一步深入研究。     

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.     

DNA mini‐barcoding of leporids using noninvasive fecal DNA samples and its significance for monitoring an invasive species

Introduced in South America at the end of the 19th century, the European hare population has expanded dramatically and now represents a risk to native Brazilian forest rabbits. Monitoring the invasive Lepus europaeus and its coexistence with native Sylvilagus brasiliensis is a challenge that can be efficiently addressed by the use of molecular tools. This work describes a set of primers useful for amplifying three mini‐barcodes for the molecular identification of both invasive and native leporid species using degraded fecal DNA. In addition, tests in silico indicate that these mini‐barcodes can successfully amplify the DNA sequences of a number of leporids. These mini‐barcodes constitute a powerful tool for the monitoring and management of the invasive L. europaeus and the conservation of native rabbits.     

DNA barcoding highlights a cryptic species of grenadier Macrourus in the Southern Ocean

Although three species of the genus Macrourus are recognized in the Southern Ocean, DNA sequencing of the mitochondrial COI gene revealed four well-supported clades. These barcode data suggest the presence of an undescribed species, a conclusion supported by meristic and morphometric examination of specimens.     

Pliocene isolation of a north‐west Saharan cichlid fish

The evolutionary origin of the only north‐west Saharan haplochromine cichlid, Astatotilapia desfontainii, was explored using mitochondrial DNA sequences. Phylogenetic analyses revealed that this species belonged to the main East African–Nilotic haplochromine clade, but was a distinct lineage that diverged from modern haplochromines in the Pliocene. The results suggest that A. desfontainii is a relict haplochromine lineage that has endured major climate fluctuations in North Africa.     

Applications of DNA barcoding to fish landings: authentication and diversity assessment

DNA barcoding methodologies are being increasingly applied not only for scientific purposes but also for diverse real-life uses. Fisheries assessment is a potential niche for DNA barcoding, which serves for species authentication and may also be used for estimating within-population genetic diversity of exploited fish. Analysis of single-sequence barcodes has been proposed as a shortcut for measuring diversity in addition to the original purpose of species identification. Here we explore the relative utility of different mitochondrial sequences (12S rDNA, COI, cyt b, and D-Loop) for application as barcodes in fisheries sciences, using as case studies two marine and two freshwater catches of contrasting diversity levels. Ambiguous catch identification from COI and cyt b was observed. In some cases this could be attributed to duplicated names in databases, but in others it could be due to mitochondrial introgression between closely related species that may obscure species assignation from mtDNA. This last problem could be solved using a combination of mitochondrial and nuclear genes. We suggest to simultaneously analyze one conserved and one more polymorphic gene to identify species and assess diversity in fish catches.     

Molecular diversity of Germany's freshwater fishes and lampreys assessed by DNA barcoding

This study represents the first comprehensive molecular assessment of freshwater fishes and lampreys from Germany. We analysed COI sequences for almost 80% of the species mentioned in the current German Red List. In total, 1056 DNA barcodes belonging to 92 species from all major drainages were used to (i) build a reliable DNA barcode reference library, (ii) test for phylogeographic patterns, (iii) check for the presence of barcode gaps between species and (iv) evaluate the performance of the barcode index number (BIN) system, available on the Barcode of Life Data Systems. For over 78% of all analysed species, DNA barcodes are a reliable means for identification, indicated by the presence of barcode gaps. An overlap between intra‐ and interspecific genetic distances was present in 19 species, six of which belong to the genus Coregonus. The Neighbour‐Joining phenogram showed 60 nonoverlapping species clusters and three singleton species, which were related to 63 separate BIN numbers. Furthermore, Barbatula barbatula, Leucaspius delineatus, Phoxinus phoxinus and Squalius cephalus exhibited remarkable levels of cryptic diversity. In contrast, 11 clusters showed haplotype sharing, or low levels of divergence between species, hindering reliable identification. The analysis of our barcode library together with public data resulted in 89 BINs, of which 56% showed taxonomic conflicts. Most of these conflicts were caused by the use of synonymies, inadequate taxonomy or misidentifications. Moreover, our study increased the number of potential alien species in Germany from 14 to 21 and is therefore a valuable groundwork for further faunistic investigations.     

Tripleurospermum ziganaense (Asteraceae,Anthemideae), a new species from north‐east Anatolia,Turkey

A new species of Tripleurospermum Sch.Bip., Tripleurospermum ziganaense Inceer & Hay?rl?oglu‐Ayaz (Asteraceae, Anthemideae), is described and illustrated. The species grows in open places, on rocky slopes and on roadsides in north‐east Anatolia, Turkey. The diagnostic morphological characters that distinguish it from closely related taxa are discussed, and its conservation status is indicated. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158 , 696–700.     

Utility of DNA taxonomy and barcoding for the inference of larval community structure in morphologically cryptic Chironomus (Diptera) species

Biodiversity studies require species level analyses for the accurate assessment of community structures. However, while specialized taxonomic knowledge is only rarely available for routine identifications, DNA taxonomy and DNA barcoding could provide the taxonomic basis for ecological inferences. In this study, we assessed the community structure of sediment dwelling, morphologically cryptic Chironomus larvae in the Rhine-valley plain/Germany, comparing larval type classification, cytotaxonomy, DNA taxonomy and barcoding. While larval type classification performed poorly, cytotaxonomy and DNA-based methods yielded comparable results: detrended correspondence analysis and permutation analyses indicated that the assemblages are not randomly but competitively structured. However, DNA taxonomy identified an additional species that could not be resolved by the traditional method. We argue that DNA-based identification methods such as DNA barcoding can be a valuable tool to increase accuracy, objectivity and comparability of the taxonomic assessment in biodiversity and community ecology studies.     

Evaluation of candidate DNA barcoding loci for economically important timber species of the mahogany family (Meliaceae)

There has been considerable debate regarding locus choice for DNA barcoding land plants. This is partly attributable to a shortage of comparable data from proposed candidate loci on a common set of samples. In this study, we evaluated main candidate plastid regions (rpoC1, rpoB, accD) and additional plastid markers (psbB, psbN, psbT exons and the trnS-trnG spacer) as well as the nuclear ribosomal spacer region (ITS1-5.8S-ITS2) in a group of land plants belonging to the mahogany family, Meliaceae. Across these samples, only ITS showed high levels of resolvability. Interspecific sharing of sequences from individual plastid loci was common. The combination of multiple loci did not improve performance. DNA barcoding with ITS alone revealed cryptic species and proved useful in identifying species listed in Convention on International Trade of Endangered Species appendixes.     

Genetic diversity of trout (genus Salmo) from its most eastern native range based on mitochondrial DNA and nuclear gene variation

Russia and western Asia harbour trout populations that have been classified as distinct species and subspecies, most often on the basis of morphological and ecological variation. In order to assess their origins and to verify whether traditional taxonomy reflects their evolutionary distinctiveness, we documented their genetic relationships on the basis of mitochondrial DNA (mtDNA) RFLP, mtDNA sequence analysis, and allozyme variation. Both mtDNA and nuclear gene variation defined two ancient phylogenetic assemblages of populations distributed among northern (Baltic, White, Barents), and southern (Black, Caspian, Aral) sea basins, between which gene flow has been possible but limited in postglacial times. These results supported the traditional taxonomic differentiation between populations of these two regions. They provided weak support for the taxonomic distinction of southern brown trout (Salmo trutta) populations based on their basin of origin. They also refuted the hypothesis that L. Sevan trout (Salmo ischchan) diverged from a primitive brown trout ancestor. Nevertheless, all trout populations from southern sea basins possessed private alleles or mtDNA genotypes and were genetically distinct Therefore, they represent unique gene pools that warrant individual recognition for conservation and management.     

An integrative approach to species discovery in odonates: from character‐based DNA barcoding to ecology

Modern taxonomy requires an analytical approach incorporating all lines of evidence into decision‐making. Such an approach can enhance both species identification and species discovery. The character‐based DNA barcode method provides a molecular data set that can be incorporated into classical taxonomic data such that the discovery of new species can be made in an analytical framework that includes multiple sources of data. We here illustrate such a corroborative framework in a dragonfly model system that permits the discovery of two new, but visually cryptic species. In the African dragonfly genus Trithemis three distinct genetic clusters can be detected which could not be identified by using classical taxonomic characters. In order to test the hypothesis of two new species, DNA‐barcodes from different sequence markers (ND1 and COI) were combined with morphological, ecological and biogeographic data sets. Phylogenetic analyses and incorporation of all data sets into a scheme called taxonomic circle highly supports the hypothesis of two new species. Our case study suggests an analytical approach to modern taxonomy that integrates data sets from different disciplines, thereby increasing the ease and reliability of both species discovery and species assignment.     

Evaluation of DNA barcoding and identification of new haplomorphs in Canadian deerflies and horseflies

This paper reports the first tests of the suitability of the standardized mitochondrial cytochrome c oxidase subunit I (COI) barcoding system for the identification of Canadian deerflies and horseflies. Two additional mitochondrial molecular markers were used to determine whether unambiguous species recognition in tabanids can be achieved. Our 332 Canadian tabanid samples yielded 650 sequences from five genera and 42 species. Standard COI barcodes demonstrated a strong A + T bias (mean 68.1%), especially at third codon positions (mean 93.0%). Our preliminary test of this system showed that the standard COI barcode worked well for Canadian Tabanidae: the target DNA can be easily recovered from small amounts of insect tissue and aligned for all tabanid taxa. Each tabanid species possessed distinctive sets of COI haplotypes which discriminated well among species. Average conspecific Kimura two‐parameter (K2P) divergence (0.49%) was 12 times lower than the average divergence within species. Both the neighbour‐joining and the Bayesian methods produced trees with identical monophyletic species groups. Two species, Chrysops dawsoni Philip and Chrysops montanus Osten Sacken (Diptera: Tabanidae), showed relatively deep intraspecific sequence divergences (～10 times the average) for all three mitochondrial gene regions analysed. We suggest provisional differentiation of Ch. montanus into two haplotypes, namely, Ch. montanus haplomorph 1 and Ch. montanus haplomorph 2, both defined by their molecular sequences and by newly discovered differences in structural features near their ocelli.     

甘肃省鱼类资源现状及DNA条形码在鱼类物种鉴定中的应用

为了摸清甘肃省土著鱼类资源与分布现状, 探索DNA条形码在鱼类辅助物种鉴定中的适用性, 2012年6-9月对甘肃境内黄河水系、嘉陵江水系和河西内陆河水系进行了较全面的鱼类调查。共采集鱼类标本3,087尾, 隶属于5目10科38属64种, 以鲤科种类最多, 为30种, 占总种数的46.88%。物种多样性分析表明, 在黄河水系的夏河和庄浪河多样性指数是所有调查点中最低的, 分别为1.38和1.09。嘉陵江水系各河段的多样性指数较高(H = 2.15-3.27), 其次为河西内陆河水系(H = 2.01-2.83)。在河西内陆河水系中, 疏勒河的均匀度指数最高, 为1.10, 黑河最低(0.68)。庄浪河的优势度指数最高, 为0.34, 而嘉陵江干流两当段的优势度指数在所有调查点中最低, 为0.04。利用DNA条形码分析了49种662尾标本的COI基因部分序列, 大部分种类在neighbor-joining系统树中形成各自的单系, 种内平均遗传距离0.88%, 种间平均遗传距离为9.99%, 在种内和种间COI序列遗传距离之间形成明显的条形码间隙, 斯氏高原鳅(Triplophysa stoliczkae)与达里湖高原鳅(T. dalaica), 甘肃高原鳅(T. robusta)与似鲇高原鳅(T. siluroides), 嘉陵裸裂尻鱼(Schizopygopsis kialingensis)与黄河裸裂尻鱼(S. pylzovi)之间的遗传距离低于2%, 甘肃高原鳅与似鲇高原鳅不能通过COI基因片段区分开, 其他两对物种可以采用核苷酸诊断法来进一步区分。斯氏高原鳅和拉氏鱼岁(Phoxinus lagowskii)种内遗传分歧较大, 揭示种内可能存在隐存种。结果表明, 对某些近缘种和不同地理种群差异较大的物种, 要将分子、形态和地理分布特点结合起来才能准确鉴定。     

Multilocus phylogeny and Bayesian estimates of species boundaries reveal hidden evolutionary relationships and cryptic diversity in Southeast Asian monitor lizards

Recent conceptual, technological and methodological advances in phylogenetics have enabled increasingly robust statistical species delimitation in studies of biodiversity. As the variety of evidence purporting species diversity has increased, so too have the kinds of tools and inferential power of methods for delimiting species. Here, we showcase an organismal system for a data‐rich, comparative molecular approach to evaluating strategies of species delimitation among monitor lizards of the genus Varanus. The water monitors (Varanus salvator Complex), a widespread group distributed throughout Southeast Asia and southern India, have been the subject of numerous taxonomic treatments, which have drawn recent attention due to the possibility of undocumented species diversity. To date, studies of this group have relied on purportedly diagnostic morphological characters, with no attention given to the genetic underpinnings of species diversity. Using a 5‐gene data set, we estimated phylogeny and used multilocus genetic networks, analysis of population structure and a Bayesian coalescent approach to infer species boundaries. Our results contradict previous systematic hypotheses, reveal surprising relationships between island and mainland lineages and uncover novel, cryptic evolutionary lineages (i.e. new putative species). Our study contributes to a growing body of literature suggesting that, used in concert with other sources of data (e.g. morphology, ecology, biogeography), multilocus genetic data can be highly informative to systematists and biodiversity specialists when attempting to estimate species diversity and identify conservation priorities. We recommend holding in abeyance taxonomic decisions until multiple, converging lines of evidence are available to best inform taxonomists, evolutionary biologists and conservationists.     

中国华东地区孤雌生殖的滨棘虫兆属一新种记述及DNA条形码分析（弹尾纲：棘虫兆科）

记述中国华东地区上海市的滨棘虫兆属1新种:后滩滨棘虫兆Thalassaphorura houtanensis sp.nov.。该种与T.encarpata(Denis,1931)近似,二者均具有A型下唇和相同的假眼式,但可通过爪的形状、下唇毛序、下颚须和腹管加以区分;实验室内的繁殖实验证明该种为严格孤雌生殖种类;同时,对该新种的DNA条形码序列进行了测定分析,发现该新种的种内遗传变异平均为0.18%,介于0~0.46%之间。