Phylogeny,natural groups and nemertean classification

Contemporary practice in the classification of nemerteans (phylum Nemertea) is critically discussed. It is argued that basing higher taxa on the existence of a unique combination of characters in a species (or genus) is unlikely to lead to monophyletic taxa, and that this approach should be abandoned in favour of a classification based on explicit hypotheses of phylogeny. These hypotheses should be based on all available characters and characters should not be excluded before the analysis. The classification should be based on a reconstruction of the phylogeny and reflect this phylogeny in an unambiquous way.     

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.     

A Retrospective Approach to Testing the DNA Barcoding Method

A decade ago, DNA barcoding was proposed as a standardised method for identifying existing species and speeding the discovery of new species. Yet, despite its numerous successes across a range of taxa, its frequent failures have brought into question its accuracy as a short-cut taxonomic method. We use a retrospective approach, applying the method to the classification of New Zealand skinks as it stood in 1977 (primarily based upon morphological characters), and compare it to the current taxonomy reached using both morphological and molecular approaches. For the 1977 dataset, DNA barcoding had moderate-high success in identifying specimens (78-98%), and correctly flagging specimens that have since been confirmed as distinct taxa (77-100%). But most matching methods failed to detect the species complexes that were present in 1977. For the current dataset, there was moderate-high success in identifying specimens (53-99%). For both datasets, the capacity to discover new species was dependent on the methodological approach used. Species delimitation in New Zealand skinks was hindered by the absence of either a local or global barcoding gap, a result of recent speciation events and hybridisation. Whilst DNA barcoding is potentially useful for specimen identification and species discovery in New Zealand skinks, its error rate could hinder the progress of documenting biodiversity in this group. We suggest that integrated taxonomic approaches are more effective at discovering and describing biodiversity.     

DNA barcoding and phylogenetic analysis of Pectinidae (Mollusca: Bivalvia) based on mitochondrial COI and 16S rRNA genes

DNA sequence data enable not only the inference of phylogenetic relationships but also provide an efficient method for species-level identifications under the terms DNA barcoding or DNA taxonomy. In this study, we have sequenced partial sequences of mitochondrial COI and 16S rRNA genes from 63 specimens of 8 species of Pectinidae to assess whether DNA barcodes can efficiently distinguish these species. Sequences from homologous regions of four other species of this family were gathered from GenBank. Comparisons of within and between species levels of sequence divergence showed that genetic variation between species exceeds variation within species. When using neighbour-joining clustering based on COI and 16S genes, all species fell into reciprocally monophyletic clades with high bootstrap values. These evidenced that these scallop species can be efficiently identified by DNA barcoding. Evolutionary relationships of Pectinidae were also examined using the two mitochondrial genes. The results are almost consistent with Waller’s classification, which was proposed on the basis of shell microstructure and the morphological characteristics of juveniles.     

Identifying species of moths (Lepidoptera) from Baihua Mountain,Beijing, China,using DNA barcodes

DNA barcoding has become a promising means for the identification of organisms of all life‐history stages. Currently, distance‐based and tree‐based methods are most widely used to define species boundaries and uncover cryptic species. However, there is no universal threshold of genetic distance values that can be used to distinguish taxonomic groups. Alternatively, DNA barcoding can deploy a “character‐based” method, whereby species are identified through the discrete nucleotide substitutions. Our research focuses on the delimitation of moth species using DNA‐barcoding methods. We analyzed 393 Lepidopteran specimens belonging to 80 morphologically recognized species with a standard cytochrome c oxidase subunit I (COI) sequencing approach, and deployed tree‐based, distance‐based, and diagnostic character‐based methods to identify the taxa. The tree‐based method divided the 393 specimens into 79 taxa (species), and the distance‐based method divided them into 84 taxa (species). Although the diagnostic character‐based method found only 39 so‐identifiable species in the 80 species, with a reduction in sample size the accuracy rate substantially improved. For example, in the Arctiidae subset, all 12 species had diagnostics characteristics. Compared with traditional morphological method, molecular taxonomy performed well. All three methods enable the rapid delimitation of species, although they have different characteristics and different strengths. The tree‐based and distance‐based methods can be used for accurate species identification and biodiversity studies in large data sets, while the character‐based method performs well in small data sets and can also be used as the foundation of species‐specific biochips.     

DNA barcoding in Tardigrada: the first case study on Macrobiotus macrocalix Bertolani & Rebecchi 1993 (Eutardigrada, Macrobiotidae)

Morphological and molecular studies on a tardigrade species have been carried out to verify the possibility of using a DNA barcoding approach for species identification in this phylum. Macrobiotus macrocalix Bertolani & Rebecchi, 1993 was chosen as the test species since it belongs to a group of species in which the taxonomy is quite problematic. Animals and eggs belonging to three Italian and one Swedish populations have been investigated. Both morphological and molecular analyses show that all the populations belong to the same species. The low genetic distances recorded among the studied populations (0.3-1.0%) and the high genetic distance (15.9-16.3%) between these populations and a closely related species confirm the possibility of identifying a specimen of this species by its cytochrome oxidase subunit I sequence. Data from other authors support our results indicating that DNA barcoding can be applied to tardigrades. With our protocols, we have obtained voucher specimens that enable us to show a correspondence between morphology and molecular data.     

Using DNA barcodes for assessing diversity in the?family Hybotidae (Diptera,Empidoidea)

Empidoidea is one of the largest extant lineages of flies, but phylogenetic relationships among species of this group are poorly investigated and global diversity remains scarcely assessed. In this context, one of the most enigmatic empidoid families is Hybotidae. Within the framework of a pilot study, we barcoded 339 specimens of Old World hybotids belonging to 164 species and 22 genera (plus two Empis as outgroups) and attempted to evaluate whether patterns of intra- and interspecific divergences match the current taxonomy. We used a large sampling of diverse Hybotidae. The material came from the Palaearctic (Belgium, France, Portugal and Russian Caucasus), the Afrotropic (Democratic Republic of the Congo) and the Oriental realms (Singapore and Thailand). Thereby, we optimized lab protocols for barcoding hybotids. Although DNA barcodes generally well distinguished recognized taxa, the study also revealed a number of unexpected phenomena: e.g., undescribed taxa found within morphologically very similar or identical specimens, especially when geographic distance was large; some morphologically distinct species showed no genetic divergence; or different pattern of intraspecific divergence between populations or closely related species. Using COI sequences and simple Neighbour-Joining tree reconstructions, the monophyly of many species- and genus-level taxa was well supported, but more inclusive taxonomical levels did not receive significant bootstrap support. We conclude that in hybotids DNA barcoding might be well used to identify species, when two main constraints are considered. First, incomplete barcoding libraries hinder efficient (correct) identification. Therefore, extra efforts are needed to increase the representation of hybotids in these databases. Second, the spatial scale of sampling has to be taken into account, and especially for widespread species or species complexes with unclear taxonomy, an integrative approach has to be used to clarify species boundaries and identities.     

Evaluating DNA barcoding criteria using African duiker antelope (Cephalophinae) as a test case

African duikers in the subfamily Cephalophinae (genera Cephalophus, Philantomba and Sylvicapra) constitute an important target for DNA barcoding efforts because of their importance to the bushmeat trade and protection under the Convention for International Trade in Endangered Species (CITES). Duikers also make a challenging test case of barcoding methods due to their recent diversification, substantial intra-specific genetic variation and high species richness. However, no study to date has evaluated how well DNA barcoding methods can be used to delineate all of the taxa within this group. To address this question, cytochrome c oxidase subunit 1 (COX1) sequences from all eighteen species within this subfamily and an outgroup taxon (genus Tragelaphus) were used to build a neighbor-joining tree, identify species-specific diagnostic synapomorphies, and determine whether species exceed a given pair-wise genetic distance threshold commonly employed in DNA barcoding studies. Tree-based analyses of the data indicate that several species within two clusters of closely related taxa consistently failed to form reciprocally monophyletic clades and similarly lack species-specific synapomorphies. Furthermore, one additional taxon failed to constitute a diagnosable clade and another occupied an unresolved position in the tree. Of the two genetic distance criteria evaluated, the 3% threshold was far more effective in delimiting species than a threshold level based on the ratio of inter- to intra-specific distances. However, neither approach could effectively delineate all sister species. While the taxonomy of this group might be open to question, the fact that barcodes consistently failed to differentiate several currently recognized sister taxa challenges the routine application of this approach in forensic studies of duiker species. Future barcoding work of this group should always include a complete taxonomic sampling and strive to include a broader geographic sampling of sequence diversity than has been carried out to date. Lastly, this work highlights the need to re-examine the taxonomy of this group, which may illuminate why some barcoding criteria fail to reliably differentiate species.     

Testing the utility of mitochondrial cytochrome oxidase subunit 1 sequences for phylogenetic estimates of relationships between crane (Grus) species

Morphology and biogeography are widely used in animal taxonomy. Recent study has suggested that a DNA-based identification system, using a 648-bp portion of the mitochondrial gene cytochrome oxidase subunit 1 (CO1), also known as the barcoding gene, can aid in the resolution of inferences concerning phylogenetic relationships and for identification of species. However, the effectiveness of DNA barcoding for identifying crane species is unknown. We amplified and sequenced 894-bp DNA fragments of CO1 from Grus japonensis (Japanese crane), G. grus (Eurasian crane), G. monacha (hooded crane), G. canadensis (sandhill crane), G. leucogeranus (Siberian crane), and Balearica pavonina (crowned crane), along with those of 15 species obtained from GenBank and DNA barcoding, to construct four algorithms using Tringa stagnatilis, Scolopax rusticola, and T. erythropus as outgroups. The four phylum profiles showed good resolution of the major taxonomic groups. We concluded that reconstruction of the molecular phylogenetic tree can be helpful for classification and that CO1 sequences are suitable for studying the molecular evolution of cranes. Although support for several deeper branches was limited, CO1 data gave remarkably good separations, especially considering that our analysis was based on just a fragment of the gene and that CO1 has generally been viewed as useful only for resolving shallow divergences.     

Cryptic biodiversity and phylogenetic relationships revealed by DNA barcoding of Oriental black flies in the subgenus Gomphostilbia (Diptera: Simuliidae)

Understanding the medical, economic, and ecological importance of black flies relies on correct identification of species. However, traditional taxonomy of black flies is impeded by a high degree of morphological uniformity, especially the presence of cryptic biodiversity, historically recognized by details of chromosomal banding patterns. We assess the utility of DNA barcoding, based on cytochrome c oxidase subunit 1 (COI) sequences, for identifying 13 species of Oriental black flies in the subgenus Gomphostilbia. Samples of larvae fixed in Carnoy's solution were used to gather molecular and chromosomal data from the same individual. We found that larvae refrigerated in Carnoy's fixative for as long as 11 years can be used for DNA study. Levels of intraspecific genetic divergence, based on the Kimura-2 parameter, range from 0% to 9.28%, with a mean of 2.75%, whereas interspecific genetic divergence ranges from 0.34% to 16.05%. Values of intraspecific and interspecific genetic divergence overlap in seven species owing to incomplete lineage sorting and imperfect taxonomy, implying that DNA barcoding to identify these species will be ambiguous. Despite a low level of success, we found that DNA barcoding is useful in revealing cryptic biodiversity, potentially facilitating traditional taxonomy. Phylogenetic analyses indicate that species groups currently recognized on morphological criteria are not monophyletic, suggesting a need to reevaluate the classification of the subgenus Gomphostilbia.     

A new approach to an old conundrum--DNA barcoding sheds new light on phenotypic plasticity and morphological stasis in microsnails (Gastropoda, Pulmonata, Carychiidae)

The identification of microsnail taxa based on morphological characters is often a time-consuming and inconclusive process. Aspects such as morphological stasis and phenotypic plasticity further complicate their taxonomic designation. In this study, we demonstrate that the application of DNA barcoding can alleviate these problems within the Carychiidae (Gastropoda, Pulmonata). These microsnails are a taxon of the pulmonate lineage and most likely migrated onto land independently of the Stylommatophora clade. Their taxonomical classification is currently based on conchological and anatomical characters only. Despite much confusion about historic species assignments, the Carychiidae can be unambiguously subdivided into two taxa: (i) Zospeum species, which are restricted to karst caves, and (ii) Carychium species, which occur in a broad range of environmental conditions. The implementation of discrete molecular data (COI marker) enabled us to correctly designate 90% of the carychiid microsnails. The remaining cases were probably cryptic Zospeum and Carychium taxa and incipient species, which require further investigation into their species status. Because conventional reliance upon mostly continuous (i.e. nondiscrete) conchological characters is subject to fallibility for many gastropod species assignments, we highly recommend the use of DNA barcoding as a taxonomic, cutting-edge method for delimiting microsnail taxa.     

DNA条形码技术在河北保定、廊坊地区鳞翅目昆虫上的应用及小型区域数据库的构建

【目的】为了探究DNA条形码技术和小型区域数据库在蛾类鉴定上的可行性,本研究利用条形码通用引物扩增了采自河北保定、廊坊地区10种夜蛾82个样本的线粒体细胞色素c氧化酶亚基I(Mitochondrial cytochrome c oxidase subunit I,COⅠ)基因序列。【方法】基于进化树、距离、阈值和特征的方法。【结果】虽然整体分类效果较好,但基于进化树、距离、阈值的方法都无法将二点委夜蛾Athetis lepigone进行较好的分类;样本LF110802.008总是被分入标瑙夜蛾Maliattha signifera类群,与形态学分类结果发生分歧。基于特征的方法运用核基因28S进行分析,结果与形态分类一致。同时还探讨了基于特征方法得到的诊断特征数目与样本数量之间的关系,发现两者密切相关;基于特征的方法对小样本量的鉴定也比较有效。本研究建立了小型区域的DNA条形码数据库,使物种识别具有更强的针对性,有利于提高地区性蛾类病虫害防治效果。【结论】在蛾类鉴定中,DNA条形码有很好的分类效果,小型区域数据库很有实际应用价值。     

The Necessity of DNA Taxonomy to Reveal Cryptic Diversity and Spatial Distribution of Meiofauna,with a Focus on Nemertea

Meiofauna represent one of the most abundant and diverse communities in marine benthic ecosystems. However, an accurate assessment of diversity at the level of species has been and remains challenging for these microscopic organisms. Therefore, for many taxa, especially the soft body forms such as nemerteans, which often lack clear diagnostic morphological traits, DNA taxonomy is an effective means to assess species diversity. Morphological taxonomy of Nemertea is well documented as complicated by scarcity of unambiguous character states and compromised by diagnoses of a majority of species (and higher clades) being inadequate or based on ambiguous characters and character states. Therefore, recent studies have advocated for the primacy of molecular tools to solve the taxonomy of this group. DNA taxonomy uncovers possible hidden cryptic species, provides a coherent means to systematize taxa in definite clades, and also reveals possible biogeographic patterns. Here, we analyze diversity of nemertean species by considering the barcode region of the mitochondrial gene Cytochrome Oxidase subunit I (COI) and different species delineation approaches in order to infer evolutionarily significant units. In the aim to uncover actual diversity of meiofaunal nemerteans across different sites in Central America, COI sequences were obtained for specimens assigned here to the genera Cephalothrix, Ototyphlonemertes, and Tetrastemma-like worms, each commonly encountered in our sampling. Additional genetic, taxonomic, and geographic data of other specimens belonging to these genera were added from GenBank. Results are consistent across different DNA taxonomy approaches, and revealed (i) the presence of several hidden cryptic species and (ii) numerous potential misidentifications due to traditional taxonomy. (iii) We additionally test a possible biogeographic pattern of taxonomic units revealed by this study, and, except for a few cases, the putative species seem not to be widely distributed, in contrast to what traditional taxonomy would suggest for the recognized morphotypes.     

After 7 years and 1000 citations: comparative assessment of the DNA barcoding and the DNA taxonomy proposals for taxonomists and non-taxonomists

In 2003, two different approaches-DNA taxonomy and DNA barcoding-were simultaneously proposed to overcome some of the perceived intrinsic weaknesses of the traditional morphology-based taxonomical system, and to help non-taxonomists to resolve their crucial need for accurate and rapid species identification tools. After 7 years, it seems unlikely that a completely new taxonomical system based on molecular characters only (DNA taxonomy) will develop in the future. It is more likely that both morphological and molecular data will be simultaneously analyzed, developing what has been coined as "integrative taxonomy". Concerning DNA barcoding, it is now clear that it does not focus on building a tree-of-life nor to perform DNA taxonomy, but rather to produce a universal molecular identification key based on strong taxonomic knowledge that is collated in the barcode reference library. The indisputable success of the DNA barcoding project is chiefly due to the fact that DNA barcoding standards considerably enhance current practices in the molecular identification field, and standardization offers virtually endless applications for various users.     

DNA barcoding reveals a cryptic nemertean invasion in Atlantic and Mediterranean waters

For several groups, like nemerteans, morphology-based identification is a hard discipline, but DNA barcoding may help non-experts in the identification process. In this study, DNA barcoding is used to reveal the cryptic invasion of Pacific Cephalothrix cf. simula into Atlantic and Mediterranean coasts. Although DNA barcoding is a promising method for the identification of Nemertea, only 6 % of the known number of nemertean species is currently associated with a correct DNA barcode. Therefore, additional morphological and molecular studies are necessary to advance the utility of DNA barcoding in the characterisation of possible nemertean alien invasions.     

Italian odonates in the Pandora's box: A comprehensive DNA barcoding inventory shows taxonomic warnings at the Holarctic scale

The Odonata are considered among the most endangered freshwater faunal taxa. Their DNA‐based monitoring relies on validated reference data sets that are often lacking or do not cover important biogeographical centres of diversification. This study presents the results of a DNA barcoding campaign on Odonata, based on the standard 658‐bp 5′ end region of the mitochondrial COI gene, involving the collection of 812 specimens (409 of which barcoded) from peninsular Italy and its main islands (328 localities), belonging to all the 88 species (31 Zygoptera and 57 Anisoptera) known from the country. Additional BOLD and GenBank data from Holarctic samples expanded the data set to 1,294 DNA barcodes. A multi‐approach species delimitation analysis involving two distance (OT and ABGD) and four tree‐based (PTP, MPTP, GMYC and bGMYC) methods was used to explore these data. Of the 88 investigated morphospecies, 75 (85%) unequivocally corresponded to distinct molecular operational units, whereas the remaining ones were classified as ‘warnings’ (i.e. showing a mismatch between morphospecies assignment and DNA‐based species delimitation). These results are in contrast with other DNA barcoding studies on Odonata showing up to 95% of identification success. The species causing warnings were grouped into three categories depending on if they showed low, high or mixed genetic divergence patterns. The analysis of haplotype networks revealed unexpected intraspecific complexity at the Italian, Palearctic and Holarctic scale, possibly indicating the occurrence of cryptic species. Overall, this study provides new insights into the taxonomy of odonates and a valuable basis for future DNA and eDNA‐based monitoring studies.     

An evaluation of candidate plant DNA barcodes and assignment methods in diagnosing 29 species in the genus Agalinis (Orobanchaceae)

? Premise of the study: DNA barcoding has been proposed as a useful technique within many disciplines (e.g., conservation biology and forensics) for determining the taxonomic identity of a sample based on nucleotide similarity to samples of known taxonomy. Application of DNA barcoding to plants has primarily focused on evaluating the success of candidate barcodes across a broad spectrum of evolutionary divergence. Less attention has been paid to evaluating performance when distinguishing congeners or to differential success of analytical techniques despite the fact that the practical application and utility of barcoding hinges on the ability to distinguish closely related species. ? Methods: We tested the ability to distinguish among 92 samples representing 29 putative species in the genus Agalinis (Orobanchaceae) using 13 candidate barcodes and three analytical methods (i.e., threshold genetic distances, hierarchical tree-based, and diagnostic character differences). Due to questions regarding evolutionary distinctiveness of some taxa, we evaluated success under two taxonomic hypotheses. ? Key results: The psbA-trnH and trnT-trnL barcodes in conjunction with the "best close match" distance-based method best met the objectives of DNA barcoding. Success was also a function of the taxonomy used. ? Conclusions: In addition to accurately identifying query sequences, our results showed that DNA barcoding is useful for detecting taxonomic uncertainty; determining whether erroneous taxonomy or incomplete lineage sorting is the cause requires additional information provided by traditional taxonomic approaches. The magnitude of differentiation within and among the Agalinis species sampled suggests that our results inform how DNA barcoding will perform among closely related species in other genera.     

DNA条形码技术及其在微生物资源鉴别保护中的应用研究

DNA条形码作为一种新型的物种鉴定、分类、鉴别和溯源方法,通过生物信息学分析将标准DNA片段进行分析比对实现。经过多年的发展,该技术现已成为物种鉴定和分类的研究热点。回顾十余年来DNA条形码技术的发展历程,介绍了这一技术的进展与成果,分析了该技术的优势及局限,并展望了DNA条形码的应用前景,为后续研究提供参考。     

Testing evolutionary hypotheses for DNA barcoding failure in willows

The goal of DNA barcoding is to enable the rapid identification of taxa from short diagnostic DNA sequence profiles. But how feasible is this objective when many evolutionary processes, such as hybridization and selective sweeps, cause alleles to be shared among related taxa? In this issue of Molecular Ecology, Percy et al. (2014) test the full suite of seven candidate plant barcoding loci in a broad geographic sample of willow species. They show exceptional plastid haplotype sharing between species across continents, with most taxa not possessing a unique barcode sequence. Using population genetic and molecular dating analyses, they implicate hybridization and selective sweeps, but not incomplete lineage sorting, as the historical processes causing widespread haplotype sharing among willow taxa. This study represents an exceptional case of how poorly barcoding can perform, and highlights methodological issues using universal organellar regions for species identification.