Toward a global DNA barcode reference library of the intolerant nonbiting midge genus Rheocricotopus Brundin, 1956

Environmental DNA metabarcoding is becoming a predominant tool in biodiversity assessment, as this time‐ and cost‐efficient tactics have the ability to increase monitoring accuracy. As a worldwide distributed genus, Rheocricotopus Brundin, 1956 still does not possess a complete and comprehensive global DNA barcode reference library for biodiversity monitoring. In the present study, we compiled a cytochrome c oxidase subunit 1 (COI) DNA barcode library of Rheocricotopus with 434 barcodes around the world, including 121 newly generated DNA barcodes of 32 morphospecies and 313 public barcodes. Automatic Barcode Gap Discovery (ABGD) was applied on the 434 COI barcodes to provide a comparison between the operational taxonomic units (OTU) number calculated from the Barcode Index Number (BIN) with the “Barcode Gap Analysis” and neighbor‐joining (NJ) tree analysis. Consequently, these 434 COI barcodes were clustered into 78 BINs, including 42 new BINs. ABGD yielded 51 OTUs with a prior intraspecific divergence of Pmax = 7.17%, while NJ tree revealed 52 well‐separated clades. Conservatively, 14 unknown species and one potential synonym were uncovered with reference to COI DNA barcodes. Besides, based on our ecological analysis, we discovered that annual mean temperature and annual precipitation could be considered as key factors associated with distribution of certain members from this genus. Our global DNA barcode reference library of Rheocricotopus provides one fundamental database for accurate species delimitation in Chironomidae taxonomy and facilitates the biodiversity monitoring of aquatic biota.     

Cryptic diversity and discordance in single‐locus species delimitation methods within horned lizards (Phrynosomatidae: Phrynosoma)

Biodiversity reduction and loss continues to progress at an alarming rate, and thus, there is widespread interest in utilizing rapid and efficient methods for quantifying and delimiting taxonomic diversity. Single‐locus species delimitation methods have become popular, in part due to the adoption of the DNA barcoding paradigm. These techniques can be broadly classified into tree‐based and distance‐based methods depending on whether species are delimited based on a constructed genealogy. Although the relative performance of these methods has been tested repeatedly with simulations, additional studies are needed to assess congruence with empirical data. We compiled a large data set of mitochondrial ND4 sequences from horned lizards (Phrynosoma) to elucidate congruence using four tree‐based (single‐threshold GMYC, multiple‐threshold GMYC, bPTP, mPTP) and one distance‐based (ABGD) species delimitation models. We were particularly interested in cases with highly uneven sampling and/or large differences in intraspecific diversity. Results showed a high degree of discordance among methods, with multiple‐threshold GMYC and bPTP suggesting an unrealistically high number of species (29 and 26 species within the P. douglasii complex alone). The single‐threshold GMYC model was the most conservative, likely a result of difficulty in locating the inflection point in the genealogies. mPTP and ABGD appeared to be the most stable across sampling regimes and suggested the presence of additional cryptic species that warrant further investigation. These results suggest that the mPTP model may be preferable in empirical data sets with highly uneven sampling or large differences in effective population sizes of species.     

Integrative taxonomy reveals hidden diversity in the southern African darters genus Nannocharax Günther 1867 (Characiformes: Distichodontidae)

The present study explored the diversity of Nannocharax within southern Africa by implementing three species delimitation methods for a data set consisting of 37 mitochondrial cytochrome oxidase subunit I sequences. Two unilocus coalescent methods, the General Mixed Yule Coalescent (GMYC) and the Bayesian implementation of the Poisson Tree Processes (bPTP), and a genetic distance method, the Automatic Barcode Gap Discovery (ABGD), were applied. Both GMYC and bPTP delimited the same operational taxonomic units (OTUs), revealing a higher diversity for the genus in the region than previously recognised, whereas the ABGD failed to delimit the same candidate species. All methods delimited two species groups, and these are supported based on colouration patterning and morphology; the Nannocharax multifasciatus and the Nannocharax macropterus species groups and the delimited OTUs were assigned to each. Two putative new species were identified, Nannocharax cf. lineostriatus “Okavango” from the Okavango River in Angola and N. cf. lineostriatus “Kwanza” from the Kwanza River system in Angola. The distribution of Nannocharax dageti was confirmed for the Upper Zambezi and extended to the Okavango system, and an identification key for the southern Africa Nannocharax species is provided.     

Exploring species boundaries with multiple genetic loci using empirical data from non‐biting midges

Over the past decade, molecular approaches to species delimitation have seen rapid development. However, species delimitation based on a single locus, for example, DNA barcodes, can lead to inaccurate results in cases of recent speciation and incomplete lineage sorting. Here, we compare the performance of Automatic Barcode Gap Discovery (ABGD), Bayesian Poisson tree processes (PTP), networks, generalized mixed Yule coalescent (GMYC) and Bayesian phylogenetics and phylogeography (BPP) models to delineate cryptic species previously detected by DNA barcodes within Tanytarsus (Diptera: Chironomidae) non‐biting midges. We compare the results from analyses of one mitochondrial (cytochrome c oxidase subunit I [COI]) and three nuclear (alanyl‐tRNA synthetase 1 [AATS1], carbamoyl phosphate synthetase 1 [CAD1] and 6‐phosphogluconate dehydrogenase [PGD]) protein‐coding genes. Our results show that species delimitation based on multiple nuclear DNA markers is largely concordant with morphological variation and delimitations using a single locus, for example, the COI barcode. However, ABGD, GMYC, PTP and network models led to conflicting results based on a single locus and delineate species differently than morphology. Results from BPP analyses on multiple loci correspond best with current morphological species concept. In total, 10 lineages of the Tanytarsus curticornis species complex were uncovered. Excluding a Norwegian population of Tanytarsus brundini which might have undergone recent hybridization, this suggests six hitherto unrecognized species new to science. Five distinct species are well supported in the Tanytarsus heusdensis species complex, including two species new to science.     

中国北方常见芫菁分子物种界定(鞘翅目: 芫菁科)

【目的】应用线粒体COI和核CAD基因片段探讨自动条形码间隔探索(automatic barcode gapdiscovery, ABGD)、广义混合Yule溯祖模型(generalized mixed Yule coalescent, GMYC)、贝叶斯泊松树进程(Bayesian Poisson tree processes, bPTP)和贝叶斯系统发育和系统地理分析(Bayesianphylogenetics and phylogeography, BPP) 4种分析方法在芫菁科(Meloidae)昆虫分子物种界定中的适用性。【方法】分别基于COI, CAD和COI+CAD串联序列数据集,应用ABGD, GMYC, bPTP和BPP 4种方法对中国北方芫菁科常见的6属(沟芫菁属Hycleus、斑芫菁属Mylabris、豆芫菁属Epicauta、绿芫菁属Lytta、星芫菁属Megatrachelus和短翅芫菁属Meloe)18个形态种进行分子物种界定,并与形态学鉴定结果进行比较。【结果】利用COI+CAD串联序列数据集所得物种界定结果与形态鉴定结果一致;COI数据集使用ABGD和GMYC方法的界定结果与形态鉴定结果一致,而bPTP划分的物种数较形态鉴定结果多;基于CAD序列在3种单基因物种界定方法的结果中,除GMYC与形态划分一致外,其余均显示部分结果与形态划分不同。【结论】在芫菁科分子物种界定中,多基因联合序列、多种界定方法分析所得结果优于单一基因片段和界定方法的分析结果。本研究的结果为芫菁科昆虫的分子物种界定和整合分类提供了数据支持和参考。     

Exploring Genetic Divergence in a Species-Rich Insect Genus Using 2790 DNA Barcodes

DNA barcoding using a fragment of the mitochondrial cytochrome c oxidase subunit 1 gene (COI) has proven to be successful for species-level identification in many animal groups. However, most studies have been focused on relatively small datasets or on large datasets of taxonomically high-ranked groups. We explore the quality of DNA barcodes to delimit species in the diverse chironomid genus Tanytarsus (Diptera: Chironomidae) by using different analytical tools. The genus Tanytarsus is the most species-rich taxon of tribe Tanytarsini (Diptera: Chironomidae) with more than 400 species worldwide, some of which can be notoriously difficult to identify to species-level using morphology. Our dataset, based on sequences generated from own material and publicly available data in BOLD, consist of 2790 DNA barcodes with a fragment length of at least 500 base pairs. A neighbor joining tree of this dataset comprises 131 well separated clusters representing 121 morphological species of Tanytarsus: 77 named, 16 unnamed and 28 unidentified theoretical species. For our geographically widespread dataset, DNA barcodes unambiguously discriminate 94.6% of the Tanytarsus species recognized through prior morphological study. Deep intraspecific divergences exist in some species complexes, and need further taxonomic studies using appropriate nuclear markers as well as morphological and ecological data to be resolved. The DNA barcodes cluster into 120–242 molecular operational taxonomic units (OTUs) depending on whether Objective Clustering, Automatic Barcode Gap Discovery (ABGD), Generalized Mixed Yule Coalescent model (GMYC), Poisson Tree Process (PTP), subjective evaluation of the neighbor joining tree or Barcode Index Numbers (BINs) are used. We suggest that a 4–5% threshold is appropriate to delineate species of Tanytarsus non-biting midges.     

Diversity investigation by application of DNA barcoding: A case study of lepidopteran insects in Xinjiang wild fruit forests,China

To investigate the species diversity of lepidopteran insects in Xinjiang wild fruit forests, establish insect community monitoring systems, and determine the local species pool, we test the applicability of DNA barcoding based on cytochrome c oxidase subunit I (COI) gene for accurate and rapid identification of insect species. From 2017 to 2019, a total of 212 samples with ambiguous morphological identification were selected for DNA barcoding analysis. Five sequence‐based methods for species delimitation (ABGD, BINs, GMYC, jMOTU, and bPTP) were conducted for comparison to traditional morphology‐based identification. In total, 2,422 samples were recorded, representing 143 species of 110 genera in 17 families in Lepidoptera. The diversity analysis showed that the richness indices for Noctuidae was the highest (54 species), and for Pterophoridae, Cossidae, Limacodidae, Lasiocampidae, Pieridae, and Lycaenidae were the lowest (all with 1 species). The Shannon–Wiener species diversity index (H′) and Pielou''s evenness (J′) of lepidopteran insects first increased and then decreased across these 3 years, while the Simpson diversity index showed a trend of subtracted then added. For molecular‐based identification, 67 lepidopteran species within 61 genera in 14 families were identified through DNA barcoding. Neighbor‐joining (NJ) analysis showed that conspecific individuals were clustered together and formed monophyletic groups with a high support value, except for Lacanobia contigua (Denis & Schiffermüller, 1775) (Noctuidae: Hadeninae). Sixty‐seven morphospecies were classified into various numbers of MOTUs based on ABGD, BINs, GMYC, jMOTU, and bPTP (70, 96, 2, 71, and 71, respectively). In Xinjiang wild fruit forests, the family with the largest number of species is Noctuidae, followed by Geometridae, Crambidae, and the remaining families. The highest Shannon diversity index is observed for the family Noctuidae. Our results indicate that the distance‐based methods (ABGD and jMOTU) and character‐based method (bPTP) outperform GMYC. BINs is inclined to overestimate species diversity compared to other methods.     

Molecular identification of selected bees from the Indian Himalaya: A preliminary effort

DNA barcoding has largely been tested for a wide range of taxa and evidenced as a reliable and rapid molecular tool for species-level identification. The present study lends to generate 156 DNA barcodes, of which 141 belonged to 30 morphologically identified bees from the Indian Himalayan Regions (IHRs). The generated barcode data along with 84 sequences of global database distinctly discriminated all the studied species with sufficient genetic distances and cohesive monophyletic clustering in Bayesian analysis (BA) phylogeny. The species delimitation methods, Automatic Barcode Gap Discovery (ABGD), Bayesian Poisson-Tree-Processes (bPTP), and General Mixed Yule-coalescent (GMYC) yielded 68, 70, and 71 molecular operational taxonomic units (MOTUs) respectively. The present DNA barcode-based examination detected the possible cryptic diversity in two Apis species (A. cerana and A. dorsata), Bombus hypnorum, Lepidotrigona arcifera, and Ceratina sutepensis. The present study also evidenced the species complexes within Bombus albopleuralis and Bombus trifasciatus in the IHRs. The species delimitation methods also detected an additional seven putative species from the IHRs, which were identified up to the genus level. In conclusion, this preliminary effort helps to develop a reliable barcode database of bees from the Indian IHRs to facilitate the future systematics study. These molecular data can be utilized to evaluate the population structures and assist to formulate the effective plans for bee conservation.     

Multiple species delimitation approaches with COI barcodes poorly fit each other and morphospecies – An integrative taxonomy case of Sri Lankan Sericini chafers (Coleoptera: Scarabaeidae)

DNA taxonomy including barcoding and metabarcoding is widely used to explore the diversity in biodiversity hotspots. In most of these hotspot areas, chafers are represented by a multitude of species, which are well defined by the complex shape of male genitalia. Here, we explore how well COI barcode data reflect morphological species entities and thus their usability for accelerated species inventorization. We conducted dedicated field surveys in Sri Lanka to collect the species‐rich and highly endemic Sericini chafers (Coleoptera: Scarabaeidae). Congruence among results of a series of protocols for de novo species delimitation and with morphology‐based species identifications was investigated. Different delimitation methods, such as the Poisson tree processes (PTP) model, Statistical Parsimony Analysis (TCS), Automatic Barcode Gap Discovery (ABGD), Assemble Species by Automatic Partitioning (ASAP), and Barcode Index Number (BIN) assignments, resulted in different numbers of molecular operational taxonomic units (MOTUs). All methods showed both over‐splitting and lumping of morphologically identified species. Only 18 of the observed 45 morphospecies perfectly matched MOTUs from all methods. The congruence of delimitation between MOTUs and morphospecies expressed by the match ratio was low, ranging from 0.57 to 0.67. TCS and multirate PTP (mPTP) showed the highest match ratio, while (BIN) assignment resulted in the lowest match ratio and most splitting events. mPTP lumped more species than any other method. Principal coordinate analysis (PCoA) on a match ratio‐based distance matrix revealed incongruent outcomes of multiple DNA delimitation methods, although applied to the same data. Our results confirm that COI barcode data alone are unlikely to correctly delimit all species, in particular, when using only a single delimitation approach. We encourage the integration of various approaches and data, particularly morphology, to validate species boundaries.     

Barcoding rotifer biodiversity in Mediterranean ponds using diapausing egg banks

The biodiversity of Mediterranean freshwater bodies is among the most threatened worldwide; therefore, its accurate estimation is an urgent issue. However, traditional methods are likely to underestimate freshwater zooplankton biodiversity due to its high species seasonality and cryptic diversity. We test the value of applying DNA barcoding to diapausing egg banks, in combination with the creation of a reference collection of DNA barcodes using adult individual samples, to characterize rotifer communities. We use monogonont rotifers from two lakes in Doñana National Park and one from Ruidera Natural Park in Spain as models to create a reference collection of DNA barcodes for taxonomically diagnosed adult individuals sampled from the water column, to compare with the sequences obtained from individual eggs from the diapausing egg banks. We apply two different approaches to carry out DNA taxonomy analyses, the generalized mixed Yule coalescent method (GMYC) and the Automatic Barcode Gap Discovery (ABGD), to the obtained sequences and to publicly available rotifer sequences. We obtained a total of 210 new rotifer COI sequences from all three locations (151 diapausing eggs and 59 adults). Both GMYC and ABGD generated the same 35 operational taxonomic units (OTUs), revealing four potential cryptic species. Most sequences obtained from diapausing eggs (85%) clustered with sequences obtained from morphologically diagnosed adults. Our approach, based on a single sediment sample, retrieved estimates of rotifer biodiversity higher than or similar to those of previous studies based on a number of seasonal samples. This study shows that DNA barcoding of diapausing egg banks is an effective aid to characterize rotifer diversity in Mediterranean freshwater bodies.     

江西新岗山森林昆虫种内遗传距离研究

DNA条形码目前广泛用于昆虫多样性研究。本研究采用DNA条形码（即线粒体细胞色素c氧化酶亚基I基因COI 5′端）,通过比较所获分子分类操作单元（Molecular operational taxonomic units,MOTU）的种内遗传距离,探究DNA条形码在亚热带森林（位于我国江西省新岗山）不同昆虫类群中的物种鉴定和界定效用。数据分析中结合数据库比对信息,采用jMOTU、ABGD、bPTP、GMYC 这4种物种界定方法获得MOTU,从而开展种内遗传距离分析。本研究共挑选出479个昆虫样本,获得475条COI序列,经NCBI、BOLD在线数据库比对属于6个目,与形态初步划分一致;物种界定分析获得288个MOTU,其中鳞翅目最多,达85个,膜翅目、双翅目、半翅目、鞘翅目次之,分别为80、74、21和20个,直翅目最少,仅8个。膜翅目和双翅目的种内遗传距离均值及标准偏差较大（膜翅目：0.89%±0.87%;双翅目：0.73%±0.58%）,鳞翅目的最小（0.28%±0.20%）。研究表明：不同昆虫类群的种内遗传距离虽然整体在一定范围,但仍然存在一定的差异,因此不能笼统地依靠遗传距离的距离阈值进行物种划分;现有数据库需要补充足够的昆虫物种信息,才能提升物种鉴定效率。本研究丰富了亚热带森林昆虫分子数据库,同时也为进一步探索基于分子分类学开展昆虫多样性研究提供了基础数据和参考。     

One species in eight: DNA barcodes from type specimens resolve a taxonomic quagmire

Each holotype specimen provides the only objective link to a particular Linnean binomen. Sequence information from them is increasingly valuable due to the growing usage of DNA barcodes in taxonomy. As type specimens are often old, it may only be possible to recover fragmentary sequence information from them. We tested the efficacy of short sequences from type specimens in the resolution of a challenging taxonomic puzzle: the Elachista dispunctella complex which includes 64 described species with minuscule morphological differences. We applied a multistep procedure to resolve the taxonomy of this species complex. First, we sequenced a large number of newly collected specimens and as many holotypes as possible. Second, we used all >400 bp examine species boundaries. We employed three unsupervised methods (BIN, ABGD, GMYC) with specified criteria on how to handle discordant results and examined diagnostic bases from each delineated putative species (operational taxonomic units, OTUs). Third, we evaluated the morphological characters of each OTU. Finally, we associated short barcodes from types with the delineated OTUs. In this step, we employed various supervised methods, including distance‐based, tree‐based and character‐based. We recovered 658 bp barcode sequences from 194 of 215 fresh specimens and recovered an average of 141 bp from 33 of 42 holotypes. We observed strong congruence among all methods and good correspondence with morphology. We demonstrate potential pitfalls with tree‐, distance‐ and character‐based approaches when associating sequences of varied length. Our results suggest that sequences as short as 56 bp can often provide valuable taxonomic information. The results support significant taxonomic oversplitting of species in the Elachista dispunctella complex.     

The unknown diversity of the genus Characidium (Characiformes: Crenuchidae) in the Chocó biogeographic region,Colombian Andes: Two new species supported by morphological and molecular data

Two new species of Characidium, C. tatama and C. dule, are described from the biogeographic Chocó region in western Colombia. Both new species are supported by both morphological and molecular data. C. tatama from the San Juan River in the Pacific and C. dule from the Atrato River in the Caribbean portion of Colombia are both distributed in the upper and lower portions of these basins. An extensive comparison with other trans- and cis-Andean species of Characidium was made, in addition to species delimitation, using COI sequences by distinct methods (GMYC, ABGD, bPTP).     

Algorithmic single‐locus species delimitation: effects of sampling effort,variation and nonmonophyly in four methods and 1870 species of beetles

The vast number of undescribed species and the fast rate of biodiversity loss call for new approaches to speed up alpha taxonomy. A plethora of methods for delimiting species or operational taxonomic units (OTUs) based on sequence data have been published in recent years. We test the ability of four delimitation methods (BIN, ABGD, GMYC, PTP) to reproduce established species boundaries on a carefully curated DNA barcode data set of 1870 North European beetle species. We also explore how sampling effort, intraspecific variation, nearest neighbour divergence and nonmonophyly affect the OTU delimitations. All methods produced approximately 90% identity between species and OTUs. The effects of variation and sampling differed between methods. ABGD was sensitive to singleton sequences, while GMYC showed tendencies for oversplitting. The best fit between species and OTUs was achieved using simple rules to find consensus between discordant OTU delimitations. Using several approaches simultaneously allows the methods to compensate for each other's weaknesses. Barcode‐based OTU‐picking is an efficient way to delimit putative species from large data sets where the use of more sophisticated methods based on multilocus or genomic data is not feasible.     

A Good Compromise: Rapid and Robust Species Proxies for Inventorying Biodiversity Hotspots Using the Terebridae (Gastropoda: Conoidea)

Devising a reproducible approach for species delimitation of hyperdiverse groups is an ongoing challenge in evolutionary biology. Speciation processes combine modes of passive and adaptive trait divergence requiring an integrative taxonomy approach to accurately generate robust species hypotheses. However, in light of the rapid decline of diversity on Earth, complete integrative approaches may not be practical in certain species-rich environments. As an alternative, we applied a two-step strategy combining ABGD (Automated Barcode Gap Discovery) and Klee diagrams, to balance speed and accuracy in producing primary species hypotheses (PSHs). Specifically, an ABGD/Klee approach was used for species delimitation in the Terebridae, a neurotoxin-producing marine snail family included in the Conoidea. Delimitation of species boundaries is problematic in the Conoidea, as traditional taxonomic approaches are hampered by the high levels of variation, convergence and morphological plasticity of shell characters. We used ABGD to analyze gaps in the distribution of pairwise distances of 454 COI sequences attributed to 87 morphospecies and obtained 98 to 125 Primary Species Hypotheses (PSHs). The PSH partitions were subsequently visualized as a Klee diagram color map, allowing easy detection of the incongruences that were further evaluated individually with two other species delimitation models, General Mixed Yule Coalescent (GMYC) and Poisson Tree Processes (PTP). GMYC and PTP results confirmed the presence of 17 putative cryptic terebrid species in our dataset. The consensus of GMYC, PTP, and ABGD/Klee findings suggest the combination of ABGD and Klee diagrams is an effective approach for rapidly proposing primary species proxies in hyperdiverse groups and a reliable first step for macroscopic biodiversity assessment.     

COI DNA barcoding for molecular identification of the genus Aphidius (Braconidae: Aphidiinae) in South Korea,with diagnoses of two newly recorded species

The genus Aphidius is one of the most commercially important species in greenhouse for controlling pests, especially aphids. Although 13 Aphidius species had been recorded in South Korea, it was not carefully surveyed yet. In this study, we surveyed and collected 8 Aphidius spp. including two unrecorded species. Using the DNA barcode region of mitochondrial cytochrome c oxidase subunit I (COI), sequences were newly generated for 16 molecular operational taxonomic units (MOTUs) of 8 species, except for 15 duplicates, in addition of 58 MOTUs of 18 species referred from GenBank for comparison and identification, which were reconstructed in a barcode phylogeny with Neighbor-Joining method. Then, to define each species, both Automatic Barcode Gap Discovery (ABGD) and Bayesian Poisson Tree Processes (bPTP) analyses were performed. As results, although most of Aphidius spp. could be identified by clustering into a clade, we recognized that three Aphdius species were placed in a clade, of which each species was not separated as an independent species. Our data suggested that those species demonstrating a species complex were able to be hypothesized as one species. In addition, diagnoses and illustrations of the two newly recorded species in South Korea were provided.     

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.     

Integrative taxonomy of the primitively segmented spider genus Ganthela (Araneae: Mesothelae: Liphistiidae): DNA barcoding gap agrees with morphology

Species delimitation is difficult for taxa in which the morphological characters are poorly known because of the rarity of adult morphs or sexes, and in cryptic species. In primitively segmented spiders, family Liphistiidae, males are often unknown, and female genital morphology – usually species‐specific in spiders – exhibits considerable intraspecific variation. Here, we report on an integrative taxonomic study of the liphistiid genus Ganthela Xu & Kuntner, 2015, endemic to south‐east China, where males are only available for two of the seven morphological species (two known and five undescribed). We obtained DNA barcodes (cytochrome c oxidase subunit I gene, COI) for 51 newly collected specimens of six morphological species and analysed them using five species‐delimitation methods: DNA barcoding gap, species delimitation plugin [P ID(Liberal)], automatic barcode gap discovery (ABGD), generalized mixed Yule‐coalescent model (GMYC), and statistical parsimony (SP). Whereas the first three agreed with the morphology, GMYC and SP indicate several additional species. We used the consensus results to delimit and diagnose six Ganthela species, which in addition to the type species Ganthela yundingensis Xu, 2015, completes the revision of the genus. Although multi‐locus phylogenetic approaches may be needed for complex taxonomic delimitations, our results indicate that even single‐locus analyses based on the COI barcodes, if integrated with morphological and geographical data, may provide sufficiently reliable species delimitation. © 2015 The Linnean Society of London     

Identification of gobies (Teleostei: Perciformes: Gobiidae) from the North and Baltic Seas combining morphological analysis and DNA barcoding

Gobies are difficult to identify, as they are very similar in appearance. Here, we identified (sub)adult specimens of 12 goby species from the North Sea and the Baltic Sea by carefully analysing meristic characters, coloration patterns, papillae row patterns and morphometric measurements. The results of the morphological identifications were congruent with those obtained with the analysis of COI DNA barcodes; sequences from morphological conspecific specimens were clustered together in clades with bootstrap values ≥ 99%. Mean intra‐ and interspecific distance (uncorrected p) was 0.37 and 18.97%, respectively. A gap between the maximum intraspecific distance and the distance to the nearest neighbour was apparent in every species and ranged from 2.35 to 16.11%. The Barcode Index Number (BIN) analysis performed on the Barcode of Life Data Systems (BOLD) web platform, assigned the DNA barcodes to 12 separate clusters corresponding to sequence‐ and morphology‐based identification. In 25% of the investigated species, the BIN clusters showed taxonomic discordances, as they contained sequences assigned to more than one species. This result demonstrates the importance of accurate morphological species identification at the beginning of the barcoding pipeline. © 2014 The Linnean Society of London