Species diversity of freshwater shrimp in Henan Province,China, based on morphological characters and COI mitochondrial gene

Freshwater shrimp are a rich species group, with a long and problematic taxonomic history attributed to their wide distribution and similar morphological characteristics. Shrimp diversity and species identification are important cornerstones for fisheries management. However, identification based on morphological characteristics is a difficult task for a nonspecialist. Abundant freshwater shrimp species are distributed in the waters of Henan Province, but investigations of freshwater shrimp are limited in this region, especially concerning molecular features. Here, we combined morphology and DNA barcodes to reveal the species diversity of freshwater shrimp in Henan province. A total of 1,200 freshwater shrimp samples were collected from 46 sampling sites, and 222 samples were chosen for further microscopic examination and molecular delimitation. We used tree‐based methods (NJ, ML, and bPTP) and distance‐based methods (estimation of the paired genetic distances and ABGD) to delimit species. The results showed that there were nine morphospecies based on morphological characteristics; all could effectively be defined by molecular methods, among which bPTP and ABGD defined 13 and 8 MOTUs, respectively. The estimation of the paired genetic distances of K2P and the p‐distances had similar results. Mean K2P distances and p‐distances within species were both equal to 1.2%. The maximum intraspecific genetic distances of all species were less than 2%, with the exception of Palaemon modestus and M. maculatum. Various analyses have shown that P. modestus and M. maculatum have a large genetic differentiation, which may indicate the existence of cryptic species. By contrast, DNA barcoding could unambiguously discriminate 13 species and detect cryptic diversity. Our results demonstrate the high efficiency of DNA barcoding to delimit freshwater shrimp diversity and detect the presence of cryptic species.     

The efficacy of DNA barcoding in the classification,genetic differentiation,and biodiversity assessment of benthic macroinvertebrates

Macroinvertebrates have been recognized as key ecological indicators of aquatic environment and are the most commonly used approaches for water quality assessment. However, species identification of macroinvertebrates (especially of aquatic insects) proves to be very difficult due to the lack of taxonomic expertise in some regions and can become time‐consuming. In this study, we evaluated the feasibility of DNA barcoding for the classification of benthic macroinvertebrates and investigated the genetic differentiation in seven orders (Insecta: Ephemeroptera, Plecoptera, Trichoptera, Diptera, Hemiptera, Coleoptera, and Odonata) from four large transboundary rivers of northwest China and further explored its potential application to biodiversity assessment. A total of 1,144 COI sequences, belonging to 176 species, 112 genera, and 53 families were obtained and analyzed. The barcoding gap analysis showed that COI gene fragment yielded significant intra‐ and interspecific divergences and obvious barcoding gaps. NJ phylogenetic trees showed that all species group into monophyletic species clusters whether from the same population or not, except two species (Polypedilum. laetum and Polypedilum. bullum). The distance‐based (ABGD) and tree‐based (PTP and MPTP) methods were utilized for grouping specimens into Operational Taxonomic Units (OTUs) and delimiting species. The ABGD, PTP, and MPTP analysis were divided into 177 (p = .0599), 197, and 195 OTUs, respectively. The BIN analysis generated 186 different BINs. Overall, our study showed that DNA barcoding offers an effective framework for macroinvertebrate species identification and sheds new light on the biodiversity assessment of local macroinvertebrates. Also, the construction of DNA barcode reference library of benthic macroinvertebrates in Eurasian transboundary rivers provides a solid backup for bioassessment studies of freshwater habitats using modern high‐throughput technologies in the near future.     

DNA barcodes successfully delimit morphospecies in a superdiverse insect genus

Polypedilum Kieffer (Diptera: Chironomidae), with 520 currently known species worldwide, can be extremely difficult to identify species level based on the morphology. We used 3,670 cytochrome c oxidase subunit I (COI) barcodes to explore the efficiency of the COI barcodes to differentiate between species in a superdiverse aquatic insect genus. The Barcode of Life Data System (BOLD) presented 286 BIN clusters in Polypedilum, representing 163 morphospecies, of which 93 were contributed from our laboratory. Molecular operational taxonomic units (OTUs) ranged from 158 to 345, based on Automatic Barcode Gap Discovery (ABGD), the Barcode Index Number (BIN), Bayesian Poisson tree processes (bPTP), generalized mixed Yule coalescent (GMYC), jMOTU, multi‐rate Poisson tree processes (mPTP), neighbor‐joining (NJ) tree and prethreshold clustering. In comparison, GMYC, bPTP, mPTP and BIN suggested more species than warranted by morphology, while ABGD, jMOTU, NJ, prethreshold clustering and ABGD yielded a conservative number of species when setting higher thresholds. Nine species complexes with deep intraspecific divergences indicated 18 potentially cryptic species, which require further taxonomic research including complete life histories and nuclear genetic data to be resolved. The discrimination of Polypedilum species by DNA barcodes proved to be successful in 94.4% of all studied morphological species.     

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.     

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。     

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。     

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.     

Excluding spatial sampling bias does not eliminate oversplitting in DNA‐based species delimitation analyses

DNA barcoding and DNA‐based species delimitation are major tools in DNA taxonomy. Sampling has been a central debate in this context, because the geographical composition of samples affects the accuracy and performance of DNA barcoding. Performance of complex DNA‐based species delimitation is to be tested under simpler conditions in absence of geographic sampling bias. Here, we present an empirical dataset sampled from a single locality in a Southeast‐Asian biodiversity hotspot (Laos: Phou Pan mountain). We investigate the performance of various species delimitation approaches on a megadiverse assemblage of herbivorous chafer beetles (Coleoptera: Scarabaeidae) to infer whether species delimitation suffers in the same way from exaggerate infraspecific variation despite the lack of geographic genetic variation that led to inconsistencies between entities from DNA‐based and morphology‐based species inference in previous studies. For this purpose, a 658 bp fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) was analyzed for a total of 186 individuals of 56 morphospecies. Tree‐based and distance‐based species delimitation methods were used. All approaches showed a rather limited match ratio (max. 77%) with morphospecies. Poisson tree process (PTP) and statistical parsimony network analysis (TCS) prevailingly over‐splitted morphospecies, while 3% clustering and Automatic Barcode Gap Discovery (ABGD) also lumped several species into one entity. ABGD revealed the highest congruence between molecular operational taxonomic units (MOTUs) and morphospecies. Disagreements between morphospecies and MOTUs have to be explained by historically acquired geographic genetic differentiation, incomplete lineage sorting, and hybridization. The study once again highlights how important morphology still is in order to correctly interpret the results of molecular species delimitation.     

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.     

A Multilocus Species Delimitation Reveals a Striking Number of Species of Coralline Algae Forming Maerl in the OSPAR Maritime Area

Maerl beds are sensitive biogenic habitats built by an accumulation of loose-lying, non-geniculate coralline algae. While these habitats are considered hot-spots of marine biodiversity, the number and distribution of maerl-forming species is uncertain because homoplasy and plasticity of morphological characters are common. As a result, species discrimination based on morphological features is notoriously challenging, making these coralline algae the ideal candidates for a DNA barcoding study. Here, mitochondrial (COI-5P DNA barcode fragment) and plastidial (psbA gene) sequence data were used in a two-step approach to delimit species in 224 collections of maerl sampled from Svalbard (78°96’N) to the Canary Islands (28°64’N) that represented 10 morphospecies from four genera and two families. First, the COI-5P dataset was analyzed with two methods based on distinct criteria (ABGD and GMYC) to delineate 16 primary species hypotheses (PSHs) arranged into four major lineages. Second, chloroplast (psbA) sequence data served to consolidate these PSHs into 13 secondary species hypotheses (SSHs) that showed biologically plausible ranges. Using several lines of evidence (e.g. morphological characters, known species distributions, sequences from type and topotype material), six SSHs were assigned to available species names that included the geographically widespread Phymatolithon calcareum, Lithothamnion corallioides, and L. glaciale; possible identities of other SSHs are discussed. Concordance between SSHs and morphospecies was minimal, highlighting the convenience of DNA barcoding for an accurate identification of maerl specimens. Our survey indicated that a majority of maerl forming species have small distribution ranges and revealed a gradual replacement of species with latitude.     

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.     

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.     

High‐throughput sequencing outperforms traditional morphological methods in Blue Catfish diet analysis and reveals novel insights into diet ecology

Blue Catfish Ictalurus furcatus are an invasive, yet economically important species in the Chesapeake Bay. However, their impact on the trophic ecology of this system is not well understood. In order to provide in‐depth analysis of predation by Blue Catfish, we identified prey items using high‐throughput DNA sequencing (HTS) of entire gastrointestinal tracts from 134 samples using two genetic markers, mitochondrial cytochrome c oxidase I (COI) and the nuclear 18S ribosomal RNA gene. We compared our HTS results to a more traditional “hybrid” approach that coupled morphological identification with DNA barcoding. The hybrid study was conducted on additional Blue Catfish samples (n = 617 stomachs) collected from the same location and season in the previous year. Taxonomic representation with HTS vastly surpassed that achieved with the hybrid methodology in Blue Catfish. Significantly, our HTS study identified several instances of at‐risk and invasive species consumption not identified using the hybrid method, supporting the hypothesis that previous studies using morphological methods may greatly underestimate consumption of critical species. Finally, we report the novel finding that Blue Catfish diet diversity inversely correlates to daily flow rates, perhaps due to higher mobility and prey‐seeking behaviors exhibited during lower flow.     

Assessing diversity of King Crab Lithodes spp. in the south‐eastern pacific using phylogeny and molecular species delimitation methods

The purpose of this study was to test the hypothesis that the genetic diversity of commercially significant species of King Crabs (Lithodes spp.) along the south‐eastern Pacific (SEP) comprises different independent evolutionary units (IEUs) with spatially isolated distribution. Nine localities from inner and open waters along the SEP Chilean coast (39°S‐55°S) were sampled. We analyzed sequences from 173 individuals for the mitochondrial gene Cytochrome oxidase I (COX‐I), 151 individuals for the Internal Transcribed Spacer 1 (ITS) and 135 for the structural ribosomal RNA (28S). Genetic delimitation was performed through three analytical methods: ABGD, GMYC, and its Bayesian implementation, bGMYC. Bayesian phylogenetic analyses and haplotype networks were also performed. Divergence time between clades was assessed for the COX‐I marker and estimated from known evolutionary rates for this marker in other crustacean species and fossil calibration from other Anomuran species. Delimitation analyses, phylogenetic analyses, and mitochondrial haplotype networks suggested the presence of two deeply divergent mitochondrial lineages of Lithodes in the SEP, referred to as Clade1 and Clade 2. Nuclear markers showed low phylogenetic resolution and therefore were unsuitable for molecular species delimitation. Divergence time analysis of the mitochondrial lineages suggests a separation between Clades of approximately 2.3 Mya. The divergence time obtained suggested that Pliocene glaciations and deglaciations cycles could be involved in hybridization events between Lithodes IEUs at southern tip of South American coasts. The different frequencies of Lithodes haplotypes in inner and open water environments along SEP coasts could be explained by events such as the last glacial maximum or by differences in the adaptation of each clade to different environments. These findings support the necessity of evaluating the taxonomic status of Lithodes individuals found along SEP coasts under an integrative taxonomy approach or through markers with other evolution rates than those already used.     

High diversity and local endemism in Aotearoa New Zealand's groundwater crustacean fauna

We used DNA barcoding to assess the diversity and distribution of New Zealand''s groundwater amphipods and isopods (Crustacea) and to determine whether biodiversity and endemism within tectonically active New Zealand are similar to those of more tectonically stable continents. Sixty‐five wells were sampled in seven aquifers across four regions within the North and South islands of New Zealand, and resident invertebrates were morphologically identified and then assessed using sequencing of the mitochondrial DNA cytochrome c oxidase subunit one (COI) gene. Invertebrates were found in 54 wells. Of the 228 individual amphipods and isopods found in 36 of the wells, 154 individuals were successfully sequenced for COI (68% success rate) from 25 wells, with at least one well in each aquifer containing sequenced individuals. Of the 45 putative species identified using Barcode Index Numbers (BINs), 30 BINs (78% of all taxa and 83% of amphipods) were previously unrecorded. Substantial morphologically cryptic, species‐level diversity was revealed, particularly within the amphipod Family Paraleptamphopidae. Similarly, one isopod taxon morphologically identified as Cruregens fontanus was assigned to five well‐separated BINs based on COI sequences. Endemism appeared high, with all taxa regionally endemic; 87% of species were restricted to one aquifer and more than 50% restricted to one well. Non‐saturated species accumulation curves indicated that, while additional sampling may increase the range of some currently identified taxa, additional range‐restricted taxa are also likely to be discovered. Patterns of diversity and short‐range endemism were similar to those found elsewhere, including locations which are more tectonically stable. The predominance of local endemism within New Zealand''s groundwater fauna suggests that land‐use activities and groundwater extraction require careful evaluation to minimize threats to groundwater biodiversity.     

Genetic differentiation and connectivity of morphological types of the broadcast‐spawning coral Galaxea fascicularis in the Nansei Islands,Japan

Population connectivity resulting from larval dispersal is essential for the maintenance or recovery of populations in marine ecosystems, including coral reefs. Studies of species diversity and genetic connectivity within species are essential for the conservation of corals and coral reef ecosystems. We analyzed mitochondrial DNA sequence types and microsatellite genotypes of the broadcast‐spawning coral, Galaxea fascicularis, from four regions in the subtropical Nansei Islands in the northwestern Pacific Ocean. Two types (soft and hard types) of nematocyst morphology are known in G. fascicularis and are significantly correlated with the length of a mitochondrial DNA noncoding sequence (soft type: mt‐L; hard type: mt‐S type). Using microsatellites, significant genetic differentiation was detected between the mitochondrial DNA sequence types in all regions. We also found a third genetic cluster (mt‐L+), and this unexpected type may be a cryptic species of Galaxea. High clonal diversity was detected in both mt‐L and mt‐S types. Significant genetic differentiation, which was found among regions within a given type (F _ST = 0.009–0.024, all Ps ≤ 0.005 in mt‐L; 0.009–0.032, all Ps ≤ 0.01 in mt‐S), may result from the shorter larval development than in other broadcast‐spawning corals, such as the genus Acropora. Nevertheless, intraspecific genetic diversity and connectivity have been maintained, and with both sexual and asexual reproduction, this species appears to have a potential for the recovery of populations after disturbance.     

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 and habitat association of medium and large mammals in Gibe Sheleko National Park,Southern Ethiopia

Complete documentation on the status of mammals is indispensable for appropriate conservation measures in protected areas. However, there is inadequate information on mammalian resources in the ecosystem of Gibe Sheleko National Park (GSNP). Thus, the study aimed to assess species diversity, abundance, and habitat association of medium‐ and large‐sized mammals in GSNP. We stratified the study area into five dominant habitat types, namely dense forest, wooded grassland, grassland, riverine forest, and farmland habitat types based on land cover and vegetation structures and further employed stratified random sampling technique across each habitat type. The sample transects covered 20% of the study area. Transect width ranged from 50 m to 400 m based on vegetation cover and visibility of mammals. The main data were collected via direct observation. Data were analyzed via chi‐square test and species diversity indexes. We recorded the total of 20 mammals species'' those belong to 10 families of which 8 species were large‐sized and 12 species medium‐sized mammals. There were two IUCN vulnerable species, namely Hippopotamus amphibious and Panthera pardus, and two globally near‐threatened species, particularly Litocranius walleri and Caracal caracal in the study area. Dense forest held the highest species diversity of medium‐ and large‐sized mammals (H′ = 2.28) with the highest evenness index (J = 0.84). Riverine forest had the least diversity with uneven population distribution. Papio anubis was the most abundance species, whereas Caracal caracal was the least abundant in the study area. GSNP is home for threatened and spectacular mammals species''; hence, an appropriate conservation measure is mandatory to keep existing mammals species''.     

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.