Automatic Barcode Gap Discovery Research Articles

In Silico Evaluation of the Taxonomic Resolution and Coverage of the COI Region and Alternative Barcode Markers for Echinoderms

DNA barcoding methods are potentially useful for the detection and identification of species difficult to identify using morphology-based taxonomy, such as the larvae of marine taxa. Combining barcoding methods with massively parallel sequencing and low-cost DNA library preparation improves the feasibility of processing several samples but limits the taxonomic resolution of resulting sequences. In light of these limitations, we looked for suitable barcode regions for echinoderm larvae, first by examining the cytochrome c oxidase subunit I (COI) region, the most commonly used barcode for echinoderms. Using a dataset containing over 4000 COI sequences representing nearly 400 echinoderm species across all five extant classes, the COI gene was found to lack a sufficiently conserved region for barcoding. Taxonomic resolution was also found lacking, with no clear barcode gap across classes and the results of Automatic Barcode Gap Discovery (ABGD) failing to reflect current taxonomic assignments. While sequences may be mislabeled, the aggregation of multiple species in one ABGD partition suggests that course-grained taxonomic resolution may still result from using COI alone. The search for short alternative mitochondrial regions across 110 genomes of distinct species using ecoPrimers software revealed that class-specific primers targeting 12S and 16S ribosomal RNA (rRNA) regions are prime candidates for barcoding echinoderm sequences. The results of in silico PCR across the 110 mitochondrial genomes indicate that compared to existing COI primers, the candidate 12S and 16S primers yield similar taxonomic coverage, with slightly lower resolution. Taking into consideration the previous analysis of COI, we suggest the use of multiple markers (COI, 12S, 16S) to adequately capture the understudied diversity of echinoderm larvae. In vitro PCR of the 12S and 16S primers is also needed to validate the results of the in silico analyses.

High-level taxonomic splitting in allopatric taxa causes confusion downstream: a revision of the nudibranch family Сoryphellidae

Abstract Conformity of trans-Arctic Coryphellidae genera with the most recent revised taxonomy for the group was tested. Both morphological and molecular data were used to compare more than 200 specimens from various localities in the North Pacific, North Atlantic and Arctic Oceans. The molecular study included four markers (COI, 16S, H3 and 28S) and implemented Bayesian and maximum likelihood phylogenetic approaches. Species delimitation was investigated with automatic barcode gap discovery (ABGD), Poisson tree processes (PTP) and generalized mixed Yule coalescent (GMYC) methods. The inter- and intraspecific divergence were studied using the TCS-based haplotype analysis and calculation of p-distances. Morphological analysis included standard anatomical techniques and scanning electron microscopy. Based on integrative analysis, we conclude that Coryphella verrucosa and C. pseudoverrucosa represent a single species (C. verrucosa), which is highly heterogeneous at morphological and molecular levels. For Microchlamylla gracilis and M. amabilis, their synonymization under the name M. gracilis is suggested, but increased sample size is needed for further clarification of their identities. Finally, the amphiboreal species Borealea nobilis and the North Pacific B. sanamyanae are distinct species displaying secondary sympatry in the north-west Pacific. Based on these results, we propose a new classification system for the family Coryphellidae uniting all currently considered genera in the single genus Coryphella, and suggest a new approach for further revision of Flabellinidae s.l. taxa.

Shedding light on species boundaries in small endogeic animals through an integrative approach: species delimitation in the centipedeClinopodes carinthiacus(Chilopoda: Geophilidae) in the south-eastern Alps

AbstractThe investigation of species boundaries in strictly endogeic animals is challenging because they are prone to fine-scale genetic and phenotypic geographical differentiation owing to low dispersal ability. An integrative approach exploiting different sources of information has seldom been adopted in these animals and even more rarely by treating all data sources equally. We investigated species boundaries in the endogeic centipede Clinopodes carinthiacus across the south-eastern Alps by studying genetic and morphological differentiation in a sample of 66 specimens from 27 sites, complemented by the morphological examination of more than 1100 specimens from other sites. Hypotheses of species delimitation were obtained independently from the molecular sequences of three markers (mitochondrial 16S and COI and nuclear 28S) by means of different species discovery methods (automatic barcode gap discovery, assemble species by automatic partitioning, general mixed Yule coalescent and the Poisson tree process) and from ten morphological characters by means of a model-based cluster analysis and Bayesian model selection. We found strong support for the existence of at least two species: C. carinthiacus s.s. and Clinopodes strasseri, which was formerly described as a subspecies of another species, and later placed in synonymy with C. carinthiacus. The two species coexist in syntopy in at least one site.

Discovery of the male of Loxaulus hyalinus, and implications for the sympatric species Loxaulus laeta (Hymenoptera: Cynipidae: Cynipini): an integrative taxonomical case of species delimitation

ABSTRACT Loxaulus hyalinus Pujade-Villar & Melika, 2014 was originally described as an asexual form but the recent collection of male samples confirms that it is, actually, a sexual form. The male of L. hyalinus is here morphologically described. With L. hyalinus being a sexual form, the reasonable question arises whether it may be the alternate generation of the only other Loxaulus species currently known from Mexico, L. laeta Pujade-Villar, 2014 (asex). An analysis of the phylogenetic relationships between L. hyalinus (sex) and L. laeta (asex) shows an uncorrected genetic distance variation between sympatric and allopatric individuals averaging 6.4% (range 5.9–6.9%) between the two species independently of the sampled locality. Four different species delimitation models – Automatic Barcode Gap Discovery (ABGD), Bayesian implementation of the Poisson tree processes (bPTP), Bayesian Phylogenetics and Phylogeography (BPP), and Bayesian Factor Delimitation (BFD) – support the distinctiveness of the two Loxaulus species.

DNA barcoding of the soft coral, Clavularia inflata, shows two major groups across Indonesian coral reefs

Clavularia inflata was first described from Ternate Island, Indonesia in 1896 and later reported appeared from Japan and Taiwan in 1953.Clavularia (Blainville 1830) soft corals exhibit complex morphological traits that are difficult to differentiate, thus complicating their identification. DNA barcoding has been envisioned and actively pushed as a credible method for assigning unidentified specimens to known species by comparison to a molecular reference data database. Thus, the purpose of this study was to use molecular methods to confirm the identity of 25 colonies taken from 13 Indonesian coral reef sites and putatively identified as Clavularia inflata. All specimens were identified as Clavularia inflata molecularly using the mitochondrial DNA mtMuts gene. Although a comparison of the nucleotide base chains to Genbank data indicates that the samples belong to a single species, two clades in the phylogenetic tree and data from the Automatic Barcode Gap Discovery (ABGD) indicate that there are two major groups of C. inflata in Indonesia, implying cryptic species.

Contribution to the lady beetle fauna of the Yucatan Peninsula and integrative taxonomy for species delimitation

Lady beetles (Coleoptera: Coccinellidae) are among the most familiar insects; many species are of economic importance, but their diversity in the tropics is poorly known. We aimed to contribute to the knowledge of the lady beetle fauna of the Yucatan Peninsula, particularly for Quintana Roo state. We used an integrative approach for species identification, comparing classical morphological identifications and quick automated methods for species delimitation using DNA barcode sequences. Through a literature review and a survey of lady beetles in gardens in Quintana Roo, we further provide an updated list of the species found on the Yucatan Peninsula. Out of the 40 species delimited in our study, 34 are new reports for the peninsula, and 36 are new for Quintana Roo state. Overall, 62 species of lady beetles are now recorded for the entire region, including three exotics: the invasive Harmonia axyridis, Chilocorus nigrita, and Delphastus catalinae. Our study also contributed to public reference libraries with 110 barcode sequences for the tropics belonging to 34 delineated species. We showed that cytochrome oxidase 1 (COI) sequences can be useful for lady beetle species delimitation and that the Automatic Barcode Gap Discovery algorithm (ABGD) was the best method, complementing the number of initially delineated morphospecies. The Barcode Index Number (BIN) approach overestimated seven putative species due to the splitting of conspecifics, while the ABGD method suggested two additional MOTUs at a prior intraspecific distance of 0.059. Combined molecular and morphological data in our study revealed one additional putative species of Diomus, initially considered a tentative colour variation. Our study exemplifies how molecular methods paired with classical taxonomy can efficiently assist in delineating species when descriptions and identification keys are unavailable and highlights the possible great richness of coccinellid species awaiting exploration and description on the Yucatan Peninsula.

In Silico Study of the Effectiveness of 16S rRNA Gene as a Universal Genetic Marker to Identify Closely Related Burkholderia Spp. in Panicle Blight of Rice

The 16S rRNA gene is a housekeeping genetic marker that is available in almost all bacterial species and it is used in bacterial phylogeny and taxonomy studies. In many studies, the 16S rRNA gene is used in identification of certain bacterial species. Being a less conserved genetic marker, certain studies found it is a useful tool to infer the genome-wide similarity levels among the closely related prokaryotic organisms. Thus, this study aimed to compare the variation in the 16S rRNA partial region of Burkholderia spp. that infect the panicle of rice from eight different geographical areas. 58 sequences with total of 688 base pairs (bp) of 16S rRNA gene in B. glumae and B. gladioli were retrieved from public database based on several countries namely United State, Panama, Ecuador, Thailand, China, India, Korea and Malaysia. Then, the data sequences were analysed and validated using MEGAX and ABGD software respectively. The result of phylogenetic tree confirmed that B. glumae and B. gladioli were species that present in the panicle blight of rice. However, Data Analysis in Molecular Biology and Evolution (DAMBE) and Automatic Barcode Gap Discovery (ABGD) software were not able to detect substitution saturation and divergence between B. glumae and B. gladioli respectively based on the 58 sequences of the 16S rRNA partial region. Hence, it proves that 16S rRNA gene is an ineffective genetic marker to be used to differentiate the closely related species of bacteria from similar genus.

DNA Barcoding of Key Storage Pests Using Mitochondrial Cytochrome Oxidase I

Stored grain insect pests are of economic importance, and they spread rapidly through the grain trade. Hence, it is important to identify these pests accurately. Although several morphological methods exist, it is tedious and time-consuming. DNA barcoding using mitochondrial COI is an alternative approach that aids in precise species identification. In this study, 13 stored grain insect pests belonging to the order Coleoptera and Lepidoptera were collected from different storage structures in Coimbatore. A fragment of mt-COI was amplified and sequenced. Sequence analyses were carried out with BOLD (Barcode of Life Data System) and ABGD (Automatic Barcode Gap Discovery) tools. The barcoding gap analysis revealed that the inter-species genetic distance is greater than the intra-species genetic distance. The ABGD analysis for species delimitation partitioned the Coleopteran and Lepidopteran datasets into 9 and 4 putative species, respectively. The barcode gap was absent in more closely related species. However, analysis of their sequences revealed significant variations. Our results showed the ability of the mt-COI to discriminate between the species, thus provide a complementary technique for the diagnosis of stored grain insect pests.

Molecular Validation of Aulacophora Species Complex within the Geographical Limits of Tamil Nadu

The present study aimed to the delimitation of Aulacophora spp. from Tamil Nadu through DNA-barcoding. Samples were collected from three districts of Tamil Nadu and cytochrome c oxidase subunit I (COI) gene ofthree species (A. foveicollis, A. lewisii and A. cincta) was sequenced. Eight specimens were sequenced and was submitted to the Genbank. Molecular delineation of species was done using tree-based (phylogenetic tree), and distance-based (Automatic Barcode Gap Discovery) approaches. The data set consists of 35 sequences, including an out-group. Two phylogenetic trees were constructed using neighborhood joining and maximum likelihood method. Both the trees resulted in six distinct species groups, and the branching topology was also similar for both the trees. A. foveicollis, A. abdominalis and A. relicta had branched into three distinct groups from a single node, and A. cincta and A. lewisii sub-branched into different clades from another node. ABGD method gave six identical species groups similar to that of morphological data. The p-value for the grouping ranges from 0.001–0.0359, which gave satisfactory results for species delineation. Both tree-based and distance-based approachesenabled the rapid delineation of species with accurate species identification.

Morphological and Molecular Evidence Reveals the Longnose Skate Zearaja brevicaudata (Marini, 1933) to be a Senior Synonym of Dipturus lamillai Concha, Caira, Ebert & Pompert 2019.

Longnose skates have great economic importance in South American fisheries, and in order to preserve them, it is important to have a well-defined taxonomic status of their species. Dipturus lamillai was recently described for Malvinas Islands waters based on morphological and molecular comparisons with Zearaja chilensis. Although D. lamillai has been compared with several congeneric species, it was not properly compared with the morphologically similar Zearaja brevicaudata, the most abundant longnose skate in the Southwest Atlantic. Here, these species were compared by morphological and molecular analyses in order to evaluate their conspecificity. Linear morphometric variables of holotype and paratypes of D. lamillai and 69 specimens of Z. brevicaudata were compared and investigated using Principal Component Analysis. In addition, thorn patterns, denticle distributions, color, and clasper morphology were compared. No body proportions or other single character that could differentiate D. lamillai from Z. brevicaudata were found. Molecular analyses comprised of the comparison of the Cytochrome oxidase subunit I (COI) and the NADH dehydrogenase subunit 2. The results of the Maximum Likelihood (ML) carried out for each molecular marker showed that sequences from D. lamillai clustered together with those of Z. brevicaudata, and the molecular distance determined by Kimura two-parameter were lower than the expected for different species. Additionally, the Automatic Barcode Gap Discovery method and the Bayesian implementation of the Poisson tree processes were carried out with COI sequences to explore species limits, and their results were consistent with ML analyses. In summary, the results obtained showed that there are no morphological or molecular differences between these nominal species of the valid skate genus Zearaja, leading to the conclusion that they are conspecific. Therefore, we designated Z. brevicaudata as a senior synonym of D. lamillai.

DNA barcoding of Naididae (Annelida, Oligochaeta), based on cytochrome C oxidase gene and ITS2 region in China.

Exploring the effectiveness of DNA barcoding in species identification is a prerequisite for biodiversity conservation and environmental monitoring. Aquatic oligochaetes could serve as excellent indicators in aquatic monitoring programmes. However, few studies have examined the effectiveness of DNA barcoding in these specific organisms. The mitochondrial cytochrome C oxidase (COI) gene of 83 specimens belonging to 40 species of 18 genera were sequenced in this study. The results showed that there was a barcode gap between species of Naididae and the intraspecific genetic distances of each species were smaller than interspecific genetic distances. The classification results of ABGD (Automatic Barcode Gap Discovery) were consistent with those of morphological identification, except for Tubifextubifex and Lumbriculusvariegatus. All species were successfully distinguished in the phylogenetic tree, based on the ITS2 region, which was coincident with the morphological result. Our results provided evidence that DNA barcoding can be used as an effective and convenient tool for species identification of the family Naididae and even for other aquatic oligochaetes.

Integrative taxonomy-based discovery of Dussumieria modakandai sp. nov. from India.

This study is based on integrative taxonomy and reports a new fish species Dussumieria modakandai sp. nov. from India. The new species differs from three valid species within the genus by a combination of characters such as longer maxilla (9.1%-9.9% standard length vs. 8.7% in Dussumieria elopsoides, 6.3%-8.5% in Dussumieria acuta) and one or two rows of small conical teeth on palatine (vs. several rows in D. elopsoides and Dussumieria albulina). It also differs by the absence of longitudinal striae on the posterior side of body scales (vs. presence in D. acuta and D. albulina) and the absence of parasphenoid teeth (vs. presence in D. acuta). The maxilla length of D. modakandai sp. nov. is greater than snout length, which distinguishes it from other congeners. The multivariate analysis of morphometric characters using PCA differentiated the new species from D. elopsoides and D. acuta samples collected in this study. The molecular analysis, based on cytochrome c oxidase I, distinguished the new species from D. acuta, D. albulina and D. elopsoides with a high genetic distance of 13.73%, 12.22% and 12.74%, respectively. The maximum-likelihood phylogenetic tree and automatic barcode gap discovery analysis showed the existence of six putative species in Dussumieria. Even the exhaustive sub-clade formation within species and high intra-species genetic distance in D. acuta (1.59) and D. modakandai (1.95) indicate the possibility of a few more cryptic species. This warrants comprehensive sample collection across the distribution range and integrative taxonomic study of the genus Dussumieria.

Two New Species of Fibrodontia (Trechisporales, Basidiomycota) with a Key to Worldwide Species.

Fibrodontia is a genus of wood-inhabiting fungi consisting of four species so far, including F. gossypina as generic type. Two new species, Fibrodontia austrosinensis and F. subalba, are described and illustrated from China. Fibrodontia austrosinensis from southwestern China is characterized by a grandinioid to odontioid hymenophore with numerous small aculei, a dimitic hyphal system with scattered, smooth skeletal hyphae and ellipsoid basidiospores measuring 4.2–5.2 × 3.5–4.5 μm. Fibrodontia subalba from the West Tianshan Mountain in northwestern China is distinguished by an odontioid to hydnoid hymenophore, a dimitic hyphal system, and ellipsoid basidiospores measuring 3.7–4.4 × 2.8–3.4 μm. The phylogenies inferred from the data set of nuc rDNA ITS1-5.8S-ITS2 (ITS) and D1–D2 domains of nuc 28S rDNA (28S), and that of ITS, 28S, translation elongation factor (tef1α), and RNA polymerase II second largest subunit (rpb2) supported Fibrodontia as a monophyletic genus in the Trechisporales, and F. austrosinensis and F. subalba as separate lineages within Fibrodontia. Multi-rate Poisson Tree Processes, Automatic Barcode Gap Discovery and genetic distance methods based on ITS sequences of Fibrodontia also supported F. austrosinensis and F. subalba as distinct species. The taxonomic status of F. fimbriata that was recently transferred from Cystidiodendron, is briefly discussed. A key to all six known species of Fibrodontia is provided.

Elevational Diversity Patterns of Green Lacewings (Neuroptera: Chrysopidae) Uncovered With DNA Barcoding in a Biodiversity Hotspot of Southwest China

Elevational diversity patterns can reflect the responses of biodiversity to climate change spatially. We investigate the species diversity patterns of green lacewings (an important predatory group of insects) along the gradient of elevation from the Shaluli Mountains (Mts. Shaluli), which belong to the Hengduan Mountains in southwestern China, one of the important hotspots of global biodiversity. We combined multiple approaches, including Automatic Barcode Gap Discovery (ABGD), Assemble Species by Automatic Partitioning analysis (ASAP), General Mixed Yule Coalescent (GMYC), Poisson tree processes (bPTP), multi-rate Poisson tree processes (mPTP), to delimit the green lacewings species based on the standard barcoding region of cytochrome c oxidase subunit I (COI). The α-diversity and β-diversity patterns of green lacewings from the Mts. Shaluli along the gradient of elevation were analyzed, with further exploration on how the temperature effect elevational-diversity pattern on broad-scale (county scale) elevational gradients. The DNA barcoding reference library consisted of 40 green lacewing species from the Mts. Shaluli. The α-diversity of green lacewings decreased with the increasing elevation. The temperature was found to have a significant effect on the abundance and Shannon-Wiener diversity index but not on the species richness. Nestedness replaced turnover as the main component of Sørensen’s dissimilarity with the increasing elevation, and greater nestedness occurred at low temperature areas. The combination of a reliable DNA barcoding database could improve the accuracy and efficiency to investigate the species diversity patterns of green lacewings. Temperature, resource, and resultant interspecific competitions may have important roles in explaining the species diversity patterns of green lacewings from the Mts. Shaluli. Priority of conservation should be given to the species at low elevation, middle elevation, and relatively high temperature regions under the background of global climate warming.

COI mtDNA barcoding and morphology for the description of a new species of ricinuleid of the genus Pseudocellus (Arachnida: Ricinulei: Ricinoididae) from El Triunfo Biosphere Reserve, Chiapas, Mexico

A new species of epigean ricinuleid of the genus Pseudocellus Platnick, 1980 from El Triunfo Biosphere Reserve, Chiapas, Mexico is described. DNA barcoding utilizing mitochondrial cytochrome c oxidase subunit 1 (CO1) and morphology were used for species delimitation. Molecular analyses and species delimitation included four methods: 1) General Mixed Yule Coalescent model (GMYC), 2) Automatic Barcode Gap Discovery (ABGD), 3) Bayesian Poisson Tree Process (bPTP), and 4) Assemble Species by Automatic Partitioning (ASAP). All molecular methods and morphology were consistent in delimiting and recognizing the new species described herein. The average interspecific genetic distance (p-distance) among analyzed species of Pseudocellus was 11.6%. The species is described based on adult males and females: Pseudocellus giribeti sp. nov. This is the seventh species described from Chiapas, which holds the highest number of ricinuleids species for the country. The total number of described species of Pseudocellus from Mexico increases to 21, having the highest species diversity of known ricinuleids worldwide.

Cryptic diversity in the subgenus Oxyphortica (Diptera, Drosophilidae, Stegana).

Phylogenetic relationships of the subgenus Oxyphortica were reconstructed based on two mitochondrial genes (COI and ND2). The results revealed the paraphyly of Oxyphortica and supported high levels of cryptic diversity within this subgenus. By integrating morphological characteristics and molecular evidence, we identified 17 new species as members of Oxyphortica: S. (O.) amphigya sp. nov., S. (O.) armillata sp. nov., S. (O.) ashima sp. nov., S. (O.) bawo sp. nov., S. (O.) crypta sp. nov., S. (O.) gelea sp. nov., S. (O.) hengduanmontana sp. nov., S. (O.) jinmingi sp. nov., S. (O.) mengbalanaxi sp. nov., S. (O.) mouig sp. nov., S. (O.) setipes sp. nov., S. (O.) shangrila sp. nov., S. (O.) tsauri sp. nov., S. (O.) valleculata sp. nov., S. (O.) wanhei sp. nov., S. (O.) yangjin sp. nov. and S. (O.) hypophaia sp. nov. To test the early morphological identifications and confirm the species boundaries, different species delimitation methods, including Automatic Barcode Gap Discovery (ABGD) and Bayesian Phylogenetics and Phylogeography (BP&P), were used, together with traditional distance. All species boundaries were clearly defined. As Oxyphortica species are mainly distributed across Southwest China (e.g., 20 spp. from the Hengduan Mountains), the complex climate and topographic landforms of the area may be responsible for the high levels of species diversity and endemism.

Automatic barcode gap discovery reveals diverse clades of Rhipicephalus spp. and Haemaphysalis spp. ticks from small mammals in 'Asir, Saudi Arabia

BackgroundThe ixodid tick genera Rhipicephalus and Haemaphysalis contain several species of medical and/or veterinary importance, but their diversity in some regions of the world remains under-explored. For instance, very few modern studies have been performed on the taxonomy of these genera on the Arabian Peninsula.MethodsIn this study, we trapped small mammals in the 'Asir Mountains of south-western Saudi Arabia and collected tick specimens for morphological examination and molecular barcoding, targeting three mitochondrial loci: cox1, 16S rRNA and 12S rRNA.ResultsWe obtained a total of 733 ticks (608 Haemaphysalis spp. and 125 Rhipicephalus spp.) from 75 small mammal hosts belonging to six species. All tick specimens were immature except for nine adults recovered from a hedgehog (Paraechinus aethiopicus). Morphologically, the Rhipicephalus ticks resembled R. camicasi, but the Haemaphysalis ticks showed differences in palp morphology compared with species previously described from Saudi Arabia. Phylogenetic analysis and automatic barcode gap discovery identified a novel clade of Rhipicephalus sp. representing most of the nymphs. This was most closely related to R. leporis, R. guilhoni and R. linnaei. The adult ticks and a small proportion of nymphs clustered with R. camicasi sequences from a previous study. Finally, the Haemaphysalis nymphs formed two distinct clades that were clearly separated from all reference sequences but closest to some African species.ConclusionsThis apparent high level of tick diversity observed in a single study site of only ~ 170 km2, on a relatively small number of hosts, highlights the potential for the discovery of new tick species on the Arabian Peninsula.Graphical

Barcoding of South African forest‐dwelling snails (Mollusca: Gastropoda) reveals widespread cryptic diversity

AbstractSouth Africa harbors a rich and diverse terrestrial snail fauna. Despite their importance in ecosystem functioning and structure, their alpha taxonomic diversity is poorly understood, and most groups have not been the subject of molecular systematic scrutiny. During the present study, we used the mitochondrial cytochrome c oxidase subunit I (COI) locus to start a DNA barcode reference library, to examine intra‐ and interspecific genetic divergence, and to explore the presence of cryptic diversity among terrestrial gastropods from forested regions in the country. We generated 399 COI barcodes comprising 70 morphospecies in 26 genera and 15 families, representing nearly 10% of the described gastropod diversity in South Africa. Our results showed a substantial overlap between mean intraspecific and interspecific genetic variation and no barcoding gap. Three species delimitation methods—Poisson Tree Processes (PTP), Refined Single Linkage (RESL) algorithm in Barcode of Life Data Systems, and the Automatic Barcode Gap Discovery (ABGD)—applied to selected widespread species retrieved incongruent operational taxonomic units (OTUs). These results, coupled with high intraspecific variation, suggested cryptic diversity among several terrestrial gastropod genera in the country. We further demonstrated that DNA barcoding analysis holds the potential for highlighting cryptic lineages in snails and for revealing taxa in need of taxonomic revision. We recommend that future taxonomic studies of South African terrestrial snails incorporate multiple sources of information for species delimitation.

The genusJorunna(Nudibranchia: Discodorididae) in Europe: a new species and a possible case of incipient speciation

ABSTRACTTo investigate the conspecificity of different morphotypes of Jorunna tomentosa (Cuvier, 1804) (type species of genus Jorunna Bergh, 1876), we studied specimens sampled from across part of the geographical distribution of the species, using a combination of morphoanatomical characters and molecular phylogenetics. Bayesian and maximum likelihood phylograms were inferred based on the mitochondrial genes cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA, and the nuclear gene histone H3. We used the automatic barcode gap discovery method to aid in species delimitation. COI genetic uncorrected p-distances were estimated between and within species. Animals were dissected and the reproductive system, radulae and labial cuticles were examined; scanning electron microscopy was employed to study ultrastructural elements of anatomical characters. The results revealed the presence of a new species (Jorunna artsdatabankia n. sp.) and a possible case of incipient speciation in J. tomentosa with our COI data indicating the presence of two morphoanatomically indistinct lineages that are separated from each other by distances of 3.2–5.0%. The genetic distance between J. artsdatabankia n. sp. and its sister species J. tomentosa was 9.0–12.3%; the former species is characterized by a plain white to yellow background colour with irregularly placed small brown spots, smooth radular teeth and a longer vas deferens, wider vagina and a longer copulatory spine (up to 600 μm longer) than the latter. A diagnostic comparison of all species of European Jorunna is included, as well as a discussion of the assignment of J. lemchei to the genus Gargamella.

A DNA barcode library for mangrove gastropods and crabs of Hong Kong and the Greater Bay Area reveals an unexpected faunal diversity associated with the intertidal forests of Southern China

BackgroundMangroves are tropical and subtropical intertidal forests colonising sheltered coasts across the world. They host a unique faunal community, dominated by brachyuran crabs and gastropods. These invertebrates strongly contribute to the functionality of the entire forest. The reliable assessment of mangrove faunal diversity is, thus, a crucial step for efficient management and conservation plans, but it is hindered by difficulties in species identification. Here we provide a verified DNA barcode library for brachyuran crabs and gastropods inhabiting the mangroves of the Greater Bay Area, Southern China. In particular, we collected and morphologically identified 1100 specimens of mangrove associated brachyuran crabs and gastropods. The partial sequences of the mtDNA cytochrome oxidase subunit I gene were obtained from 275 specimens. Barcode sequences were then used to delineate Molecular Operational Taxonomic Units (MOTUs), employing three different delimitation methods: the automatic barcode gap discovery (ABGD) method, the general mixed Yule coalescent (GMYC) model and a Bayesian implementation of the Poisson tree processes (bPTP) model.ResultsBy integrating DNA barcodes with morphology, we identified 44 gastropod species and 58 brachyuran species associated with Hong Kong mangroves, with five and seven new records, for gastropods and crabs, respectively, for the Greater Bay Area. The delineation of MOTUs based on barcode sequences revealed a strong congruence between morphological and molecular identification for both taxa, showing the high reliability of the barcode library.ConclusionsThis study provides the first reference barcode library for mangrove-associated macrobenthic fauna in the Greater Bay Area and represents a reliable tool to management and conservation plans. Our molecular analyses resolved long lasting taxonomic misidentifications and inconsistencies and updated the knowledge on the geographical distribution of Asian mangrove associated fauna, ultimately highlighting a level of biodiversity higher than previously thought for Southern China.