Automatic Barcode Gap Discovery Research Articles

Species boundaries of the whiptail catfish Rineloricaria (Siluriformes: Loricariidae) from the Paraíba do Sul River drainage, southeastern Brazil, with species redescriptions and description of a new species.

Species of the catfish genus Rineloricaria are common in the Paraíba do Sul River basin, in southeastern Brazil; here we present a revision of the taxonomic diversity and geographic distribution of the species of the genus inhabiting the basin, based on novel morphologic and molecular data. Five species delimitation methods based on cytochrome C oxidase subunit 1 nucleotide sequences yielded comparable molecular operational taxonomic units. The automatic barcode gap discovery, assemble species by automatic partitioning, barcode index number, and Bayesian implementation of the Poisson tree process methods supported the recognition of five evolutionary lineages. These taxonomic units were assigned to the previously described Rineloricaria nigricauda, Rineloricaria steindachneri, Rineloricaria zawadzkii, and Rineloricaria nudipectoris, and an additional undescribed species. R. zawadzkii was further divided into two intraspecific geographically structured lineages using the generalized mixed Yule coalescent delimitation method. A maximum likelihood phylogenetic analysis revealed that the five lineages from the Paraíba do Sul have closer relationships to different species from southern and southeastern Brazil (Ribeira de Iguape, Lagoa dos Patos, Uruguay, Paraguay, and Parana river basins) than to each other. Based on the analysis of lectotypes, recently collected material, and specimens from ichthyological collections, the poorly described R. nigricauda and R. steindachneri are redescribed following current descriptive standards. The undescribed species from the middle and upper Paraíba do Sul River basin is formally described. The description of a new species, along with the description of species boundaries in R. nigricauda and R. steindachneri, contributes to the knowledge of the ichthyofauna of the Paraíba do Sul River basin and adjacent coastal drainages of southeastern Brazil. An identification key for the species of Rineloricaria occurring in the Paraíba do Sul River basin is provided.

Towards a Comprehensive DNA Barcode Library of Stenochironomus Kieffer, 1919 (Diptera: Chironomidae) from China

The adoption of DNA-based assessments for biodiversity monitoring has been on the rise. However, the effectiveness of DNA-based taxonomic assignments heavily relies on the availability and reliability of DNA barcode libraries. There is growing demand for a comprehensive understanding of aquatic biodiversity and the critical role of Chironomidae, specifically Stenochironomus in freshwater ecosystems. Therefore, our objective is to develop a reference barcode library for Stenochironomus in China. From 2016 to 2021, we collected Stenochironomus specimens in diverse Chinese landscapes using malaise traps, light traps, and sweep nets. These specimens were carefully preserved for DNA extraction and barcode sequencing. Our analysis unveiled 36 unique operational taxonomic units from 180 COI barcode sequences through a Neighbor-Joining tree and Automatic Barcode Gap Discovery program, highlighting a significant diversity within the Stenochironomus species. The findings emphasize the constraints of conventional morphological identification methods, especially for species with ambiguous morphologies. It also underscores the effectiveness of DNA barcoding in revealing hidden species diversity, known as cryptic species. Consequently, this study advocates for an integrated taxonomic approach, combining morphological and molecular data, to refine species identification and conservation strategies.

Refining taxonomic identification of microalgae through molecular and genetic evolution: a case study of Prorocentrum lima and Prorocentrum arenarium.

Species delimitation based on lineage definition has become increasingly popular. However, these methods have been limited, especially for species that lack genomic data and are morphologically similar. The trickiest part for the species identification is that the interspecific and intraspecific boundaries are vague. Taking Prorocentrum (Dinophyta) as an example, analysis of cell morphology, growth, and toxin synthesis in both species of P. lima and P. arenarium does not provide a reliable basis for species delineation. However, through phylogenetic and genetic distance analyses of their ITS and LSU sequences, establishment of evolutionary tree based on orthologous gene sequences, and combining the results of automatic barcode gap discovery and Poisson tree processes models, it was sustained that P. arenarium does not belong to the P. lima complex and should be considered as an independent species. Interspecies genetic evolution analysis revealed that P. lima and P. arenarium may contribute to evolutionary direction that favors combating reverse environmental factors. In P. lima, viral invasion may be one of the reasons for its large genome size. In the study, P. lima complex has been selected as an example to enhance the taxonomic identification of microalgae through molecular and genetic evolution, offering valuable insights into refining taxonomic identification and promoting microbial biodiversity research in other species.IMPORTANCEMicroalgae, especially the species known as Prorocentrum, have received significant attention due to their ability to trigger harmful algal blooms and produce toxins. However, the boundaries between species and within species are ambiguous. Clear and comprehensive species delineation indicates that Prorocentrum arenarium should be considered as an independent species, separate from the Prorocentrum lima complex. Improving the classification and identification of microalgae through molecular and genetic evolution will provide reference points for other cryptic species. Prorocentrum occupy multiple ecological niches in marine environments, and studying their evolutionary direction contributes to understanding their ecological adaptations and community succession.

Barcoding Fails to Delimit Species in Mongolian Oedipodinae (Orthoptera, Acrididae).

Mongolia, a country in central Asia, with its vast grassland areas represents a hotspot for Orthoptera diversity, especially for the Acrididae. For Mongolia, 128 Acrididae species have been documented so far, of which 41 belong to the subfamily Oedipodinae (band-winged grasshoppers). Yet, few studies concerning the distribution and diversity of Oedipodinae have been conducted in this country. Molecular genetic data is almost completely absent, despite its value for species identification and discovery. Even, the simplest and most used data, DNA barcodes, so far have not been generated for the local fauna. Therefore, we generated the first DNA barcode data for Mongolian band-winged grasshoppers and investigated the resolution of this marker for species delimitation. We were able to assemble 105 DNA barcode (COI) sequences of 35 Oedipodinae species from Mongolia and adjacent countries. Based on this data, we reconstructed maximum likelihood and Bayesian inference phylogenies. We, furthermore, conducted automatic barcode gap discovery and used the Poisson tree process (PTP) for species delimitation. Some resolution was achieved at the tribe and genus level, but all delimitation methods failed to differentiate species by using the COI region. This lack of resolution may have multiple possible reasons, which likely differ between taxa: the lack of resolution in the Bryodemini may be partially explained by their massive genomes, implying the potential presence of large numbers of pseudogenes, while within the Sphingonotini incomplete lineage sorting and incorrect taxonomy are more likely explanations for the lack of signal. Further studies based on a larger number of gene fragments, including nuclear DNA, are needed to distinguish the species also at the molecular level.

Species richness in Myotis (Chiroptera: Vespertilionidae): species delimitation and expanded geographic sampling reveal high Neotropical diversity

Abstract With up to 137 species worldwide, Myotis,— a genus of small, insectivorous bats are an example for understanding biogeographic and evolutionary processes such as intercontinental colonization, diversification, speciation, and convergent adaptive evolution. Species limits and associated distributions, however, remain poorly delineated across significant portions of this diverse group and encompassing a vast geographic range. Here, we explore how diversity is partitioned across this radiation with a focus on Neotropical species of Myotis, using a barcoding approach (706 bp of the mitochondrial DNA gene cytochrome-b) that includes 544 individual sequences (116 newly generated sequences and 428 sequences from GenBank, including outgroups). Single-locus molecular barcoding is useful for identifying cryptic diversity and establishing hypotheses that can guide investigations of species boundaries and phylogenetic relationships, an especially important initial step in assessing poorly characterized, diverse clades. The global phylogeny produced from these data is consistent with the hypothesized monophyly of the New World clade and places the evolutionary relationships of Neotropical species of Myotis within the context of the Palearctic and Nearctic clades. We then explore species boundaries for 25 of the 36 nominal species of Neotropical Myotis, using 3 alternative species delimitation approaches (1 distance-based ABGD [Automatic Barcode Gap Discovery]; and 2 coalescent-based mPTP [multi-rate Poisson Tree Processes], GMYC [Generalized Mixed Yule Coalescent] model). The mPTP (n = 29 species) and ABGD (n = 26) approaches inferred different numbers of species than that recognized by current Neotropical Myotis taxonomy, whereas GMYC (n = 25) inferred the same number of species. In addition to the original 36 Neotropical nominal species currently recognized, we suggest that taxonomic revisions are needed to assess whether M. oxyotus gardneri and M. nigricans osculatii should be elevated to the species level and whether M. dinellii is distinct from M. levis. With the 7 potentially cryptic species identified herein that require further study, the number of Neotropical Myotis may approach 45 species. Taken together, our results suggest that rather than a classic example of a temperate radiation, there may be more species of Myotis in the Neotropics than Nearctic, Afrotropic, Indomalayan, and Oceanian Realms, with only the Palearctic region showing higher species richness.

A comprehensive DNA barcoding of Indian freshwater fishes of the Indus River system, Beas

The Beas River is one of the important rivers of the Indus River system located in Himachal Pradesh, India, that harbors a diverse range of freshwater fish species. The present study employed COI gene to investigate the ichthyofaunal diversity of river Beas. Through the sequencing of 203 specimens from Beas River, we identified 43 species, belonging to 31 genera, 16 families, and 10 orders. To analyze the genetic divergence and phylogeny of identified species, 485 sequences of Indian origin were retrieved from BOLD, resulting in a dataset of 688 sequences. Our findings consistently revealed a hierarchical increase in the mean K2P genetic divergence within species (0.80%), genus (9.06%), and families (15.35%). Automated Barcode Gap discovery, Neighbour Joining, and Bayesian inference consensus tree methodologies were employed to determine the putative species and their phylogeny, successfully delimiting most of the species with only a few exceptions. The results unveiled six species exhibiting high intra-species divergence (> 2%), suggesting the presence of sibling species and falsely identified sequences on online databases. The present study established the first DNA barcoding-based inventory of freshwater fish species in the Beas River providing comprehensive insights into economically exploited endangered and vulnerable species. In order to ensure the sustainable use of aquatic resources in the Beas River, we recommend the implementation of species measures to protect biodiversity and genetic resources.

Delimiting Species with Single-Locus DNA Sequences.

DNA sequences are increasingly used for large-scale biodiversity inventories. Because these genetic data avoid the time-consuming initial sorting of specimens based on their phenotypic attributes, they have been recently incorporated into taxonomic workflows for overlooked and diverse taxa. Major statistical developments have accompanied this new practice, and several models have been proposed to delimit species with single-locus DNA sequences. However, proposed approaches to date make different assumptions regarding taxon lineage history, leading to strong discordance whenever comparisons are made among methods. Distance-based methods, such as Automatic Barcode Gap Discovery (ABGD) and Assemble Species by Automatic Partitioning (ASAP), rely on the detection of a barcode gap (i.e., the lack of overlap in the distributions of intraspecific and interspecific genetic distances) and the associated threshold in genetic distances. Network-based methods, as exemplified by the REfined Single Linkage (RESL) algorithm for the generation of Barcode Index Numbers (BINs), use connectivity statistics to hierarchically cluster-related haplotypes into molecular operational taxonomic units (MOTUs) which serve as species proxies. Tree-based methods, including Poisson Tree Processes (PTP) and the General Mixed Yule Coalescent (GMYC), fit statistical models to phylogenetic trees by maximum likelihood or Bayesian frameworks.Multiple webservers and stand-alone versions of these methods are now available, complicating decision-making regarding the most appropriate approach to use for a given taxon of interest. For instance, tree-based methods require an initial phylogenetic reconstruction, and multiple options are now available for this purpose such as RAxML and BEAST. Across all examined species delimitation methods, judicious parameter setting is paramount, as different model parameterizations can lead to differing conclusions. The objective of this chapter is to guide users step-by-step through all the procedures involved for each of these methods, while aggregating all necessary information required to conduct these analyses. The "Materials" section details how to prepare and format input files, including options to align sequences and conduct tree reconstruction with Maximum Likelihood and Bayesian inference. The Methods section presents the procedure and options available to conduct species delimitation analyses, including distance-, network-, and tree-based models. Finally, limits and future developments are discussed in the Notes section. Most importantly, species delimitation methods discussed herein are categorized based on five indicators: reliability, availability, scalability, understandability, and usability, all of which are fundamental properties needed for any approach to gain unanimous adoption within the DNA barcoding community moving forward.

Species delimitation by DNA barcoding reveals undescribed diversity in Stelliferinae (Sciaenidae).

Stelliferinae is the third most speciose subfamily of Sciaenidae, with 51 recognized species arranged in five genera. Phylogenies derived from both morphological and molecular data support the monophyly of this subfamily, although there is no general consensus on the intergeneric relationships or the species diversity of this group. We used the barcoding region of the cytochrome oxidase C subunit I (COI) gene to verify the delimitation of Stelliferinae species based on the Automatic Barcode Gap Discovery (ABGD), Generalized Mixed Yule Coalescence (GMYC), and Bayesian Poisson Tree Process (bPTP) methods. In general, the results of these different approaches were congruent, delimiting 30-32 molecular operational taxonomic units (MOTUs), most of which coincided with valid species. Specimens of Stellifer menezesi and Stellifer gomezi were attributed to a single species, which disagrees with the most recent review of this genus. The evidence also indicated that Odontoscion xanthops and Corvula macrops belong to a single MOTU. In contrast, evidence also indicates presence of distinct lineages in both Odontoscion dentex and Bairdiella chrysoura. Such results are compatible with the existence of cryptic species, which is supported by the genetic divergence and haplotype genealogy. Therefore, the results of the present study indicate the existence of undescribed diversity in the Stelliferinae, which reinforces the need for an ample taxonomic review of the fish in this subfamily.

Molecular and morphometric data provide evidence of intraspecific variation in shape and pigmentation pattern in Otocinclus cocama (Siluriformes: Loricariidae) across major river drainages.

Otocinclus cocama, a uniquely colored species of the loricariid catfish genus Otocinclus described solely from the type locality in the lower Ucayali River in northern Peru, is reported occurring in the Tigre River, a tributary to the Marañón River that drains a different section of the Andean Mountain range in the western Amazon. Both populations differ in the number of dark bars spanning the flanks of the body, and we investigated whether these morphotypes constitute distinct species. The body shapes of populations from the Tigre and Ucayali rivers were compared using geometric morphometrics. Although principal component analysis detected a broad overlap between populations, multivariate analysis of variance and linear driscriminat analysis revealed a subtle differentiation between the populations of the two hydrographic basins. Average body shape of the Ucayali River population tend to be slightly higher than that of the Tigre River, with the caudal peduncle stretched vertically in the Ucayali population. Multivariate regression of shape and centroid size revealed an allometric effect of 10.7% (p < 0.001), suggesting that the variation between Tigre and Ucayali populations was purely shape variation. Molecular data of coI, cytb, nd2, and 16S mitochondrial genes indicated a nucleotide diversity range from 0.001 to 0.003, and haplotypic diversity range from 0.600 ± 0.11 to 0.79 ± 0.07. The median-joining haplotype network for the concatenated matrix exhibited two divergent haplogroups related to the geographic area and separated by <10 mutational steps. The molecular species delimitation methods based on distance (automatic barcode gap discovery and assemble species by automatic partitioning) recovered two molecular lineages evolving independently, being one of the lineages formed by individuals from both populations. Tree-based methods (generalized mixed Yule coalescent and Bayesian implementation of the Poisson tree process) recovered similar topologies and supported single lineage recognition. Methods of molecular delimitation of species disclosed the high similarity between the two populations of Otocinclus cocama, further supported by the presence of old haplotypes common to both groups which could indicate that the populations still maintain gene flow. Although the morphological data reveal a subtle variation between both river basins, the molecular data suggest a weak population structuration based on hydrographic areas, but not different species lineages, therefore Otocinclus cocama is composed of a single lineage with two distinct morphotypes.

Genetic diversity and population differentiation in Earliella scabrosa, a pantropical species of Polyporales

Earliella scabrosa is a pantropical species of Polyporales (Basidiomycota) and well-studied concerning its morphology and taxonomy. However, its pantropical intraspecific genetic diversity and population differentiation is unknown. We initiated this study to better understand the genetic variation within E. scabrosa and to test if cryptic species are present. Sequences of three DNA regions, the nuclear ribosomal internal transcribed spacer (ITS), the large subunit ribosomal DNA (LSU), and the translation elongation factor (EF1α) were analysed for 66 samples from 15 geographical locations. We found a high level of genetic diversity (haplotype diversity, Hd = 0.88) and low nucleotide diversity (π = 0.006) across the known geographical range of E. scabrosa based on ITS sequences. The analysis of molecular variance (AMOVA) indicates that the genetic variability is mainly found among geographical populations. The results of Mantel tests confirmed that the genetic distance among populations of E. scabrosa is positively correlated with the geographical distance, which indicates that geographical isolation is an important factor for the observed genetic differentiation. Based on phylogenetic analyses of combined dataset ITS-LSU-EF1α, the low intraspecific divergences (0–0.3%), and the Automated Barcode Gap Discovery (ABGD) analysis, E. scabrosa can be considered as a single species with five different geographical populations. Each population might be in the process of allopatric divergence and in the long-term they may evolve and become distinct species.

Wing geometric morphometrics and DNA barcoding to distinguish three closely related species of Armigeres mosquitoes (Diptera: Culicidae) in Thailand

Armigeres subalbatus, a mosquito species widely found in Thailand and other Asian countries, serves as a vector for filarial parasites, affecting both humans and animals. However, the surveillance of this vector is complicated because of its morphological similarity to two other species, Armigeres dohami and Armigeres kesseli. To differentiate these morphologically similar species, our study employed both wing geometric morphometrics (GM) and DNA barcoding, offering a comprehensive approach to accurately identify these closely related Armigeres species in Thailand. Our GM analyses based on shape demonstrated significant accuracy in differentiating Armigeres species. Specifically, the outline-based GM method focusing on the 3rd posterior cell exhibited an accuracy rate of 82.61%, closely followed by the landmark-based GM method with 81.54%. Both these GM techniques effectively distinguished Ar. subalbatus from Ar. dohami and Ar. kesseli. Regarding DNA barcoding, our investigation of pairwise intra- and interspecific divergences revealed a "barcoding gap". Furthermore, the results of species confirmation using both species delimitation methods including the automatic barcode gap discovery method (ABGD) and the Multi-rate Poisson tree process (mPTP) were consistent with those of morphological identification, sequence comparisons with the GenBank and Barcode of Life Data System (BOLD) databases, and the neighbor-joining tree construction. These consistent results emphasize the efficacy of DNA barcoding in the precise identification of Armigeres species.

Species delimitation and molecular phylogeny of the grasshopper subfamily Gomphocerinae (Orthoptera: Acrididae) from Algeria based on mitochondrial and nuclear DNA markers

Gomphocerinae grasshoppers (Orthoptera: Acrididae) are generally characterised by their polymorphism and cryptic diversity, which can be confusing when relying on morphological identification alone. DNA taxonomy serves as a powerful molecular tool for species identification and biodiversity assessment. In the context of zoogeography and the biodiversity conservation of animal resources in ecosystems, DNA barcoding data for Algerian Gomphocerinae fauna remains limited in global databases, despite their agroeconomic and environmental importance. Therefore, in this study, we collected various species of this subfamily from the Biskra region in Algeria and conducted DNA barcoding analysis, employing different molecular species delimitation methods (the automatic barcode gap discovery, assemble species by automatic partitioning, single-threshold general mixed Yule coalescent model, Bayesian Poisson tree process and multi-rate Poisson tree process methods), as well as phylogenetic analyses (maximum-likelihood and Bayesian inference) based on two molecular markers (COI and ITS). The results of the 163 newly generated sequences demonstrated that DNA barcoding technology is highly efficient and valuable for species identification in the subfamily Gomphocerinae, showing strong congruence with morphological evidence for 12 species, including eight species that were sequenced for the first time. This study also reported a new record of the genus Stenohippus Uvarov, 1926 in Algeria, comprising two species. Additionally, taxonomic revision allowed the species Dociostaurus biskrensis Moussi & Petit, 2014, to be transferred to the genus Stenohippus. Molecular tree analyses revealed the phylogenetic positions of the newly sequenced species within the subfamily Gomphocerinae and provided insights into their evolutionary relationships. These new data serve as a starting point for future research in other geographical areas, enabling a better understanding of the biodiversity of this insect group in Algeria.

Unveiling Cryptic Species Diversity and Genetic Variation of Lasiodiplodia (Botryosphaeriaceae, Botryosphaeriales) Infecting Fruit Crops in Taiwan.

The genus Lasiodiplodia, a member of the family Botryosphaeriaceae, is an important fungal disease genus in agriculture. However, the Lasiodiplodia species survey and genetic diversity in Taiwan remain unclear. This study aimed to investigate the Lasiodiplodia species associated with various fruit species to explore the cryptic Lasiodiplodia species diversity, validate species delimitation, and unveil cryptic genetic diversity. Overall, six Lasiodiplodia species were identified, with several new records of infection identified. Additionally, phylogenetic analyses indicated that the relations of all isolates of L. theobromae might be paraphyletic. They were grouped with L. brasiliense based on Automatic Barcode Gap Discovery (ABGD), Automatic Partitioning (ASAP) and structure-based clustering analyses. These analyses did not provide conclusive evidence for L. brasiliensis as a stable species. It may be necessary to gather more information to clarify the species delineation. The multiple new records of Lasiodiplodia species with high genetic diversity and differentiation revealed that the diversity of Lasiodiplodia in Taiwan was underestimated in the past. We found that L. theobromae has the highest number of haplotypes but the lowest number of haplotype and nucleotide diversities, indicating a recent population expansion. This was supported by the significant negative Tajima's D and Fu and Li's D* tests. The high genetic diversity, low gene flow, and host-associated differentiation of Lasiodiplodia species indicate that they might harbour powerful evolutionary potential in Taiwan. This study provided critical insights into genetic variation, host-associated differentiation, and demography of Lasiodiplodia species, which would be helpful for disease management of related pathogens.

Two new species of the genus Laena (Coleoptera, Tenebrionidae, Lagriinae) from northern Sichuan in China based on morphological and molecular data.

In this study, the Laena species from northern Sichuan Province were reviewed using a combination of molecular and morphological datasets. Three molecular methods for species delimitation were firstly used in the genus Laena to explore species boundaries. The results show that the number of morphospecies and putative species using Automatic Barcode Gap Discovery (ABGD) is consistent. Based on the results, two new species are described and illustrated: Laenamounigouicasp. nov. and Laenadentithoraxasp. nov. New materials and distribution of 10 Laena species are also provided in this study. This work also provides valuable molecular data for species identification and phylogenetic analyses of the genus Laena and subfamily Lagriinae.

DNA Barcoding and Phylogenetics Relationship of Pangasiid Catfishes in Peninsular Malaysia Revealed the Impacts of Aquaculture on the Native Species Conservation

Pangasiids are an economically significant group of catfish, and many pangasiids are threatened in the wild from anthropogenic pressures, including increases in fishing pressure, habitat degradation, and improperly managed aquaculture practices. This study demonstrates the usage of DNA barcoding of the Cytochrome Oxidase subunit I (COI) gene as an identification tool in detecting potentially threatening invasive pangasiid species by establishing the diversity and phylogenetic relationship of Pangasiidae catfishes in Peninsular Malaysia. A neighbour-joining (NJ) dendrogram (Kimura-2-parameter model) generated five clades to represent distinct genera. Pangasius was further subdivided into two clades (Clade A: Pangasius bocourti-P. djambal and Clade B: P. nasutus-P. conchophilus). Given the marginal genetic divergence, indigenous and non-native species should be treated cautiously in allopatrically distributed species. The analysis used Automatic Barcode Gap Discovery (ABGD) and revealed barcode gaps between the intraspecific and interspecific distances. The sequences were partitioned into five groupings, corresponding with the species delineation based on the distribution of pairwise differences, which could not be differentiated using the NJ dendrogram. ABGD allows the recognition of one or two additional species using the recursive approach, but other taxonomic methods should be considered for a solid conclusion. DNA barcoding demonstrates the identification of closely related species, thus justifying its application towards the conservation of these fish.

Comparison between p-distance and single-locus species delimitation models for delineating reproductively tested strains of pennate diatoms (Bacillariophyceae) using cox1, rbcL and ITS.

Several automated molecular methods have emerged for distinguishing eukaryote species based on DNA sequence data. However, there are knowledge gaps around which of these single-locus methods is more accurate for the identification of microalgal species, such as the highly diverse and ecologically relevant diatoms. We applied genetic divergence, Automatic Barcode Gap Discovery for primary species delimitation (ABGD), Assemble Species by Automatic Partitioning (ASAP), Statistical Parsimony Network Analysis (SPNA), Generalized Mixed Yule Coalescent (GMYC) and Poisson Tree Processes (PTP) using partial cox1, rbcL, 5.8S + ITS2, ITS1 + 5.8S + ITS2 markers to delineate species and compare to published polyphasic identification data (morphological features, phylogeny and sexual reproductive isolation) to test the resolution of these methods. ASAP, ABGD, SPNA and PTP models resolved species of Eunotia, Seminavis, Nitzschia, Sellaphora and Pseudo-nitzschia corresponding to previous polyphasic identification, including reproductive isolation studies. In most cases, these models identified diatom species in similar ways, regardless of sequence fragment length. GMYC model presented smallest number of results that agreed with previous published identification. Following the recommendations for proper use of each model presented in the present study, these models can be useful tools to identify cryptic or closely related species of diatoms, even when the datasets have relatively few sequences.

Revealing cryptic diversity and population divergence in subtropical aphids through DNA barcoding

AbstractAphids are worldwide distributed pests, which feed on plant sap and cause serious losses to agricultural and forestry industries. Relatively few comprehensive studies have been conducted on aphid diversity in subtropics, although these regions may harbour hidden aphid diversity due to high plant diversity. In this study, we conducted DNA barcoding analysis for aphids in subtropical regions based on a comprehensive DNA barcode reference library including 5821 sequences and related ecological information (e.g. geography, host plant) of 379 morphospecies. In the reference library, 2140 sequences and related data of 151 morphospecies belonging to 77 genera and 11 subfamilies were newly produced by the current study. The average Kimura 2‐parameter distances within species, genera and subfamilies were 0.92%, 3.55% and 8.3%, respectively. The minimum interspecific distances were greater than the maximum intraspecific distances in 79.8% species, suggesting that barcode gaps existed in most aphid species. Automatic barcode gap discovery, Barcode Index Number, Bayesian Poisson tree processes and generalized mixed Yule‐coalescent revealed 419, 401, 462 and 455 single species‐representing clusters, respectively. A total of 32 morphospecies were assigned to more than one subclade respectively, suggesting geography or host plant‐mediated divergences and the existence of cryptic diversity. This study demonstrates that subtropical regions should have a higher species diversity of aphids, and cryptic species in certain morphospecies indicated in this study need to be investigated based on integrative taxonomic practices in the future. The DNA barcode reference library constructed herein provides a robust baseline data set to support future research in taxonomy, phylogenetics, ecology and evolution of aphids.

The phylogenetic structure and coalescent species delimitation of an endemic trapdoor spider genus, Stasimopus (Araneae, Mygalomorphae, Stasimopidae) in the Karoo region of South Africa

The Karoo region of South Africa is a unique and sensitive ecosystem which is facing pressure for development due to economic incentives such as mining, farming and shale gas exploration. The species diversity of many taxa in the area is largely unknown. A phylogenetic analysis of the cork-lid trapdoor spider genus, Stasimopus (Stasimopidae) was undertaken in order to gain insight into the relationships between the species that may be present in the area. The species within Stasimopus are challenging to identify and define using traditional morphological methods due to a high degree of morphological conservatism within the genus. For this reason, multiple coalescent based species delimitation methods were used to attempt to determine the species present for Stasimopus in the region which was tested against the morphological identifications and genetic clades (based on CO1, 16S and EF-1ɣ). We tested single-locus methods Automatic Barcode Gap Discovery (ABGD), Bayesian implementation of Poisson Tree Processes (bPTP) and General Mixed Yule- Coalescent (GMYC), as well as multi-locus Brownie. The phylogenetic analysis of Stasimopus in the Karoo showed that there is a high degree of genetic diversity within the genus. The species delimitation results proved unfruitful for the genus, as they appear to delimit population structure rather than species for most methods. Alternative methods should be investigated to aid in the identification of the species in order truly understand the species diversity of the genus.

Diversity of Pholcus Spiders (Araneae: Pholcidae) in China’s Lüliang Mountains: An Integrated Morphological and Molecular Approach

Spiders of the genus Pholcus were collected for the first time during an expedition to the Lüliang Mountains in Shanxi Province, North China. Phylogenetic analyses of DNA sequence data from COI, H3, wnt, and 28S genes allowed us to group them into nine well-supported clades. We used morphology and four methods of molecular species delimitation, namely Automatic Barcode Gap Discovery (ABGD), the Generalized Mixed Yule Coalescent (GMYC), Bayesian Poisson Tree Processes (bPTP), and Bayesian Phylogenetics and Phylogeography (BPP), to investigate species boundaries. These integrative taxonomic analyses identified the nine clades as nine distinct species, comprising Pholcus luya Peng & Zhang, 2013 and eight other species new to science: Pholcus jiaocheng sp. nov., Pholcus linfen sp. nov., Pholcus lishi sp. nov., Pholcus luliang sp. nov., Pholcus wenshui sp. nov., Pholcus xiangfen sp. nov., Pholcus xuanzhong sp. nov., and Pholcus zhongyang sp. nov. The species occur in geographic proximity and show many morphological similarities. All of them belong to the P. phungiformes species group. The records from the Lüliang Mountains represent the westernmost distribution limit of this species group.

Species diversity and distribution of the calcareous green macroalgae Halimeda in Taiwan, Spratly Island, and Dongsha Atoll, with the proposal of Halimeda taiwanensis sp. nov.

The calcified green algal genus Halimeda is one of the most ecologically important but morphologically diverse seaweeds in sub-tropical and tropical waters. Because of its high morphological plasticity, the identification of Halimeda species based on morphological characters is challenging without the assistance of molecular analysis. To date, the species diversity of Halimeda in Taiwan and its overseas territories has not been investigated with the assistance of DNA sequencing, and this taxonomic knowledge gap should be filled. The present study initiates a systematic examination of the species diversity and distribution of Halimeda in Taiwan, Spratly Island, and Dongsha Atoll in the South China Sea, using DNA sequence data (plastid tufA gene and rbcL) and morphological data. Our DNA analyses revealed the presence of 10 Halimeda species (Halimeda borneensis, Halimeda cylindracea, Halimeda discoidea, Halimeda distorta, Halimeda macroloba, Halimeda minima, Halimeda opuntia, Halimeda renschii, Halimeda taiwanensis sp. nov., and Halimeda velasquezii) in the waters around Taiwan, Spratly Island, and Dongsha Atoll. The majority of the species could be readily distinguished by their morphological and anatomical characters. The proposed new species, H. taiwanensis, was differentiated not only by our algorithmic species delimitation analyses (statistical parsimony network analysis and automated barcode gap discovery), but also by its morphological features. The proposed new species differs from two externally resembled species, H. cuneata and H. discoidea, in having an undulated segment margin, the complete fusion of medullary siphons at the node, the lack of segment stalk, and the presence of a large primary utricle. Here, we present the up-to-date taxonomic account, molecular diversity, and geographical distribution of Halimeda spp. in Taiwan and associated areas of the South China Sea. Environmental factors that might drive the occurrence and latitudinal distribution of the species are also discussed.