General Mixed Yule Coalescent Research Articles

Oxynoemacheilus fatmae, a new species from the Güzelhisar Stream in the Aegean Sea basin, Türkiye (Teleostei: Nemacheilidae).

Oxynoemacheilus fatmae, a new species, is found in the Güzelhisar Stream in the northern Aegean Sea basin. It is differentiated from all other species of Oxynoemacheilus in the northern Aegean Sea and adjacent basins by having four to eight irregularly shaped narrow black bars on the posterior part of flank, and anterior parts of the flank with a marbled pattern. O. fatmae is differentiated from the closest species Oxynoemacheilus theophilii by having 14 fixed diagnostic nucleotide substitution sites, and the pair-wise genetic distance is 2.22%. It further differs from O. theophilii by having a slenderer body (body at dorsal-fin origin: 15%-17% standard length [SL] vs. 17%-18%), a slenderer caudal peduncle (10%-12% SL vs. 12%-13%), a more forked caudal fin (length of middle caudal-fin lope: 16%-19% SL vs. 19%-23%), and the absence the dorsal and ventral adipose crests on the caudal peduncle behind the vertical of the posterior anal-fin base (vs. present). Three species delimitation tests (assemble species by automatic partitioning (ASAP), automatic barcode gap discovery (ABGD) and generalized mixed yule-coalescent (GMYC)) and phylogenetic analyses reinforce the validity of O. fatmae as a distinct species.

A description of Aliichlorella ignota gen. et sp. nov. and a comparison of the efficiency of species delimitation methods in the Chlorella‐clade (Trebouxiophyceae, Chlorophyta)

AbstractThe present study reports a new representative of Chlorella‐clade that was newly isolated from the Lake Vos'merka (Samara region, Russian Federation). 18S–ITS1–5.8S–ITS2 sequence analyses indicated that the studied strain ACSSI 368 and related Chlorella chlorelloides, Chlorella pulchelloides, formed an separate cluster in the Chlorella‐clade, unrelated to the holotype of the genus Chlorella – Chlorella vulgaris. The independent place of the genus was also confirmed by genetic distances. The individual species status of the studied strain ACSSI 368, compared with the sister C. chlorelloides, was confirmed by morphological differences (cell number per colony, type of chloroplast, number of autospores), genetic distances of the interspecific level by 18S–ITS1–5.8S–ITS2 fragment, alone by internal transcribed spacer 1 (ITS1) and internal transcribed spacer 2 (ITS2) sequences, one complementary base change in ITS1, and the results of species delimitation using five methods. A description of studied strain ACSSI 368 was provided as an authentic strain of the type species Aliichlorella ignota. Currently, the new genus Aliichlorella also includes Aliichlorella chlorelloides comb. nov. and Aliichlorella pulchelloides comb. nov. In general, six different delimitation algorithms were used in the present study. The ASAP (i.e. assemble species by automatic partitioning) and LocMin (i.e. ‘local minimum’ function) algorithms showed synchronous results, but their accuracy with respect to closely related species was somewhat lower. The mlPTP algorithm simultaneously aimed to combine closely related species in one and at the same time divide slightly distinguishing strains within a species into several molecular operational taxonomic units. The bPTP algorithm separated excessively Chlorella‐clade representatives into individual species. The generalized mixed Yule coalescent (GMYC) algorithm results were consistent the most with the modern understanding of the Chlorella‐clade taxonomy. However, the same method is one of the most time‐consuming because, for its implementation, it is necessary to carry out long preparatory work. The KoT results were less accurate than the GMYC results, although this is less time‐consuming because it does not require the preliminary construction of ultrametric trees.

Identification of fish specimens of the Tocantins River, Brazil, using DNA barcoding.

The fish fauna of the Tocantins River possesses many endemic species; however, it is little studied in molecular terms and is quite threatened by the construction of several hydroelectric dams. Therefore, the objective of this study was to identify the ichthyofauna of the Tocantins River using DNA barcoding. For this, collections were carried out in five points of this river, which resulted in the capture of 725 individuals from which partial sequences of the cytochrome oxidase subunit I (COI) gene were obtained for genetic analysis. A total of 443 haplotypes were recovered with the mean intraspecific K2P genetic distance of 1.82%. Altogether, 138 species were identified based on morphological criteria, which was a quantity that was much lower than that indicated by the four molecular methods (assemble species by automatic partitioning [ASAP], barcode index number [BIN], generalized mixed Yule coalescent (GMYC), and Bayesian Poisson tree processes [bPTP]) through which 152-157 molecular entities were identified. In all, 41 unique BINs were obtained based on the data generated in the BOLDSystems platform. According to the result indicated by ASAP (species delimitation approach considered the most appropriate in the present study), there was an increase of 17 molecular entities (12.32%), when compared to the number of species identified through their morphological criteria, as it can show cryptic diversity, candidates for new species, and misidentifications. There were 21 incongruities indicated between the different identification approaches for species. Therefore, it is suggested that these taxonomic problems be cautiously evaluated by experts to solve such taxonomic issues.

Molecular characterisation of Amblyomma integrum circulating in southern India

Amblyomma integrum is a large gooseberry sized longirostrate tick (when fully repleted) found in India and Sri Lanka. In Kerala (India), this tick is commonly found in the forest and its fringe areas frequently infesting deer and hence it is locally known as “maan chellu / maanunny” (deer tick). In the present study, molecular characterisation and phylogenetic analysis of A. integrum collected from the area grazed by the sambar deer (Rusa unicolor) of Kerala, south India was performed using three molecular markers viz., the mitochondrial cytochrome c oxidase subunit 1 (COI), mitochondrial 16S ribosomal RNA, and nuclear 18S ribosomal RNA genes. Cytochrome c oxidase subunit 1 (COI) gene showed better resolving ability for elucidating the evolutionary relationship of A. integrum and identified two distinct clades, viz., A and B. The Tamil Nadu isolates of south India and Marayoor isolate 1 (from Idukki district of Kerala bordering with Tamil Nadu) belonged to clade A. Majority of Wayanad isolates from Kerala, occupied clade B. The intraspecific genetic distance among the A. integrum species ranged from 0.00 to 13.34%. Between clades A and B, the genetic distance observed was 11.49%. The clade B isolates were genetically close to A. geoemydae (GD: 1.22%). Morphological variations between the clades included darker exoskeletal coloration in clade A and distinct differences in the shape of basis capitulum. Further analysis using Assemble Species by Automatic Partitioning (ASAP) and Generalized Mixed Yule Coalescent (GMYC) provided additional insights. Assemble Species by Automatic Partitioning (ASAP) identified 26 Molecular Operational Taxonomic Units (MOTUs) at a threshold distance of 5.38%, supporting the species partition of A. integrum clade B. Generalized Mixed Yule Coalescent (GMYC) analysis retained the same species complex (A. integrum-geoemydae Complex) inferred from the ASAP analyses. It could be inferred from the present study that the A. integrum clades A and B could be two different putative pseudocryptic species.

DNA barcoding of the genus Gampsocleis (Orthoptera, Tettigoniidae) from China.

DNA barcoding is a useful addition to the traditional morphology-based taxonomy. A ca. 650 bp fragment of the 5' end of mitochondrial cytochrome c oxidase subunit I (hereafter COI-5P) DNA barcoding was sued as a practical tool for Gampsocleis species identification. DNA barcodes from 889 specimens belonging to 8 putative Gampsocleis species was analyzed, including 687 newly generated DNA barcodes. These barcode sequences were clustered/grouped into Operational Taxonomic Units (OTUs) using the criteria of five algorithms, namely Barcode Index Number (BIN) System, Assemble Species by Automatic Partitioning (ASAP), a Java program uses an explicit, determinate algorithm to define Molecular Operational Taxonomic Unit (jMOTU), Generalized Mixed Yule Coalescent (GMYC), and Bayesian implementation of the Poisson Tree Processes model (bPTP). The Taxon ID Tree grouped sequences of morphospecies and almost all MOTUs in distinct nonoverlapping clusters. Both long- and short-winged Gampsocleis species are reciprocally monophyletic in the Taxon ID Tree. In BOLD, 889 barcode sequences are assigned to 17 BINs. The algorithms ASAP, jMOTU, bPTP and GMYC clustered the barcode sequences into 6, 13, 10, and 23 MOTUs, respectively. BIN, ASAP, and bPTP algorithm placed three long-winged species, G. sedakovii, G. sinensis and G. ussuriensis within the same MOTU. All species delimitation algorithms split two short-winged species,G. fletcheri and G. gratiosa into at least two MOTUs each, except for ASAP algorithm. More detailed molecular and morphological integrative studies are required to clarify the status of these MOTUs in the future.

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.

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.

Occurrence of five distinct clades of mermithid nematodes (Nematoda: Mermithidae) infecting black fly larvae (Diptera: Simuliidae) in tropical streams in Malaysia.

Mermithids are the most common parasites of black flies and are associated with host feminization and sterilization in infected hosts. However, information on the species / lineage of black fly mermithids in Southeast Asia, including Malaysia requires further elucidation. In this study, mermithids were obtained from black fly larvae collected from 138 freshwater stream sites across East and West Malaysia. A molecular approach based on nuclear-encoded 18S ribosomal RNA (18S rRNA) gene was used to identify the species identity / lineage of 77 nematodes successfully extracted and sequenced from the specimens collected. Maximum likelihood and neighbor-joining phylogenetic analyses demonstrated five distinct mermithid lineages. Four species delimitation analyses: automated simultaneous analysis phylogenetics (ASAP), maximum likelihood Poisson tree processes with Bayesian inferences (bPTP_ML), generalized mixed yule coalescent (GMYC) and single rate Poisson tree processes (PTP) were applied to delimit the species boundaries of mermithid lineages in this data set along with genetic distance analysis. Data analysis supports five distinct lineages or operational taxonomic units for mermithids in the present study, with two requiring further investigation as they may represent intraspecific variation or closely related taxa. One mermithid lineage was similar to that previously observed in Simulium nigrogilvum from Thailand. Co-infection with two mermithids of different lineages was observed in one larva of Simulium trangense. This study represents an important first step towards exploring other aspects of host - parasite interactions in black fly mermithids.

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.

A new species of the red alga Erythrotrichia (Erythropeltales, Rhodophyta) from Korea: Erythrotrichia johnawestii sp. nov. and observations in culture

Abstract Four species of Erythrotrichia (E. biseriata, E. reflexa, E. tetraseriata, E. carnea) have been reported in Korea, but phylogenetic studies have not been conducted on them. We analyzed two newly collected Erythrotrichia isolates using rbcL DNA sequences and determined their species status using four DNA-based species delimitation methods. One isolate (KNU-1) grouped with E. carnea in three methods: Assemble Species by Automatic Partitioning, both single-threshold and multiple-threshold general mixed yule coalescent, but grouped as a distinct species in the statistical parsimony network analysis. A second isolate (KNU-2) was considered a distinct species in all the above species-delimitation methods. We propose the name, Erythrotrichia johnawestii sp. nov., for this new species. The new species was different from E. carnea in lacking a thick outer cell wall layer and with a smaller cell size. Mature monosporangia produced raised openings that released monospores. Cell walls were not detected in newly released monospores but began to appear when the spore attached to the substratum and produced rhizoids. Sexual reproduction was not observed during the four-year culture period.

New Color-Patterned Species of Microtendipes Kieffer, 1913 (Diptera: Chironomidae) and a Deep Intraspecific Divergence of Species by DNA Barcodes.

The genus Microtendipes Kieffer (Diptera: Chironomidae) has a nearly worldwide distribution, comprising more than 60 species, which are further divided into two species groups based on larval stage. However, species delimitation and identification among the adults of this genus are controversial and uncertain. For instance, previous studies have provided many synonymies based on conspecific color pattern variations in Microtendipes species. Here, we used DNA barcode data to address Microtendipes species delimitation as well as to test whether color pattern variations can be diagnostic characters for interspecific identification. The 151 DNA barcodes used, 51 of which were contributed by our laboratory, represent 21 morphospecies. Species with specific color patterns could be accurately separated based on DNA barcodes. Consequently, the color patterns of adult males could be important diagnostic characters. The average intraspecific and interspecific sequence divergences were 2.8% and 12.5%, respectively, and several species exhibited deep intraspecific divergences higher than 5%. Molecular operational taxonomic units (OTUs) ranged from 21 to 73, based on methods including phylogenetic trees, the assemble species by automatic partitioning method, the Poisson tree process (PTP), and the general mixed Yule-coalescent (GMYC) method. As a result of these analyses, five new species were recognized (M. baishanzuensis sp. nov., M. bimaculatus sp. nov., M. nigrithorax sp. nov., M. robustus sp. nov., and M. wuyiensis sp. nov.).

A complete COI library of Samoan butterflies reveals layers of endemic diversity on oceanic islands

AbstractWe investigated the entire butterfly fauna of the Samoan Archipelago (Pacific Ocean) by combining COI barcode sequences for specimens from these islands with those available in repositories at larger biogeographic scale. Haplotype networks and a generalized mixed Yule‐coalescent (GMYC) model were applied to identify evolutionary significant units (ESUs). The ESUs from Samoan islands were compared with ESUs of the same or sister taxa regionally and worldwide to explore the level of endemicity and of congruence between established taxonomy and COI barcodes. The level of ESUs endemicity was similar to that shown by species and subspecies. Australia was the most frequent origin for Samoan lineages, followed by Orient‐Asia. When comparing the agreement and mismatch between established taxonomy and ESUs between the Australia‐Oceania region and Europe and North America, the COI molecular marker revealed a similar performance in taxonomic identification. Despite this overall convergent pattern, the degree of mtDNA divergence and the analysis of functional traits suggested that the mechanisms producing patterns of genetic differentiation in temperate butterflies over ancient continental lands differ to those occurring across a vast ocean into geologically young islands. Mechanisms on Samoan islands include relatively recent and exceptional oceanic dispersal, possibly followed by repeated extinction events. In the Australia‐Oceania region we found a similar fraction of species showing introgression with the maintenance of phenotypic differences as it occurs on the mainland, but the phenomenon was limited to sectors of each species distribution area. Regular gene flow among the Samoan islands seems to prevent allopatric speciation within the archipelago.

Khargia gen. nov., a new genus of simple trichal Cyanobacteria from the Persian Gulf

Information regarding the diversity of Cyanobacteria in many parts of the world is still minimal. One example of a region that has not yet been widely studied is south-western Asia, including the region of the Persian Gulf. A culture-dependent study of cyanobacterial diversity in a rainwater basin on Kharg Island enabled the isolation of a novel taxon, previously unnamed, from a simple trichal cyanobacterium. Further comparisons showed the existence of closely related strains/taxa from other parts of the world, namely the strains CCALA 945 isolated from South Italy and "<i>Leptolyngbya india" </i>from India. Herein, we have thus described the new genus <i>Khargia</i> and the new species <i>Khargia iranica. </i>Other strains, isolated by other authors, were included in the <i>Khargia</i> genus as additional species: <i>Khargia italica</i> and <i>Khargia indica</i>. The recognition of the new genus was based on morphological evaluations (identification by both light and electron microscopy), the phylogenetic analyses based on the 16S rRNA gene, and species delimitation based on Automatic Barcode Gap Definition (ABGD), Generalized Mixed Yule Coalescent (GMYC), bayesian version of Poisson Tree Processes (bPTP), and the secondary structure of 16S-23S rRNA ITS region of the studied strains.

DNA Barcoding of Fish Species Diversity in Guizhou, China

Guizhou is an important ecological barrier in the upper reaches of the Yangtze River and the Pearl River basins with abundant fish species. However, fish from these regions are threatened by anthropogenic activities, including overfishing and habitat destruction. Here, we assessed the fish diversity including more than half of the species from the region using DNA barcoding (partial sequence of cytochrome c oxidase subunit I (COI) gene). We obtained 800 mitochondrial COI barcode sequences from 82 genera, 18 families and 8 orders of fishes. The average Kimura two-parameter (K2P) distances within species and genera were 0.35% and 5.44%, respectively. The average interspecific distance was 15.54 times higher than the mean intraspecific distance. Moreover, DNA barcodes revealed 175 operational taxonomic units (OTUs) based on consensus demarcation schemes. Barcoding gaps were detected in 94.81% of morphospecies. Three fish species (Schistura fasciolata, Vanmanenia pingchowensis, and Misgurnus dabryanus) have considerable intraspecific variability, and each was divided into multiple molecular operational taxonomic units (MOTUs) using molecular definition methods (Automatic Barcode Gap Discovery, Refined Single Linkage, General Mixed Yule Coalescent, and Poisson Tree Processes), possibly indicating the occurrence of cryptic species. Altogether, our study reveals the complex diversity of fish species in Guizhou Province, serving as a reference for the conservation and monitoring of fish species in this region.

Assessing Species Boundaries in the Freshwater Snail Family Physidae Using Coalescent-Based Delimitation Methods

A common issue for several freshwater snail families is the fact that, for some genera, the criteria for species delimitation are scarce, subjected to intraspecific and ontogenetic variation, or insufficiently discrete to allow a clear distinction between species. For these genera, the use of molecular approaches is decisive in recognizing valid species. Herein, we applied coalescent methods to delimit evolutionary significant units (ESUs) in Physidae. We used 496 sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene available in GenBank, ascribed to six genera and 25 nominal species, and the algorithms GMYC (General Mixed Yule-Coalescent) with two criteria (single-threshold and multiple-threshold), PTP (Poisson Tree Process), and mPTP (multi-rate Tree Poisson Process). The method that presented the best performance was the mPTP. This method showed a higher frequency of taxonomic species recovery and has delimited a lower number of ESUs (25 ESUs from a dataset attributed to 25 nominal species). Physidae is a family of freshwater snails that presents several issues related to the recognition of species boundaries. Herein, the use of coalescent delimitation methods allowed us to address important taxonomic questions, opening new avenues for future taxonomic research. Our findings have shown that the morphological operational criteria used to delimit several valid species fall within the limits of intraspecific variability, demonstrating taxonomic inflation within this family. Our results also demonstrated the presence of cryptic diversity under the nominal species Physella acuta (Draparnaud, 1805), Beringophysa jennessi (Dall, 1919), Physella pomilia (Conrad, 1834), and Physella gyrina (Say, 1821).

Cucullanus pinnai pinnai and C. pinnai pterodorasi (Nematoda Cucullanidae): what does the integrative taxonomy tell us about these species and subspecies classification?

Cucullanus pinnai has been divided in two subspecies (C. pinnai pinnai and C. pinnai pterodorasi) based on the morphology of oesophastome. While C. pinnai pinnai apparently shows low host specificity and broad geographic occurrence, with certain morphological variations, C. pinnai pterodorasi was reported once, parasitizing Pterodoras granulosus. We used an integrative taxonomic approach to evaluate whether or not populations of C. pinnai pinnai from Trychomycterus spegazzinii (Escoipe River, Argentina) and Pimelodus fur (Miranda River, Brazil), and of C. pinnai pterodorasi from Pterodoras granulosus (Miranda River, Brazil) are conspecific. Parasites were observed using light microscopy and genetically characterized based on partial sequences of the 18S and 28S rDNA, ITS1-5.8S-ITS2, and COI mtDNA. Phylogenies were reconstructed and the Generalized Mixed Yule Coalescent (GMYC), Poisson Tree Process (bPTP), and Automatic Barcode Gap Discovery (ABGD) were used for species delimitation purposes. The present samples formed well-supported monophyletic assemblages, corroborating in part the results of morphological analyses; however, they grouped according to geographic origin. Species delimitation suggested conspecificity of C. pinnai pinnai with C. pinnai pterodorasi from Brazil; consequently, the morphology of oesophastome may be an intraspecific variation. Results also indicated that C. pinnai may represent a species complex as samples from Argentina were suggestive of an independent specific entity. However, definitive affirmations are premature, since there is no autapomorphy for separating C. pinnai from Brazil and Argentina and sampling was limited to three host species from two river basins. The phylogenetic reconstructions also confirmed the artificiality of some genera within Cucullanidae.

OTU Delimitation with Earthworm DNA Barcodes: A Comparison of Methods

Although DNA barcodes-based operational taxonomic units (OTUs) are increasingly used in earthworm research, the relative efficiency of the different methods available to delimit them has not yet been tested on a comprehensive dataset. For this study, we used three datasets containing 651, 2304 and 4773 COI barcodes of earthworms from French Guiana, respectively, to compare five of these methods: two phylogenetic methods—namely Poisson Tree Processes (PTP) and General Mixed Yule Coalescence (GMYC)—and three distance matrix methods—namely Refined Single Linkage (RESL, used for assigning Barcode Index Numbers in the Barcode of Life Data systems), Automatic Barcode Gap Discovery (ABGD), and Assemble Species by Automatic Partitioning (ASAP). We found that phylogenetic approaches are less suitable for delineating OTUs from DNA barcodes in earthworms, especially for large sets of sequences. The computation times are unreasonable, they often fail to converge, and they also show a strong tendency to oversplit species. Among distance-based methods, RESL also has a clear tendency to oversplitting, while ABGD and ASAP are less prone to mismatches and have short computation times. ASAP requires less a priori knowledge for model parameterisation than AGBD, provides efficient graphical outputs, and has a much lower tendency to generate mismatches.

DNA Barcoding and Species Delimitation for Dogfish Sharks Belonging to the Squalus Genus (Squaliformes: Squalidae)

The Squalus genus comprises a group of small demersal sharks occurring circumglobally, popularly known as dogfish sharks. This genus exhibits a conserved morphology, thus making correct morphological identification difficult. Considering these taxonomic problems and the scarcity of molecular data, the present study aimed to identify Squalus genus MOTUs, using DNA barcoding for species delimitation via ABGD (automatic barcode gap discovery), PTP (Poisson tree process), and GMYC (general mixed Yule coalescent) employing the mitochondrial COI gene. A total of 69 sequences were generated from samples obtained from the American coast in both the Atlantic and Pacific Oceans. The ABGD analysis was the most conservative among the three applied delimitations, indicating three taxonomic units, while the PTP analysis revealed nine MOTUs, with two conflicting units noted between S. clarkae + S. mitsukurii and S. albicaudus + S. cubensis. The GMYC analysis indicated an excessive division, with S. acanthias and S. mitsukurii subdivided into six MOTUs each and S. blainville, into four. These findings demonstrated that Squalus presents a complex of previously defined species, with misidentified samples deposited in databases leading to difficulties in analyzing the real distribution and diversity of species belonging to this genus. Thus, further efforts to highlight possible new species are recommended.

Coalescent-based species delimitation in North American pinyon pines using low-copy nuclear genes and plastomes.

PremiseAccurate species delimitation is essential for evolutionary biology, conservation, and biodiversity management. We studied species delimitation in North American pinyon pines, Pinus subsection Cembroides, a natural group with high levels of incomplete lineage sorting.MethodsWe used coalescent‐based methods and multivariate analyses of low‐copy number nuclear genes and nearly complete high‐copy number plastomes generated with the Hyb‐Seq method. The three coalescent‐based species delimitation methods evaluated were the Generalized Mixed Yule Coalescent (GMYC), Poisson Tree Process (PTP), and Trinomial Distribution of Triplets (Tr2). We also measured admixture in populations with possible introgression.ResultsOur results show inconsistencies among GMYC, PTP, and Tr2. The single‐locus based GMYC analysis of plastid DNA recovered a higher number of species (up to 24 entities, including singleton lineages and clusters) than PTP and the multi‐locus coalescent approach. The PTP analysis identified 10 species whereas Tr2 recovered 13, which agreed closely with taxonomic treatments.ConclusionsWe found that PTP and GMYC identified species with low levels of ILS and high morphological divergence (P. maximartinezii, P. pinceana, and P. rzedowskii). However, GMYC method oversplit species by identification of more divergent samples as singletons. Moreover, both PTP and GMYC were incapable of identifying some species that are readily identified morphologically. We suggest that the divergence times between lineages within North American pinyon pines are so disparate that GMYC results are unreliable. Results of the Tr2 method coincided well with previous delimitations based on morphology, DNA, geography, and secondary chemistry.