General Mixed Yule-coalescent Analysis Research Articles

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.

Species delimitation of the northeastern Anatolian Symphytum (Boraginaceae) taxa

Symphytum is regarded one of the most complicated genera in terms of the classification of its members among the Boraginaceae. In addition to different infrageneric classification methods, several species complex or aggregates have been proposed to deal with the taxonomical problem of genus members. Symphytum asperum aggregate was first introduced by Kurtto, who proposed six taxa within this aggregate. However, according to further studies by different researchers based on morphological data, total number of species of the complex was variable. The number of species was reduced to three, comprising S. asperum, S. savvalense, and S. sylvaticum, after the phylogenetic and morphological studies of Tarıkahya-Hacıoğlu and Erik. However, the taxonomical status of some of these species (i.e., S. savvalense and S. sylvaticum, and S. sepulcrale), which was assigned as a member of this complex by Kurtto, has been regarded as unresolved. To solve this uncertainty, different species delimitation methods were used, including statistical parsimony network analysis (TCS), generalized mixed Yule coalescent (GMYC), and Bayesian Phylogenetics and Phylogeography (BPP) of the ITS, trnL-F and trnS-G sequence data. In addition to members of this complex, S. ibericum, which is phylogenetically nested within the S. asperum aggregate, was also used. The TCS and GMYC analyses demonstrated more complicated clusters, whereas high posterior probabilities of BPP clusters were more compatible with the morphological data. In accordance with the morphological approach of Tarıkahya-Hacıoğlu and Erik, the species delimitation analyses based on molecular data support the recognition of S. asperum, S. ibericum, S. savvalense, and S. sylvaticum as different species.

Species delimitation of Margattea cockroaches from China, with seven new species (Blattodea, Ectobiidae, Pseudophyllodromiinae).

Nearly 450 Margattea specimens were collected from 27 locations in China and their morphology was examined. Then 68 Margattea COI sequences were obtained and used to carry out phylogenetic analyses as well as species delimitation analyses using General Mixed Yule Coalescent (GMYC), Automatic Barcode Gap Discovery (ABGD), and Poisson Tree Processes (bPTP). GMYC analysis resulted in 21 molecular operational taxonomic units (MOTUs) (confidence interval: 20–22), which was completely consistent with the result of the bPTP. There were 15 MOTUs using the ABGD method. The number of MOTUs was slightly different from the assigned morphospecies (16). As to the incongruence between molecular and morphological results, we checked the specimens again and made sure that most morphological differences were determined to be intraspecific differences (except the difference between M. angusta and M. mckittrickae), although a large genetic distance existed. Finally, 16 Margattea species from China were defined in this study, of which, seven new species are established, i.e. Margattea deltodonta J-J He & Z-Q Wang, sp. nov., Margattea cuspidata J-J He & Z-Q Wang, sp. nov., Margattea caudata J-J He & Z-Q Wang, sp. nov., Margattea paratransversa J-J He & Z-Q Wang, sp. nov., Margattea disparilis J-J He & Z-Q Wang, sp. nov., Margattea transversa J-J He & Z-Q Wang, sp. nov., and Margattea bicruris J-J He & Z-Q Wang, sp. nov.

DNA barcoding elucidates the population genetic diversity of venomous cobra species (Reptilia: Elapidae) in Indo-Bangladesh region

The DNA barcode data of venomous cobra species (Naja naja and Naja kaouthia) are limited in the global database, especially from India and Bangladesh. Owing to the rapid success of DNA barcoding for discriminating a variety of species around the world, the present study aimed to generate the mitochondrial cytochrome c oxidase subunit I (COI) gene information of two morphologically identified deadly elapid species from the Mizoram state in northeast India and Rangpur in northern Bangladesh. The multiple species delimitation methods: Automatic Barcode Gap Discovery (ABGD), General Mixed Yule-Coalescent (GMYC), and Poisson-Tree-Processes (bPTP) revealed 14, 16, and 18 molecular operational taxonomic units (MOTUs) for 12 Naja species. The binocellate cobra, N. naja showed monophyletic clustering in both maximum-likelihood (ML) and Bayesian (BA) trees, single MOTU in ABGD and GMYC, and negligible intra-species genetic distance (0.2%) with two haplotypes. However, the monocellate cobra, N. kaouthia showed paraphyletic and polytomy in ML and BA phylogenies respectively; more than one MOTUs in ABGD, GMYC, and bPTP analyses; and sufficient intra-species genetic distances (0.6–2.3%) with five haplotypes related to the diverse geographical locations in Bangladesh, China, India, and Thailand. By superimposing the executed species delimitation criteria, the present molecular-based investigation concludes the presence of cryptic diversity of N. kaouthia in Indo-Bangladesh, China, and Thailand.

Morphometrical and molecular evidence suggests cryptic diversity among hookworms (Nematoda: Uncinaria) that parasitize pinnipeds from the south-eastern Pacific coasts

Hookworms of the genus Uncinaria parasitize pinniped pups in various locations worldwide. Four species have been described, two of which parasitize pinniped pups in the southern hemisphere: Uncinaria hamiltoni parasitizes Otaria flavescens and Arctocephalus australis from the South American coast, and Uncinaria sanguinis parasitizes Neophoca cinerea from the Australian coast. However, their geographical ranges and host specificity are unknown. Uncinaria spp. are morphologically similar, but molecular analyses have allowed the recognition of new species in the genus Uncinaria. We used nuclear genetic markers (internal transcribed spacer (ITS) and large subunit (LSU) rDNA) and a mitochondrial genetic marker (cytochrome c oxidase subunit I (COI)) to evaluate the phylogenetic relationships of Uncinaria spp. parasitizing A. australis and O. flavescens from South American coasts (Atlantic and Pacific coasts). We compared our sequences with published Uncinaria sequences. A Generalized Mixed Yule Coalescent (GMYC) analysis was also used to delimit species, and principal component analysis was used to compare morphometry among Uncinaria specimens. Parasites were sampled from A. australis from Peru (12°S), southern Chile (42°S), and the Uruguayan coast, and from O. flavescens from northern Chile (24°S) and the Uruguayan coast. Morphometric differences were observed between Uncinaria specimens from both South American coasts and between Uncinaria specimens from A. australis in Peru and southern Chile. Phylogenetic and GMYC analyses suggest that south-eastern Pacific otariid species harbour U. hamiltoni and an undescribed putative species of Uncinaria. However, more samples from A. australis and O. flavescens are necessary to understand the phylogenetic patterns of Uncinaria spp. across the South Pacific.

Ancient landscapes of the Namib Desert harbor high levels of genetic variability and deeply divergent lineages for Collembola.

AimTo assess spatial patterns of genetic and species‐level diversity for Namib Desert Collembola using mitochondrial DNA cytochrome c oxidase subunit I (COI) gene sequences.LocationNamib Desert gravel plains.TaxonCollembola (springtails).MethodsA total of 77 soil samples were collected along NE‐SW (60 km) and E‐W (160 km) transects from within a 4,000 km2 area of the Namib Desert gravel plains. We extracted 434 springtails from the 37 samples which contained Collembola and sequenced them at the COI gene locus. In the absence of specific taxonomic keys and previous genetic data for these taxa, we used Generalized Mixed Yule Coalescent (GMYC) analyses to provide putative species‐level designations.ResultsWe obtained 341 successful COI sequences, 175 of which were unique haplotypes. GMYC analyses identified 30 putative species, with up to 28% sequence divergence (uncorrected p‐distance). The distribution of genetic variants was disjunct, with 97% of haplotypes and 70% of “GMYC species” found only at single sites.Main conclusionsDispersal events, although rare, may be facilitated by environmental events such as prevailing onshore winds or occasional flow of rainwater to the coast. We conclude that the high genetic diversity we observed is the result of ancient springtail lineages, patchy distribution of suitable habitats, and limited dispersal (gene flow) among habitable locations.

High-efficiency 18S microalgae barcoding by coalescent, distance and character-based approaches: a test in Chlorella and Scenedesmus

The relatively conserved 18S is often used in the phylogenetic analysis of microalgae. However, whether it can really help in barcoding microalgae needs to be evaluated. In this study the multiple approaches of coalescent, distance and character-based barcoding are first employed in Chlorella and Scenedesmus to test the efficiency of 18S sequences for barcoding green microalgae. We show that most Chlorella and Scenedesmus species, including the cryptic species, can be distinguished by 18S sequences with all coalescent General Mixed Yule-coalescent (GMYC), poisson tree process (PTP), and P ID, distance (ABGD) and character-based approaches. Both GMYC and PTP analyses produce more genetic groups. The P ID and ABGD analyses only cluster some species. All species (apart from a few of lineages) can be separated in character-based barcoding analysis with more than three character attributes. In comparison with previous barcoding results with rbcL, tufA, ITS and 16S, 18S produces good resolution in identifying Chlorella and Scenedesmus. Our results reveal that 18S is highly efficient in identifying taxa of green microalgae at species level, based on a combination of multiple barcoding approaches. Combining 18S with other gene markers may be useful in barcoding microalgae.

Multi-approach analysis of the diversity in Colletotrichum cliviae sensu lato.

Colletotrichum cliviae is a fungal species reported both as pathogen and endophyte with broad geographical distribution. Some purported isolates of this species have been assigned to different taxa, including Colletotrichum aracearum, Colletotrichum orchidearum and Colletotrichum. sichuanensis, for which a preliminary analysis of extensive multilocus (ACT, GAPDH, ITS, TUB2) data in this study revealed high sequence similarity with C. cliviae. We further reassessed the species delineation by using the coalescent method of the generalized mixed Yule-coalescent (GMYC) and Poisson Tree Processes (PTP). Single and multilocus gene trees strongly supported a C. cliviae s. lat. clade including the four species. This clade unfolded eight subclades grouped into three distinct lineages, but no monophyly of any of the four species. GMYC and PTP analyses confidently supported the evolutionary independence of these lineages. C. sichuanensis and C. cliviae, except one isolate, formed the largest lineage. The second lineage was made up of isolates named C. aracearum and some of C. orchidearum sharing the haplotype and the third lineage accommodated two isolates named C. cliviae and C. orchidearum. This finding suggests the synonymization of C. sichuanensis with C. cliviae whereas the taxonomic status of C. aracearum and C. orchidearum still needs clarification. This study lays great stress upon the use of comprehensive data for sequence-based characterisation of species in the C. cliviae s. lat. It also presents the first report of C. cliviae in tropical Africa and on citrus host.

Impact of tree priors in species delimitation and phylogenetics of the genus Oligoryzomys (Rodentia: Cricetidae)

The use of genetic data and tree-based algorithms to delimit evolutionary lineages is becoming an important practice in taxonomic identification, especially in morphologically cryptic groups. The effects of different phylogenetic and/or coalescent models in the analyses of species delimitation, however, are not clear. In this paper, we assess the impact of different evolutionary priors in phylogenetic estimation, species delimitation, and molecular dating of the genus Oligoryzomys (Mammalia: Rodentia), a group with complex taxonomy and morphological cryptic species. Phylogenetic and coalescent analyses included 20 of the 24 recognized species of the genus, comprising of 416 Cytochrome b sequences, 26 Cytochrome c oxidase I sequences, and 27 Beta-Fibrinogen Intron 7 sequences. For species delimitation, we employed the General Mixed Yule Coalescent (GMYC) and Bayesian Poisson tree processes (bPTP) analyses, and contrasted 4 genealogical and phylogenetic models: Pure-birth (Yule), Constant Population Size Coalescent, Multiple Species Coalescent, and a mixed Yule-Coalescent model. GMYC analyses of trees from different genealogical models resulted in similar species delimitation and phylogenetic relationships, with incongruence restricted to areas of poor nodal support. bPTP results, however, significantly differed from GMYC for 5 taxa. Oligoryzomys early diversification was estimated to have occurred in the Early Pleistocene, between 0.7 and 2.6 MYA. The mixed Yule-Coalescent model, however, recovered younger dating estimates for Oligoryzomys diversification, and for the threshold for the speciation-coalescent horizon in GMYC. Eight of the 20 included Oligoryzomys species were identified as having two or more independent evolutionary units, indicating that current taxonomy of Oligoryzomys is still unsettled.

Species Delimitation and Phylogenetic Relationships in Ectobiid Cockroaches (Dictyoptera, Blattodea) from China.

We collected Ectobiidae cockroach specimens from 44 locations in the south of the Yangtze valley. We obtained 297 COI sequences specimens and carried out phylogenetic and divergence dating analyses, as well as species delimitation analysis using a General Mixed Yule Coalescent (GMYC) framework. The intraspecific and interspecific sequence divergence in Ectobiidae cockroaches ranged from 0.0 to 7.0% and 4.6 to 30.8%, respectively. GMYC analysis resulted in 53 (confidence interval: 37–65) entities (likelihood ratio = 103.63) including 14 downloaded species. The COI GMYC groups partly corresponded to the ectobiid species and 52 ectobiid species were delimited successfully based on the combination of GMYC result with morphological information. We used the molecular data and 6 cockroach fossil calibrations to obtain a preliminary estimate of the timescale of ectobiid evolution. The major subfamilies in the group were found to have diverged between ~125–110 Ma, and morphospecies pairs were found to have diverged ~10 or more Ma.

Phylogenetic relationships of freshwater fishes of the genus Capoeta (Actinopterygii, Cyprinidae) in Iran.

The Middle East contains a great diversity of Capoeta species, but their taxonomy remains poorly described. We used mitochondrial history to examine diversity of the algae‐scraping cyprinid Capoeta in Iran, applying the species‐delimiting approaches General Mixed Yule‐Coalescent (GMYC) and Poisson Tree Process (PTP) as well as haplotype network analyses. Using the BEAST program, we also examined temporal divergence patterns of Capoeta. The monophyly of the genus and the existence of three previously described main clades (Mesopotamian, Anatolian‐Iranian, and Aralo‐Caspian) were confirmed. However, the phylogeny proposed novel taxonomic findings within Capoeta. Results of GMYC, bPTP, and phylogenetic analyses were similar and suggested that species diversity in Iran is currently underestimated. At least four candidate species, Capoeta sp4, Capoeta sp5, Capoeta sp6, and Capoeta sp7, are awaiting description. Capoeta capoeta comprises a species complex with distinct genetic lineages. The divergence times of the three main Capoeta clades are estimated to have occurred around 15.6–12.4 Mya, consistent with a Mio‐Pleistocene origin of the diversity of Capoeta in Iran. The changes in Caspian Sea levels associated with climate fluctuations and geomorphological events such as the uplift of the Zagros and Alborz Mountains may account for the complex speciation patterns in Capoeta in Iran.

Inferring phylogeny and speciation of Gymnosporangium species, and their coevolution with host plants.

Gymnosporangium species (Pucciniaceae, Pucciniales) cause serious diseases and significant economic losses to apple cultivars. Most of the reported species are heteroecious and complete their life cycles on two different plant hosts belonging to two unrelated genera, i.e. Juniperus and Malus. However, the phylogenetic relationships among Gymnosporangium species and the evolutionary history of Gymnosporangium on its aecial and telial hosts were still undetermined. In this study, we recognized species based on rDNA sequence data by using coalescent method of generalized mixed Yule-coalescent (GMYC) and Poisson Tree Processes (PTP) models. The evolutionary relationships of Gymnosporangium species and their hosts were investigated by comparing the cophylogenetic analyses of Gymnosporangium species with Malus species and Juniperus species, respectively. The concordant results of GMYC and PTP analyses recognized 14 species including 12 known species and two undescribed species. In addition, host alternations of 10 Gymnosporangium species were uncovered by linking the derived sequences between their aecial and telial stages. This study revealed the evolutionary process of Gymnosporangium species, and clarified that the aecial hosts played more important roles than telial hosts in the speciation of Gymnosporangium species. Host switch, losses, duplication and failure to divergence all contributed to the speciation of Gymnosporangium species.

Testing the potential of DNA barcoding in vertebrate radiations: the case of the littoral cichlids (Pisces, Perciformes, Cichlidae) from Lake Tanganyika

We obtained 398 cytochrome c oxidase subunit I barcodes of 96 morphospecies of Lake Tanganyika (LT) cichlids from the littoral zone. The potential of DNA barcoding in these fishes was tested using both species identification and species delineation methods. The best match (BM) and best close match (BCM) methods were used to evaluate the overall identification success. For this, three libraries were analysed in which the specimens were categorized into Operational Taxonomic Units (OTU) in three alternative ways: (A) morphologically distinct, including undescribed, species, (B) valid species and (C) complexes of morphologically similar or closely related species. For libraries A, B and C, 73, 73 and 96% (BM) and 72, 70 and 94% (BCM) of the specimens were correctly identified. Additionally, the potential of two species delineation methods was tested. The General Mixed Yule Coalescent (GMYC) analysis suggested 70 hypothetical species, while the Automatic Barcode Gap Discovery (ABGD) method revealed 115 putative species. Although the ABGD method had a tendency to oversplit, it outperformed the GMYC analysis in retrieving the species. In most cases where ABGD suggested oversplitting, this was due to intraspecific geographical variation. The failure of the GMYC method to retrieve many species could be attributed to discrepancies between mitochondrial gene trees and the evolutionary histories of LT cichlid species. Littoral LT cichlids have complex evolutionary histories that include instances of hybridization, introgression and rapid speciation. Nevertheless, although the utility of DNA barcoding in identification is restricted to the level of complexes, it has potential for species discovery in cichlid radiations.

DNA sequencing reveals unexpected Recent diversity and an ancient dichotomy in the American marsupial genusMarmosops(Didelphidae: Thylamyini)

To assess species-level diversity in the didelphid marsupial genus Marmosops, we obtained sequence data from the mitochondrial cytochrome b (CYTB) gene from > 200 specimens, including exemplars of every currently recognized species together with multiple specimens of all geographically widespread forms. Analyses of these data using the general mixed Yule coalescent (GMYC) model suggest that the genus could be twice as speciose as currently recognized, but putative species identified by the GMYC criterion require careful evaluation using other data. To assess phylogenetic relationships within Marmosops, we additionally sequenced a large fragment of the breast cancer activating 1 (BRCA1) gene from one specimen each of the putative species identified by the GMYC analyses of CYTB. Maximum likelihood and Bayesian analyses of a concatenated gene (CYTB + BRCA1) matrix revealed a basal dichotomy between two ancient, morphologically diagnosable clades with apparently distinct distributions and adaptive phenotypes. We describe those clades as subgenera and assign 12 nominal taxa to Sciophanes subgen. nov. (with type species Marmosops parvidens) and 27 nominal taxa to the nominotypical subgenus (with type species Marmosops incanus).

Molecular phylogenetics and biogeography of the Neotropical skink genus Mabuya Fitzinger (Squamata: Scincidae) with emphasis on Colombian populations

Understanding the phylogenetic and geographical history of Neotropical lineages requires having adequate geographic and taxonomic sampling across the region. However, Colombia has remained a geographical gap in many studies of Neotropical diversity. Here we present a study of Neotropical skinks of the genus Mabuya, reptiles that are difficult to identify or delimit due to their conservative morphology. The goal of the present study is to propose phylogenetic and biogeographic hypotheses of Mabuya including samples from the previously under-studied territory of Colombia, and address relevant biogeographic and taxonomic issues. We combined molecular and morphological data sampled densely by us within Colombia with published data representing broad sampling across the Neotropical realm, including DNA sequence data from two mitochondrial (12S rRNA and cytochrome b) and three nuclear genes (Rag2, NGFB and R35).To evaluate species boundaries we employed a general mixed Yule-coalescent (GMYC) model applied to the mitochondrial data set. Our results suggest that the diversity of Mabuya within Colombia is higher than previously recognized, and includes lineages from Central America and from eastern and southern South America. The genus appears to have originated in eastern South America in the Early Miocene, with subsequent expansions into Central America and the Caribbean in the Late Miocene, including at least six oceanic dispersal events to Caribbean Islands.We identified at least four new candidate species for Colombia and two species that were not previously reported in Colombia. The populations of northeastern Colombia can be assigned to M. zuliae, while specimens from Orinoquia and the eastern foothills of the Cordillera Oriental of Colombia correspond to M. altamazonica. The validity of seven species of Mabuya sensu lato was not supported due to a combination of three factors: (1) non-monophyly, (2) <75% likelihood bootstrap support and <0.95 Bayesian posterior probability, and (3) GMYC analysis collapsing named species. Finally, we suggest that Mabuya sensu stricto may be regarded as a diverse monophyletic genus, widely distributed throughout the Neotropics.

Phylogeny and species delimitations in European Dicranum (Dicranaceae, Bryophyta) inferred from nuclear and plastid DNA

DNA sequences have been widely used for taxonomy, inferring phylogenetic relationships and identifying species boundaries. Several specific methods to define species delimitations based on molecular phylogenies have appeared recently, with the generalized mixed Yule coalescent (GMYC) method being most popular. However, only few studies on land plants have been published so far and GMYC analyses of bryophytes are missing. Dicranum is a large genus of mosses whose (morpho-)species are partly ill-defined and frequently confused. To infer molecular species delimitations, we reconstructed phylogenetic trees based on five chloroplast markers and nuclear ribosomal ITS sequences from 27 out of 30 species occurring in Europe. We applied the species delimitation methods GMYC and Poisson tree processes (PTP) in order to compare their discriminatory power with species boundaries inferred from the molecular phylogenetic reconstructions and with the morphological species concept. Phylogenetic circumscriptions were congruent with the morphological concept for 19 species, while eight species were molecularly not well delimited, mostly forming closely related species pairs. The automated species delimitation methods achieved similar results but tended to overestimate the number of potential species and exposed several incongruences between the morphological concept and inference from molecular phylogenetic reconstructions. It is concluded that GMYC and PTP methods potentially provide a useful and objective way of delimiting bryophyte species, but studies on further bryophyte data sets are necessary to infer whether incongruences might ensue from evolutionary processes and to test the suitability of these approaches.

DNA barcoding of common soft scales (Hemiptera: Coccoidea: Coccidae) in China.

The soft scales (Hemiptera: Coccoidea: Coccidae) are a group of sap-sucking plant parasites, many of which are notorious agricultural pests. The quarantine and economic importance of soft scales necessitates rapid and reliable identification of these taxa. Nucleotide sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene (barcoding region) and 28S rDNA were generated from 340 individuals of 36 common soft scales in China. Distance-based [(best match, Automated Barcode Gap Discovery (ABGD)], tree-based (neighbor-joining, Bayesian inference), Klee diagrams, and general mixed Yule coalescent (GMYC) models were used to evaluate barcoding success rates in the data set. Best match showed that COI and 28S sequences could provide 100 and 95.52% correct identification, respectively. The average interspecific divergences were 19.81% for COI data and 20.38% for 28S data, and mean intraspecific divergences were 0.56 and 0.07%, respectively. For COI data, multiple methods (ABGD, Klee, and tree-based methods) resulted in general congruence with morphological identifications. However, GMYC analysis tended to provide more molecular operational taxonomic units (MOTUs). Twelve MOTUs derived from five morphospecies (Rhodococcus sariuoni, Pulvinaria vitis, Pulvinaria aurantii, Parasaissetia nigra, and Ceroplastes rubens) were observed using the GMYC approach. In addition, tree-based methods showed that 28S sequences could be used for species-level identification (except for Ceroplastes ceriferus - Ceroplastes pseudoceriferus), even with low genetic variation (<1%). This report demonstrates the robustness of DNA barcoding for species discrimination of soft scales with two molecular markers (COI and 28S) and provides a reliable barcode library and rapid diagnostic tool for common soft scales in China.

Factors affecting species delimitations with the GMYC model: insights from a butterfly survey

Summary The generalized mixed Yule‐coalescent (GMYC) model has become one of the most popular approaches for species delimitation based on single‐locus data, and it is widely used in biodiversity assessments and phylogenetic community ecology. We here examine an array of factors affecting GMYC resolution (tree reconstruction method, taxon sampling coverage/taxon richness and geographic sampling intensity/geographic scale). We test GMYC performance based on empirical data (DNA barcoding of the Romanian butterflies) on a solid taxonomic framework (i.e. all species are thought to be described and can be determined with independent sources of evidence). The data set is comprehensive (176 species), and intensely and homogeneously sampled (1303 samples representing the main populations of butterflies in this country). Taxonomy was assessed based on morphology, including linear and geometric morphometry when needed. The number of GMYC entities obtained constantly exceeds the total number of morphospecies in the data set. We show that c. 80% of the species studied are recognized as entities by GMYC. Interestingly, we show that this percentage is practically the maximum that a single‐threshold method can provide for this data set. Thus, the c. 20% of failures are attributable to intrinsic properties of the COI polymorphism: overlap in inter‐ and intraspecific divergences and non‐monophyly of the species likely because of introgression or lack of independent lineage sorting. Our results demonstrate that this method is remarkably stable under a wide array of circumstances, including most phylogenetic reconstruction methods, high singleton presence (up to 95%), taxon richness (above five species) and the presence of gaps in intraspecific sampling coverage (removal of intermediate haplotypes). Hence, the method is useful to designate an optimal divergence threshold in an objective manner and to pinpoint potential cryptic species that are worth being studied in detail. However, the existence of a substantial percentage of species wrongly delimited indicates that GMYC cannot be used as sufficient evidence for evaluating the specific status of particular cases without additional data. Finally, we provide a set of guidelines to maximize efficiency in GMYC analyses and discuss the range of studies that can take advantage of the method.

A new species of &lt;i&gt;Margotrema&lt;/i&gt; (Digenea, Allocreadiidae) from the leopard splitfin &lt;i&gt;Xenotaenia resolanae&lt;/i&gt; (Cyprinodontiformes, Goodeidae) from west-central Mexico

A new species of Margotrema is herein described from the intestine of the freshwater fish Xenotaenia resolanae from the Cuzalapa River, Jalisco State, on the Pacific slope of west-central Mexico. The new species was first recognized after a molecular phylogenetic analysis which explored the genetic variation at the intraspecific level of Margotrema spp across central Mexico. Sequences of mitochondrial (COI) and nuclear (ITS1) genes were obtained for 125 individuals, and a general mixed Yule-coalescent analysis (GMYC) for species delimitation of both genes allowed the recognition of an independent evolutionary lineage, representing an undescribed species of Margotrema. Detailed morphological observations allowed the species description. The new species is easily distinguished from the other valid species of the genus, M. bravoae, by having vitelline follicles restricted to the region between the pharynx and the ventral sucker a wider and more prominent ventral sucker, larger eggs, and symmetrical testes.

Extensive cryptic species diversity and fine-scale endemism in the marine red alga Portieria in the Philippines

We investigated species diversity and distribution patterns of the marine red alga Portieria in the Philippine archipelago. Species boundaries were tested based on mitochondrial, plastid and nuclear encoded loci, using a general mixed Yule-coalescent (GMYC) model-based approach and a bayesian multilocus species delimitation method. The outcome of the GMYC analysis of the mitochondrial encoded cox2-3 dataset was highly congruent with the multilocus analysis. In stark contrast with the current morphology-based assumption that the genus includes a single, widely distributed species in the Indo-West Pacific (Portieria hornemannii), DNA-based species delimitation resulted in the recognition of 21 species within the Philippines. Species distributions were found to be highly structured with most species restricted to island groups within the archipelago. These extremely narrow species ranges and high levels of intra-archipelagic endemism contrast with the wide-held belief that marine organisms generally have large geographical ranges and that endemism is at most restricted to the archipelagic level. Our results indicate that speciation in the marine environment may occur at spatial scales smaller than 100 km, comparable with some terrestrial systems. Our finding of fine-scale endemism has important consequences for marine conservation and management.