Mixed Yule Coalescent Model Research Articles

Violins we see, species we don't… Species delimitation of the spider genus Loxosceles Heineken & Lowe (Araneae: Sicariidae) from North America using morphological and molecular evidence.

In modern systematics, different sources of evidence are commonly used for the discovery, identification, and delimitation of species, especially when morphology fails to delineate between species or in underestimated species complexes or cryptic species. In this study, morphological data and two DNA barcoding markers-cytochrome c oxidase subunit I (COI) and internal transcribed spacer 2 (ITS2)-were used to delimit species in the spider genus Loxosceles from North America. The molecular species delimitation analyses were carried out using three different methods under the corrected p-distance Neighbor-Joining (NJ) criteria: 1) Assemble Species by Automatic Partitioning (ASAP), 2) General Mixed Yule Coalescent model (GMYC), and 3) Bayesian Poisson Tree Processes (bPTP). The analyses incorporated 192 terminals corresponding to 43 putative species of Loxosceles, of which 15 are newly recognized herein, as putative new species, based on morphology and congruence between molecular methods with COI. The average intraspecific genetic distance (p-distance) was <2%, whereas the average interspecific genetic distance was 15.6%. The GMYC and bPTP molecular methods recovered 65-79 and 69 species respectively, overestimating the diversity in comparison with morphology, whereas the ASAP method delimited 60 species. The morphology of primary sexual structures (males palps and female seminal receptacles) was congruent with most of the molecular methods mainly with COI, showing that they are robust characters for identification at the species level. For species delimitation COI was more informative than ITS2. The diversity of Loxosceles species is still underestimated for North America, particularly in Mexico which holds the highest diversity of this genus worldwide.

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.

To be or not to be… Integrative taxonomy and species delimitation in the daddy long-legs spiders of the genus Physocyclus (Araneae, Pholcidae) using DNA barcoding and morphology.

Integrative taxonomy is crucial for discovery, recognition, and species delimitation, especially in underestimated species complex or cryptic species, by incorporating different sources of evidence to construct rigorous species hypotheses. The spider genus Physocyclus Simon, 1893 (Pholcidae, Arteminae) is composed of 37 species, mainly from North America. In this study, traditional morphology was compared with three DNA barcoding markers regarding their utility in species delimitation within the genus: 1) Cytochrome c Oxidase subunit 1 (CO1), 2) Internal Transcribed Spacer 2 (ITS2), and 3) Ribosomal large subunit (28S). The molecular species delimitation analyses were carried out using four methods under the corrected p-distances Neighbor-Joining (NJ) criteria: 1) Automatic Barcode Gap Discovery (ABGD), 2) Assemble Species by Automatic Partitioning (ASAP), 3) General Mixed Yule Coalescent model (GMYC), and 4) Bayesian Poisson Tree Processes (bPTP). The analyses incorporated 75 terminals from 22 putative species of Physocyclus. The average intraspecific genetic distance (p-distance) was found to be < 2%, whereas the average interspecific genetic distance was 20.6%. The ABGD, ASAP, and GMYC methods were the most congruent, delimiting 26 or 27 species, while the bPTP method delimited 33 species. The use of traditional morphology for species delimitation was congruent with most molecular methods, with the male palp, male chelicerae, and female genitalia shown to be robust characters that support species-level identification. The barcoding with CO1 and 28S had better resolution for species delimitation in comparison with ITS2. The concatenated matrix and traditional morphology were found to be more robust and informative for species delimitation within Physocyclus.

Diversity of interstitial nemerteans of the genus Ototyphlonemertes (Nemertea: Monostilifera: Ototyphlonemertidae) in the South China Sea, with a comment on the distribution pattern of the genus

The genus Ototyphlonemertes Diesing, 1863, consisting of 33 named species and numerous unnamed morphospecies/molecular entities, is a unique group of nemerteans that possess cerebral statocysts and specifically live in coarse-grained sands. Only eight named species of this genus have yet been recorded from the Indo-Polynesian biogeographic province, which harbors the highest marine biodiversity in the world. In recent years, Ototyphlonemertes were collected from eight sites along the South China Sea coasts. Nine species/entities were revealed by four phylogenetic markers (COI, 16S, 18S, 28S) analyzed by three species delimitation methods: Automatic Barcode Gap Discovery (ABGD), Poisson Tree Process (PTP), and Generalized Mixed Yule Coalescent model (GMYC). Six entities are described as new species based on integration of morphological and molecular species delimitations: Ototyphlonemertes conicobasis sp. nov., Ototyphlonemertes coralli sp. nov., Ototyphlonemertes similis sp. nov., Ototyphlonemertes sinica sp. nov., Ototyphlonemertes subrubra sp. nov., and Ototyphlonemertes yingge sp. nov. No morphological differences were detected between two entities and Ototyphlonemertes chernyshevi Kajihara et al., 2018, despite large genetic differences, so are treated as candidate species. Ototyphlonemertes ani Chernyshev, 2007 is first recorded in China. Based mostly on results of phylogenetic analyses, two previously established subgenera are re-defined, and a new subgenus, Procso subgen. nov., is established. Through reviewing the existing studies, we recognize 101 species/entities of Ototyphlonemertes, which are distributed in 18 marine biogeographic provinces. Most (88.1%) of them are endemic to a single biogeographic province, and evolutionary lineages endemic to a geographic area are not uncommon. Maximum diversity has been recorded in the Indo-Polynesian Province (22 species), though sampling to date has covered only a small part of the biogeographic province.

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

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

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

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

DNA Barcoding of Penaeidae (Decapoda; Crustacea): Non-Distance-Based Species Delimitation of the Most Economically Important Shrimp Family

The Penaeidae family includes some of the most economic and ecological important marine shrimp, comprising hundreds of species. Despite this importance and diversity, the taxonomic classification for penaeid shrimp has constantly been revised, and issues related to the species identification are common. In this study, we implemented DNA barcoding analyses in addition to single-gene species delimitation analyses in order to identify molecular operational taxonomy units (MOTUs) and to generate robust molecular information for penaeid shrimp based on the cytochrome oxidase subunit I (COI) mitochondrial gene. Our final data set includes COI sequences from 112 taxa distributed in 23 genera of penaeids. We employed the general mixed Yule coalescent (GMYC) model, the Poisson tree processes (PTP), and the Bayesian PTP model (bPTP) for MOTUs delimitation. Intraspecific and interspecific genetic distances were also calculated. Our findings evidenced a high level of hidden diversity, showing 143 MOTUs, with 27 nominal species not agreeing with the genetic delimitation obtained here. These data represent potential new species or highly structured populations, showing the importance of including a non-distance-based species delimitation approach in biodiversity studies. The results raised by this study shed light on the Penaeidae biodiversity, addressing important issues about taxonomy and mislabeling in databases and contributing to a better comprehension of the group, which can certainly help management policies for shrimp fishery activity in addition to conservation programs.

Species Delimitation of Southeast Pacific Angel Sharks (Squatina spp.) Reveals Hidden Diversity through DNA Barcoding

Angel sharks are distributed worldwide in tropical to subtropical waters. Across the Eastern Pacific Ocean (EPO), two valid species are reported: The Pacific angelshark Squatina californica and the Chilean angelshark Squatina armata; however, there is still uncertainty about their geographic distribution, mainly along the northern Peru coast where the species have been reported to be sympatric. The aim of this study is to describe the genetic differences between the genus Squatina from the EPO, including samples from northern Peru, and using DNA barcoding and three species delimitation models: Poisson tree processes (PTP) model, Bayesian implementation of the PTP (bPTP) model and the general mixed Yule coalescent (GMYC) model. The three approaches summarized 19 nominal Squatina species in 23 consensus Molecular Taxonomic Units (MOTU). Only 16 of them were in accordance with taxonomic identifications. From the EPO, four Squatina MOTUs were identified, one from North America (S. californica USA/Mexico) and three sampled in northern Peru, S. californica Peru, S. armata and Squatina sp. (a potential new species). This study contributes to the management and conservation policies of angel sharks in Peru, suggesting the presence of an undescribed species inhabiting the northern Peruvian coast. The use of molecular approaches, such as DNA barcoding, has the potential to quickly flag undescribed species in poorly studied regions, including the Southeast Pacific, within groups of ecologically and economically important groups like angel sharks.

Use of species delimitation approaches to tackle the cryptic diversity of an assemblage of high Andean butterflies (Lepidoptera: Papilionoidea).

Cryptic biological diversity has generated ambiguity in taxonomic and evolutionary studies. Single-locus methods and other approaches for species delimitation are useful for addressing this challenge, enabling the practical processing of large numbers of samples for identification and inventory purposes. This study analyzed an assemblage of high Andean butterflies using DNA barcoding and compared the identifications based on the current morphological taxonomy with three methods of species delimitation (automatic barcode gap discovery, generalized mixed Yule coalescent model, and Poisson tree processes). Sixteen potential cryptic species were recognized using these three methods, representing a net richness increase of 11.3% in the assemblage. A well-studied taxon of the genus Vanessa, which has a wide geographical distribution, appeared with the potential cryptic species that had a higher genetic differentiation at the local level than at the continental level. The analyses were useful for identifying the potential cryptic species in Pedaliodes and Forsterinaria complexes, which also show differentiation along altitudinal and latitudinal gradients. This genetic assessment of an entire assemblage of high Andean butterflies (Papilionoidea) provides baseline information for future research in a region characterized by high rates of endemism and population isolation.

P2C2M.GMYC: An R package for assessing the utility of the Generalized Mixed Yule Coalescent model

Abstract In clades that contain large numbers of undescribed species, DNA barcoding methods can serve as a first pass at identifying species limits. The General Mixed Yule Coalescent (GMYC) approach to species delimitation is accurate under certain conditions that are difficult to verify in the very clades for which the GMYC holds the greatest appeal. To circumvent this challenge, we have developed an R package for assessing how well the statistical model implemented in the GMYC fits empirical data. Our approach uses either a parametric bootstrap or a posterior predictive simulation to evaluate model fit. Computational requirements are modest for our package, and most analyses can be completed within minutes to an hour on a typical laptop, depending on whether a user selects a maximum likelihood or Bayesian framework. Results of simulation testing indicate that our approach is effective for assessing the utility of the GMYC model and suggest that it should be included in the analytical pipeline whenever researchers apply the GMYC to clades with unknown species boundaries.

Species delimitation in the cyanolichen genus Rostania

BackgroundIn this study, we investigate species limits in the cyanobacterial lichen genus Rostania (Collemataceae, Peltigerales, Lecanoromycetes). Four molecular markers (mtSSU rDNA, β-tubulin, MCM7, RPB2) were sequenced and analysed with two coalescent-based species delimitation methods: the Generalized Mixed Yule Coalescent model (GMYC) and a Bayesian species delimitation method (BPP) using a multispecies coalescence model (MSC), the latter with or without an a priori defined guide tree.ResultsSpecies delimitation analyses indicate the presence of eight strongly supported candidate species. Conclusive correlation between morphological/ecological characters and genetic delimitation could be found for six of these. Of the two additional candidate species, one is represented by a single sterile specimen and the other currently lacks morphological or ecological supporting evidence.ConclusionsWe conclude that Rostania includes a minimum of six species: R. ceranisca, R. multipunctata, R. occultata 1, R. occultata 2, R. occultata 3, and R. occultata 4,5,6. Three distinct Nostoc morphotypes occur in Rostania, and there is substantial correlation between these morphotypes and Rostania thallus morphology.

Phylogeny and species delimitation of the genus Longgenacris and Fruhstorferiola viridifemorata species group (Orthoptera: Acrididae: Melanoplinae) based on molecular evidence.

Phylogenetic positions of the genus Longgenacris and one of its members, i.e. L. rufiantennus are controversial. The species boundaries within both of L. rufiantennus+Fruhstorferiola tonkinensis and F. viridifemorata species groups are unclear. In this study, we explored the phylogenetic positions of the genus Longgenacris and the species L. rufiantennus and the relationships among F. viridifemorata group based on the 658-base fragment of the mitochondrial gene cytochrome c oxidase subunit I (COI) barcode and the complete sequences of the internal transcribed spacer regions (ITS1 and ITS2) of the nuclear ribosomal DNA. The phylogenies were reconstructed in maximum likelihood framework using IQ-TREE. K2P distances were used to assess the overlap range between intraspecific variation and interspecific divergence. Phylogenetic species concept and NJ tree, K2P distance, the statistical parsimony network as well as the generalized mixed Yule coalescent model (GMYC) were employed to delimitate the species boundaries in L. rufiantennus+F. tonkinensis and F. viridifemorata species groups. The results demonstrated that the genus Longgenacris should be placed in the subfamily Melanoplinae but not Catantopinae, and L. rufiantennus should be a member of the genus Fruhstorferiola but not Longgenacris. Species boundary delimitation confirmed the presence of oversplitting in L. rufiantennus+F. tonkinensis and F. viridifemorata species groups and suggested that each group should be treated as a single species.

Hidden diversity in Prochilodus nigricans: A new genetic lineage within the Tapajós River basin.

Highly spread through the Amazon River basin, Prochilodus nigricans have had its taxonomic validity recently questioned, when genetic differences between Western and Eastern Amazon populations from the Brazilian shield were detected. This area has been seeing as a region of high ichthyofaunal diversity and endemism, in which the hybrid origin of the Tapajós River basin has been raised. In this paper, we report a new molecular lineage within P. nigricans of Tapajós River, highlighting this region still hides taxonomically significant diversity. Haplotype networks were reconstructed using the mitochondrial COI and ATP6/8 markers, which were also used to calculate genetic distances among clusters. We additionally conducted a delimiting species approach by employing a Generalized Mixed Yule-Coalescent model (GMYC) with COI sequences produced here, and previous ones published for individuals sampled across the Amazon River basin. In addition to the genetic differentiation within P. nigricans, our findings favor the hypothesis of hybrid origin of the Tapajós River basin and reaffirm the importance of studies aiming to investigate hidden diversity to address taxonomic and biogeographic issues, that certainly benefit better biodiversity conservation actions.

Description of a new species of Loxosceles Heineken &amp; Lowe (Araneae, Sicariidae) recluse spiders from Hidalgo, Mexico, under integrative taxonomy: morphological and DNA barcoding data (CO1 + ITS2)

Based on an integrative taxonomic approach, a new species of the genus Loxosceles Heineken &amp; Lowe, 1832, is described from the state of Hidalgo, Mexico. Loxosceles tolantongo sp. nov. is described based on DNA barcoding using cytochrome c oxidase subunit 1 (CO1) and internal transcribed spacer 2 (ITS2), and morphology. For species delimitation, four molecular methods were implemented: 1) corrected p-distances under neighbor joining (NJ); 2) automatic barcode gap discovery (ABGD); 3) general mixed yule coalescent model (GMYC) and 4) Bayesian Poisson tree processes (bPTP). The new species morphologically resembles L. jaca, another species from Hidalgo, but there are morphological differences mainly in the tibiae of the male palp, the seminal receptacles of the females and also the high genetic p-distances. CO1 was more informative than ITS2 for the genetic separation; however, both concatenated genes (CO1 + ITS2) present robust evidence for species delimitation. Loxosceles tolantongo sp. nov. is considered a unique species for four reasons: 1) it can be diagnosed and distinguished by morphological characters (of the male palps mainly, but also of the seminal receptacles of the females); 2) the genetic p-distances with CO1 were high (&gt;10%); 3) the molecular species delimitation methods were congruent under CO1 and CO1 + ITS2; and 4) under CO1 and CO1 + ITS2, the new species is a putative sister group of L. jaca + L. tenango.

COI mtDNA barcoding and morphology for species delimitation in the spider genus Ixchela Huber (Araneae: Pholcidae), with the description of two new species from Mexico.

The spider genus Ixchela Huber 2000, is comprised of 22 species distributed from north-eastern Mexico to Central America, including the two new species described herein from Mexico: Ixchela panchovillai sp. nov. and Ixchela zapatai sp. nov., both from the state of Oaxaca and described for both sexes. DNA barcoding utilizing mitochondrial cytochrome c oxidase subunit 1 (COI) and morphology were used for species delimitation. Molecular analyses and species delimitation included four methods: 1) corrected p-distances under neighbor-joining (NJ), 2) general mixed yule coalescent model (GMYC), 3) automatic barcode gap discovery (ABGD), and 4) Poisson tree processes (bPTP). All molecular methods and morphology were consistent in delimiting and recognizing the two new species describing herein. The average inter-specific genetic distance (p-distance) within the genus Ixchela is 12%. Ixchela panchovillai sp. nov. is closely related in the NJ analysis with I. placida, with an average p-distance of 7.9%, whereas I. zapatai sp. nov. is closely related to I. taxco, with an average p-distance of 8.4% between both species. Additionally, identification keys for males and females of the genus Ixchela are presented.

A molecular assessment of species diversity in Tympanopleura and Ageneiosus catfishes (Auchenipteridae: Siluriformes).

In order to test the congruence of genetic data to the morphologically defined Neotropical catfish genera Tympanopleura and Ageneiosus and explore species diversity, we generated 17 DNA barcodes from five of six species of Tympanopleura and 12 of 13 species of Ageneiosus. To discriminate limits between species, an automatic barcode gap discovery (ABGD), a generalised mixed yule-coalescent model (GYMC) and fixed distance thresholds Kimura two-parameter (K2P; 3%) were used to discriminate putative species limits from the DNA barcodes. The ABGD, GMYC and K2P methods agreed by each generating 13 clusters: six in Tympanopleura (five nominal plus one undescribed species) and seven in Ageneiosus. These clusters corresponded broadly to the described species, except in the case of the Ageneiosus ucayalensis group (A. akamai, A. dentatus, A. intrusus, A. ucayalensis, A. uranophthalmus and A. vittatus). Haplotype sharing and low divergences may have prevented molecular methods from distinguishing these species. We hypothesise that this is the result of a recent radiation of a sympatric species group distributed throughout the Amazon Basin. One putative new species of Tympanopleura was also supported by the molecular data. These results taken together highlight the utility of molecular methods such as DNA barcoding in understanding patterns of diversification across large geographic areas and in recognising overlooked diversity.

Under an integrative taxonomic approach: the description of a new species of the genus Loxosceles (Araneae, Sicariidae) from Mexico City

A new species of the spider genus Loxosceles Heineken & Lowe, 1832, Loxosceles tenochtitlan Valdez-Mondragón & Navarro-Rodríguez, sp. nov., is described based on adult male and female specimens from the states of Mexico City, Estado de Mexico and Tlaxcala. Integrative taxonomy including traditional morphology, geometric and lineal morphology, and molecules (DNA barcodes of cytochrome c oxidase subunit 1 (CO1) and internal transcribed spacer 2 (ITS2)), were used as evidence to delimit the new species. Four methods were used for molecular analyses and species delimitation: 1) corrected p-distances under neighbor joining (NJ), 2) automatic barcode gap discovery (ABGD), 3) general mixed yule coalescent model (GMYC), and 4) poisson tree processes (bPTP). All molecular methods, traditional, geometric and lineal morphology were consistent in delimiting and recognizing the new species. Loxosceles tenochtitlansp. nov. is closely related to L. misteca based on molecular data. Although both species are morphologically similar, the average p-distance from CO1 data was 13.8% and 4.2% for ITS2 data. The molecular species delimitation methods recovered well-supported monophyletic clusters for samples of L. tenochtitlansp. nov. from Mexico City + Tlaxcala and for samples of L. misteca from Guerrero. Loxosceles tenochtitlansp. nov. is considered a unique species for three reasons: (1) it can be distinguished by morphological characters (genitalic and somatic); (2) the four different molecular species delimitation methods were congruent to separate both species; and (3) there is variation in leg I length of males between both species, with the males of L. misteca having longer legs than males of L. tenochtitlansp. nov., also morphometrically, the shape of tibiae of the palp between males of both species is different.

Historical museum collections clarify the evolutionary history of cryptic species radiation in the world's largest amphibians.

Inaccurate taxonomic assessment of threatened populations can hinder conservation prioritization and management, with human‐mediated population movements obscuring biogeographic patterns and confounding reconstructions of evolutionary history. Giant salamanders were formerly distributed widely across China, and are interpreted as a single species, Andrias davidianus. Previous phylogenetic studies have identified distinct Chinese giant salamander lineages but were unable to associate these consistently with different landscapes, probably because population structure has been modified by human‐mediated translocations for recent commercial farming. We investigated the evolutionary history and relationships of allopatric Chinese giant salamander populations with Next‐Generation Sequencing methods, using historical museum specimens and late 20th‐century samples, and retrieved partial or near‐complete mitogenomes for 17 individuals. Samples from populations unlikely to have been affected by translocations form three clades from separate regions of China, spatially congruent with isolation by either major river drainages or mountain ranges. Pliocene–Pleistocene divergences for these clades are consistent with topographic modification of southern China associated with uplift of the Qinghai‐Tibet Plateau. General Mixed Yule Coalescent model analysis indicates that these clades represent separate species: Andrias davidianus (Blanchard, 1871) (northern Yangtze/Sichuan), Andrias sligoi (Boulenger, 1924) (Pearl/Nanling), and an undescribed species (Huangshan). Andrias sligoi is possibly the world's largest amphibian. Inclusion of additional reportedly wild samples from areas of known giant salamander exploitation and movement leads to increasing loss of biogeographic signal. Wild Chinese giant salamander populations are now critically depleted or extirpated, and conservation actions should be updated to recognize the existence of multiple species.

Hidden species diversity in Pachyhynobius: A multiple approaches species delimitation with mitogenomes

The lack of distinct morphological features of cryptic species is a hard problem for taxonomy, especially when the taxa are closely related with considerable amounts of ancestral polymorphism. Lately, intensive coalescent-based analyses involving multiple loci have become the preferred method to assess the extent of genetic distinctiveness in otherwise phenotypically similar populations. Previously, phylogenetic studies on Pachyhynobius shangchengensis uncovered five extremely deeply divergent clades, which suggested that this species may be a cryptic species complex. In this study, we used the complete mitochondrial genome data and samples from the entire range of stout salamander (Pachyhynobius), as well as publicly available mitochondrial genomes to assess species boundaries within this genus using a suite of diverse methodologies (e.g. general mixed Yule coalescent model, Automatic Barcode Gap Discovery). The phylogenetic relationships recovered two major groups within P. shangchengensis, with one group formed by four of the six extant populations and corresponding to the central and eastern range of the Dabie mountains, while the other group encompassed two other lineages in the north west of the Dabie mountain range. The species delimitation comparison within Pachyhynobius supported the presence of recognized species within the genus, and consensus was observed across methods for the existence of up to five cryptic species within what has been traditionally considered to be P. shangchengensis. While this implies the existence of four taxa in addition to the described P. shangchengensis species, morphological data and life history information are further required to contribute to the species definition. The observed pattern of genetic variation is likely the outcome of a discontinuous habitat combined with niche conservatism, which produced the sky-island effect observed in Pachyhynobius, and which led to formation of a hidden species diversity in this genus.

COI-based species delimitation in Indochinese Tetraserica chafers reveal hybridisation despite strong divergence in male copulation organs

Species of Tetraserica chafers (Coleoptera: Scarabaeidae: Sericini) can easily be distinguished by the distinct shape of strongly divergent male genitalia; however, it was so far unclear if such divergent copulation organs may avoid hybridisation. Here, we analysed an 826 bp fragment of the mitochondrial cytochrome c oxidase subunit 1 (COI) for a total of 113 Tetraserica individuals out of 38 morphospecies from 33 localities in Southeast Asia. DNA-based species delimitation was performed using statistical parsimony network analysis (TCS), Poisson tree processes (PTP) model, Automatic Barcode Gap Discovery (ABGD) and the generalised mixed Yule-coalescent (GMYC) model. The different approaches for DNA-based species delimitation revealed different numbers of molecular operational taxonomic units (MOTUs) and showed low congruence with a priori assigned morphospecies. GMYC and PTP generally tended to lump morphospecies while ABGD and TCS more often split morphospecies. TCS revealed the highest congruence between MOTUs and morphospecies. While incongruence of species hypotheses was discussed in the context of incomplete lineage sorting and introgression, we could identify two unexpected cases in which introgression occurred between species with highly dissimilar morphology of the male genitalia and that resulted in lumping with DNA-based species delimitation. Most other cases of not matching morphospecies and MOTUs represent young speciation in which standard markers (CO1) do not yet fit the morphological signal of divergence, as reflected by male copulation organs.