Disintegrating over space and time: Paraphyly and species delimitation in the Wehrle's Salamander complex

Accurate species delimitation is critical for biodiversity studies. However, species complexes characterized by introgression, high levels of population structure and subtle phenotypic differentiation can be challenging to delimit. Here, we report on a molecular systematic investigation of the woodland salamanders Plethodon wehrlei and Plethodon punctatus, which traditionally have been placed in the Plethodon wehrlei species group. To quantify patterns of genetic variation, we collected genetic samples from throughout the range of both species, including 22 individuals from nine populations of P. punctatus, and 60 individuals from 26 populations of P. wehrlei. From these samples, we sequenced three mtDNA loci (5596 base pairs) and five nuclear loci (3377 base pairs). We inferred time‐calibrated gene trees and species trees using BEAST 2.4.6, and we delimited putative species using a Bayesian implementation of the general mixed Yule‐coalescent model (bGMYC) and STRUCTURE. Finally, we validated putative species using the multispecies coalescent as implemented in Bayesian Phylogenetics and Phylogeography (BPP). We found substantial phylogeographic diversity in P. wehrlei, including multiple geographically cohesive clades and an inferred mitochondrial common ancestor at 11.5 myr (95% HPD: 9.6–13.6 myr) that separated populations formerly assigned to P. dixi from all other populations. We also found that P. punctatus is deeply nested within P. wehrlei, rendering the latter paraphyletic. After discussing the challenges faced by modern species delimitation methods, we recommend retaining P. punctatus because it is ecologically and phenotypically distinct. We further recommend that P. dixi be recognized as a valid species.     

Integrative species delimitation reveals cryptic diversity in the southern Appalachian Antrodiaetus unicolor (Araneae: Antrodiaetidae) species complex

Although species delimitation can be highly contentious, the development of reliable methods to accurately ascertain species boundaries is an imperative step in cataloguing and describing Earth's quickly disappearing biodiversity. Spider species delimitation remains largely based on morphological characters; however, many mygalomorph spider populations are morphologically indistinguishable from each other yet have considerable molecular divergence. The focus of our study, the Antrodiaetus unicolor species complex containing two sympatric species, exhibits this pattern of relative morphological stasis with considerable genetic divergence across its distribution. A past study using two molecular markers, COI and 28S, revealed that A. unicolor is paraphyletic with respect to A. microunicolor. To better investigate species boundaries in the complex, we implement the cohesion species concept and use multiple lines of evidence for testing genetic exchangeability and ecological interchangeability. Our integrative approach includes extensively sampling homologous loci across the genome using a RADseq approach (3RAD), assessing population structure across their geographic range using multiple genetic clustering analyses that include structure , principal components analysis and a recently developed unsupervised machine learning approach (Variational Autoencoder). We evaluate ecological similarity by using large‐scale ecological data for niche‐based distribution modelling. Based on our analyses, we conclude that this complex has at least one additional species as well as confirm species delimitations based on previous less comprehensive approaches. Our study demonstrates the efficacy of genomic‐scale data for recognizing cryptic species, suggesting that species delimitation with one data type, whether one mitochondrial gene or morphology, may underestimate true species diversity in morphologically homogenous taxa with low vagility.     

Assessing the diversity and distribution of Cephalothrix species (Nemertea: Palaeonemertea) in European waters by comparing different species delimitation methods

Soft‐bodied marine taxa, like ribbon worms (Nemertea), often lack clear diagnostic morphological characters impeding traditional species delimitation. Therefore, recent studies concentrated on molecular genetic methods to solve taxonomic issues. Different delimitation methods were employed to explore species boundaries and the presence of cryptic species. However, the performance of the different delimitation methods needs to be tested. A particularly promising nemertean genus in this regard is the palaeonemertean genus Cephalothrix that is commonly found in European waters. In order to gain information on the number and distribution of European cephalotrichids and to test different tree‐based and non‐tree‐based delimitation methods, we analyzed a dataset comprising the barcoding region of the mitochondrial cytochrome c oxidase subunit I (COI) of 215 European Cephalothrix specimens, of which 78 were collected for this study. Our results show the presence of 12–13 European lineages of which several can be assigned to known European species. Analyzing a second dataset comprising 74 additional sequences from the Pacific and the Atlantic Oceans helped identify some of the unassigned European specimens. One resulting clade seems to represent a non‐native introduced Cephalothrix species, while another has never been recorded from Europe before. In our analysis, especially the tree‐based methods and the phylogenetic analysis proved to be a useful tool when delimiting species. It remains unclear whether the different identified clades result from cryptic speciation or from a high genetic variability of the COI gene.     

The influence of locus number and information content on species delimitation: an empirical test case in an endangered Mexican salamander

Perhaps the most important recent advance in species delimitation has been the development of model‐based approaches to objectively diagnose species diversity from genetic data. Additionally, the growing accessibility of next‐generation sequence data sets provides powerful insights into genome‐wide patterns of divergence during speciation. However, applying complex models to large data sets is time‐consuming and computationally costly, requiring careful consideration of the influence of both individual and population sampling, as well as the number and informativeness of loci on species delimitation conclusions. Here, we investigated how locus number and information content affect species delimitation results for an endangered Mexican salamander species, Ambystoma ordinarium. We compared results for an eight‐locus, 137‐individual data set and an 89‐locus, seven‐individual data set. For both data sets, we used species discovery methods to define delimitation models and species validation methods to rigorously test these hypotheses. We also used integrated demographic model selection tools to choose among delimitation models, while accounting for gene flow. Our results indicate that while cryptic lineages may be delimited with relatively few loci, sampling larger numbers of loci may be required to ensure that enough informative loci are available to accurately identify and validate shallow‐scale divergences. These analyses highlight the importance of striking a balance between dense sampling of loci and individuals, particularly in shallowly diverged lineages. They also suggest the presence of a currently unrecognized, endangered species in the western part of A. ordinarium's range.     

Multilocus phylogeny and Bayesian estimates of species boundaries reveal hidden evolutionary relationships and cryptic diversity in Southeast Asian monitor lizards

Recent conceptual, technological and methodological advances in phylogenetics have enabled increasingly robust statistical species delimitation in studies of biodiversity. As the variety of evidence purporting species diversity has increased, so too have the kinds of tools and inferential power of methods for delimiting species. Here, we showcase an organismal system for a data‐rich, comparative molecular approach to evaluating strategies of species delimitation among monitor lizards of the genus Varanus. The water monitors (Varanus salvator Complex), a widespread group distributed throughout Southeast Asia and southern India, have been the subject of numerous taxonomic treatments, which have drawn recent attention due to the possibility of undocumented species diversity. To date, studies of this group have relied on purportedly diagnostic morphological characters, with no attention given to the genetic underpinnings of species diversity. Using a 5‐gene data set, we estimated phylogeny and used multilocus genetic networks, analysis of population structure and a Bayesian coalescent approach to infer species boundaries. Our results contradict previous systematic hypotheses, reveal surprising relationships between island and mainland lineages and uncover novel, cryptic evolutionary lineages (i.e. new putative species). Our study contributes to a growing body of literature suggesting that, used in concert with other sources of data (e.g. morphology, ecology, biogeography), multilocus genetic data can be highly informative to systematists and biodiversity specialists when attempting to estimate species diversity and identify conservation priorities. We recommend holding in abeyance taxonomic decisions until multiple, converging lines of evidence are available to best inform taxonomists, evolutionary biologists and conservationists.     

Discordance between morphological and molecular species boundaries among Caribbean species of the reef sponge Callyspongia

Sponges are among the most species‐rich and ecologically important taxa on coral reefs, yet documenting their diversity is difficult due to the simplicity and plasticity of their morphological characters. Genetic attempts to identify species are hampered by the slow rate of mitochondrial sequence evolution characteristic of sponges and some other basal metazoans. Here we determine species boundaries of the Caribbean coral reef sponge genus Callyspongia using a multilocus, model‐based approach. Based on sequence data from one mitochondrial (COI), one ribosomal (28S), and two single‐copy nuclear protein‐coding genes, we found evolutionarily distinct lineages were not concordant with current species designations in Callyspongia. While C. fallax, C. tenerrima, and C. plicifera were reciprocally monophyletic, four taxa with different morphologies (C. armigera, C. longissima, C. eschrichtii, and C. vaginalis) formed a monophyletic group and genetic distances among these taxa overlapped distances within them. A model‐based method of species delimitation supported collapsing these four into a single evolutionary lineage. Variation in spicule size among these four taxa was partitioned geographically, not by current species designations, indicating that in Callyspongia, these key taxonomic characters are poor indicators of genetic differentiation. Taken together, our results suggest a complex relationship between morphology and species boundaries in sponges.     

Long forsaken species diversity in the Middle American lizard Holcosus undulatus (Teiidae)

Numerous reptile species have been divided into subspecies. Although this classification may capture the morphological variation within species, it often conceals significant species diversity because many subspecies actually represent species under lineage‐based species concepts. The lizard Holcosus undulatus is a common, widely distributed, monotypic species in Middle America. However, 12 subspecies of this taxon were recognized until the early 1970s. We used two lineage‐based methods for species delimitation to re‐evaluate species limits within H. undulatus with DNA sequence and morphological data. We included all the previously recognized subspecies of H. undulatus except H. u. miadis. Holcosus undulatus was exclusive. In addition, H. u. amphigrammus, H. u. gaigeae, H. u. hartwegi, H. u. parvus, H. u. pulcher, H. u. sinister, H. u. stuarti, H. u. thomasi and H. u. undulatus were supported as distinct evolutionary lineages based on the molecular and morphological evidence. We therefore elevate all of these subspecies to species rank. In addition, two separate mitochondrial lineages may represent cryptic, undescribed species within H. undulatus. The morphological distinctness and allopatry of H. u. miadis and H. u. pulcher, as well as the high genetic divergence of the latter species, suggest that they also represent distinct evolutionary species. Our results also suggest that additional species diversity may still be hidden within the H. u. amphigrammus, H. u. parvus, H. u. sinister and H. u. undulatus lineages. This work supports resurrection of overlooked diversity within Holcosus, which has important implications for the conservation of this genus in Middle America. © 2015 The Linnean Society of London     

Cryptic diversity and discordance in single‐locus species delimitation methods within horned lizards (Phrynosomatidae: Phrynosoma)

Biodiversity reduction and loss continues to progress at an alarming rate, and thus, there is widespread interest in utilizing rapid and efficient methods for quantifying and delimiting taxonomic diversity. Single‐locus species delimitation methods have become popular, in part due to the adoption of the DNA barcoding paradigm. These techniques can be broadly classified into tree‐based and distance‐based methods depending on whether species are delimited based on a constructed genealogy. Although the relative performance of these methods has been tested repeatedly with simulations, additional studies are needed to assess congruence with empirical data. We compiled a large data set of mitochondrial ND4 sequences from horned lizards (Phrynosoma) to elucidate congruence using four tree‐based (single‐threshold GMYC, multiple‐threshold GMYC, bPTP, mPTP) and one distance‐based (ABGD) species delimitation models. We were particularly interested in cases with highly uneven sampling and/or large differences in intraspecific diversity. Results showed a high degree of discordance among methods, with multiple‐threshold GMYC and bPTP suggesting an unrealistically high number of species (29 and 26 species within the P. douglasii complex alone). The single‐threshold GMYC model was the most conservative, likely a result of difficulty in locating the inflection point in the genealogies. mPTP and ABGD appeared to be the most stable across sampling regimes and suggested the presence of additional cryptic species that warrant further investigation. These results suggest that the mPTP model may be preferable in empirical data sets with highly uneven sampling or large differences in effective population sizes of species.     

Exploring species boundaries with multiple genetic loci using empirical data from non‐biting midges

Over the past decade, molecular approaches to species delimitation have seen rapid development. However, species delimitation based on a single locus, for example, DNA barcodes, can lead to inaccurate results in cases of recent speciation and incomplete lineage sorting. Here, we compare the performance of Automatic Barcode Gap Discovery (ABGD), Bayesian Poisson tree processes (PTP), networks, generalized mixed Yule coalescent (GMYC) and Bayesian phylogenetics and phylogeography (BPP) models to delineate cryptic species previously detected by DNA barcodes within Tanytarsus (Diptera: Chironomidae) non‐biting midges. We compare the results from analyses of one mitochondrial (cytochrome c oxidase subunit I [COI]) and three nuclear (alanyl‐tRNA synthetase 1 [AATS1], carbamoyl phosphate synthetase 1 [CAD1] and 6‐phosphogluconate dehydrogenase [PGD]) protein‐coding genes. Our results show that species delimitation based on multiple nuclear DNA markers is largely concordant with morphological variation and delimitations using a single locus, for example, the COI barcode. However, ABGD, GMYC, PTP and network models led to conflicting results based on a single locus and delineate species differently than morphology. Results from BPP analyses on multiple loci correspond best with current morphological species concept. In total, 10 lineages of the Tanytarsus curticornis species complex were uncovered. Excluding a Norwegian population of Tanytarsus brundini which might have undergone recent hybridization, this suggests six hitherto unrecognized species new to science. Five distinct species are well supported in the Tanytarsus heusdensis species complex, including two species new to science.     

A polyphasic approach to the delimitation of diatom species: a case study for the genus Pinnularia (Bacillariophyta)

Diatoms are one of the most abundant and arguably the most species‐rich group of protists. Diatom species delimitation has often been based exclusively on the recognition of morphological discontinuities without investigation of other lines of evidence. Even though DNA sequences and reproductive experiments have revealed several examples of (pseudo)cryptic diversity, our understanding of diatom species boundaries and diversity remains limited. The cosmopolitan pennate raphid diatom genus Pinnularia represents one of the most taxon‐rich diatom genera. In this study, we focused on the delimitation of species in one of the major clades of the genus, the Pinnularia subgibba group, based on 105 strains from a worldwide origin. We compared genetic distances between the sequences of seven molecular markers and selected the most variable pair, the mitochondrial cox1 and nuclear encoded LSU rDNA, to formulate a primary hypothesis on the species limits using three single‐locus automated species delimitation methods. We compared the DNA‐based primary hypotheses with morphology and with other available lines of evidence. The results indicate that our data set comprised 15 species of the P. subgibba group. The vast majority of these taxa have an uncertain taxonomic identity, suggesting that several may be unknown to science and/or members of (pseudo)cryptic species complexes within the P. subgibba group.     

Considering gene flow when using coalescent methods to delimit lineages of North American pitvipers of the genus Agkistrodon

Examining species diversity and mechanisms of speciation using coalescent models provides a framework for how regional diversity is accrued, even in well‐studied areas such as the Nearctic. It is likely, that gene flow among closely‐related species with adjacent distributions may be common. However, the absence of gene flow is a primary assumption of many phylogeographical methods that produce species trees and delimit species using Bayesian or likelihood functions in a coalescent framework. In the present study, we examine delimitation when gene flow between species is present using empirical datasets from two species of North American pitvipers of the genus Agkistrodon. We also use niche modelling to determine whether these young lineages occur in distinct environmental niches. To manage the problem of gene flow between species, we first identify admixed individuals, demonstrate that gene flow has occurred, and then identify the impact of alternative population assignments of admixed individuals on delimitation posterior probabilities. In addition, we examine the influence of mitochondrial genes relative to other loci combined in coalescent analyses that delimit species. Here, we find that the copperheads (Agkistrodon contortrix) and the cottonmouths (Agkistrodon piscivorus) are each composed of two distinct species, with each occupying different niches. Importantly, we find that species can be delimited when the amount of gene flow between lineages is low, although the methods are acutely sensitive to population assignment of individuals. © 2014 The Linnean Society of London     

Phylogeny and species delimitation of the C‐genome diploid species in Oryza

Abstract The diploid Oryza species with C‐genome type possesses abundant genes useful for rice improvement and provides parental donors of many tetraploid species with the C‐genome (BBCC, CCDD). Despite extensive studies, the phylogenetic relationship among the C‐genome species and the taxonomic status of some taxa remain controversial. In this study, we reconstructed the phylogeny of three diploid species with C‐genome (Oryza officinalis, O. rhizomatis, and O. eichingeri) based on sequences of 68 nuclear single‐copy genes. We obtained a fully resolved phylogenetic tree, clearly indicating the sister relationship of O. officinalis and O. rhizomatis, with O. eichingeri being the more divergent lineage. Incongruent phylogenies of the C‐genome species found in previous studies might result from lineage sorting, introgression/hybridization and limited number of genetic markers used. We further applied a recently developed Bayesian species delimitation method to investigate the species status of the Sri Lankan and African O. eichingeri. Analyses of two datasets (68 genes with a single sample, and 10 genes with multiple samples) support the distinct species status of the Sri Lankan and African O. eichingeri. In addition, we evaluated the impact of the number of sampled individuals and loci on species delimitation. Our simulation suggests that sampling multiple individuals is critically important for species delimitation, particularly for closely related species.     

Cryptic species in the Neotropical fish genus Curimatopsis (Teleostei,Characiformes)

Detritivores of the fish family Curimatidae are assigned to eight genera, one of which, the Curimatopsis, with only five species, is the least speciose genus and sister to other seven genera in the family. Ongoing morphological investigations reveal, however, the likely existence of additional species. In this study, fifty‐one specimens of Curimatopsis from multiple rivers of the Amazon, Paraguay and Suriname drainages were identified morphologically according to the present species concepts and then barcoded using the universal cytochrome c oxidase subunit I (COI) mitochondrial marker. Species delimitation analyses were conducted using Bayesian methods through the general mixed Yule‐coalescent analysis combined with conventional likelihood, genetic distance and haplotypic diversity approaches. We found eleven well‐supported clusters that represent four of the named species and seven cryptic, undescribed species of Curimatopsis. Our results show a clear delimitation of species boundaries constrained by distinct Amazonian river ecotones that may have promoted intrageneric lineage diversification. This is the first of a series of genetic studies applicable to future taxonomic, phylogenetic and evolutionary studies across the Curimatidae.     

Multilocus species delimitation in Mesoamerican Scaptotrigona stingless bees (Apidae: Meliponini) supports the existence of cryptic species

Accelerating taxonomic knowledge and making accurate species identifications are critically important given the current biodiversity crisis, particularly in biodiversity hotspots such as Mesoamerica. Objective species delimitation that reduces investigator‐driven bias is fundamental to the establishment of appropriate conservation strategies, above all in managed species. Previous morphological and molecular studies on three managed stingless bee species of the genus Scaptotrigona distributed in Mexico (S. mexicana, S. pectoralis and S. hellwegeri) suggested that both S. mexicana and S. hellwegeri are cryptic species complexes. Herein we tested species delimitation by analysing sequence information of five markers (two mitochondrial: cox1 and 16S, and three nuclear: ITS1, EF1‐α, ArgK) within a Bayesian coalescent framework to test the putative species. We obtained two different hypotheses using a Generalized Mixed Yule Coalescent (GMYC) model: four (cox1) and six (16S) species. After the species validation step with the Bayesian species‐delimitation analysis (BPP), we suggest that only S. mexicana is a complex of two species with different distribution (along the Pacific and the Atlantic coasts, respectively). We highly recommend avoiding colony exchange between geographical regions in order to conserve the genetic integrity of both taxa.     

Integrative taxonomy reveals hidden diversity in the southern African darters genus Nannocharax Günther 1867 (Characiformes: Distichodontidae)

The present study explored the diversity of Nannocharax within southern Africa by implementing three species delimitation methods for a data set consisting of 37 mitochondrial cytochrome oxidase subunit I sequences. Two unilocus coalescent methods, the General Mixed Yule Coalescent (GMYC) and the Bayesian implementation of the Poisson Tree Processes (bPTP), and a genetic distance method, the Automatic Barcode Gap Discovery (ABGD), were applied. Both GMYC and bPTP delimited the same operational taxonomic units (OTUs), revealing a higher diversity for the genus in the region than previously recognised, whereas the ABGD failed to delimit the same candidate species. All methods delimited two species groups, and these are supported based on colouration patterning and morphology; the Nannocharax multifasciatus and the Nannocharax macropterus species groups and the delimited OTUs were assigned to each. Two putative new species were identified, Nannocharax cf. lineostriatus “Okavango” from the Okavango River in Angola and N. cf. lineostriatus “Kwanza” from the Kwanza River system in Angola. The distribution of Nannocharax dageti was confirmed for the Upper Zambezi and extended to the Okavango system, and an identification key for the southern Africa Nannocharax species is provided.     

Phylogeny and species delimitation of North European Lumbricillus (Clitellata,Enchytraeidae)

The enchytraeid genus Lumbricillus comprises about 80 described species of clitellate worms, which are up to a few centimetres long, and they mostly inhabit the littoral zone of non‐tropical marine and brackish waters world‐wide. The phylogeny of this genus is poorly studied, but previous work has suggested that Lumbricillus is a non‐monophyletic group. In this study, species boundaries and the phylogeny of this genus is re‐assessed using more than 300 DNA‐barcoded specimens (using COI mtDNA), part of which was also sequenced for two additional mitochondrial and four nuclear molecular markers. Statistical and coalescent based applications were used for the delimitation of a total of 24 species, of which 20 were identified as belonging to 17 described morphospecies; one morphospecies was found to be a complex of four delimited species, and another four delimited species could not be matched with any described species. Furthermore, gene trees, concatenation and multispecies coalescent based species trees were estimated using Bayesian inference. The estimated phylogenies confirm a non‐monophyletic Lumbricillus as L. semifuscus is clearly excluded from the genus. Furthermore, the placement of a monophyletic clade consisting of L. arenarius, L. dubius, and an unidentified species varies between analyses; they are either found as the sister‐group to the genus Grania or as sister‐group to the remaining Lumbricillus, where the latter relationship is supported by the multispecies coalescent, which we consider as the most reliable method.     

Phylogeny,phylogeography and geographical variation in the Crocidura monax (Soricidae) species complex from the montane islands of Tanzania,with descriptions of three new species

We assess morphological and multilocus genetic variation among 11 isolated montane populations of white‐toothed shrews from Tanzania that have been referred to either Crocidura monax Thomas or C. montis Thomas. The montane sites we sampled represent ‘sky‐islands’ from two geologically distinct archipelagos (Northern Highlands and the Eastern Arc Mountains) and are a significant component of the Eastern Afromontane Biodiversity Hotspot. We used multivariate analyses of morphometric traits and phylogenetic and species‐delimitation analyses of multilocus DNA sequence data to assess species‐level diversity. Our species delimitation analyses included a novel, pairwise validation approach that avoids potential biases associated with specifying a guide tree. These analyses reveal several distinct lineages, which we treat as six allopatric species. Each species is restricted to one, two or four mountains. We use available names to recognize C. monax, C. tansaniana Hutterer and C. usambarae Dippenaar, while naming and describing three new species. Our results demonstrate the effectiveness of combining morphological and genetic data to uncover and describe hidden diversity in a cryptic mammalian system. © 2015 The Linnean Society of London