Single-locus Species Delimitation Research Articles

Molecular Evidence Reveals Taxonomic Uncertainties and Cryptic Diversity in the Neotropical Catfish of the Genus Pimelodus (Siluriformes: Pimelodidae).

Pimelodus is the most speciose genus of the family Pimelodidae, and is amply distributed in the Neotropical region. The species-level taxonomy and phylogenetic relationships within this genus are still poorly resolved, however. These taxonomic problems and the general lack of data have generated major uncertainties with regard to the identification of specimens from different localities. In the present study, we applied a single-locus species delimitation approach to identify the MOTUs found within the genus Pimelodus and provide sound evidence for the evaluation of the species richness of this genus in the different river basins of the Neotropical region. The study was based on the analysis of sequences of the mitochondrial COI gene of 13 nominal species, which resulted in the identification of 24 consensus MOTUs. Only six nominal species were recovered as well-defined molecular entities by both the traditional barcoding analysis and the molecular delimitation methods, while the other seven presented cryptic diversity or persistent taxonomic uncertainties. The lineages identified from the Parnaíba ecoregions, Amazonas Estuary and Coastal Drainages may represent a much greater diversity of Pimelodus species than that recognized currently, although a more detailed study of this diversity will be necessary to provide a more definitive classification of the genus.

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

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

Comparison of the Efficiency of Single-Locus Species Delimitation Methods: A Case Study of a Single Lake Fish Population in Comparison against the Barcodes from International Databases

To date, a rather large set of both mathematical theories for species delimitation, based on single-locus genetic data, and their implementations as software products, has been accumulated. Comparison of the efficiencies of different delineation methods in the task of accumulating and analyzing data with reference to different taxa in different regions, is vital. The aim of this study was to compare the efficiency of fifteen single-locus species delimitation methods using the example of a fish species found in a single lake in European Russia (Lake Plescheyevo) with reference to other sequences of revealed taxa deposited in international databases. We analyzed 186 original COI sequences belonging to 24 haplotypes, and 101 other sequences previously deposited in GenBank and BOLD. Comparison of all 15 alternative taxonomies demonstrated that all methods adequately separate only the genera, while the number of delimited mOTUs differed from 16 (locMin) to 43 (HwM/CoMa). We can assume that the effectiveness of each method is correlated with the number of matches based on Ctax and MatchRatio criteria. The most comparable results were provided by bGMYC, mPTP, STACEY, KoT and ASAP and the most synchronous results were obtained from bGMYC, mPTP, STACEY and ASAP. We believe that these results are maximally realistic in the number of revealed mOTUs. A high genetic diversity, resulting in the existence of several mOTUs and phylogenetic lineages within many species, demonstrates the usefulness of the “polymorphic species” concept, which does not underestimate species richness and does not prevent the rational use and protection of biodiversity.

An integrative approach reveals high species diversity in the primitively segmented spider genus

Accurate species delimitation is crucial for our understanding of evolution, biodiversity and conservation. However, morphology-based species delimitation alone appears to be prone to taxonomic errors and ineffective for taxa with high interspecific morphological homogeneity or intraspecific morphological variations, as is the case for mesothele and mygalomorph spiders. Combined molecular–morphology species delimitation has shown great potential to delimit species boundaries in such ancient lineages. In the present study, molecular and morphological evidence were integrated to delimit species of the primitively segmented spider genus Songthela Ono, 2000. The cytochrome c oxidase subunit I gene (COI) was sequenced for 192 novel specimens belonging to 12 putative morphospecies. The evolutionary relationships within Songthela and the 12-morphospecies hypothesis were tested in two steps – species discovery and species validation – using four single-locus species delimitation approaches. All species delimitation analyses supported the 12-species hypothesis. Phylogenetic analyses yielded three major clades in Songthela, which are consistent with morphology. Accordingly, we assigned 19 known and 11 new species (S. aokoulong, sp. nov., S. bispina, sp. nov., S. dapo, sp. nov., S. huayanxi, sp. nov., S. lianhe, sp. nov., S. lingshang, sp. nov., S. multidentata, sp. nov., S. tianmen, sp. nov., S. unispina, sp. nov., S. xiujian, sp. nov., S. zizhu, sp. nov.) of Songthela to three species-groups: the bispina-group, the multidentata-group and the unispina-group. Another new species, S. zimugang, sp. nov., is not included in any species groups, but forms a sister lineage to the bispina- and unispina-groups. These results elucidate a high species diversity of Songthela in a small area and demonstrate that integrating morphology with COI-based species delimitation is fast and cost-effective in delimiting species boundaries. http://zoobank.org/urn:lsid:zoobank.org:pub:AF0F5B31-AFAF-4861-9844-445AE8678B67

Species boundaries in the messy middle-A genome-scale validation of species delimitation in a recently diverged lineage of coastal fog desert lichen fungi.

Species delimitation among closely related species is challenging because traditional phenotype‐based approaches, for example, using morphology, ecological, or chemical characteristics, may not coincide with natural groupings. With the advent of high‐throughput sequencing, it has become increasingly cost‐effective to acquire genome‐scale data which can resolve previously ambiguous species boundaries. As the availability of genome‐scale data has increased, numerous species delimitation analyses, such as BPP and SNAPP+Bayes factor delimitation (BFD*), have been developed to delimit species boundaries. However, even empirical molecular species delimitation approaches can be biased by confounding evolutionary factors, for example, hybridization/introgression and incomplete lineage sorting, and computational limitations. Here, we investigate species boundaries and the potential for micro‐endemism in a lineage of lichen‐forming fungi, Niebla Rundel & Bowler, in the family Ramalinaceae by analyzing single‐locus and genome‐scale data consisting of (a) single‐locus species delimitation analysis using ASAP, (b) maximum likelihood‐based phylogenetic tree inference, (c) genome‐scale species delimitation models, e.g., BPP and SNAPP+BFD, and (d) species validation using the genealogical divergence index (gdi). We specifically use these methods to cross‐validate results between genome‐scale and single‐locus datasets, differently sampled subsets of genomic data and to control for population‐level genetic divergence. Our species delimitation models tend to support more speciose groupings that were inconsistent with traditional taxonomy, supporting a hypothesis of micro‐endemism, which may include morphologically cryptic species. However, the models did not converge on robust, consistent species delimitations. While the results of our analysis are somewhat ambiguous in terms of species boundaries, they provide a valuable perspective on how to use these empirical species delimitation methods in a nonmodel system. This study thus highlights the challenges inherent in delimiting species, particularly in groups such as Niebla, with complex, relatively recent phylogeographic histories.

Molecular species delimitation refines the taxonomy of native and nonnative physinine snails in North America

Being able to associate an organism with a scientific name is fundamental to our understanding of its conservation status, ecology, and evolutionary history. Gastropods in the subfamily Physinae have been especially troublesome to identify because morphological variation can be unrelated to interspecific differences and there have been widespread introductions of an unknown number of species, which has led to a speculative taxonomy. To resolve uncertainty about species diversity in North America, we targeted an array of single-locus species delimitation methods at publically available specimens and new specimens collected from the Snake River basin, USA to generate species hypotheses, corroborated using nuclear analyses of the newly collected specimens. A total-evidence approach delineated 18 candidate species, revealing cryptic diversity within recognized taxa and a lack of support for other named taxa. Hypotheses regarding certain local endemics were confirmed, as were widespread introductions, including of an undescribed taxon likely belonging to a separate genus in southeastern Idaho for which the closest relatives are in southeast Asia. Overall, single-locus species delimitation was an effective first step toward understanding the diversity and distribution of species in Physinae and to guiding future investigation sampling and analyses of species hypotheses.

Single-locus species delimitation and ecological niche modelling provide insights into the evolution, historical distribution and taxonomy of the Pacific chorus frogs

Abstract The Pacific chorus frogs are a complex of three wide-ranging species (i.e. Hyliola hypochondriaca, Hyliola regilla, Hyliola sierra) whose current taxonomy remains unresolved. We conducted species delimitation analyses of these taxa using fragments of the cytochrome b and 12S–16S mtDNA genes to assess the species diversity. Importantly, we included samples from new locations throughout the range to better understand species distributions and identify potential contact zones among clades. Our analyses revealed three slightly parapatric but distinct species-level clades. Molecular dating revealed that these species began diverging in the Pleistocene c. 1.4 Mya with H. hypochondriaca and H. sierra diverging more recently c. 0.8 Mya. We found that populations from western Montana and Idaho originated recently from populations to the southwest that belong to H. sierra, rather than from H. regilla populations directly to the west. Population sizes of each species expanded c. 130–80 Kya with H. hypochondriaca exhibiting a more pronounced expansion beginning c. 100 Kya than the more gradual expansion of the other two species. The climatic niche models suggest that distributions of the three species were similar during the last interglacial (LIG) as they are today. During the Last Glacial Maximum (LGM), H. hypochondriaca and H. sierra occupied a larger range than they do today whereas H. regilla occupied a smaller refugium, shifted south from the current distribution. This study highlights the continued effectiveness of utilizing single-locus data sets for species delimitation and biogeographic analyses.

Mitochondrial sequence data clarify species concepts in the Cyclocephala mafaffa species complex (Coleoptera: Scarabaeidae: Dynastinae: Cyclocephalini)

The speciose genus Cyclocephala Dejean (Coleoptera: Scarabaeidae: Dynastinae: Cyclocephalini) has attracted research attention due to their diversity, agroeconomic importance, and floral visitation habits. Uniquely among Cyclocephala species, C. mafaffa Burmeister and C. deceptor (Casey), two nearly identical species, are diagnosed by a pronotal character: beaded or not beaded basal pronotal margin. We evaluated these morphological species hypotheses with a phylogenetic analysis of 12S and COI, neighbor-joining analysis, and several single-locus species delimitation procedures (automatic barcode gap analysis and three Poisson tree processes analyses). Together, these analyses supported the species concepts for C. deceptor and C. mafaffa. Delimitation procedures supported several distinct molecular operational taxonomic units among these taxa. We consider the separation of C. deceptor and C. mafaffa to be valid. We conservatively synonymize the West Indian subspecies C. mafaffa grandis Burmeister under C. mafaffa and offer a discussion on subspecific concepts in Cyclocephalini. We designate the lectotype of Stigmalia deficiens Casey. Implications of this study for other geographically widespread cyclocephalines or species with variable pronotal morphology are discussed.

Dwarf geckos and giant rivers: the role of the São Francisco River in the evolution of Lygodactylus klugei (Squamata: Gekkonidae) in the semi-arid Caatinga of north-eastern Brazil

AbstractSpecies diversification can be strongly influenced by geomorphological features, such as mountains, valleys and rivers. Rivers can act as hard or soft barriers to gene flow depending on their size, speed of flow, historical dynamics and regional topographical characteristics. The São Francisco River (SFR) is the largest perennial river in the Caatinga biome in north-eastern Brazil and has been considered a barrier to gene flow and dispersal. Herein, we evaluated the role of the SFR on the evolution of Lygodactylus klugei, a small gecko from the Caatinga. Using a single-locus species delimitation method (generalized mixed Yule coalescent), we defined lineages (haploclades). Subsequently, we evaluated the role of the SFR in structuring genetic diversity in this species using a multilocus approach to quantify migration across margins. We also evaluated genetic structure based on nuclear markers, testing the number of populations found through an assignment test (STRUCTURE) across the species distribution. We recovered two mitochondrial lineages structured with respect to the SFR, but only a single population was inferred from nuclear markers. Given that we detected an influence of the SFR only on mitochondrial markers, we suggest that the current river course has acted as a relatively recent geographical barrier for L. klugei, for ~450 000 years.

Speciation in a biodiversity hotspot: Phylogenetic relationships, species delimitation, and divergence times of Patagonian ground frogs from the Eupsophus roseus group (Alsodidae).

The alsodid ground frogs of the Eupsophus genus are divided into two groups, the roseus (2n = 30) and vertebralis (2n = 28), which are distributed throughout the temperate Nothofagus forests of South America. Currently, the roseus group is composed by four species, while the vertebralis group consists of two. Phylogenetic relationships and species delimitation within each group are controversial. In fact, previous analyses considered that the roseus group was composed of between four to nine species. In this work, we evaluated phylogenetic relationships, diversification times, and species delimitation within the roseus group using a multi-locus dataset. For this purpose, mitochondrial (D-loop, Cyt b, and COI) and nuclear (POMC and CRYBA1) partial sequences from 164 individuals were amplified, representing all species. Maximum Likelihood (ML) and Bayesian approaches were used to reconstruct phylogenetic relationships. Species tree was estimated using BEAST and singular value decomposition scores for species quartets (SVDquartets). Species limits were evaluated with six coalescent approaches. Diversification times were estimated using mitochondrial and nuclear rates with LogNormal relaxed clock in BEAST. Nine well-supported monophyletic lineages were recovered in Bayesian, ML, and SVDquartets, including eight named species and a lineage composed by specimens from the Villarrica population (Bootstrap:>70, PP:> 0.99). Single-locus species delimitation analyses overestimated the species number in E. migueli, E. calcaratus, and E. roseus lineages, while multi-locus analyses recovered as species the nine lineages observed in phylogenetic analyses (Ctax = 0.69). It is hypothesized that Eupsophus diversification occurred during Mid-Pleistocene (0.42–0.14 Mya), with most species having originated after the Last Southern Patagonian Glaciation (0.18 Mya). Our results revitalize the hypothesis that the E. roseus group is composed of eight species and support the Villarrica lineage as a new putative species.

Diversity of the green algal genus Spirogyra (Conjugatophyceae) in the Hawaiian Islands

:Identification of species in the green algal genus Spirogyra is complicated by cryptic morphological diversity, seasonality of critical features of sexual reproduction necessary for morphological identification, the presence of polyploid species groups and a lack of molecular sequence data tied to authentic specimens. Yet the prevalence and importance of these species in freshwater ecosystems drives researchers to advance knowledge of this lineage in various regions around the world. In this study, 180 specimens of Spirogyra from five different Hawaiian Islands were collected and analysed using both morphological and molecular approaches. Comparisons to published data on the genus were also performed to determine the species identification and to estimate the number of unique species in the Hawaiian Islands. Phylogenetic analyses of the rbcL marker revealed 16 clusters of Hawaiian Spirogyra specimens, of which eight were unique to the Hawaiian Islands and recognised as putative endemics, and five were confidently named to species with taxonomic names from other parts of the world (S. communis, S. fluviatilis, S. maxima, S. setiformis and S. submaxima). Ten clusters of Hawaiian Spirogyra were revealed in the SSU phylogenetic analyses, of which only one was unique to the Hawaiian Islands; interpretation of the SSU results was limited to assessment of molecular diversity due to a paucity of named reference sequences. A DNA bar-coding approach was used to examine species boundaries and identification for the Hawaiian Spirogyra specimens with the UPA marker, resulting in 27 unique haplotypes, the vast majority of which were from Hawai'i. Five single-locus species delimitation methods were compared within the different marker data sets to explore congruence in interpretation of species boundaries, and although the number of estimated species varied by method, clusters of specimens were identified based on consensus of these results as well as tree topology and identification criteria used in previous publications. This study revealed a remarkable level of genus-level diversity for Hawaiian Spirogyra, which may be interpreted cautiously as potential endemism for this widespread genus of common freshwater green algae, and which emphasises the importance of continued support for survey-style research into biological diversity of distinct regions.

Genetic and Morphological Analyses Demonstrate That Schizolecis guntheri (Siluriformes: Loricariidae) Is Likely to Be a Species Complex.

Schizolecis is a monotypic genus of Siluriformes widely distributed throughout isolated coastal drainages of southeastern Brazil. Previous studies have shown that fish groups found in isolated river basins tend to differentiate over time in the absence of gene flow, resulting in allopatric speciation. In this study, we used partial sequences of the mitochondrial gene COI with the analysis of the General Mixed Yule Coalescent model (GMYC) and the Automatic Barcode Gap Discovery (ABGD) for single locus species delimitation, and a Principal Component Analysis (PCA) of external morphology to test the hypothesis that Schizolecis guntheri is a complex of species. We analyzed 94 samples of S. guntheri for GMYC and ABGD, and 82 samples for PCA from 22 coastal rivers draining to the Atlantic in southeastern Brazil from the Paraná State to the north of the Rio de Janeiro State. As a result, the GMYC model and the ABGD delimited five operational taxonomy units (OTUs – a nomenclature referred to in the present study of the possible new species delimited for the genetic analysis), a much higher number compared to the traditional alfa taxonomy that only recognizes S. guntheri across the isolated coastal rivers of Brazil. Furthermore, the PCA analysis suggests that S. guntheri is highly variable in aspects of external body proportions, including dorsal-fin spine length, pectoral-fin spine length, pelvic-fin spine length, lower caudal-fin spine length, caudal peduncle depth, anal width and mandibular ramus length. However, no exclusive character was found among the isolated populations that could be used to describe a new species of Schizolecis. Therefore, we can conclude, based on our results of PCA contrasting with the results of GMYC and ABGD, that S. guntheri represents a complex of species.

Multi-rate Poisson tree processes for single-locus species delimitation under maximum likelihood and Markov chain Monte Carlo.

MotivationIn recent years, molecular species delimitation has become a routine approach for quantifying and classifying biodiversity. Barcoding methods are of particular importance in large-scale surveys as they promote fast species discovery and biodiversity estimates. Among those, distance-based methods are the most common choice as they scale well with large datasets; however, they are sensitive to similarity threshold parameters and they ignore evolutionary relationships. The recently introduced “Poisson Tree Processes” (PTP) method is a phylogeny-aware approach that does not rely on such thresholds. Yet, two weaknesses of PTP impact its accuracy and practicality when applied to large datasets; it does not account for divergent intraspecific variation and is slow for a large number of sequences.ResultsWe introduce the multi-rate PTP (mPTP), an improved method that alleviates the theoretical and technical shortcomings of PTP. It incorporates different levels of intraspecific genetic diversity deriving from differences in either the evolutionary history or sampling of each species. Results on empirical data suggest that mPTP is superior to PTP and popular distance-based methods as it, consistently yields more accurate delimitations with respect to the taxonomy (i.e., identifies more taxonomic species, infers species numbers closer to the taxonomy). Moreover, mPTP does not require any similarity threshold as input. The novel dynamic programming algorithm attains a speedup of at least five orders of magnitude compared to PTP, allowing it to delimit species in large (meta-) barcoding data. In addition, Markov Chain Monte Carlo sampling provides a comprehensive evaluation of the inferred delimitation in just a few seconds for millions of steps, independently of tree size.Availability and ImplementationmPTP is implemented in C and is available for download at http://github.com/Pas-Kapli/mptp under the GNU Affero 3 license. A web-service is available at http://mptp.h-its.org.Supplementary information Supplementary data are available at Bioinformatics online.

Delimiting Species-Poor Data Sets using Single Molecular Markers: A Study of Barcode Gaps, Haplowebs and GMYC.

Most single-locus molecular approaches to species delimitation available to date have been designed and tested on data sets comprising at least tens of species, whereas the opposite case (species-poor data sets for which the hypothesis that all individuals are conspecific cannot by rejected beforehand) has rarely been the focus of such attempts. Here we compare the performance of barcode gap detection, haplowebs and generalized mixed Yule-coalescent (GMYC) models to delineate chimpanzees and bonobos using nuclear sequence markers, then apply these single-locus species delimitation methods to data sets of one, three, or six species simulated under a wide range of population sizes, speciation rates, mutation rates and sampling efforts. Our results show that barcode gap detection and GMYC models are unable to delineate species properly in data sets composed of one or two species, two situations in which haplowebs outperform them. For data sets composed of three or six species, bGMYC and haplowebs outperform the single-threshold and multiple-threshold versions of GMYC, whereas a clear barcode gap is only observed when population sizes and speciation rates are both small. The latter conditions represent a "sweet spot" for molecular taxonomy where all the single-locus approaches tested work well; however, the performance of these methods decreases strongly when population sizes and speciation rates are high, suggesting that multilocus approaches may be necessary to tackle such cases.

Single-locus species delimitation: a test of the mixed Yule–coalescent model, with an empirical application to Philippine round-leaf bats

Prospects for a comprehensive inventory of global biodiversity would be greatly improved by automating methods of species delimitation. The general mixed Yule-coalescent (GMYC) was recently proposed as a potential means of increasing the rate of biodiversity exploration. We tested this method with simulated data and applied it to a group of poorly known bats (Hipposideros) from the Philippines. We then used echolocation call characteristics to evaluate the plausibility of species boundaries suggested by GMYC. In our simulations, GMYC performed relatively well (errors in estimated species diversity less than 25%) when the product of the haploid effective population size (N(e)) and speciation rate (SR; per lineage per million years) was less than or equal to 10(5), while interspecific variation in N(e) was twofold or less. However, at higher but also biologically relevant values of N(e) × SR and when N(e) varied tenfold among species, performance was very poor. GMYC analyses of mitochondrial DNA sequences from Philippine Hipposideros suggest actual diversity may be approximately twice the current estimate, and available echolocation call data are mostly consistent with GMYC delimitations. In conclusion, we consider the GMYC model useful under some conditions, but additional information on N(e), SR and/or corroboration from independent character data are needed to allow meaningful interpretation of results.