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Factors affecting species delimitations with the GMYC model: Insights from a butterfly survey
Methods in Ecology and Evolution
Abstract
1. 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).
2. 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 dataset 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.
3. The number of GMYC entities obtained constantly exceeds the total number of morphospecies in the dataset. We show that approximately 80% of the species studied are recognised as entities by GMYC. Interestingly, we show that this percentage is practically the maximum that a single threshold method can provide for this dataset. Thus the ca. 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.
4. 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 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.
5. 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.
Supplementary resource (1)
... One of the species delimitation methods we used with the COI data (see below) requires equivalent sampling of multiple closely related species (Talavera et al. 2013) and a less closely related outgroup species to train the model (as well as the unresolved taxa) to perform reliably. To achieve equivalent sampling, we downloaded all publicly available COI barcode sequences from genbank (Sayers et al. 2022) for B. occidentalis, B. terricola, B. hypocrita Pérez 1905, and B. jacobsoni Skorikov 1912, which are closely related, and B. lucorum Linnaeus 1761, which is not as closely related but still within the sub-genus, as an outgroup. ...
... We applied both PTP and mPTP to our COI barcode dataset in this study. We did not apply these methods to the UCE dataset because they require a minimum of five well-sampled, related species to train their algorithms (similar to GMYC as described by Talavera et al. 2013). COI sequences for species closely related to our taxa of interest were publicly available via BOLD, but we did not have a comparable dataset for our UCE analyses (UCE data for closely related but non-target species). ...
... GMYC classifies the branches of an ultrametric gene or species phylogenetic tree by maximizing the likelihood of a GMYC model, i.e., speciation rates held constant among species without extinction and panmixia within species (Talavera et al. 2013). GMYC is the only analysis included in this study that requires an ultrametric tree in which the terminal taxa are equidistant from the root. ...
Accurate species delimitation is critical to identifying the conservation status of species. Molecular species delimitation methods have revealed previously unrecognized cryptic species across the taxonomic spectrum. However, studies vary in the molecular markers selected, analytical approaches used, and taxon sampling, which sometimes results in conflicting conclusions. One example of such a conflict is seen in the species delimitation analyses of the western bumble bee, Bombus occidentalis. This species was once an abundant insect pollinator in western North America but has declined severely since the mid 1990s and is predicted to continue to diminish under even optimistic future climate scenarios. Complicating this conservation crisis, the species status of B. occidentalis has varied over time, with most recent studies recognizing one or two species. Previous studies that used molecular methods to address this question focused on a Bayesian phylogeny of the mitochondrial cytochrome oxidase I (COI) gene. Phylogenetic studies that focus on a single gene are criticized for misrepresenting the evolutionary history of species because nuclear and mitochondrial genomes, and even some genes within them, may have different evolutionary patterns. We tested a two species hypothesis of the B. occidentalis complex using nuclear (ultraconserved elements) and mitochondrial (COI) markers to infer maximum likelihood and Bayesian phylogenies for the taxa. We present our results and conclusions from eight species delimitation methods. Based on the genomic, morphological and geographic differences between the taxa we find support for the two species hypothesis, with B. occidentalis and B. mckayi as separate species. We discuss the strengths and limitations of each genetic dataset and delimitation method, make recommendations for best practices, and highlight opportunities for equitable knowledge and technology development for phylogenomics in conservation biology.
... To obtain the ultrametric trees required by this method, we used BEAST as it provides a powerful statistical framework to interpret the chronological context of molecular variation [45]. Despite some investigations of the effects of priors on the number of species estimated by GMYC ( [38,44,46]), we here revisit the impact of specifying a strict or a log-normal relaxed clock, as well as the type of tree prior used (coalescent with constant population size [47], coalescent with exponentially growing population size [48], and Yule [49]). The type of clock used will affect branch lengths, it is however difficult to predict a priori how the misspecification of this parameter will influence the results. ...
... To our knowledge few studies have investigated the effect of retaining identical haplotypes in the analysis (but see [46]). In other studies, identical sequences were removed as they were thought to make the task of estimating the transition problematic [38], as the GMYC method cannot accommodate for the null branch lengths associated with identical sequences [44]. ...
... Simulated data have been used to test the influence of diversification rates, effective population sizes, and migration rates, on the accuracy and precision of GMYC estimates [62,63]. Some of the effects of the methodological choices have been evaluated by the authors of the method [37,38,44] and by others [46], but the conclusions might be difficult to generalize as they depend in part on the nature of the dataset. Other species delineation methods using sequence data are also available (e.g., RESL [64], ABGD [65], bPTP [66], bGMYC [67]), and it would be interesting to investigate how they perform compared to the results obtained here. ...
Identifying accurately species is critical for our understanding of patterns of diversity and speciation. However, for many organisms with simple and variable morphological traits, the characters traditionally used by taxonomists to identify species might lead to a considerable under appreciation of their diversity. Recent advances in molecular-data based computational methods have considerably improved our ability to identify and test species limits. Here, we use an integrative approach to delineate species in a complex of sea cucumbers. We used a three-step approach to show that “ Holothuria impatiens ”, a common, shallow-water species, occurring across the Indo-Pacific, the Western Atlantic and the Mediterranean Sea, targeted locally by fisheries, is a complex of at least 13 species. (1) We used the Generalized Mixed Yule Coalescent (GMYC) model to identify putative species without a priori hypotheses. In the process, we also show that the number of putative species estimated with GMYC can be affected considerably by the priors used to build the input tree. (2) We assessed based on coloration patterns and distributional information, the most relevant hypothesis. This approach allowed us to identify unambiguously 9 species. However, some of the lineages consistently assigned to belong to different species using GMYC, are occurring in sympatry and are not differentiated morphologically. (3) We used Bayes factors to compare competing models of species assignment using the multispecies coalescent as implemented in *BEAST. This approach allowed us to validate that the species identified using GMYC were likely reproductively isolated. Estimates of the timing of diversification also showed that these species diverged less than 2 Ma, which is the fastest case of closely related species occurring in sympatry for a marine metazoan. Our study demonstrates how clarifying species limits contribute to refining our understanding of speciation.
... However, compared to other approaches, it often produces more OTUs. [73]. Dysmicoccus brevipes, M. hirsutus, and Ps. ...
... annonae (Pacheco da Silva & Kaydan) [38,75]. The bPTP approach was eliminated from our study because it estimated too many singletons, which might have led to an incorrect interpretation of the data [73]. As input, bPTP does not require an ultrametric tree or a sequence-similarity threshold [65]. ...
... However, compared to other approaches, it often produces more OTUs. [73]. The use of genetic/molecular databases has been shown to increase the accuracy of identification [79]. ...
Mealybugs are insects belonging to the family Pseudococcidae. This family includes many plant-pest species with similar morphologies, which may lead to errors in mealybug identification and delimitation. In the present study, we employed molecular-species-delimitation approaches based on distance (ASAP) and coalescence (GMYC and mPTP) methods to identify mealybugs collected from coffee and other plant hosts in the states of Espírito Santo, Bahia, Minas Gerais, and Pernambuco, Brazil. We obtained 171 new COI sequences, and 565 from the BOLD Systems database, representing 26 candidate species of Pseudococcidae. The MOTUs estimated were not congruent across different methods (ASAP-25; GMYC-30; mPTP-22). Misidentifications were revealed in the sequences from the BOLD Systems database involving Phenacoccus solani × Ph. solenopsis, Ph. tucumanus × Ph. baccharidis, and Planacoccus citri × Pl. minor species. Ten mealybug species were collected from coffee plants in Espírito Santo. Due to the incorrect labeling of the species sequences, the COI barcode library of the dataset from the database needs to be carefully analyzed to avoid the misidentification of species. The systematics and taxonomy of mealybugs may be improved by integrative taxonomy which may facilitate the integrated pest management of these pests.
... Nevertheless, simulations of the GMYC method showed that if the effective population size remains low relative to species divergence (that is, relatively low withinspecies variation) the threshold is optimized at the correct point of the tree and the effect of geographic structuring is minimal [59]. Moreover, Talavera et al. [68] observed that the removal of intermediate haplotypes had little effect in the results and did not delimit the most extreme haplotypes as different species. The analyses have been conducted by both including repeated sequences and pruning repeated sequences. ...
... However, when the tree is produced using a genealogy-based inference, as is the case of BEAST, the program does not assign null lengths to identical terminals, since they are treated as different haplotypes coalescing to a MRCA node, which allows GMYC to handle them. Talavera et al. [68] did not observe differences in GMYC results when using repeated sequences from a tree inferred with BEAST relative to when they were removed, although they recommend collapsing all repeated sequences to only keep different haplotypes to reduce computational times. Conversely, Michonneau [10] advocated the use of repeated sequences in GMYC analysis using a tree inferred by BEAST. ...
... When one haplotype was found in only one population, it was represented only once in the dataset, whether it was the only haplotype in the population or not. The possibility that one species could be represented by only one sequence in the dataset (i.e., a singleton) has demonstrated no negative effects in the GMYC analysis [68]. ...
Groups with morphological stasis are an interesting framework to address putative cryptic species that may be hidden behind traditional taxonomic treatments, particularly when distribution ranges suggest disjunct and environmentally heterogeneous biogeographic patterns. New hypotheses of delimitation of evolutionary independent units can lead to the identification of different biogeographic processes, laying the foundation to investigate their historical and ecological significance. Jasione is a plant genus with a distribution centered in the Mediterranean basin, characterized by significant morphological stasis. Within the western Mediterranean J. gr. crispa species complex, J. sessiliflora s.l. and allied taxa form a distinct group, occupying environmentally diverse regions. At least two ploidy levels, diploid and tetraploid, are known to occur in the group. The internal variability is assessed with phylogenetic tools, viz. GMYC and ASAP, for species delimitation. The results are compared with other lines of evidence, including morphology and cytology. The fitting of distribution patterns of the inferred entities to chorological subprovinces is also used as a biogeographical and environmental framework to test the species hypothesis. Despite the scarcity of diagnostic morphological characters in the group, phylogenetic delimitation supports the description of at least one cryptic species, a narrow endemic in the NE Iberian Peninsula. Moreover, the results support the segregation of a thermophilic group of populations in eastern Iberia from J. sessiliflora. Ploidy variation from a wide geographical survey supports the systematic rearrangement suggested by species delimitation. Taxonomic reorganization in J. sessiliflora s.l. would allow ecological interpretations of distribution patterns in great accordance with biogeographical regionalization at the subprovince level, supporting geobotanical boundaries as a framework to interpret species ecological coherence of cryptic lineages. These results suggest that species differentiation, together with geographic isolation and polyploidization, is associated with adaptation to different environments, shifting from more to less thermophilic conditions. Thus, the recognition of concealed evolutionary entities is essential to correctly interpret biogeographical patterns in regions with a complex geologic and evolutionary history, such as the Mediterranean basin, and biogeographical units emerge as biologically sound frameworks to test the species hypothesis.
... This step is important because the accumulation of short branches can lead to an overestimate of candidate species by the GMYC algorithms (see section 2.4.1) (Reid and Carstens, 2012;Talavera et al., 2013). ...
... For this analysis, the substitution model was identified using bModelTest, as above, with the Yule tree and log-normal clock models. This combination of priors resulted in a high percentage of correct hits in the GMYC performance test, based on empirical data from butterflies (Talavera et al., 2013). The analysis was run twice, with 1 × 10 7 generations in each case, and thinning to 1 × 10 3 . ...
... This analysis was conducted in the bGMYC v.1.0.2 package of the R software (Reid and Carstens, 2012;R Core Team, 2020), using the single-threshold method and limiting the boundaries for the number of putative species to a threshold between one and the maximum value of the credibility interval of the entities delimited by the ML-GMYC analysis. Talavera et al. (2013) reported an improvement in precision when the taxonomic operational units are represented by only a single or a few haplotypes. Given this, we ran the discovery step twice. ...
Despite their limited vagility and pronounced habitat heterogeneity in the tropics, many anuran species have unexpectedly extensive geographic ranges. One prominent example of this phenomenon is Pithecopus hypochondrialis, which is found in the Cerrado, Guianan savanna, and Llanos domains, as well as isolated tracts of savanna and open habitat within the Amazon Forest. The present study employs an integrative species delimitation approach to test the hypothesis that P. hypochondrialis is in fact a species complex. We also reconstruct the relationships among the lineages delimited here and other Pithecopus species. In this study, we employ Ecological Niche Modelling (ENM) and spatiotemporal phylogeographic reconstruction approaches to evaluate a multitude of scenarios of connectivity across the Neotropical savannas. We identified three divergent lineages, two of which have been described previously. The lineages were allocated to a lowland Pithecopus clade, although the relationships among these lineages are weakly supported. Both the ENM and the phylogeographic reconstruction highlight the occurrence of periods of connectivity among the Neotropical savannas over the course of the Pliocene and Pleistocene epochs. These processes extended from eastern Amazonia to the northern coast of Brazil. The findings of the present study highlight the presence of hidden diversity within P. hypochondrialis, and reinforce the need for a comprehensive taxonomic review. These findings also indicate intricate and highly dynamic patterns of connectivity across the Neotropical savannas that date back to the Pliocene.
... Due to the lack of consensus on the most appropriate clock and tree priors for reconstructing gene trees in species delimitation (Monaghan et al., 2009;Ratnasingham & Hebert, 2013;Talavera et al., 2013;Tang et al., 2014), we conducted a formal test to compare two different clock models (strict and relaxed lognormal) and two different tree priors (coalescent constant population and Yule) (Rodrigues et al., 2020). The aim was to determine the most suitable models for our dataset using the nested sampling (NS) algorithm (Maturana et al., 2019), implemented as a package for BEAST. ...
... The sGMYC analysis based on a single gene revealed the presence of 378 MOTUs. This species-delimitation algorithm relies on the priors and parameters used to construct the ultrametric tree (Ceccarelli et al., 2012), and tends to overestimate species diversity compared to other methods (Kekkonen & Hebert, 2014;Miralles & Vences, 2013;Paz & Crawford, 2012;Talavera et al., 2013). In our study, the sGMYC method seems to be the most accurate since it recovered substantially fewer putative species than the bPTP and sPTP analyses despite its hypothesized oversplitting. ...
... In our study, the sGMYC method seems to be the most accurate since it recovered substantially fewer putative species than the bPTP and sPTP analyses despite its hypothesized oversplitting. Moreover, the sGMYC approach has been suggested to suit datasets with large numbers of singleton taxa (Talavera et al., 2013), which is what we observe for Polypedilum. Based on the aforementioned considerations, we chose the putative species delimited by the sGMYC method as the basis for the biogeographical analyses. ...
South America, particularly within its tropical belt, is renowned for its unparalleled high levels of species richness, surpassing other major biomes. Certain neotropical areas harbor fragmented knowledge of insect diversity and face imminent threats from biodiversity loss and climate change. Hence, there is an urgent need for rapid estimation methods to complement slower traditional taxonomic approaches. A variety of algorithms for delimiting species through single‐locus DNA barcodes have been developed and applied for rapid species diversity estimates across diverse taxa. However, tree‐based and distance‐based methods may yield different group assignments, leading to potential overestimation or underestimation of putative species. Here, we investigate the performance of different DNA‐based species delimitation approaches to rapidly estimate the diversity of Polypedilum (Chironomidae, Diptera) in South America. Additionally, we test the hypothesis that significant differences exist in the community structure of Polypedilum fauna between South America and its neighboring regions, particularly the Nearctic. Our analysis encompasses a dataset of 1492 specimens from 598 locations worldwide, with a specific focus on South America. Within this region, we analyzed a subset of 247 specimens reported from 37 locations. Using various methods including the Barcode Index Number (BIN), Bayesian Poisson tree processes (bPTP), multi‐rate Poisson tree processes (mPTP), single‐rate Poisson tree processes (sPTP), and generalized mixed Yule coalescent (sGMYC), we identify molecular operational taxonomic units (MOTUs) ranging from 267 to 520. Our results indicate that the sGMYC method is the most suitable for estimating putative species in our dataset, resulting in the identification of 75 species in the Neotropical region, particularly in South America. Notably, this region exhibited higher species richness in comparison to the Palearctic and Oriental realms. Additionally, our findings suggest potential differences in species composition of Polypedilum fauna between the Neotropical and the adjacent Nearctic realms, highlighting high levels of endemism and species richness in the first. These results support our hypothesis that there are substantial differences exist in species composition between the Polypedilum fauna in South America and the neighboring regions.
... The method has become very popular in ecology because it does not require prior knowledge of the target study group; which makes it a particularly useful tool for studies involving species for which taxonomic knowledge is limited or non-existent (Talavera et al., 2013). ...
... The performance of the GMYC model has been predominantly tested on simulated data where the effects of factors are controlled, and the model's assumptions are not violated (Papadopoulou et al., 2009;Esselstyn et al., 2012;Fujisawa and Barraclough, 2013;Talavera et al., 2013). Most applications of the GMYC using empirical data will, however, likely violate these assumptions. ...
... Generally, however, a single-threshold approach is recommended as it is less likely to oversplit (Fujisawa and Barraclough, 2013;Talavera et al., 2013;Blair and Bryson Jr, 2017). ...
... However, compared to other approaches, it often produces more OTUs. [73]. Dysmicoccus brevipes, M. hirsutus, and Ps. ...
... annonae (Pacheco da Silva & Kaydan) [38,75]. The bPTP approach was eliminated from our study because it estimated too many singletons, which might have led to an incorrect interpretation of the data [73]. As input, bPTP does not require an ultrametric tree or a sequence-similarity threshold [65]. ...
... However, compared to other approaches, it often produces more OTUs. [73]. The use of genetic/molecular databases has been shown to increase the accuracy of identification [79]. ...
Mealybugs are insects belonging to the family Pseudococcidae. This family includes many plant-pest species with similar morphologies, which may lead to errors in mealybug identification and delimitation. In the present study, we employed molecular-species-delimitation approaches based on distance (ASAP) and coalescence (GMYC and mPTP) methods to identify mealybugs collected from coffee and other plant hosts in the states of Espírito Santo, Bahia, Minas Gerais, and Pernambuco, Brazil. We obtained 171 new COI sequences, and 565 from the BOLD Systems database, representing 26 candidate species of Pseudococcidae. The MOTUs estimated were not congruent across different methods (ASAP-25; GMYC-30; mPTP-22). Misidentifications were revealed in the sequences from the BOLD Systems database involving Phenacoccus solani × Ph. solenopsis, Ph. tucumanus × Ph. baccharidis, and Planacoccus citri × Pl. minor species. Ten mealybug species were collected from coffee plants in Espírito Santo. Due to the incorrect labeling of the species sequences, the COI barcode library of the dataset from the database needs to be carefully analyzed to avoid the misidentification of species. The systematics and taxonomy of mealybugs may be improved by integrative taxonomy which may facilitate the integrated pest management of these pests.
... PtP analysis depends on fitting models to a tree for a single gene in order to model: (1) few changes between branches (short branching lengths) on the tree within species; and (2) many changes between branches (long branch lengths) on the tree between species (zhang et al. 2013). For the PtP technique to perform properly, the models in each analysis need to be fitted and calibrated to data representing at least five separate, verified, most-closely-related species (dellicour & Flot, 2015;Fujisawa & Barraclough, 2013;leliaert et al., 2014;reid & Carstens, 2012;talavera, dinca, & vila, 2013;j.-j. zhang et al., 2013). ...
... these three nodes are also the nodes selected by the optimal PtP maximum-likelihood solution for coalescents identifying candidate species. this tree still includes five widely-accepted species in addition to the weisi-complex being revised, which is considered an adequate number for the training of the PtP models (dellicour & Flot, 2015;Fujisawa & Barraclough, 2013;leliaert et al., 2014;reid & Carstens, 2012;talavera et al., 2013;j.-j. zhang et al., 2013). ...
COI-barcode-like sequences appear to show substantially more species diversity among Mesoamerican bumblebees than had been reported previously from morphological studies. Closer examination shows that some of this apparent diversity may be pseudospecies (groups falsely misinterpreted as separate species), often supported by paralogous ‘numts’ (nuclear copies of mitochondrial sequences). For the well-sampled weisi-complex, we seek to filter out pseudogenes in order to use the orthologous COI-barcode sequences for identifying estimates of evolutionary relationships and likely species’ gene coalescents for candidate species. Even after this filtering, in contrast to recent purely morphological studies our results from an integrative assessment of species’ gene coalescents together with skeletal morphology support that ‘Bombus weisi’ Friese in its recent broad sense consists of two species: B. weisi (which includes the taxon montezumae Cockerell); and B. nigrodorsalis Franklin. Our interpretation rejects likely numts-based pseudospecies and a candidate species that are unsupported by skeletal morphology. This shows that careful attention needs to be paid to both barcode analysis and to skeletal morphology, to avoid describing pseudospecies.
... Study design , which includes sampling strategy (Chambers & Hillis, 2020;Pentinsaari et al., 2017;Talavera et al., 2013), may influence species delimitation outcomes as much as data type and analytical framework. Singlelocus approaches based on mitochondrial data routinely yield inflated lineage/species numbers, particularly in sedentary taxa with allopatric or fragmented population distributions (Hamilton et al., 2014;Opatova & Arnedo, 2014b). ...
... Obtaining sufficient taxon sampling is important in the species delimitation process (Chambers & Hillis, 2020;Pentinsaari et al., 2017;Talavera et al., 2013); however, it might be difficult in endangered or rare taxa. In order to avoid overexploitation, the sampling strategy usually relies on non-lethal tissue collection (Hamm et al., 2010;Machkour M'Rabet et al., 2009;Ožana et al., 2020) or alternatively on completely non-invasive approaches such as analyses of shed hair and faeces (Beja-Pereira et al., 2009;Dufresnes et al., 2019;Waits & Paetkau, 2005). ...
The outcome of species delimitation depends on many factors, including conceptual framework, study design, data availability, methodology employed and subjective decision making. Obtaining sufficient taxon sampling in endangered or rare taxa might be difficult, particularly when non‐lethal tissue collection cannot be utilized. The need to avoid overexploitation of the natural populations may thus limit methodological framework available for downstream data analyses and bias the results. We test species boundaries in rare North American trapdoor spider genus Cyclocosmia Ausserer (1871) inhabiting the Southern Coastal Plain biodiversity hotspot with the use of genomic data and two multispecies coalescent model methods. We evaluate the performance of each methodology within a limited sampling framework . To mitigate the risk of species over splitting, common in taxa with highly structured populations, we subsequently implement a species validation step via genealogical diversification index ( gdi ), which accounts for both genetic isolation and gene flow. We delimited eight geographically restricted lineages within sampled North American Cyclocosmia, suggesting that major river drainages in the region are likely barriers to dispersal. Our results suggest that utilizing BPP in the species discovery step might be a good option for datasets comprising hundreds of loci, but fewer individuals, which may be a common scenario for rare taxa. However, we also show that such results should be validated via gdi , in order to avoid over splitting.
... Both jMOTU and sGMYC algorithms produce more reliable partitions with the sampled morphospecies, while the bPTP and BIN algorithms typically resulted in an "overrated" solution. We used only the single-threshold version of GMYC because it has been shown to outperform the multi-threshold version (Fujisawa and Barraclough 2013;Talavera et al. 2013). Most of the MOTU splits detected in this study, such as A. sinensis, A. zhengi, S. occipitalis, were divided into at least two MOTUs, each using different species delimitation algorithms, except for the ASAP algorithm. ...
DNA barcoding has been proposed as a rapid and reliable tool for animal identification and species delineation. The 5’ end of the mitochondrial cytochrome c oxidase I gene (COI-5P) was sequenced for 318 specimens of 55 mantis species. Of these, 44 species had not been sequenced before, thus being new COI-5P barcode sequences to science. Another 61 COI-5P barcode sequences comprising five species were retrieved from the Barcode of Life Database (BOLD; www.boldsystems.org). Five species delimitation algorithms were employed to sort barcode sequences into Molecular Operational Taxonomic Units (MOTUs), namely the distance-based Barcode Index Number (BIN) System, Generalized Mixed Yule Coalescent (GMYC), a Java program that uses an explicit, determinate algorithm to define Molecular Operational Taxonomic Unit (jMOTU), Assemble Species by Automatic Partitioning (ASAP), and Bayesian implementation of the Poisson Tree Processes model (bPTP). All species, except Hierodula chinensis Werner, 1929, were recovered as monophyletic on the neighbor-joining (NJ) tree. For the final dataset, 379 COI-5P barcode sequences were assigned to 68 BINs. Fifty-five out of 68 BINs obtained were new to BOLD. The low level of BIN overlap with other nations highlights the importance of constructing a regional DNA barcode reference library. The algorithms ASAP, jMOTU, bPTP, and GMYC clustered barcode sequences into 32, 58, 68, and 60 MOTUs, respectively. All species delimitation algorithms (except ASAP analysis) split Anaxarcha sinensis Beier, 1933, Anaxarcha zhengi Ren & Wang, 1994, H. chinensis, Spilomantis occipitalis (Westwood, 1889), Titanodula formosana Giglio-Tos, 1912 into more than one MOTUs. All algorithms merged Hierodula sp. BCM-2019 and H. chinensis into the same MOTU, as for Tenodera aridifolia Stoll, 1813 and Tenodera sinensis Saussure, 1871. More accurate identification results need to be supplemented by detailed morphological classification.
... Lakeplain prairie (Globally imperiled(G2)/State critically imperiled(S1)) and prairie fen (Globally vulnerable (G3)/State vulnerable (S3)) habitats serve as a refuge for numerous at-risk wildlife species, including threatened and endangered species and multiple rare insect species, such as Karner blue butterfly, Plebujus samuelis (Nabokov, 1943;Talavera et al. 2013) and ...
Michigan’s lakeplain prairie and prairie fen natural communities contain refugia
for many at-risk species of insects, including species that are listed at the federal and state level. Wild bees are a group of insects crucial for maintaining robust plant-pollinator communities. Numerous species of bees are presumed to be in decline. However, baseline community surveys are lacking and needed to document the status of species occupying these natural communities. In 2021, Michigan Natural Features Inventory completed wild bee surveys in lakeplain prairie (lakeplain wet-mesic prairie and lakeplain wet prairie) and prairie fens in Michigan using a combination of aerial netting and bowl trapping. A total of 1,118 wild bees, representing 104 unique species or morphospecies, were collected during
these surveys, including new state records for Dufourea marginata (Cresson, 1878) and Sphecodes nigricorpus Mitchell, 1956 (Hymenoptera: Anthophila). The baseline inventory of wild bees in 2021 provides valuable information on species presence at these locations and suggests that they may act as refuges for rare species of native bees in the state. Continued management of lakeplain prairie and prairie fen natural communities should take into consideration wild bee communities and strive to ensure populations maintain stable numbers.
... Apparently, this phenomenon is quite rare and is discovered accidentally (see, for example, genus Bothrocara in Turanov et al. 2016). Despite the absence of shared haplotypes in such species and clear genetic isolation, standard methods of species delimitation can give ambiguous assessments, and paraphyly can introduce misleading estimates into other methods that rely solely on topological information (e.g., the Yule-coalescent model; see Talavera et al. 2013). Given that uncorrected p-distances yield estimates comparable to those from more complex models for species identification (Collins et al. 2012) and, moreover, according to our data, does not induce topological artifacts, it is appropriate to recommend using this measure in future work in this area. ...
This study provides an updated assessment of the taxonomic and molecular-genetic diversity of fish species in the western Chukchi Sea, employing both traditional and modern research methodologies. The research was conducted aboard the Research Vessel Akademik Oparin in 2016, utilizing various collection methods including trawling and remotely operated vehicles (ROVs). A total of 486 specimens representing 26 species across 8 families were collected and identified. Among them, 97 specimens were partially genotyped using mitochondrial markers COI and 12S rRNA, with some specimens genotyped for one marker only and others for both. Our results confirm that the Arctic Staghorn Goby, Gymnocanthus tricuspis, remains a dominant species with a high frequency of occurrence. High occurrence was also noted for the Hamecon, Artediellus scaber, and the Shorthorn Sculpin, Myoxocephalus scorpius. The study further investigates the genetic distances within and among species, highlighting the efficacy of DNA barcoding in species identification. Phylogenetic analysis revealed distinct clusters that correspond to taxonomic relationships. Both 12S rRNA and COI markers were effective in identifying species within closely related groups, but 12S demonstrated superior performance in cases of degraded DNA samples. The efficacy of this marker highlights its utility in augmenting the global DNA barcode library and enhancing large-scale biodiversity assessments, potentially facilitating the transition to next-generation sequencing technologies. Additionally, the use of ROVs marked a non-intrusive advancement in observing fish distribution, complementing traditional capture methods. This comprehensive approach not only enhances our understanding of Arctic biodiversity, but also contributes to monitoring ecological impacts due to climatic changes.
... Here, GMYC clearly oversplitted our dataset into 41 unrealistic MOTUs. This method is widely disseminated and used in several taxa to discover cryptic diversity using ultrametric gene trees 43 , but these are generally more prone to inference errors than the maximum likelihood ones, and GMYC seems to overestimate the real number of MOTUs with discontinued sampling efforts of populations/species [70][71][72][73] . Not to the same extent, but RESL and TCS also overestimated the real number of MOTUs in our dataset. ...
The Monticola series comprises two anthropophilic and widely distributed species in Brazil: Pintomyia (Pifanomyia) monticola (Costa Lima, 1932) and Pintomyia (Pifanomyia) misionensis (Castro, 1959). They mainly occur in the Atlantic Rainforest, and it is known that Pi. monticola comprises at least two well-structured genetic lineages regarding a fragment of the cytochrome c oxidase subunit I (COI) gene. Here, we aim to elucidate the taxonomic status of this group using integrative taxonomy tools. Collections were performed in nine localities of four Brazilian states, and COI fragments were sequenced and merged with publicly available data. Several single-locus species delimitation algorithms, genetic distance metrics, phylogenetic trees, and haplotype networks were used to uncover cryptic diversity and population structure within Pi. monticola and Pi. misionensis. The resulting genetic clusters were then tested for morphological differences through linear and geometric morphometry of several characters. We analyzed 152 COI sequences, comprising 48 haplotypes. The maximum intraspecific p distances were 8.21% (mean 4.17%) and 9.12% (mean 4.4%) for Pi. monticola and Pi. misionensis, respectively, while interspecific ones ranged from 10.94 to 14.09% (mean 12.33%). Phylogenetic gene trees showed well-supported clades for both species, with clear structuring patterns within them. Species-delimitation algorithms split our dataset into at least three putative species for each taxon. Moreover, population structure analysis showed a strong correlation between Atlantic Forest areas of endemism as sources of molecular variation in Pi. monticola. Morphometric analyses were significant for wing shape variation and some linear measurements (mainly of the head) when comparing specimens of different genetic clusters for both taxa. These results indicate strong genetic structuring of Monticola series species, confirmed by morphometry, indicating two possible cryptic species complexes.
... We specified a constantsize coalescent tree prior under the best models and partitioning scheme suggested by PartitionFinder. Ultrametric trees constructed in BEAST using a strict clock model and a constant-size prior generally perform well in GMYC (Monaghan et al. 2009;Talavera et al. 2013). We ran two independent runs with a length of 100 million generations each and sampling every 1000 generations. ...
... These are often linked to mitochondrial hybrid introgression, incomplete lineage sorting, and malebiased gene flow (e.g. Despres, 2019;McGuire et al., 2007;Moritz & Cicero, 2004;Talavera et al., 2013;Wallis et al., 2017), but can also stem from human factors such as tree inference methods, inaccurate reference taxonomy, under-sampling, or other operational factors (Bergsten et al., 2012;Lim et al., 2012;Meier et al., 2008;Mutanen et al., 2016;Wiemers & Fiedler, 2007). The development of integrative taxonomy concepts and techniques serves to improve the issue of species misidentification caused by reliance on single data sources (Borges et al., 2016;Collins & Cruickshank, 2013;Orr et al., 2022;Spiers et al., 2022;Will et al., 2005;Wright et al., 2019). ...
Recent advances in DNA barcoding have immeasurably advanced global biodiversity research in the last two decades. However, inherent limitations in barcode sequences, such as hybridization, introgression or incomplete lineage sorting can lead to misidentifications when relying solely on barcode sequences.
Here, we propose a new Niche‐model‐Based Species Identification (NBSI) method based on the idea that species distribution information is a potential complement to DNA barcoding species identifications. NBSI performs species membership inference by incorporating niche modelling predictions and traditional DNA barcoding identifications.
Systematic tests across diverse scenarios show significant improvements in species identification success rates under the newly proposed NBSI framework, where the largest increase is from 4.7% (95% CI: 3.51%–6.25%) to 94.8% (95% CI: 93.19%–96.06%). Additionally, obvious improvements were observed when using NBSI on potentially ambiguous sequences whose genetic nearest neighbours belongs to another species or more than two species, which occurs commonly with species represented by single or short DNA barcodes.
These results support our assertion that environmental factors/variables are valuable complements to DNA sequence data for species identification by avoiding potential misidentifications inferred from genetic information alone. The NBSI framework is currently implemented as a new R package, ‘NicheBarcoding’, that is open source under GNU General Public Licence and freely available from https://CRAN.R‐project.org/package=NicheBarcoding.
... For both GMYC and PTP, the initial methods paved the way to subsequent refinements. In the case of GMYC, a multiple-threshold approach was developed that allowed variability in the depth of transition between coalescent speciation events (75), but subsequent tests generally revealed that this model did not outperform the single-threshold method (29,120). In the case of PTP, a multi-rate model (mPTP) was developed that allowed for variability in the distribution of branching events in different species units (47). ...
Most insects encountered in the field are initially entomological dark matter in that they cannot be identified to species while alive. This explains the enduring quest for efficient ways to identify collected specimens. Morphological tools came first but are now routinely replaced or complemented with DNA barcodes. Initially too expensive for widespread use, these barcodes have since evolved into powerful tools for specimen identification and sorting, given that the evolution of sequencing approaches has dramatically reduced the cost of barcodes, thus enabling decentralized deployment across the planet. In this article, we review how DNA barcodes have become a key tool for accelerating biodiversity discovery and analyzing insect communities through both megabarcoding and metabarcoding in an era of insect decline. We predict that DNA barcodes will be particularly important for assembling image training sets for deep learning algorithms, global biodiversity genomics, and functional analysis of insect communities.
... One common application of feature extraction is the problem of automatic classification of individuals to species (Fassnacht et al., 2016;Spampinato et al., 2010). The individuals grouped into self-similar clusters, and then often mapped back to manual taxonomic classifications as species, comprise distinctive sets of genetic (Pons et al., 2006;Talavera et al., 2013) or phenotypic traits (Pearson & Ezard, 2014). ...
The rapid and repeatable characterization of individual morphology has advanced automated taxonomic classification. The most direct study of evolutionary processes is, however, not from taxonomic description, but rather of the evolution of the traits that comprise individuals and define species. Repeatable signatures of individual morphology are crucial for analysing the response to selection at scale, and thus tracking evolutionary trajectories through time and across species boundaries.
Here, we introduce our 3DKMI—an open‐source MATLAB package designed for the study of morphology using three‐dimensional (3D) Krawtchouk moment invariants. The volumetric features derived from the 3D images remain stable under translation, scaling and rotation and, for an image of size 128 × 128 × 128 can be computed in less than 0.1 s.
We applied our package as a case study on a collection of 300 X‐ray computed tomography scans of planktonic foraminifera specimens across five species to (1) assess the invariance of the features under different transformations and (2) analyse morphological differences among species based on the extracted characteristics.
We show that 3DKMI has the capacity to efficiently and repeatedly characterize the signatures of individual morphology. In the future, we hope that the 3D feature extraction technique 3DKMI will be widely applied to digital collections to advance research in ecology and evolution.
... This over-splitting phenomenon has been previously observed in Gaidropsarus (Barros- . Collapsing sequences to haplotypes is a common approach for GMYC analyses for less-demanding computational methods, and there is no evidence of different results when noncollapsed and collapsed datasets are compared (Talavera et al., 2013). ...
Gaidropsarus mauritanicus sp. nov. is described from one specimen collected using a grab sample from the Tanoûdêrt Canyon (ca. 20° N) off Mauritania at a depth of 595 m. The new species was further observed during eight remotely operated vehicle (ROV) dives along the Mauritanian slope southwards down to the Tiguent Coral Mound Complex (~17° N) in a bathymetric range between 613 and 416 m. It can be distinguished from congeners by a combination of characteristics, including large eyes (38.1% head length [HL]), large head (25.8% standard length [SL]), elongated pelvic fins (35.7% SL), low number of vertebrae (44), and coloration (pinkish with a dorsal darker brownish hue and bright blotches along the dorsal‐fin base). A species‐delimitation analysis performed with available cytochrome c oxidase subunit 1 (COI) sequences affiliated to the genus Gaidropsarus additionally supported the validity of the new species. Video analyses showed a deep‐water coral‐associated and protection‐seeking behavior, which may explain why the species has remained undescribed until now. Additional ROV footage from separate deep‐water coral sites in the North Atlantic and Mediterranean Sea further highlights the ecological behavior and hidden diversity of bathyal three‐bearded rocklings. Here, we additionally discuss the biogeographical distribution of all genetically verified Gaidropsarus spp. in combination with genetic data and morphological characters. G. mauritanicus sp. nov. is closely related to a species from Tasmania (43° S), a geographical point furthest among the studied samples, which may hint to an important influence of (paleo‐) oceanography on the distributions of Gaidropsarus species.
... The results of molecular species delimitation analyses (ASAP and PTP) showed an overall tendency towards over-splitting in the latter method. Previous studies have shown a decrease in delimitation accuracy for PTP method when applied to the unevenly sampled datasets [47,51,52], and it appears that the sampling imbalances across taxa in our dataset has similarly resulted in an over-estimation of number of taxa. ...
Even though the high plateaus of Qinghai-Tibet and Iran share many faunal elements, the historical biogeography of the species present in this area are not very well understood. We present a complete COI barcode library for Aporia Hübner and a first comprehensive phylogeny for the genus including all known species and majority of subspecies using ten available genes (COI-COII, ND1, ND5, Cytb, EF-1a, Wg, 16S, 28S-D2/D3 and 28S-D8). We then focus on A. leucodice (Eversmann, 1843) and related taxa in order to resolve some long-standing taxonomic issues in this species-group. Based on DNA sequence data as well as morphology, we raise Aporia illumina (Grum-Grshimailo 1890) stat. nov. (= pseudoillumina Tshikolovets 2021 syn. nov.) as a distinct species and designate a lectotype; synonymize Aporia leucodice leucodice Eversmann, 1843 (= A. l. morosevitshae Sheljuzhko, 1908 syn. nov.); and describe a new species, Aporia ahura sp. nov., from the Central Alborz Mountains in northern Iran.
... The molecular operational taxonomic units (MOTUs) obtained by the GMYC method are more numerous than those determined by ABGD method and ASAP method. Empirical studies have revealed that the GMYC approach tends to over-split species compared to alternative methods of species delimitation (Esselstyn et al. 2012;Paz & Crawford 2012;Sauer & Hausdorf 2012;Talavera et al. 2013;Siddiqui et al. 2019). Despite occupying right positions in the phylogenetic tree and exhibiting high node support, certain species (e.g. ...
Oribatid mites (Acari, Oribatida) are a large group of soil animals, with small body sizes and complex morphological structures. At present, oribatid mite species identification is essentially based on morphological characters. In this study, we collected 124 specimens of oribatid mites belonging to 110 species of 72 genera in Russia, and extracted their genomic DNA subsequently. In order to investigate whether the molecular data (DNA-barcoding marker, COI gene) and morphological classification results are consistent, we used three methods, ASAP, ABGD and GMYC, to delimit species, and calculate a maximum likelihood tree. The results indicate that COI gene fragments can be effectively used for species delimitation. There are some cryptic species identified in oribatid mites, which do not have distinct morphological differences, but diverge considerably in molecular data. Meanwhile, some formerly unidentified species could be identified through molecular means. The study enriches the molecular data of oribatid mites worldwide and provides the basis for more detailed taxonomic studies of oribatid mites in Russia. In the future, more analyses and mining of molecular data, combined with morphological analyses, will help to reveal and further understand the phylogenetic relationships of Oribatida.
... Briefly, we partitioned the ITS into the ITS1, 5.8S and ITS2 regions and the remaining markers into their intronic and exonic regions and carried out the BEAST analysis using a lognormal uncorrelated relaxed clock, the GTR + I + G substitution model and a random starting tree. We selected the 'Coalescent: Constant Size' Tree Prior, following Talavera et al. (2013) and having previously compared different tree priors with Bayes Factors. bPTP was run on https://species.h-its.org/ ...
Oceanic islands have been recognized as natural laboratories in which to study a great variety of evolutionary processes. One such process is evolutionary radiations, the diversification of a single ancestor into a number of species that inhabit different environments and differ in the traits that allow them to exploit those environments. The factors that drive evolutionary radiations have been studied for decades in charismatic organisms such as birds or lizards, but are lacking in lichen-forming fungi, despite recent reports of some lineages showing diversification patterns congruent with radiation. Here we propose the Ramalina decipiens group as a model system in which to carry out such studies. This group is currently thought to be comprised of five saxicolous species, all of them endemic to the Macaronesian region (the Azores, Madeira, Selvagens, Canary and Cape Verde islands). Three species are single-island endemics (a rare geographic distribution pattern in lichens), whereas two are widespread and show extreme morphological variation. The latter are suspected to harbor unrecognized species-level lineages. In order to use the Ramalina decipiens group as a model system it is necessary to resolve the group's phylogeny and to clarify its species boundaries. In this study we attempt to do so following an integrative taxonomy approach. We constructed a phylogenetic tree based on six molecular markers, four of which are newly developed and generated competing species hypotheses based on molecular (species discovery strategies based on both single locus and multilocus datasets) and phenotypic data (unsupervised clustering algorithms based on morphology, secondary chemistry and geographic origin). We found that taxonomic diversity in the Ramalina decipiens group has been highly underestimated in previous studies. In consequence, we describe six new species, most of them single-island endemics and provide a key to the group. Phylogenetic relationships among species have been reconstructed with almost full support which, coupled with the endemic character of the group, makes it an excellent system for the study of island radiations in lichen-forming fungi.
... We used three automatic delimitation methods to infer species boundaries among bluefish haplogroups observed in this research. The methods were: automatic barcode gap discovery -ABGD test (Puillandre et al., 2012) which computation is based on gap-distance between intra-and interspecific lineages, and the Bayesian Phylogenetics and Phylogeography test -BP&P (Yang, 2015), and the Generalized Mixed Yule Coalescent model -GMYC (Michonneau, 2016) which are likelihood methods based on the detection of shifts between Yule (interspecific) and coalescence (intraspecific) processes to define whether branches on a tree denotes species or populations (Talavera & Dinca, 2013). ...
Neglected cryptic diversity can lead to the permanent loss of locally adapted alleles, which can reduce resilience to rapid environmental change. It can also result in overestimation of fisheries stock sizes that can result from treating different species as if they belonged to one. Bluefish ( Pomatomus saltatrix ) is considered a circumtropical and subtropical species and an important fishery resource all over the world. Differences in ecologically relevant traits are observed among isolated populations. Also, in the Southwestern Atlantic, molecular data suggest multiple populations, but these are treated as a single fish stock by regulatory agencies due to a lack of definitive information. We used whole mitogenome sequences, nuclear ( rho ) and mitochondrial ( coxI and cytb ) genes, as well as microsatellites to investigate historical and current genetic population structure and parameters of bluefish in the Western Atlantic. A total of 263 samples were collected along the Brazilian coast and in the USA (New Jersey, Northwest Atlantic). Data revealed the existence of two evolutionarily significant units (ESU) of bluefish along the South American coast, later confirmed by whole mitogenome sequencing of both haplogroups. These two ESUs have a mostly parapatric distribution, with some areas of overlap, which vary along the year. We also conducted seasonal sampling in Brazil to investigate migration patterns. ESUs occur mostly north and south of parallel 23° 40′ S, with an overlap area that varied seasonally. The level of differentiation between those two ESUs in the SW Atlantic, even in sympatry, is as high as that found between them and those from the NW Atlantic and Europe. Parapatric distribution and restricted gene flow suggest the existence of ecological barriers and local adaptation. The splitting of an ancient population from the Southwestern Atlantic into two putative species is important to understand bluefish evolutionary diversification and has implications for fishery regulatory measures in Brazil.
... In contrast to distance-based methods, GMYC and bPTP are topology-based methods that require an estimated gene tree as input data. Factors influencing the performance of GMYC involves the effective population size and divergence times between species [58,59], phylogenetic methods, taxon level and sampling coverage [53,60,61], and migration rates among populations [62]. Some earlier studies have shown that the performance of these methods is primarily affected by the ratio of population sizes to species divergence times [63], and the two methods often over-split (false-positive) species [48,56,64,65]. ...
An accurate assessment of species diversity is a cornerstone of biology and conservation. The lynx spiders (Araneae: Oxyopidae) represent one of the most diverse and widespread cursorial spider groups, however their species richness in Asia is highly underestimated. In this study, we revised species diversity with extensive taxon sampling in Taiwan and explored species boundaries based on morphological traits and genetic data using a two-step approach of molecular species delimitation. Firstly, we employed a single COI dataset and applied two genetic distance-based methods: ABGD and ASAP, and two topology-based methods: GMYC and bPTP. Secondly, we further analyzed the lineages that were not consistently delimited, and incorporated H3 to the dataset for a coalescent-based analysis using BPP. A total of eight morphological species were recognized, including five new species, Hamataliwa cordivulva sp. nov., Hamat. leporauris sp. nov., Tapponia auriola sp. nov., T. parva sp. nov. and T. rarobulbus sp. nov., and three newly recorded species, Hamadruas hieroglyphica (Thorell, 1887), Hamat. foveata Tang & Li, 2012 and Peucetia latikae Tikader, 1970. All eight morphological species exhibited reciprocally monophyletic lineages. The results of molecular-based delimitation analyses suggested a variety of species hypotheses that did not fully correspond to the eight morphological species. We found that Hamat. cordivulva sp. nov. and Hamat. foveata showed shallow genetic differentiation in the COI, but they were unequivocally distinguishable according to their genitalia. In contrast, T. parva sp. nov. represented a deep divergent lineage, while differences of genitalia were not detected. This study highlights the need to comprehensively employ multiple evidence and methods to delineate species boundaries and the values of diagnostic morphological characters for taxonomic studies in lynx spiders.
... GMYC is a single-locus species delimitation method. It employs an ultrametric tree to detect a shift from a Yule speciation process (one lineage per species) to an intraspecific coalescent process (multiple lineages per species) (Pons et al. 2006;Talavera et al. 2013) and uses this shift to delimit species. An ultrametric tree of COI data was inferred using BEAST 2.6.3, with site models unlinked, bModeltest to infer site models and a birth-death tree prior. ...
Life history characteristics of cryptobenthic reef fishes (CRFs), such as their fast growth and reproductive rates, near-shore larval retention and high turnover, predispose these species to rapid diversification and cryptic speciation. This may be particularly true for isolated CRF populations. The sailfin blenny, Emblemaria pandionis, is widespread throughout the Caribbean and is found on the reefs of the Flower Garden Banks, an isolated reef system in the NW Gulf of Mexico. Using DNA barcoding and multi-locus delimitation, we show that E. pandionis is a species complex comprised of at least four distinct taxonomic units in the Caribbean, one at the Flower Garden Banks, a second in eastern Florida, a third in the central Caribbean and a fourth in Curaçao. The phylogenetic relationships within the E. pandionis species complex are defined by well-established phylogeographic barriers to gene flow in the Caribbean. Populations of E. pandionis from eastern Florida are genetically distinct from populations found in the Bahamas, highlighting the role of the Florida Strait as a strong barrier to gene flow and populations in Curaçao are deeply divergent from all other populations sampled in the Caribbean. Results from this study highlight the roles of isolated reefs and ocean currents in the speciation of cryptobenthic reef fishes.
... For OTU delineation, the full sequence set was collapsed to represent unique full-length sequences plus outgroups, which were trimmed to the same length (622 bp). The removal of identical haplotypes helps computational efficiency without influencing delimitation of species (Talavera et al., 2013). Two putative species delineation approaches were then used to diagnose OTUs for analysis. ...
Metacommunity theory predicts that the relative importance of regional and local processes structuring communities will change over ecological succession. Determining effects of these processes on taxonomic and evolutionary diversity in spatially structured freshwater habitats of different successional stages may greatly improve understanding of the maintenance of diversity across temporal and spatial scales. In this study, we evaluated crayfish diversity at local and regional scales in pond metacommunities undergoing secondary succession from beaver ( Castor canadensis ) disturbance. Following theoretical predictions from metacommunity ecology of the increasing importance of local processes over succession, we hypothesised a decline in crayfish local and β diversity over succession from stronger local structuring as the older ponds may provide less suitable habitat than streams.
Crayfish species and phylogenetic diversity were evaluated in beaver pond metacommunities and reference headwater streams located in three catchment regions. DNA sequences from the mitochondrial cytochrome oxidase I gene were used to assign crayfish to species for community and phylogenetic diversity tests. Local and β diversity were contrasted across beaver ponds ranging in age from 24 to 70 years and as a function of metacommunity processes.
Counter to predictions, local species diversity among streams and the successional stages of ponds categorised by age class (24–39 years; 42–57 years; 60–70 years) did not differ, but community and phylogenetic convergence occurred in the oldest pond ecosystems. Crayfish community composition differed between the youngest and oldest ponds, resulting from higher abundance in the youngest ponds and community convergence in the oldest ponds. The association between community composition and the environment was strongest in streams and decoupled with pond age. In contrast, the correlation between intraspecific haplotype composition and the environment increased over succession. Among the three metacommunities, the regional crayfish species diversity arose from a combination of the temporal and environmental drivers from beaver‐constructed ecosystems and dispersal limitation within catchments.
This study represents the first investigation of the taxonomic and phylogenetic diversity response to the successional stages of beaver pond metacommunities. The detection of differential crayfish composition and haplotype sorting to pond age suggests a role for local structuring and further indicates that future studies should acknowledge succession in shaping species diversity at local and regional scales. Dispersal limitation within catchment regions probably contributes to the evolution of crayfish species diversity in metacommunities and the overall maintenance of biodiversity.
The results support a transition in community and freshwater ecology from a recent emphasis on spatial processes towards the integration of temporal drivers to better identify regulators of taxonomic and phylogenetic diversity across scales.
... In contrast, the three methods were in agreement on the assignment of the specimen of S. microps (KP722779) to S. brasiliensis, the allocation of S. menesezi and S. gomezi to the same MOTU, and the differentiation of lineages within both O. dentex and B. chrysoura, as well as the allocation of O. xanthops and C. macrops to a single MOTU. Therefore, while a single locus delimitation analysis does have limitations in detecting incomplete lineage sorting, recent hybridization or speciation [21,41] and possible incongruities between the gene and species trees [42], the congruence of the results of the different approaches, which are based on distinct analytical assumptions and theoretical premises, are indicative of the robustness of the delimited MOTUS. In this case, the findings of the present study indicate that Stelliferinae has undescribed diversity, and that the taxonomy of the subfamily should be revised. ...
Stelliferinae is the third most speciose subfamily of Sciaenidae, with 51 recognized species arranged in five genera. Phylogenies derived from both morphological and molecular data support the monophyly of this subfamily, although there is no general consensus on the intergeneric relationships or the species diversity of this group. We used the barcoding region of the cytochrome oxidase C subunit I (COI) gene to verify the delimitation of Stelliferinae species based on the Automatic Barcode Gap Discovery (ABGD), Generalized Mixed Yule Coalescence (GMYC), and Bayesian Poisson Tree Process (bPTP) methods. In general, the results of these different approaches were congruent, delimiting 30–32 molecular operational taxonomic units (MOTUs), most of which coincided with valid species. Specimens of Stellifer menezesi and Stellifer gomezi were attributed to a single species, which disagrees with the most recent review of this genus. The evidence also indicated that Odontoscion xanthops and Corvula macrops belong to a single MOTU. In contrast, evidence also indicates presence of distinct lineages in both Odontoscion dentex and Bairdiella chrysoura. Such results are compatible with the existence of cryptic species, which is supported by the genetic divergence and haplotype genealogy. Therefore, the results of the present study indicate the existence of undescribed diversity in the Stelliferinae, which reinforces the need for an ample taxonomic review of the fish in this subfamily.
... The ultrametric tree used as input for GMYC was generated in BEAST, following the same procedure described earlier. However, a reduced dataset was utilized, in which outgroups were excluded and haplotypes were pruned (see Talavera et al. 2013) using Collapsetypes ver. 4.6 (Chesters 2013). ...
A new species belonging to the genus Habrophlebia Eaton, 1881 is described at the nymphal stage from the Rif Mountains of Morocco. This species presents unique features , such as the chorionic arrangement of the egg and the ornamentation of the posterior margin of abdominal tergites. It is compared to all west European Habrophlebia species and a table with discriminating characters is given. A phylogenetic reconstruction based on COI sequences fully supports the hypothesis of a new species in the Rif Mountains, with possible further distribution in southern Spain.
... Absolute diversity is very high, with a per-site average of 127 species for just our focal groups (that is, all arthropods excluding Acari and Myriapoda), ranging from 86 to 199. Given that most of the oversplitting by automated species delimitation methods subdivides widely distributed but weakly divergent lineages (Mason et al., 2020;Talavera et al., 2013), we would not expect single-site species counts to be particularly inflated, though instances of oversplitting across sites, where multiple populations were sampled, might be expected. ...
Developing systematic conservation plans depends on a wealth of information on a region's biodiversity. For ‘dark taxa' such as arthropods, such data are usually very incomplete and in most cases left out from assessments.
Sky islands are important and often fragile biodiversity hotspots. Southern Appalachian high‐elevation spruce–fir forests represent a particularly threatened sky‐island ecosystem, hosting numerous endemic and threatened species, but their arthropods remain understudied.
Here we use voucher‐based megabarcoding to explore genetic differentiation among leaf‐litter arthropod communities of these highlands, and to examine the extent to which they represent dispersed communities of more or less coherent species, manageable as a distributed unit. We assembled a dataset comprising more than 6000 COI sequences representing diverse arthropod groups to assess species richness and sharing across peaks and ranges. Comparisons were standardised across taxa using automated species delimitation, measuring endemism levels by putative species.
Species richness was high, with sites hosting from 86 to 199 litter arthropod species (not including mites or myriapods). Community profiles suggest that around one fourth of these species are unique to single sky islands and more than one third of all species are limited to a particular range. Across major taxa, endemicity was lowest in Araneae, and highest in neglected groups like Isopoda, Pseudoscorpionida, Protura and Diplura.
Southern Appalachian sky islands of spruce–fir habitat host significantly distinct leaf‐litter arthropod communities, with high levels of local endemicity. This is the first work to provide such a clear picture of peak and range uniqueness for a taxonomically broad sample. Ensuring the protection of a sizeable fraction of high‐elevation litter species richness will therefore require attention at a relatively fine spatial scale.
... Further sampling of the species yielding inconsistent results among delimitation methods may be needed to confirm the extent of genetic variation among populations. Differences in speciation rates, population size and sampling bias may cause over-or underestimation of the number of species (Pentinsaari et al., 2017;Renner et al., 2017;Talavera et al., 2013). The multi-locus method of BPP has been shown to be more reliable than single-locus methods for species delimitation (Leaché et al., 2016;Xu et al., 2022;Yang & Rannala, 2010) and usually better reflects observations of morphological divergence Yang & Rannala, 2017). ...
The genus Chanohirata Hayashi & Machida, including the herein synonymized monotypic genus Reticuluma Cheng & Li, is the second most speciose genus in the tribe Penthimiini after Penthimia Germar and is almost exclusively endemic to China. This study presents the first phylogeny of Chanohirata based on three mitochondrial markers (cytochrome c oxidase subunit I and II and 16S ribosomal RNA) and one nuclear fragment (28S ribosomal RNA). Reticuluma syn. n. is recovered nested within Chanohirata with strong support. Reticuluma is, therefore, proposed as a junior synonym of Chanohirata , and a new combination is proposed: Chanohirata citrana (Cheng & Li) comb. n. Molecular species delimitation analyses were conducted by combining six molecular delimitation methods with morphological evidence. The results of the species delimitation analyses confirm the status of eight described species of Chanohirata and support the recognition of three new species: Chanohirata cornicula Wang & Zhang sp. n. , Chanohirata elongata Wang & Zhang sp. n. and Chanohirata serrata Wang & Zhang sp. n. Molecular dating and biogeographic analyses suggest that Chanohirata likely originated during the late Eocene in southern China. During the late Miocene to early Pleistocene, the uplift of the Himalayas and Tibetan Plateau and climatic oscillations probably triggered several dispersal and vicariance events in Chanohirata lineages, leading to most speciation events.
... In this study, GMYC clearly oversplits P. davisi sequences, partitioning the dataset into 10 unrealistic MOTUs. This method may often overestimate the real number of species and is less conservative than PTP and ABGD (Pentinsaari et al., 2017;Talavera et al., 2013). In addition, GMYC needs ultrametric trees as input to delimit MOTUs, which are generally more prone to inference errors, making PTP a more reliable tree-based method depending on the analysed dataset (Tang et al., 2014). ...
In this study, we analysed the molecular and morphometric differences of several populations of the putative sand fly vector Psychodopygus davisi (Root, 1934) (Diptera, Psychodidae, Phlebotominae) in Brazil. We amplified the 658 base pair fragments of the DNA barcoding region-cytochrome c oxidase subunit 1 (COI) gene-for 57 specimens of P. davisi and three specimens of Psychodopygus claustrei (Abonnenc, Léger & Fauran, 1979). We merged our data with public sequences of the same species available from GenBank. Then, the combined dataset-87 sequences and 20 localities-was analysed using population structure analysis and different species delimitation approaches. Geometric morphometry of wings was performed for 155 specimens of P. davisi populations from the North, Midwest and Southeast Brazilian regions, analysing the differences in centroid sizes and canonical variates. Molecular analysis indicated high intraspecific genetic distance values for P. davisi (maximum p distance = 5.52%). All algorithms identified P. davisi and P. claustrei as distinct molecular taxonomic units, despite the low inter-specific distance (p distance to the nearest neighbour = 4.79%). P. davisi sequences were split into four genetic clusters by population structure analysis and at least five genetic lineages using intermediate scenarios of the species delimitation algorithms. The species validation analysis of BPP strongly supported the five-species model in our data-set. We found high genetic diversity in this taxon, which is in agreement with its wide geographic distribution in Brazil. Furthermore, the wing analysis showed that specimens from the Southeast Region of Brazil are different from those in the North and the Mid-west. The evolutionary patterns of P. davisi populations in Brazil suggest the presence of
... Exploratory GMYC (General Mixed Yule-Coalescent model: Pons et al., 2006;Fontaneto et al., 2007;Fujisawa & Barraclough, 2013) species delimitation analyses were previously done by G. Talavera for TEA20 (using methods described in Talavera et al. (2013) and based on the total dataset), but results remained unpublished. With kind permission from G. Talavera, these results are shown in Fig. S4. ...
The molecular data of Talavera et al. (2020) is re-analysed to provide a foundation for a taxonomic revision of Chrysoritis. A COI phylogeny recovers most of the Chrysoritis species as monophyletic, and a few as polyphyletic but supported by the CAD gene. In the thysbe clade, most species, despite occurring in sympatry with at least one other species in the clade, maintain distinctive wing facies and ecological identity without intermediate forms. Within the thysbe clade, which contains the majority of species, sister taxon comparisons based on the COI phylogeny suggest speciation has been predominantly allopatric and accompanied mostly by minor morphological change and sometimes also a change in male patrolling terrain and/or host ant species. The diversification of the thysbe clade and the taxonomic implications of our results are discussed.
... This model was then modified to include Bayesian support (bPTP) and the potential divergence in intraspecific diversity (mPTP) (Zhang et al. 2013;Kapli et al. 2017). All these coalescent single-gene species delimitation methods have been proved to be effective on simulations and empirical data (Talavera et al. 2013;Tang et al. 2014), being widely used to assess biodiversity (Pons et al. 2006;Machado et al. 2017;Hofmann et al. 2019;Ramirez et al. 2020bRamirez et al. , 2021Cañedo-Apolaya et al. 2021). ...
DNA barcoding is a powerful tool to identify species using nucleotide differences found in the cytochrome oxidase I mitochondrial gene between species. Since it was proposed, several projects are promoting the construction of a DNA barcode library for all eukaryotes. DNA barcoding in conjunction with single-gene species delimitation methods have been used to estimate diversity at species level, allowing a rapid and comprehensive assessment of biodiversity. These methods have been developed with the purpose of clustering data sets of orthologous sequences in molecular operational taxonomic unit (MOTU). The MOTUs obtained by these methods could be compared with the taxonomic data to discover hidden diversity. In this chapter we discuss the main concepts and methodologies on DNA barcoding approach and review the use of species delimitation methods in the Neotropics. The huge biodiversity, lack of taxonomic effort and scarcity of funds are still challenging the broad use of this technology in the Neotropics.KeywordsCOIMOTUBarcode of lifeHidden biodiversity
... Input BI ultra-metric tree for GMYC was generated in BEAST 1.10.4. . To avoid potential biases in threshold estimation, the identical COI haplotypes were pruned (see Talavera et al. 2013) using Collapsetypes 4.6 (Chesters 2013). Input BEAST file was created in BEAUTi 1.10.4 , implementing the best model of evolution and the partition scheme specified above, and selecting a relaxed molecular clock (uncorrelated lognormal) model, a coalescent (constant size) prior (see Monaghan et al. 2009) and a UPGMA starting tree. ...
Investigations of material collected partly in 1999 and mainly between 2006 and 2016 in New Guinea, mostly along the high, central mountain chain of the island, further increased our knowledge of the diversity of the genus Labiobaetis Novikova & Kluge on this island. Previously, 37 species were reported from New Guinea. We have identified six new species using a combination of morphology and genetic analysis (COI). They are described and illustrated based on their larvae. Five of the six new species belong to the group petersorum , which is endemic to the island. Additionally, Labiobaetis xeniolus Lugo-Ortiz & McCafferty is also assigned to this group. The morphological characterisation of the group petersorum is enhanced, and a key to all species of this group is provided. Complementary descriptions and remarks to the morphology of known species of the group petersorum are provided. Additionally, a genetic analysis (COI) including most species and several additional Molecular Operational Taxonomic Units (MOTUs) of the group petersorum is discussed. One of the new species belongs to the group vitilis . The morphological characterization of this group is slightly enhanced, and the obtained COI sequence was added to the genetic analysis of the group petersorum . The total number of Labiobaetis species worldwide is augmented to 162.
... superfamily level) (Figure 2b). Similar results have been reported for Coleoptera and Lepidoptera (Ahrens et al., 2016;Talavera et al., 2013). Alternatively, high rates of perfect matches could be explained by the nearly 19-fold higher difference between average nearest-neighbour than intraspecies divergence (Yin et al., 2022). ...
Single‐locus molecular delimitation plays a key role in meeting the need to expedite the exploration and description of the species on our planet. Multiple methods have been developed to aid data interpretation over the past 20 years, but species delimitation remains difficult due to their varying performance. In this study, we examine the accuracy of five widely used delimitation methods (i.e. BIN, ABGD, ASAP, GMYC and mPTP) in analysing 63 empirical data sets that included 1850 mitochondrial COI sequences derived from eriophyoid mites assigned to 456 morphospecies. Our results establish that all five methods resolve approximately 90% of morphospecies. We investigated some factors which might affect the species delimitation results, that is taxonomic rank, number of haplotypes per species, mean number of host plants per species, and geographical distance among sampling sites. We found complex interactions between these factors which affected delimitation effectiveness. An increase in haplotype number negatively affected delimitation accuracy, while increased geographical distance improved delimitation accuracy. BIN was influenced by the number of host plants per species as cryptic speciation linked to host plant usage might be prevalent in eriophyoid mites, while ABGD was not significantly impacted by other factors. Our results highlight multiple factors that affect molecular species delimitation and underline the value of employing multiple analytical approaches to aid species delimitation.
... GMYC has recently become one of the most frequently used delimitation methods, and just 'splits' realization is widely used in frames of the "traditional model" [34,92], because of its "just add water" approach. However, it is known that such delimitation results in excessive "splitting" [95]. Moreover, GMYC has a limitations in its objective representation of data and biological uncertainty of models [96]. ...
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.
... Using a fixed and arbitrary value for the distance thresholds between species for high taxonomic levels (e.g., 2% by Herbert et al. 2003) is considered a naive mistake for DNA barcoding uses (Collins and Cruickshank 2013). Plausible values may be obtained by accumulating data on a target taxon (e.g., Talavera et al. 2013;Gonçalves et al. 2021;Bianchi and Gonçalves 2021a), since the coalescent depths among species are variable in each lineage (Fujita et al. 2012). The 7% genetic distance between G. pulchra and G. anthracina seems acceptable to consider them different species since 6% has been considered an applicable threshold for Theraphosidae (Hamilton et al. 2011). ...
Taxonomic researchers have used multiple sources of evidence to support species hypotheses and delimitations. Grammostola Simon (Mygalomorphae: Theraphosidae) comprises 20 valid species endemic to South America, six occurring in Brazil. The classical morphological approach based mainly on genitalia may be misleading in recognizing species in this genus. Thus, we used morphology, geographical distribution, genetic distance, and phylogeny to support the redescription of Grammostola pulchra from southern Brazil, a species described a century ago. We also diagnosed and illustrated the species. Males have a developed apical keel at the apex of the embolus; for the first time, this type of structure has been reported in a species of Grammostola. The molecular analyses using the partial sequence of Cytochrome c oxidase subunit I showed 7% of genetic distance (p-distance) between G. pulchra and Grammostola anthracina. Distance and tree-based methods (ASAP and bPTP, respectively) assigned G. pulchra as a valid species. The gene-tree under Bayesian and Maximum-Likelihood recovered a similar topology, placing G. pulchra as closely related to Grammostola burzaquensis and G. anthracina. Morphological characters which could be important in the taxonomy of the genus are further discussed. Citation: Pittella RS, Bassa PG, Zefa E, Bianchi FM. 2023. Using the integrative approach to update a gap of one century: redescription and new distribution records of the South American Tarantulas Grammostola pulchra (Araneae: Mygalomorphae: Theraphosidae). Zool Stud 62:05.
... In this study, several commonly used molecular species delimitation methods were applied. Compared with morphological results, different analyses were conducted using two different barcoding fragments potentially over-or underestimating the number of putative species [78], yet here the results estimated by GMYC for the COI tree were congruent with the number of species identified morphologically. One new species was identified and four previously described species (supported by morphological diagnosis) were confirmed based on different species delimitation methods. ...
One new species of the genus Dendronotus (Nudibranchia: Dendronotidae) is described from Norway and Northern Ireland, as well as from the adjacent North Sea, and one new subspecies of Dendronotus arcticus is described from Norway by applying a combination of fine-scale morphological and molecular phylogenetic data. The present case demonstrates multilevel morphological and molecular similarities and differences considering on the one hand a grouping of three similar looking sympatric taxa (D. yrjargul, D. arcticus gartensis n. subsp. and D. keatleyae n. sp.), and on the other hand two different looking apparently allopatric subspecies (D. arcticus arcticus and D. arcticus gartensis n. subsp.). The type species of the genus, D. frondosus, which is the commonest dendronotid in Norway and the United Kingdom, consistently demonstrates substantial molecular and fine-scale morphological differences from D. keatleyae n. sp. The present study, apart from providing purely taxonomic information, also provides new data for a broad discussion of global biodiversity patterns.
... The delimitation category of a given taxon (and thus their regional incidence) may depend on the size of the region where it has been studied. In general, the same species can appear as a single entity if the assessment is made at a narrow geographic scale and as a multiple or lumped entity for wider geographic sampling areas (Talavera et al., 2013). We assessed the delimitation categories for the Samoan butterflies at two different scales: worldwide (W in Table 1) and Australian and Oceanian regions (AO in Table 1) as described by Holt et al. (2013) (Australia, New Zealand, Samoan Is., Polynesia, Papua New Guinea-Melanesia and Micronesia). ...
We investigated the entire butterfly fauna of the Samoan Archipelago (Pacific Ocean) by combining COI barcode sequences for specimens from these islands with those available in repositories at larger biogeographic scale. Haplotype networks and a generalized mixed Yule-coalescent (GMYC) model were applied to identify evolutionary significant units (ESUs). The ESUs from Samoan islands were compared with ESUs of the same or sister taxa regionally and worldwide to explore the level of endemicity and of congruence between established taxonomy and COI barcodes. The level of ESUs endemicity was similar to that shown by species and subspecies. Australia was the most frequent origin for Samoan line-ages, followed by Orient-Asia. When comparing the agreement and mismatch between established taxonomy and ESUs between the Australia-Oceania region and Europe and North America, the COI molecular marker revealed a similar performance in taxonomic identification. Despite this overall convergent pattern, the degree of mtDNA divergence and the analysis of functional traits suggested that the mechanisms producing patterns of genetic differentiation in temperate butterflies over ancient continental lands differ to those occurring across a vast ocean into geologically young islands. Mechanisms on Samoan islands include relatively recent and exceptional oceanic dispersal, possibly followed by repeated extinction events. In the Australia-Oceania region we found a similar fraction of species showing introgression with the maintenance of phenotypic differences as it occurs on the mainland, but the phenomenon was limited to sectors of each species distribution area. Regular gene flow among the Samoan islands seems to prevent allopatric speciation within the archipelago.
In this study, we reassess the phylogenetic relationships of the genus Sesamia Guenée, 1852 and examine in more detail the members of the nonagrioides species group, for which three distinct species complexes are identified. The calamistis subgroup comprises eight species, of which four new species are described: Sesamia kabirara Le Ru sp. nov., Sesamia kalale Le Ru sp. nov., Sesamia mapalense Le Ru sp. nov. and Sesamia teke Le Ru sp. nov. The incerta subgroup consists of 11 species, of which four new species are described: Sesamia kamba Le Ru sp. nov., Sesamia lalaci Le Ru sp. nov., Sesamia lusese Le Ru sp. nov. and Sesamia msowero Le Ru sp. nov. The nonagrioides subgroup comprises ten species of which two new species are described: Sesamia libode Le Ru sp. nov. and Sesamia satauensis Le Ru sp. nov. Phylogenetic and molecular species delimitation analyses of a multi-marker molecular dataset allow us to investigate and clarify the status of Sesamia species and species complexes. Our results yield a well-supported phylogenetic hypothesis for the genus, which supports the monophyletic nature of all but one species subgroup. The results of 16 distinct molecular species delimitation analyses show some levels of incongruence and, overall, a tendency towards over-splitting. We also present an updated list of species for the genus Sesamia and provide morphological keys based on male and female genitalia to determine the species group of any Sesamia species and to identify all species belonging to the nonagrioides species group.
DNA sequences are increasingly used for large-scale biodiversity inventories. Because these genetic data avoid the time-consuming initial sorting of specimens based on their phenotypic attributes, they have been recently incorporated into taxonomic workflows for overlooked and diverse taxa. Major statistical developments have accompanied this new practice, and several models have been proposed to delimit species with single-locus DNA sequences. However, proposed approaches to date make different assumptions regarding taxon lineage history, leading to strong discordance whenever comparisons are made among methods. Distance-based methods, such as Automatic Barcode Gap Discovery (ABGD) and Assemble Species by Automatic Partitioning (ASAP), rely on the detection of a barcode gap (i.e., the lack of overlap in the distributions of intraspecific and interspecific genetic distances) and the associated threshold in genetic distances. Network-based methods, as exemplified by the REfined Single Linkage (RESL) algorithm for the generation of Barcode Index Numbers (BINs), use connectivity statistics to hierarchically cluster-related haplotypes into molecular operational taxonomic units (MOTUs) which serve as species proxies. Tree-based methods, including Poisson Tree Processes (PTP) and the General Mixed Yule Coalescent (GMYC), fit statistical models to phylogenetic trees by maximum likelihood or Bayesian frameworks.
Multiple webservers and stand-alone versions of these methods are now available, complicating decision-making regarding the most appropriate approach to use for a given taxon of interest. For instance, tree-based methods require an initial phylogenetic reconstruction, and multiple options are now available for this purpose such as RAxML and BEAST. Across all examined species delimitation methods, judicious parameter setting is paramount, as different model parameterizations can lead to differing conclusions. The objective of this chapter is to guide users step-by-step through all the procedures involved for each of these methods, while aggregating all necessary information required to conduct these analyses. The “Materials” section details how to prepare and format input files, including options to align sequences and conduct tree reconstruction with Maximum Likelihood and Bayesian inference. The Methods section presents the procedure and options available to conduct species delimitation analyses, including distance-, network-, and tree-based models. Finally, limits and future developments are discussed in the Notes section. Most importantly, species delimitation methods discussed herein are categorized based on five indicators: reliability, availability, scalability, understandability, and usability, all of which are fundamental properties needed for any approach to gain unanimous adoption within the DNA barcoding community moving forward.
The Eastern Arc Mountains (EAMs) in Tanzania and Kenya are renowned for their exceptional species endemism often restricted to isolated mountain blocks. Forest fragmentation during the Pliocene-Pleistocene played a significant role in shaping the EAMs' biodiversity by facilitating allopatric speciation between different mountains. However, only a few studies focused on species diversification within the same mountain block. In this article, we investigated the taxonomy and evolution of the dung beetle genus Grebennikovius, endemic to the Uluguru Mountains. Our goal was to unravel factors promoting allopatric speciation within a confined geographic area like the Ulugurus. We used an integrative taxonomic approach, combining phenotypic data and COI barcodes to delimit species and reconstructed a timetree of the genus. Molecular and morphological evidence consistently recovered 4 distinct Grebennikovius species, of which 3 are new to science: G. armiger n. sp., G. basilewskyi (Balthasar, 1960), G. lupanganus n. sp., and G. pafelo n. sp. Grebennikovius represents a rare case of microal-lopatry, with all species occurring in close proximity within different regions of the limited Uluguru forest (∼230 km 2). We infer that speciation was driven by the interplay between climate fluctuations during the Miocene and mid-Pleistocene, Uluguru topography, and the stability of rainforest ecological conditions. The latter factor probably favored the loss of wings and a specialization for Uluguru microhabitats, reinforcing population isolation and divergence. This study highlights a unique instance of diversification resulting from small-scale speciation events, providing valuable insights into the evolution of Eastern African rainforest arthropods and underscoring the importance of microhabitats conservation.
Freshwater gastropods are one of the most species-rich and severely threatened animal groups in continental aquatic ecosystems. Unfortunately, understanding their species diversity, which is key to conservation, is often hampered by their small size, simple morphology, and restricted distribution. This is particularly the case for spring snails of the genus Pseudamnicola (family Hydrobiidae), a species-rich taxon across the Mediterranean region. Contrasting species diversities derived from molecular phylogenies and traditional taxonomy call for a holistic approach, in which molecular markers are used to identify putative taxonomic units that can then be validated morphologically. We integrated DNA sequence and morphometric data from 92 populations into a species delimitation framework to assess the taxonomic status of 25 nominal and 14 informally recognized species of Pseudamnicola across its geographical range. Although seven discovery methods delimited a varying number of putative species, taking an integrative approach generally improved species support over single datasets. The statistically favoured integrative scheme corroborated 30 of the 39 initial species as separate entities, whereas nine require taxonomic re-evaluation. Six species are described here formally. Pseudamnicola exemplifies the importance of considering multiple lines of evidence to evaluate species diversity in taxonomically complex groups, with newly developed approaches providing the needed toolbox.
The twisted‐winged parasite genus Stylops has a history of different species concepts with varying host specificity resulting in diverse species diversity estimates in different regions of the Holarctic. The adoption of a supergeneralist species concept in Europe, proposing synonymization of all Western Palaearctic Stylops species, did not facilitate taxonomic clarity and obscured the available life‐history data in the region for decades. Lack of molecular data has allowed divergent opinions on species hypotheses and little opportunity for evaluating them in this morphologically challenging genus. To solve these discrepancies and gain novel information about host associations, we applied whole‐genome sequencing to 163 specimens, representing a significant portion of putative European species. We evaluate the existing and conflicting species hypotheses with molecular species delimitation using Species bOundry Delimitation using Astral (SODA) and use a maximum likelihood phylogeny to investigate host associations of the species. Furthermore, we evaluate the effect of a number of loci used in SODA for the number of inferred species. We find justification for synonymization of multiple species and indications of undescribed species, as well as new host–parasite relationships. We show that the number of inferred species in SODA is exceedingly and positively correlated with the number of loci used, urging for cautious application. The results of our study bring clarity to the Western Palaearctic species diversity of Stylops . Furthermore, the comprehensive molecular dataset generated in this study will be a valuable resource for future studies on Stylops and the evolution of parasites in general.
The mountain bumblebees of the subgenus Alpigenobombus Skorikov, 1914, are uniquely distinctive because the females have enlarged mandibles with six large, evenly spaced teeth, which they use to bite holes in long-corolla flowers for nectar robbing. Recognition of species in this subgenus has been uncertain, with names used in various combinations. To revise the species, we examined COIlike barcodes for evidence of species’ gene coalescents using MrBayes and PTP and we compare the coalescent groups with morphological variation for integrative assessment. While we seek to include only orthologous barcodes (the ‘good’) and exclude all of the more strongly divergent barcode-like numts (the ‘bad’), for some nominal taxa only low-divergence numts could be obtained (the ‘ugly’). For taxa with no orthologous sequences available, using a minimum number of the lowest divergence numts did yield coalescent candidates for species that were consistent with morphologically diagnosable groups. These results agree in recognising 11 species within this subgenus, supporting: (1) recognising the widespread European Bombus mastrucatus Gerstaecker, 1869 stat. rev. as a species separate from the west Asian B. wurflenii Radoszkowski, 1860 s. str.; (2) the recently recognised B. rainai Williams, 2022, as a species separate from B. kashmirensis Friese, 1909, within the western Himalaya; (3) the recognition once again of B. sikkimi Friese, 1918 stat. rev. and B. validus Friese, 1905 stat. rev. as species separate from B. nobilis Friese, 1905 s. str. within the eastern Himalaya and Hengduan regions; (4) confirming the recognition of B. angustus Chiu, 1948, B. breviceps Smith, 1852 s. lat., B. genalis Friese, 1918, and B. grahami (Frison, 1933) as separate species within the Himalaya, China, and Southeast Asia; (5) recognising the conspecificity of the nominal taxa (not species) channicus Gribodo, 1892 (Southeast Asia) and dentatus Handlirsch, 1888 (Himalaya) as parts of the species B. breviceps s. lat. (southern and eastern China); and (6) recognising the conspecificity of the rare taxon beresovskii (Skorikov, 1933) syn. n. as part of the species B. grahami within China. Nectar robbing by bumblebees is reviewed briefly and prospects for future research discussed.
A new species of Alainites is described from northern of Morocco Alainites albai sp. nov. It can be separated from the other west Palearctic species by the gill number, the spination of the distal margin of tergites, the leg setation, and the paraproct shape and spination. This species is widespread in the study area but never abundant. It prefers small to medium streams with slow flow, and does not seem to be very sensitive to pollution and water logging activities.
Endeavours in species discovery, particularly the characterisation of cryptic species, have been greatly aided by the application of DNA molecular sequence data to phylogenetic reconstruction and inference of evolutionary and biogeographic processes. However, the extent of cryptic and undescribed diversity remains unclear in tropical freshwaters, where biodiversity is declining at alarming rates. To investigate how data on previously undiscovered biodiversity impacts inferences of biogeography and diversification dynamics, we generated a densely sampled species-level family tree of Afrotropical Mochokidae catfishes (220 valid species) that was ca. 70% complete. This was achieved through extensive continental sampling specifically targeting the genus Chiloglanis a specialist of the relatively unexplored fast-flowing lotic habitat. Applying multiple species-delimitation methods, we report exceptional levels of species discovery for a vertebrate genus, conservatively delimiting a staggering ca. 50 putative new Chiloglanis species, resulting in a near 80% increase in species richness for the genus. Biogeographic reconstructions of the family identified the Congo Basin as a critical region in the generation of mochokid diversity, and further revealed complex scenarios for the build-up of continental assemblages of the two most species rich mochokid genera, Synodontis and Chiloglanis. While Syndontis showed most divergence events within freshwater ecoregions consistent with largely in situ diversification, Chiloglanis showed much less aggregation of freshwater ecoregions, suggesting dispersal as a key diversification process in this older group. Despite the significant increase in mochokid diversity identified here, diversification rates were best supported by a constant rate model consistent with patterns in many other tropical continental radiations. While our findings highlight fast-flowing lotic freshwaters as potential hotspots for undescribed and cryptic species diversity, a third of all freshwater fishes are currently threatened with extinction, signifying an urgent need to increase exploration of tropical freshwaters to better characterise and conserve its biodiversity.
Although much biological research depends upon species diagnoses, taxonomic expertise is collapsing. We are convinced that the sole prospect for a sustainable identification capability lies in the construction of systems that employ DNA sequences as taxon 'barcodes'. We establish that the mitochondrial gene cytochrome c oxidase I (COI) can serve as the core of a global bioidentification system for animals. First, we demonstrate that COI profiles, derived from the low-density sampling of higher taxonomic categories, ordinarily assign newly analysed taxa to the appropriate phylum or order. Second, we demonstrate that species-level assignments can be obtained by creating comprehensive COI profiles. A model COI profile, based upon the analysis of a single individual from each of 200 closely allied species of lepidopterans, was 100% successful in correctly identifying subsequent specimens. When fully developed, a COI identification system will provide a reliable, cost-effective and accessible solution to the current problem of species identification. Its assembly will also generate important new insights into the diversification of life and the rules of molecular evolution.
DNA barcoding-type studies assemble single-locus data from large samples of individuals and species, and have provided new kinds of data for evolutionary surveys of diversity. An important goal of many such studies is to delimit evolutionarily significant species units, especially in biodiversity surveys from environmental DNA samples. The Generalized Mixed Yule Coalescent (GMYC) method is a likelihood method for delimiting species by fitting within- and between-species branching models to reconstructed gene trees. Although the method has been widely used, it has not previously been described in detail or evaluated fully against simulations of alternative scenarios of true patterns of population variation and divergence between species. Here, we present important reformulations to the GMYC method as originally specified, and demonstrate its robustness to a range of departures from its simplifying assumptions. The main factor affecting the accuracy of delimitation is the mean population size of species relative to divergence times between them. Other departures from the model assumptions, such as varying population sizes among species, alternative scenarios for speciation and extinction, and population growth or subdivision within species, have relatively smaller effects. Our simulations demonstrate that support measures derived from the likelihood function provide a robust indication of when the model performs well and when it leads to inaccurate delimitations. Finally, the so-called single threshold version of the method outperforms the multiple threshold version of the method on simulated data: we argue that this might represent a fundamental limit due to the nature of evidence used to delimit species in this approach. Together with other studies comparing its performance relative to other methods, our findings support the robustness of GMYC as a tool for delimiting species when only single-locus information is available.
Many cold adapted species occur in both montane settings and in the subarctic. Their disjunct distributions create taxonomic complexity because there is no standardized method to establish whether their allopatric populations represent single or different species. This study employs DNA barcoding to gain new perspectives on the levels and patterns of sequence divergence among populations of 122 arctic-alpine species of Lepidoptera from the Alps, Fennoscandia and North America. It reveals intraspecific variability in the barcode region ranging from 0.00-10.08%. Eleven supposedly different species pairs or groups show close genetic similarity, suggesting possible synonymy in many cases. However, a total of 33 species show evidence of cryptic diversity as evidenced by the presence of lineages with over 2% maximum barcode divergence in Europe, in North America or between the two continents. Our study also reveals cases where taxonomic names have been used inconsistently between regions and exposes misidentifications. Overall, DNA barcodes have great potential to both increase taxonomic resolution and to make decisions concerning the taxonomic status of allopatric populations more objective.
Background
Species are considered the fundamental unit in many ecological and evolutionary analyses, yet accurate, complete, accessible taxonomic frameworks with which to identify them are often unavailable to researchers. In such cases DNA sequence-based species delimitation has been proposed as a means of estimating species boundaries for further analysis. Several methods have been proposed to accomplish this. Here we present a Bayesian implementation of an evolutionary model-based method, the general mixed Yule-coalescent model (GMYC). Our implementation integrates over the parameters of the model and uncertainty in phylogenetic relationships using the output of widely available phylogenetic models and Markov-Chain Monte Carlo (MCMC) simulation in order to produce marginal probabilities of species identities.
Results
We conducted simulations testing the effects of species evolutionary history, levels of intraspecific sampling and number of nucleotides sequenced. We also re-analyze the dataset used to introduce the original GMYC model. We found that the model results are improved with addition of DNA sequence and increased sampling, although these improvements have limits. The most important factor in the success of the model is the underlying phylogenetic history of the species under consideration. Recent and rapid divergences result in higher amounts of uncertainty in the model and eventually cause the model to fail to accurately assess uncertainty in species limits.
Conclusion
Our results suggest that the GMYC model can be useful under a wide variety of circumstances, particularly in cases where divergences are deeper, or taxon sampling is incomplete, as in many studies of ecological communities, but that, in accordance with expectations from coalescent theory, rapid, recent radiations may yield inaccurate results. Our implementation differs from existing ones in two ways: it allows for the accounting for important sources of uncertainty in the model (phylogenetic and in parameters specific to the model) and in the specification of informative prior distributions that can increase the precision of the model. We have incorporated this model into a user-friendly R package available on the authors’ websites.
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.
Integrative taxonomy is a recently developed approach that uses multiple lines of evidence such as molecular, morphological, ecological and geographical data to test species limits, and it stands as one of the most promising approaches to species delimitation in taxonomically difficult groups. The Pnigalio soemius complex (Hymenoptera: Eulophidae) represents an interesting taxonomical and ecological study case, as it is characterized by a lack of informative morphological characters, deep mitochondrial divergence, and is susceptible to infection by parthenogenesis-inducing Rickettsia. We tested the effectiveness of an integrative taxonomy approach in delimiting species within the P. soemius complex. We analysed two molecular markers (COI and ITS2) using different methods, performed multivariate analysis on morphometric data and exploited ecological data such as host-plant system associations, geographical separation, and the prevalence, type and effects of endosymbiont infection. The challenge of resolving different levels of resolution in the data was met by setting up a formal procedure of data integration within and between conflicting independent lines of evidence. An iterative corroboration process of multiple sources of data eventually indicated the existence of several cryptic species that can be treated as stable taxonomic hypotheses. Furthermore, the integrative approach confirmed a trend towards host specificity within the presumed polyphagous P. soemius and suggested that Rickettsia could have played a major role in the reproductive isolation and genetic diversification of at least two species.
Eight years after DNA barcoding was formally proposed on a large scale, CO1 sequences are rapidly accumulating from around the world. While studies to date have mostly targeted local or regional species assemblages, the recent launch of the global iBOL project (International Barcode of Life), highlights the need to understand the effects of geographical scale on Barcoding's goals. Sampling has been central in the debate on DNA Barcoding, but the effect of the geographical scale of sampling has not yet been thoroughly and explicitly tested with empirical data. Here, we present a CO1 data set of aquatic predaceous diving beetles of the tribe Agabini, sampled throughout Europe, and use it to investigate how the geographic scale of sampling affects 1) the estimated intraspecific variation of species, 2) the genetic distance to the most closely related heterospecific, 3) the ratio of intraspecific and interspecific variation, 4) the frequency of taxonomically recognized species found to be monophyletic, and 5) query identification performance based on 6 different species assignment methods. Intraspecific variation was significantly correlated with the geographical scale of sampling (R-square = 0.7), and more than half of the species with 10 or more sampled individuals (N = 29) showed higher intraspecific variation than 1% sequence divergence. In contrast, the distance to the closest heterospecific showed a significant decrease with increasing geographical scale of sampling. The average genetic distance dropped from > 7% for samples within 1 km, to < 3.5% for samples up to > 6000 km apart. Over a third of the species were not monophyletic, and the proportion increased through locally, nationally, regionally, and continentally restricted subsets of the data. The success of identifying queries decreased with increasing spatial scale of sampling; liberal methods declined from 100% to around 90%, whereas strict methods dropped to below 50% at continental scales. The proportion of query identifications considered uncertain (more than one species < 1% distance from query) escalated from zero at local, to 50% at continental scale. Finally, by resampling the most widely sampled species we show that even if samples are collected to maximize the geographical coverage, up to 70 individuals are required to sample 95% of intraspecific variation. The results show that the geographical scale of sampling has a critical impact on the global application of DNA barcoding. Scale-effects result from the relative importance of different processes determining the composition of regional species assemblages (dispersal and ecological assembly) and global clades (demography, speciation, and extinction). The incorporation of geographical information, where available, will be required to obtain identification rates at global scales equivalent to those in regional barcoding studies. Our result hence provides an impetus for both smarter barcoding tools and sprouting national barcoding initiatives-smaller geographical scales deliver higher accuracy.
Large-scale sequencing of short mtDNA fragments for biodiversity inventories ('DNA barcoding') indicates that sequence variation in animal mtDNA is highly structured and partitioned into discrete genetic clusters that correspond broadly to species-level entities. Here we explore how the migration rate, an important demographic parameter that is directly related to population isolation, might affect variation in the strength of mtDNA clustering among taxa. Patterns of mtDNA variation were investigated in two groups of beetles that both contain lineages occupying habitats predicted to select for different dispersal abilities: predacious diving beetles (Dytiscidae) in the genus Bidessus from lotic and lentic habitats across Europe and darkling beetles (Tenebrionidae) in the genus Eutagenia from sand and other soil types in the Aegean Islands. The degree of genetic clustering was determined using the recently developed 'mixed Yule coalescent' (MYC) model that detects the transition from between-species to within-population branching patterns. Lineages from presumed stable habitats, and therefore displaying lower dispersal ability and migration rates, showed greater levels of mtDNA clustering and geographical subdivision than their close relatives inhabiting ephemeral habitats. Simulations of expected patterns of mtDNA variation under island models showed that MYC clusters are only detected when the migration rates are much lower than the value of Nm=1 typically used to define the threshold for neutral genetic divergence. Therefore, discrete mtDNA clusters provide strong evidence for independently evolving populations or species, but their formation is suppressed even under very low levels of dispersal.
Uncovering cryptic biodiversity is essential for understanding evolutionary processes and patterns of ecosystem functioning, as well as for nature conservation. As European butterflies are arguably the best-studied group of invertebrates in the world, the discovery of a cryptic species, twenty years ago, within the common wood white Leptidea sinapis was a significant event, and these butterflies have become a model to study speciation. Here we show that the so-called 'sibling' Leptidea actually consist of three species. The new species can be discriminated on the basis of either DNA or karyological data. Such an unexpected discovery challenges our current knowledge on biodiversity, exemplifying how a widespread species can remain unnoticed even within an intensely studied natural model system for speciation.
Singletons—species only known from a single specimen—and uniques—species that have only been collected once—are very common in biodiversity samples. Recent reviews suggest that in tropical arthropod samples, 30% of all species are represented by only one specimen (Bickel 1999; Novotny and Basset 2000; Coddington et al. 2009), with additional sampling helping little with eliminating rarity. Usually, such sampling only converts some of the singleton species to doubletons, with new singleton species being discovered in the process (Scharff et al. 2003; Coddington et al. 2009). Here, we first demonstrate that rare species are similarly common in specimen samples used for taxonomic research before we argue that the phenomenon of rarity has been insufficiently considered by the new quantitative techniques for species delimitation. Addressing this disconnect between theory and reality is pressing given that the last decade has seen a renewed interest in methods for species identification and delimitation (Sites and Marshall 2004; O’Meara 2010). Much of this interest has been fuelled by the availability of DNA sequences (Meier 2008). However, many newly proposed techniques implicitly or explicitly assume that all populations and species can be well sampled. But what is the value of these techniques if many species have only been collected once and/or are only known from one specimen? Here, we argue that all existing techniques need to be modified to accommodate the commonness of rarity and that all future techniques should be explicit about how rare species can be discovered and treated.
DNA barcoding aims to accelerate species identification and discovery, but performance tests have shown marked differences in identification success. As a consequence, there remains a great need for comprehensive studies which objectively test the method in groups with a solid taxonomic framework. This study focuses on the 180 species of butterflies in Romania, accounting for about one third of the European butterfly fauna. This country includes five eco-regions, the highest of any in the European Union, and is a good representative for temperate areas. Morphology and DNA barcodes of more than 1300 specimens were carefully studied and compared. Our results indicate that 90 per cent of the species form barcode clusters allowing their reliable identification. The remaining cases involve nine closely related species pairs, some whose taxonomic status is controversial or that hybridize regularly. Interestingly, DNA barcoding was found to be the most effective identification tool, outperforming external morphology, and being slightly better than male genitalia. Romania is now the first country to have a comprehensive DNA barcode reference database for butterflies. Similar barcoding efforts based on comprehensive sampling of specific geographical regions can act as functional modules that will foster the early application of DNA barcoding while a global system is under development.
High-throughput DNA sequencing has the potential to accelerate species discovery if it is able to recognize evolutionary entities from sequence data that are comparable to species. The general mixed Yule-coalescent (GMYC) model estimates the species boundary from DNA surveys by identifying independently evolving lineages as a transition from coalescent to speciation branching patterns on a phylogenetic tree. Applied here to 12 families from 4 orders of insects in Madagascar, we used the model to delineate 370 putative species from mitochondrial DNA sequence variation among 1614 individuals. These were compared with data from the nuclear genome and morphological identification and found to be highly congruent (98% and 94%). We developed a modified GMYC that allows for a variable transition from coalescent to speciation among lineages. This revised model increased the congruence with morphology (97%), suggesting that a variable threshold better reflects the clustering of sequence data into biological species. Local endemism was pronounced in all 5 insect groups. Most species (60-91%) and haplotypes (88-99%) were found at only 1 of the 5 study sites (40-1000 km apart). This pronounced endemism resulted in a 37% increase in species numbers using diagnostic nucleotides in a population aggregation analysis. Sample sizes between 7 and 10 individuals represented a threshold above which there was minimal increase in genetic diversity, broadly agreeing with coalescent theory and other empirical studies. Our results from > 1.4 Mb of empirical data suggest that the GMYC model captures species boundaries comparable to those from traditional methods without the need for prior hypotheses of population coherence. This provides a method of species discovery and biodiversity assessment using single-locus data from mixed or environmental samples while building a globally available taxonomic database for future identifications.
Sample size has long been one of the basic issues since the start of the DNA barcoding initiative and the global biodiversity investigation. As a contribution to resolving this problem, we propose a simple resampling approach to estimate several key sampling sizes for a DNA barcoding project. We illustrate our approach using both structured populations simulated under coalescent and real species of skipper butterflies. We found that sample sizes widely used in DNA barcoding are insufficient to assess the genetic diversity of a species, population structure impacts the estimation of the sample sizes, and hence will bias the species identification potentially.
Comparative phylogeographical studies in island archipelagos can reveal lineage-specific differential responses to the geological and climatic history. We analysed patterns of genetic diversity in six codistributed lineages of darkling beetles (Tenebrionidae) in the central Aegean archipelago which differ in wing development and habitat preferences. A total of 600 specimens from 30 islands and eight adjacent mainland regions were sequenced for mitochondrial cytochrome oxidase I and nuclear Muscular protein 20. Individual gene genealogies were assessed for the presence of groups that obey an independent coalescent process using a mixed Yule coalescent model. The six focal taxa differed greatly in the number of coalescent groups and depth of lineage subdivision, which was closely mirrored by the degree of geographical structuring. The most severe subdivision at both mitochondrial DNA and nuclear DNA level was found in flightless lineages associated with presumed stable compact-soil habitats (phrygana, maquis), in contrast to sand-obligate lineages inhabiting ephemeral coastal areas that displayed greater homogeneity across the archipelago. A winged lineage, although associated with stable habitats, showed no significant phylogenetic or geographical structuring. Patterns of nucleotide diversity and local genetic differentiation, as measured using PhiST and hierarchical AMOVA, were consistent with high levels of ongoing gene flow in the winged taxon; frequent local extinction and island recolonisation for flightless sand-obligate taxa; and very low gene flow and geographical structure largely defined by the palaeogeographical history of the region in flightless compact-soil taxa. These results show that differences in dispersal rate, mediated by habitat persistence, greatly influence the levels of phylogeographical subdivision in lineages that are otherwise subjected to the same geological events and palaeoclimatic changes.
By far the greatest challenge for diversity studies is to characterize the diversity of prokaryotes, which probably encompasses billions of species, most of which are unculturable. Recent advances in theory and analysis have focused on multi-locus approaches and on combined analysis of molecular and ecological data. However, broad environmental surveys of bacterial diversity still rely on single-locus data, notably 16S ribosomal DNA, and little other detailed information. Evolutionary methods of delimiting species from single-locus data alone need to consider population genetic and macroevolutionary theories for the expected levels of interspecific and intraspecific variation. We discuss the use of a recent evolutionary method, based on the theory of coalescence within independently evolving populations, compared with a traditional approach that uses a fixed threshold divergence to delimit species.
Phylogenies reconstructed from contemporary taxa do not contain information about lineages that have gone extinct. We derive probability models for such phylogenies, allowing real data to be compared with specified null models of evolution, and lineage birth and death rates to be estimated.
Although much biological research depends upon species diagnoses, taxonomic expertise is collapsing. We are convinced that the sole prospect for a sustainable identification capability lies in the construction of systems that employ DNA sequences as taxon 'barcodes'. We establish that the mitochondrial gene cytochrome c oxidase I (COI) can serve as the core of a global bioidentification system for animals. First, we demonstrate that COI profiles, derived from the low-density sampling of higher taxonomic categories, ordinarily assign newly analysed taxa to the appropriate phylum or order. Second, we demonstrate that species-level assignments can be obtained by creating comprehensive COI profiles. A model COI profile, based upon the analysis of a single individual from each of 200 closely allied species of lepidopterans, was 100% successful in correctly identifying subsequent specimens. When fully developed, a COI identification system will provide a reliable, cost-effective and accessible solution to the current problem of species identification. Its assembly will also generate important new insights into the diversification of life and the rules of molecular evolution.
With millions of species and their life-stage transformations, the animal kingdom provides a challenging target for taxonomy. Recent work has suggested that a DNA-based identification system, founded on the mitochondrial gene, cytochrome c oxidase subunit 1 (COI), can aid the resolution of this diversity. While past work has validated the ability of COI sequences to diagnose species in certain taxonomic groups, the present study extends these analyses across the animal kingdom. The results indicate that sequence divergences at COI regularly enable the discrimination of closely allied species in all animal phyla except the Cnidaria. This success in species diagnosis reflects both the high rates of sequence change at COI in most animal groups and constraints on intraspecific mitochondrial DNA divergence arising, at least in part, through selective sweeps mediated via interactions with the nuclear genome.
Analysis of Phylogenetics and Evolution (APE) is a package written in the R language for use in molecular evolution and phylogenetics.
APE provides both utility functions for reading and writing data and manipulating phylogenetic trees, as well as several advanced
methods for phylogenetic and evolutionary analysis (e.g. comparative and population genetic methods). APE takes advantage
of the many R functions for statistics and graphics, and also provides a flexible framework for developing and implementing
further statistical methods for the analysis of evolutionary processes.
Availability: The program is free and available from the official R package archive at http://cran.r-project.org/src/contrib/PACKAGES.html#ape. APE is licensed under the GNU General Public License.
Short DNA sequences from a standardized region of the genome provide a DNA barcode for identifying species. Compiling a public library of DNA barcodes linked to named specimens could provide a new master key for identifying species, one whose power will rise with increased taxon coverage and with faster, cheaper sequencing. Recent work suggests that sequence diversity in a 648-bp region of the mitochondrial gene, cytochrome c oxidase I (COI), might serve as a DNA barcode for the identification of animal species. This study tested the effectiveness of a COI barcode in discriminating bird species, one of the largest and best-studied vertebrate groups. We determined COI barcodes for 260 species of North American birds and found that distinguishing species was generally straightforward. All species had a different COI barcode(s), and the differences between closely related species were, on average, 18 times higher than the differences within species. Our results identified four probable new species of North American birds, suggesting that a global survey will lead to the recognition of many additional bird species. The finding of large COI sequence differences between, as compared to small differences within, species confirms the effectiveness of COI barcodes for the identification of bird species. This result plus those from other groups of animals imply that a standard screening threshold of sequence difference (10x average intraspecific difference) could speed the discovery of new animal species. The growing evidence for the effectiveness of DNA barcodes as a basis for species identification supports an international exercise that has recently begun to assemble a comprehensive library of COI sequences linked to named specimens.
This paper addresses the controversy surrounding use of mtDNA versus nDNA for phylogeography and systematics.
Unlabelled:
RAxML-VI-HPC (randomized axelerated maximum likelihood for high performance computing) is a sequential and parallel program for inference of large phylogenies with maximum likelihood (ML). Low-level technical optimizations, a modification of the search algorithm, and the use of the GTR+CAT approximation as replacement for GTR+Gamma yield a program that is between 2.7 and 52 times faster than the previous version of RAxML. A large-scale performance comparison with GARLI, PHYML, IQPNNI and MrBayes on real data containing 1000 up to 6722 taxa shows that RAxML requires at least 5.6 times less main memory and yields better trees in similar times than the best competing program (GARLI) on datasets up to 2500 taxa. On datasets > or =4000 taxa it also runs 2-3 times faster than GARLI. RAxML has been parallelized with MPI to conduct parallel multiple bootstraps and inferences on distinct starting trees. The program has been used to compute ML trees on two of the largest alignments to date containing 25,057 (1463 bp) and 2182 (51,089 bp) taxa, respectively.
Availability:
icwww.epfl.ch/~stamatak
Cataloging the very large number of undescribed species of insects could be greatly accelerated by automated DNA based approaches, but procedures for large-scale species discovery from sequence data are currently lacking. Here, we use mitochondrial DNA variation to delimit species in a poorly known beetle radiation in the genus Rivacindela from arid Australia. Among 468 individuals sampled from 65 sites and multiple morphologically distinguishable types, sequence variation in three mtDNA genes (cytochrome oxidase subunit 1, cytochrome b, 16S ribosomal RNA) was strongly partitioned between 46 or 47 putative species identified with quantitative methods of species recognition based on fixed unique ("diagnostic") characters. The boundaries between groups were also recognizable from a striking increase in branching rate in clock-constrained calibrated trees. Models of stochastic lineage growth (Yule models) were combined with coalescence theory to develop a new likelihood method that determines the point of transition from species-level (speciation and extinction) to population-level (coalescence) evolutionary processes. Fitting the location of the switches from speciation to coalescent nodes on the ultrametric tree of Rivacindela produced a transition in branching rate occurring at 0.43 Mya, leading to an estimate of 48 putative species (confidence interval for the threshold ranging from 47 to 51 clusters within 2 logL units). Entities delimited in this way exhibited biological properties of traditionally defined species, showing coherence of geographic ranges, broad congruence with morphologically recognized species, and levels of sequence divergence typical for closely related species of insects. The finding of discontinuous evolutionary groupings that are readily apparent in patterns of sequence variation permits largely automated species delineation from DNA surveys of local communities as a scaffold for taxonomy in this poorly known insect group.
Asexuals are an important test case for theories of why species exist. If asexual clades displayed the same pattern of discrete variation as sexual clades, this would challenge the traditional view that sex is necessary for diversification into species. However, critical evidence has been lacking: all putative examples have involved organisms with recent or ongoing histories of recombination and have relied on visual interpretation of patterns of genetic and phenotypic variation rather than on formal tests of alternative evolutionary scenarios. Here we show that a classic asexual clade, the bdelloid rotifers, has diversified into distinct evolutionary species. Intensive sampling of the genus Rotaria reveals the presence of well-separated genetic clusters indicative of independent evolution. Moreover, combined genetic and morphological analyses reveal divergent selection in feeding morphology, indicative of niche divergence. Some of the morphologically coherent groups experiencing divergent selection contain several genetic clusters, in common with findings of cryptic species in sexual organisms. Our results show that the main causes of speciation in sexual organisms, population isolation and divergent selection, have the same qualitative effects in an asexual clade. The study also demonstrates how combined molecular and morphological analyses can shed new light on the evolutionary nature of species.
A new method, PATHd8, for estimating ultrametric trees from trees with edge (branch) lengths proportional to the number of substitutions is proposed. The method allows for an arbitrary number of reference nodes for time calibration, each defined either as absolute age, minimum age, or maximum age, and the tree need not be fully resolved. The method is based on estimating node ages by mean path lengths from the node to the leaves but correcting for deviations from a molecular clock suggested by reference nodes. As opposed to most existing methods allowing substitution rate variation, the new method smoothes substitution rates locally, rather than simultaneously over the whole tree, thus allowing for analysis of very large trees. The performance of PATHd8 is compared with other frequently used methods for estimating divergence times. In analyses of three separate data sets, PATHd8 gives similar divergence times to other methods, the largest difference being between crown group ages, where unconstrained nodes get younger ages when analyzed with PATHd8. Overall, chronograms obtained from other methods appear smoother, whereas PATHd8 preserves more of the heterogeneity seen in the original edge lengths. Divergence times are most evenly spread over the chronograms obtained from the Bayesian implementation and the clock-based Langley-Fitch method, and these two methods produce very similar ages for most nodes. Evaluations of PATHd8 using simulated data suggest that PATHd8 is slightly less precise compared with penalized likelihood, but it gives more sensible answers for extreme data sets. A clear advantage with PATHd8 is that it is more or less instantaneous even with trees having several thousand leaves, whereas other programs often run into problems when analyzing trees with hundreds of leaves. PATHd8 is implemented in freely available software.
The evolutionary analysis of molecular sequence variation is a statistical enterprise. This is reflected in the increased use of probabilistic models for phylogenetic inference, multiple sequence alignment, and molecular population genetics. Here we present BEAST: a fast, flexible software architecture for Bayesian analysis of molecular sequences related by an evolutionary tree. A large number of popular stochastic models of sequence evolution are provided and tree-based models suitable for both within- and between-species sequence data are implemented.
BEAST version 1.4.6 consists of 81000 lines of Java source code, 779 classes and 81 packages. It provides models for DNA and protein sequence evolution, highly parametric coalescent analysis, relaxed clock phylogenetics, non-contemporaneous sequence data, statistical alignment and a wide range of options for prior distributions. BEAST source code is object-oriented, modular in design and freely available at http://beast-mcmc.googlecode.com/ under the GNU LGPL license.
BEAST is a powerful and flexible evolutionary analysis package for molecular sequence variation. It also provides a resource for the further development of new models and statistical methods of evolutionary analysis.
Genetic data are frequently used to delimit species, where species status is determined on the basis of an exclusivity criterium, such as reciprocal monophyly. Not only are there numerous empirical examples of incongruence between the boundaries inferred from such data compared to other sources like morphology -- especially with recently derived species, but population genetic theory also clearly shows that an inevitable bias in species status results because genetic thresholds do not explicitly take into account how the timing of speciation influences patterns of genetic differentiation. This study represents a fundamental shift in how genetic data might be used to delimit species. Rather than equating gene trees with a species tree or basing species status on some genetic threshold, the relationship between the gene trees and the species history is modeled probabilistically. Here we show that the same theory that is used to calculate the probability of reciprocal monophyly can also be used to delimit species despite widespread incomplete lineage sorting. The results from a preliminary simulation study suggest that very recently derived species can be accurately identified long before the requisite time for reciprocal monophyly to be achieved following speciation. The study also indicates the importance of sampling, both with regards to loci and individuals. Withstanding a thorough investigation into the conditions under which the coalescent-based approach will be effective, namely how the timing of divergence relative to the effective population size of species affects accurate species delimitation, the results are nevertheless consistent with other recent studies (aimed at inferring species relationships), showing that despite the lack of monophyletic gene trees, a signal of species divergence persists and can be extracted. Using an explicit model-based approach also avoids two primary problems with species delimitation that result when genetic thresholds are applied with genetic data -- the inherent biases in species detection arising from when and how speciation occurred, and failure to take into account the high stochastic variance of genetic processes. Both the utility and sensitivities of the coalescent-based approach outlined here are discussed; most notably, a model-based approach is essential for determining whether incompletely sorted gene lineages are (or are not) consistent with separate species lineages, and such inferences require accurate model parameterization (i.e., a range of realistic effective population sizes relative to potential times of divergence for the purported species). It is the goal (and motivation of this study) that genetic data might be used effectively as a source of complementation to other sources of data for diagnosing species, as opposed to the exclusion of other evidence for species delimitation, which will require an explicit consideration of the effects of the temporal dynamic of lineage splitting on genetic data.
1. Defining species boundaries represents a significant challenge in biodiversity studies, especially as these studies increasingly rely on high-throughput DNA sequencing technologies. A promising approach for delineating species in environmental sequence data combines phylogenetics and coalescence theory to estimate species boundaries from distributions of lineage birth rates within multispecies coalescent trees.
2. Existing methods for interpreting these models utilize hypothetico-deductive reasoning to identify thresholds associated with a mixed speciation-coalescent model that fits the data better than a null model. Here, I describe an alternative approach that ranks and assigns weights to models based on their fit to the data using information criteria and then uses model averaging to estimate parameters and species probabilities.
3. This approach is applied to data from two independent studies that address (i) patterns of cospeciation in an aphid–bacterial symbiosis and (ii) diversity of bacterial communities associated with the human gut. In both of these cases, accounting for uncertainty during model selection allowed greater flexibility to detect variable (with respect to time) speciation-coalescent thresholds among lineages.
4. The precision of the predicted species boundaries varied among the studies, and the variance-to-mean ratio for richness estimates ranged from 0.023 to 0.079. Sample-based estimates of gut bacteria richness revealed that accounting for uncertainty during species delineation increased the variance in the estimates of population means (by individual from which the samples were taken or by sex of the individuals) by up to 7.5%.
5. In ecological and evolutionary studies, conclusions are highly dependent on the classification system that is adopted; given the uncertainty in species boundaries observed here, ignoring this source of error (as is common practice) likely results in inflated type I error rates. The approach described here represents an objective, theory-based method for predicting species boundaries and explicitly incorporates uncertainty in the classification system into biodiversity estimation, thus allowing researchers to better address the causes and consequences of biodiversity.
We inferred the phylogeny and historical biogeography of Parnassiinae species from the western Palaearctic using 825 bp DNA sequence from the mitochondrial protein-coding gene cytochrome oxidase I. Investigation of genetic variation revealed several cases of overlap in extent of divergence between traditionally applied taxonomic ranks. In particular, we found deep divergences between populations of Archon apollinus (Herbst) from Turkey and Israel, Zerynthia rumina (Linnaeus) from Spain and North Africa, Zerynthia polyxena (Denis & Schiffermüller) from Italy and other parts of its range, and Hypermnestra helios (Nickerl) from Iran and Central Asia. Due to incomplete sampling and weak morphological support, we only report the possibility of more than one species within each of these four taxa. The origin of ancestral Archon and Allancastria/Zerynthia is found to lie in the Iranian region. Diversification within genera is postulated to be the result of complex tectonic interactions between Eurasia and Africa during the past 20 million years, involving multiple dispersal and vicariance events.
Migration of populations to and from glacial refugia is responsible for various cases of speciation and subspeciation in Europe. The pattern of distribution and the degree of diversification between lineages originated by isolation in different glacial refugia usually depends on ecological traits, especially to their dispersal ability. Zerynthia polyxena is a philopatric species, scattered in small populations and rarely colonizing mountain areas. These characteristics probably caused repeated isolation during the Quaternary and may have favoured diversification. Actually two studies, based on both morphological and genetic data, suggest the existence of two highly distinct lineages in Europe having in Northern Italy their contact zone. In this study, I applied geometric morphometrics to male genitalia and demonstrated that (i) two morphotypes exist in Europe approximately facing on the two sides of the Po River; (ii) the two lineages probably survived glaciations in Italy and the Balkan Peninsula, respectively; then the Balkans lineage expanded to Central and Eastern Europe; (iii) no hybrid populations seem to exist in the contact area and, in one locality at least, the two lineages live in sympatry without any evidence of intermediates. These results suggest that (i) two sister species of Zerynthia exist in Europe. Accordingly, Papilio cassandra Geyer, 1828 is reinstated, as Zerynthia cassandra stat. rev., as the species to which the Zerynthia from Italy South of the Po River belong. Male genitalia differences with Zerynthia polyxena are described.
Knowledge and estimates of species richness at all scales are biased both by our understanding of the evolutionary processes shaping diversity and by the methods used to delineate the basic units of diversity. DNA taxonomy shows that diversity may be underestimated by traditional taxonomy, especially for microscopic animals. The effects of such hidden diversity are usually overlooked in ecological studies. Here, we estimate hidden diversity in bdelloid rotifers, a group of microscopic animals. We analyse cryptic diversity using a coalescent approach to infer taxonomical units from phylogenetic trees. Cryptic diversity was measured for eight traditional species of bdelloid rotifers and the results compared to that of the monogonont rotifer Brachionus plicatilis species complex, which is well studied and for which cross-mating experiments have been performed to explicitly define some of the species boundaries. A taxonomic inflation of up to 34 potential cryptic taxa was found in bdelloids. Cryptic taxa within each traditional species may be spatially isolated, but do not have narrower ecological niches. The species deemed as generalists exhibit the highest cryptic diversity. Cryptic diversity based on molecular characterization is commonly found in animals; nevertheless, the amount of cryptic diversity in bdelloids is much higher than in other groups analysed so far, maybe because of their peculiar parthenogenetic reproduction, other than microscopic size. We discuss this hypothesis in the light of the available empirical evidence from other groups of microscopic invertebrates, such as tardigrades and mites, which share size, habitat heterogeneity, potential for dispersal, and/or parthenogenetic reproduction.
Within uncharacterized groups, DNA barcodes, short DNA sequences that are present in a wide range of species, can be used to assign organisms into species. We propose an automatic procedure that sorts the sequences into hypothetical species based on the barcode gap, which can be observed whenever the divergence among organisms belonging to the same species is smaller than divergence among organisms from different species. We use a range of prior intraspecific divergence to infer from the data a model-based one-sided confidence limit for intraspecific divergence. The method, called Automatic Barcode Gap Discovery (ABGD), then detects the barcode gap as the first significant gap beyond this limit and uses it to partition the data. Inference of the limit and gap detection are then recursively applied to previously obtained groups to get finer partitions until there is no further partitioning. Using six published data sets of metazoans, we show that ABGD is computationally efficient and performs well for standard prior maximum intraspecific divergences (a few per cent of divergence for the five data sets), except for one data set where less than three sequences per species were sampled. We further explore the theoretical limitations of ABGD through simulation of explicit speciation and population genetics scenarios. Our results emphasize in particular the sensitivity of the method to the presence of recent speciation events, via (unrealistically) high rates of speciation or large numbers of species. In conclusion, ABGD is fast, simple method to split a sequence alignment data set into candidate species that should be complemented with other evidence in an integrative taxonomic approach.
Widespread species have the potential to reveal large-scale biogeographical patterns, as well as responses to environmental changes possibly unique to habitat generalists. This study presents a continental-scale phylogeographical analysis of Polyommatus icarus, one of the most common Palaearctic butterflies, and the morphologically and ecologically similar Polyommatus celina, a recently discovered cryptic species. By combining data from mitochondrial [cytochrome c oxidase subunit I (COI)] and nuclear [internal transcribed spacer (ITS2)] molecular markers with geometric morphometrics, we document a complex phylogeographical history for the two species. Despite morphological similarities, the genetic divergence between these two species is high (more than 5% at COI) and they are not sister species. For the first time, we show that P. celina occurs not only in North Africa but also in Europe, where it inhabits several west Mediterranean islands, as well as large parts of Iberia, where it occurs in parapatry with P. icarus. The two species appear to completely exclude each other on islands, but we provide morphological and molecular evidence that introgression occurred in the Iberian Peninsula. We discovered strongly diverged lineages that seem to represent relict populations produced by past range expansions and contractions: Crete and Iberian isolates for P. icarus, Balearics-Sardinia and Sicily-Lipari for P. celina. This study shows that a combined genetic-morphometric approach can shed light on cryptic diversity while providing the necessary resolution to reconstruct a fine-scale phylogeographical history of species at both spatial and temporal levels.
Species Delimitation is a plugin to the Geneious software to support the exploration of species boundaries in a gene tree. The user assigns taxa to putative species and the plugin computes statistics relating to the probability of the observed monophyly or exclusivity having occurred by chance in a coalescent process. It also assesses the within and between species genetic distances to infer the probability with which members of a putative species might be identified successfully with tree-based methods.
The invertebrate fauna of New Zealand is of great interest as a geologically tractable model for the study of species diversification, but direct comparisons with closely related lineages elsewhere are lacking. Integrating population-level analyses with studies of taxonomy and clade diversification, we performed mtDNA analysis on Neocicindela (Cicindelidae, tiger beetles) for a broad sample of populations from 11 of 12 known species and 161 specimens (three loci, 1883 nucleotides), revealing 123 distinct haplotypes. Phylogenetic reconstruction recovered two main lineages, each composed of 5-6 Linnean species whose origin was dated to 6.66 and 7.26 Mya, while the Neocicindela stem group was placed at 10.82 ± 0.48 Mya. Species delimitation implementing a character-based (diagnostic) species concept recognized 19 species-level groups that were in general agreement with Linnean species but split some of these into mostly allopatric subgroups. Tree-based methods of species delimitation using a mixed Yule-coalescence model were inconclusive, and recognized 32-51 entities (including singletons), splitting existing species into up to 8 partially sympatric groups. These findings were different from patterns in the Australian sister genus Rivacindela, where character-based and tree-based methods were previously shown to produce highly congruent groupings. In Neocicindela, the pattern of mtDNA variation was characterized by high intra-population and intra-species haplotype divergence, the coexistence of divergent haplotypes in sympatry, and a poor correlation of genetic and geographic distance. These observations combined suggest a scenario of phylogeographic divergence and secondary contact driven by orogenetic and climatic changes of the Pleistocene/Pliocene. The complex evolutionary history of most species of Neocicindela due to the relative instability of the New Zealand biota resulted in populations of mixed ancestry but not in a general loss of genetic variation.
Systematists should distinguish between cladistic analysis, i.e., a numerical procedure, and phylogenetic analysis, i.e., the reconstruction of hierarchic descent relationships. Modern cladistic analysis uses parsimony to construct hierarchic arrangements (trees) of terminal units (terminals) that have been scored for a series of attributes. The interpretation of cladistic relationships as representative of phylogenetic relationships requires two conditions, both of which were identified by Hennig (1966, Phylogenetic systematics, Univ. Illinois Press, Urbana). First, descent relationships among the terminals must be hierarchic; that is, all terminals must have been generated by the subdivision or replication of previously existing ancestors. This is a necessary condition for phylogenetic analysis, rather than an empirical discovery of it, because the results of cladistic analysis are always hierarchically structured (however poorly resolved). Because resolution of a cladistic hierarchy does not demonstrate that a hierarchic descent system underlies the character distribution pattern it reflects, additional information is necessary, in any given case, to determine that phylogenetic analysis is appropriate. Second, to have congruence between an observable attribute hierarchy and the descent hierarchy that is to be inferred, the attribute must have been transmitted from an ancestor to all of its descendants, either in its original state or in a modified state. Both conditions are met by asexual organisms and by nonrecombining genetic elements (e.g., the chloroplast and mitochondrial genomes) but not by individual sexually reproducing organisms that bear such genetic elements. Populations of sexually reproducing organisms can meet the first condition (i.e., hierarchic descent) when new populations are founded by the division of previously existing populations and individuals do not disperse among existing populations. When the first condition is met, the second also is met for genetically fixed attributes of the populations, because populations descended from an ancestral population that was fixed for an attribute also will be fixed for that attribute (in original or modified state). In contrast, attributes that are not fixed in a population may not occur in all or any descendant populations, even if descent relationships among populations are hierarchic. The occurrence of a unique fixed character combination in an extended genealogical population (phylogenetic species sensu Nixon and Wheeler, 1990, Cladistics 6:211-223) is evidence that this population has diverged from other such populations and thus that descent relationships among such populations are hierarchic. The fixed characters of phylogenetic species therefore constitute evidence that a hierarchic descent system exists and provide the means for analyzing phylogenetic relationships among these species. Phylogenetic species can be delimited by a procedure (population aggregation analysis) that involves a search for fixed differences among local populations, followed by successive rounds of aggregation of populations and previously aggregated population groups that are not distinct from each other
Species-level phylogenies derived from molecular data provide an indirect record of the speciation events that have led to extant species. This offers enormous potential for investigating the general causes and rates of speciation within clades. To make the most of this potential, we should ideally sample all the species in a higher group, such as a genus, ensure that those species reflect evolutionary entities within the group, and rule out the effects of other processes, such as extinction, as explanations for observed patterns. We discuss recent practical and theoretical advances in this area and outline how future work should benefit from incorporating data from genealogical and phylogeographical scales.
Estimating divergence times and rates of substitution from sequence data is plagued by the problem of rate variation between lineages. R8s version 1.5 is a program which uses parametric, nonparametric and semiparametric methods to relax the assumption of constant rates of evolution to obtain better estimates of rates and times. Unlike most programs for rate inference or phylogenetics, r8s permits users to convert results to absolute rates and ages by constraining one or more node times to be fixed, minimum or maximum ages (using fossil or other evidence). Version 1.5 uses truncated Newton nonlinear optimization code with bound constraints, offering superior performance over previous versions.
Results:
Availability: The linux executable, C source code, sample data sets and user manual are available free at http://ginger.ucdavis.edu/r8s.
Contact: mjsanderson@ucdavis.edu
Loss of limb skeletal elements is a recurring theme in tetrapod evolution, but the developmental mechanisms underlying this phenomenon remain largely unknown. The Australian lizard genus Hemiergis offers an excellent model system to study limb reduction among closely related, naturally occurring populations with different numbers of digits. Evolutionary digit loss in Hemiergis does not result from simple truncation of a pentadactyl skeletal developmental program. Rather, the duration of embryonic expression of the patterning molecule Sonic hedgehog (SHH) is shortened in limbs with reduced numbers of digits, and is correlated with decreased cell proliferation in the posterior aspect of the limb. Moreover, this comparative analysis suggests an early role for SHH in specification of digit identity and later importance in maintaining cell proliferation and survival. Subtle changes in spatial or temporal regulation of SHH may alter proliferation and patterning of the developing limb, thereby effecting divergence in adult limb morphology among closely related species. In contrast, expression of MSX and Distal-less proteins were similar among embryos from different populations.
Although central to much biological research, the identification of species is often difficult. The use of DNA barcodes, short DNA sequences from a standardized region of the genome, has recently been proposed as a tool to facilitate species identification and discovery. However, the effectiveness of DNA barcoding for identifying specimens in species-rich tropical biotas is unknown. Here we show that cytochrome c oxidase I DNA barcodes effectively discriminate among species in three Lepidoptera families from Area de Conservación Guanacaste in northwestern Costa Rica. We found that 97.9% of the 521 species recognized by prior taxonomic work possess distinctive cytochrome c oxidase I barcodes and that the few instances of interspecific sequence overlap involve very similar species. We also found two or more barcode clusters within each of 13 supposedly single species. Covariation between these clusters and morphological and/or ecological traits indicates overlooked species complexes. If these results are general, DNA barcoding will significantly aid species identification and discovery in tropical settings.
• Area de Conservación Guanacaste
• cytochrome c oxidase I
• Hesperiidae
• Sphingidae
• Saturniidae
Bdelloid rotifers, darwinulid ostracods and some oribatid mites have been called 'ancient asexuals' as they speciated and survived over long-term evolutionary timescale without sexual recombination. Data on their genetic diversification are contrasting: within-species diversification is present mostly at a continental scale in a parthenogenetic oribatid mite, whereas almost no genetic diversification at all seems to occur within darwinulid ostracod species. Strangely enough, no clear data for bdelloid rotifers are available so far. In this paper, we analyse partial COI mtDNA sequences to show that a bdelloid rotifer, Philodina flaviceps, so far considered a single traditional morphological species, has actually been able to diversify into at least nine distinct evolutionary entities, with genetic distances between lineages comparable with those between different traditional species within the same genus. We discovered that local coexistence of such different independent lineages is very common: up to four lineages were found in a same stream, and up to three in a single moss sample of 5 cm(2). In contrast to the large-scale geographic pattern that has recently been reported in the oribatid mite, the spatial distribution of the bdelloid lineages provided evidence of micro-phylogeographic patterns. If the mtDNA diversity indicates that the lineages are independent and represent sympatric cryptic species within P. flaviceps, then the actual bdelloid diversity can be expected to be much greater than that recognized today.
We introduce a statistic, the genealogical sorting index (gsi), for quantifying the degree of exclusive ancestry of labeled groups on a rooted genealogy and demonstrate its application. The statistic is simple, intuitive, and easily calculated. It has a normalized range to facilitate comparisons among different groups, trees, or studies and it provides information on individual groups rather than a composite measure for all groups. It naturally handles polytomies and accommodates measures of uncertainty in phylogenetic relationships. We use coalescent simulations to explore the behavior of the gsi across a range of divergence times, with the mean value increasing to 1, the maximum value when exclusivity within a group reached monophyly. Simulations also demonstrate that the power to reject the null hypothesis of mixed genealogical ancestry increased markedly as sample size increased, and that the gsi provides a statistically more powerful measure of divergence than FST. Applications to data from published studies demonstrated that the gsi provides a useful way to detect significant exclusivity even when groups are not monophyletic. Although we describe this statistic in the context of divergence, it is more broadly applicable to quantify and assess the significance of clustering of observations in labeled groups on any tree.
& Zald? ıvar-River? on, A. (2012) Species identifica-tioninthetaxonomicallyneglected,highlydiverse,neotropicalparasitoidwasp genusNotiospathius(Braconidae:Doryctinae)basedonanintegrativemolecu-lar and morphological approach
- F S Ceccarelli
- M J Sharkey
Ceccarelli, F.S., Sharkey, M.J. & Zald? ıvar-River? on, A. (2012) Species identifica-tioninthetaxonomicallyneglected,highlydiverse,neotropicalparasitoidwasp genusNotiospathius(Braconidae:Doryctinae)basedonanintegrativemolecu-lar and morphological approach. Molecular Phylogenetics and Evolution, 62, 485–495
Splits: Species' Limits by Threshold Statistics. R package version 1.0. URL: http://R-Forge.R-project. org/projects Cryptic diversification in ancient asexuals: evidence from the bdelloid rotifer Philodina flaviceps
- T Ezard
- T Fujisawa
- T Barraclough
Ezard, T., Fujisawa, T. & Barraclough, T. (2009) Splits: Species' Limits by
Threshold Statistics. R package version 1.0. URL: http://R-Forge.R-project.
org/projects/splits/
Fontaneto, D., Boschetti, C. & Ricci, C. (2007) Cryptic diversification in ancient
asexuals: evidence from the bdelloid rotifer Philodina flaviceps. Journal of
Evolutionary Biology, 21, 580–587.