General Mixed Yule Coalescent Model Research Articles

"Forms" of water mites (Acari: Hydrachnidia): intraspecific variation or valid species?

In many groups of organisms, especially in the older literature, it has been common practice to recognize sympatrically occurring phenotypic variants of a species as “forms”. However, what these forms really represent often remains unclear, especially in poorly studied groups. With new algorithms for DNA-based species delimitation, the status of forms can be explicitly tested with molecular data. In this study, we test a number of what is now recognized as valid species of water mites (Hydrachnidia), but have in the past been treated as forms sympatrically occurring with their nominate species. We also test a form without prior taxonomical status, using DNA and morphometrics. The barcoding fragment of COI, nuclear 28S and quantitative analyses of morphological data were used to test whether these taxa merit species status, as suggested by several taxonomists. Our results confirm valid species. Genetic distances between the form and nominate species (Piona dispersa and Piona variabilis, COI 11%), as well as likelihood ratio tests under the general mixed-Yule coalescent model, supported that these are separately evolving lineages as defined by the unified species concept. In addition, they can be diagnosed with morphological characters. The study also reveals that some taxa genetically represent more than one species. We propose that P. dispersa are recognized as valid taxa at the species level. Unionicola minor (which may consist of several species), Piona stjordalensis, P. imminuta s. lat., and P. rotundoides are confirmed as species using this model. The results also imply that future studies of other water mite species complexes are likely to reveal many more genetically and morphologically distinct species.

Cryptic diversity, intraspecific phenetic plasticity and recent geographical translocations inBranchiomma(Sabellidae, Annelida)

The importance of identifying biological diversity accurately and efficiently is becoming more evident. It is therefore critical to determine the species boundaries between closely related taxa and to establish diagnostic characters that allow us to define species. This is not an easy task when species exhibit high intraspecific phenotypic plasticity or when distinct evolutionary lineages with an unusually large amount of genetic distinctiveness show no apparent morphological diversity (cryptic species). These phenomena appear to be common in the genus of fan wormsBranchiomma(Sabellidae, Annelida), and consequently, taxonomic errors are widespread in the group. Moreover, someBranchiommaspecies have been unintentionally translocated outside the area where natural range extension is expected, increasing the taxonomic problems. We have performed a range of analytical methods including genetic distances, Bayesian inference, maximum likelihood, maximum parsimony, statistical parsimony analyses and general mixed Yule coalescent model to clarify the taxonomic status and assess the species boundaries ofBranchiommain Australia. This study shows that the traditional diagnostic morphological features are greatly homoplastic. Results also indicate that the diversity ofBranchiommain Australia is higher than previously reported and evidence some cases of high phenetic plasticity (in features previously considered as stable within species), high intraspecific genetic variability, cryptic species and several unexpected cases of translocations.

Biogeography of scorpions in thePseudouroctonus minimuscomplex (Vaejovidae) from south‐western North America: implications of ecological specialization for pre‐Quaternary diversification

AbstractAimThe aim of this study was to assess the impact of pre‐Quaternary tectonics and orogeny relative to that of Pleistocene climate change on diversification within thePseudouroctonus minimuscomplex, a group of vaejovid scorpions with stenotopic habitat requirements.LocationSouth‐western North America (United States and Mexico).MethodsMultilocus sequence data (1899 base pairs from two mitochondrial and two nuclear genes) were generated from 65 samples of scorpions in theminimuscomplex. Phylogeographical structure within theminimuscomplex was explored using model‐based phylogenetic methods and a general mixed Yule coalescent model to identify independent geographical clusters. A time‐calibrated multilocus species tree was reconstructed using a multispecies coalescent approach. Ancestral areas were estimated at divergence events across the tree using a probabilistic Bayesian approach.ResultsExtensive geographical structure was evident within two well‐supported clades. These clades probably diverged over 25 million years ago (Ma), based on estimated mean divergence dates, followed by 14 divergences in the Miocene (25–5 Ma) and 4 divergences in the Pliocene and Pleistocene (< 5 Ma). The ancestral origin of theminimuscomplex was reconstructed to be across California and the Mexican Highlands. The Chihuahuan Desert was colonized twice from the Mexican Highlands, and one dispersal event occurred from the Mexican Highlands back to California.Main conclusionsSpatial and temporal patterns of evolution in theminimuscomplex support predictions that stenotopy promoted pre‐Quaternary diversification. Miocene and Pliocene geomorphology, perhaps in concert with climate change, induced allopatric divergence across the heterogeneous landscape of south‐western North America. Stenotopic scorpions such as theminimuscomplex provide a model for exploring correlations between Earth history and biological diversification.

Molecular phylogeny of the genusMargotrema(Digenea: Allocreadiidae), parasitic flatworms of goodeid freshwater fishes across central Mexico: species boundaries, host-specificity, and geographical congruence

We explored the genetic variation at the intraspecific level of two putative species of Margotrema, M. bravoae and M. guillerminae, to establish the species boundaries. Sequences of a mitochondrial (COI) and nuclear (ITS1) gene were obtained for 125 specimens distributed in 15 localities from seven hydrological systems. An alignment of 750 and 831 bp including gaps of COI and ITS1, respectively, was assembled. We analysed the gene fragments separately as well as together by using Bayesian inference for phylogenetic reconstruction. Based on the phylogenetic analyses, an ultrametric tree was built for each gene, and a general mixed Yule-coalescent model for species delimitation was carried out. A multispecies coalescent analysis was performed in *BEAST using both molecular markers. The results show four independent evolutionary lineages that we interpret as two valid species of Margotrema; the first is represented by an independent lineage, and the second is composed of three lineages. Because the species M. bravoae and M. guillerminae nested together within these three lineages, the validity of M. guillerminae as an independent species is questioned, and it is proposed to represent a junior synonym of M. bravoae. Each lineage shows a congruent geographical pattern with respect to the hydrological system where they occur. Additionally, each lineage shows congruence with respect to the hosts they parasitize, either at the species or at higher taxonomical levels (tribe). The parasite evolutionary history is congruent with the evolutionary and biogeographical history of their hosts. © 2013 The Linnean Society of London

Exploring species level taxonomy and species delimitation methods in the facultatively self-fertilizing land snail genus Rumina (gastropoda: pulmonata).

Delimiting species in facultatively selfing taxa is a challenging problem of which the terrestrial pulmonate snail genus Rumina is a good example. These snails have a mixed breeding system and show a high degree of shell and color variation. Three nominal species (R. decollata, R. saharica and R. paivae) and two color morphs within R. decollata (dark and light) are currently recognized. The present study aims at evaluating to what extent these entities reflect evolutionary diverging taxonomic units, rather than fixed polymorphisms due to sustained selfing. Therefore, a phylogenetic analysis of nuclear (ITS1, ITS2) and mitochondrial DNA (COI, CytB, 12S rDNA, 16S rDNA) sequences was performed. Putative species in Rumina, inferred from the mitochondrial DNA phylogeny, were compared with those proposed on the basis of the COI gene by (1) DNA barcoding gap analysis, (2) Automatic Barcode Gap Discovery, (3) the species delimitation plug-in of the Geneious software, (4) the Genealogical Sorting Index, and (5) the General Mixed Yule Coalescent model. It is shown that these methods produce a variety of different species hypotheses and as such one may wonder to what extent species delimitation methods are really useful. With respect to Rumina, the data suggest at least seven species, one corresponding to R. saharica and six that are currently grouped under the name R. decollata. The species-level status of R. paivae is rejected.

A time-calibrated molecular phylogeny of the precious corals: reconciling discrepancies in the taxonomic classification and insights into their evolutionary history

BackgroundSeamount-associated faunas are often considered highly endemic but isolation and diversification processes leading to such endemism have been poorly documented at those depths. Likewise, species delimitation and phylogenetic studies in deep-sea organisms remain scarce, due to the difficulty in obtaining samples, and sometimes controversial. The phylogenetic relationships within the precious coral family Coralliidae remain largely unexplored and the monophyly of its two constituent genera, Corallium Cuvier and Paracorallium Bayer & Cairns, has not been resolved. As traditionally recognized, the diversity of colonial forms among the various species correlates with the diversity in shape of their supporting axis, but the phylogenetic significance of these characters remains to be tested. We thus used mitochondrial sequence data to evaluate the monophyly of Corallium and Paracorallium and the species boundaries for nearly all named taxa in the family. Species from across the coralliid range, including material from Antarctica, Hawaii, Japan, New Zealand, Taiwan, Tasmania, the eastern Pacific and the western Atlantic were examined.ResultsThe concatenated analysis of five mitochondrial regions (COI, 16S rRNA, ND2, and ND3-ND6) recovered two major coralliid clades. One clade is composed of two subgroups, the first including Corallium rubrum, the type species of the genus, together with a small group of Paracorallium species (P. japonicum and P. tortuosum) and C. medea (clade I-A); the other subgroup includes a poorly-resolved assemblage of six Corallium species (C. abyssale, C. ducale, C. imperiale, C. laauense, C. niobe, and C. sulcatum; clade I-B). The second major clade is well resolved and includes species of Corallium and Paracorallium (C. elatius, C. kishinouyei, C. konojoi, C. niveum, C. secundum, Corallium sp., Paracorallium nix, Paracorallium thrinax and Paracorallium spp.). A traditional taxonomic study of this clade delineated 11 morphospecies that were congruent with the general mixed Yule-coalescent (GMYC) model. A multilocus species-tree approach also identified the same two well-supported clades, being Clade I-B more recent in the species tree (18.0-15.9 mya) than in the gene tree (35.2-15.9 mya). In contrast, the diversification times for Clade II were more ancient in the species tree (136.4-41.7 mya) than in the gene tree (66.3-16.9 mya).ConclusionsOur results provide no support for the taxonomic status of the two currently recognized genera in the family Coralliidae. Given that Paracorallium species were all nested within Corallium, we recognize the coralliid genus Corallium, which includes the type species of the family, and thus consider Paracorallium a junior synonym of Corallium. We propose the use of the genus Hemicorallium Gray for clade I-B (species with long rod sclerites, cylindrical autozooids and smooth axis). Species delimitation in clade I-B remains unclear and the molecular resolution for Coralliidae species is inconsistent in the two main clades. Some species have wide distributions, recent diversification times and low mtDNA divergence whereas other species exhibit narrower allopatric distributions, older diversification times and greater levels of mtDNA resolution.

Pitfalls of establishing DNA barcoding systems in protists: the cryptophyceae as a test case.

A DNA barcode is a preferrably short and highly variable region of DNA supposed to facilitate a rapid identification of species. In many protistan lineages, a lack of species-specific morphological characters hampers an identification of species by light or electron microscopy, and difficulties to perform mating experiments in laboratory cultures also do not allow for an identification of biological species. Thus, testing candidate barcode markers as well as establishment of accurately working species identification systems are more challenging than in multicellular organisms. In cryptic species complexes the performance of a potential barcode marker can not be monitored using morphological characters as a feedback, but an inappropriate choice of DNA region may result in artifactual species trees for several reasons. Therefore a priori knowledge of the systematics of a group is required. In addition to identification of known species, methods for an automatic delimitation of species with DNA barcodes have been proposed. The Cryptophyceae provide a mixture of systematically well characterized as well as badly characterized groups and are used in this study to test the suitability of some of the methods for protists. As species identification method the performance of blast in searches against badly to well-sampled reference databases has been tested with COI-5P and 5′-partial LSU rDNA (domains A to D of the nuclear LSU rRNA gene). In addition the performance of two different methods for automatic species delimitation, fixed thresholds of genetic divergence and the general mixed Yule-coalescent model (GMYC), have been examined. The study demonstrates some pitfalls of barcoding methods that have to be taken care of. Also a best-practice approach towards establishing a DNA barcode system in protists is proposed.

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

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

Cryptic Species in Putative Ancient Asexual Darwinulids (Crustacea, Ostracoda)

BackgroundFully asexually reproducing taxa lack outcrossing. Hence, the classic Biological Species Concept cannot be applied.Methodology/Principal FindingsWe used DNA sequences from the mitochondrial COI gene and the nuclear ITS2 region to check species boundaries according to the evolutionary genetic (EG) species concept in five morphospecies in the putative ancient asexual ostracod genera, Penthesilenula and Darwinula, from different continents. We applied two methods for detecting cryptic species, namely the K/θ method and the General Mixed Yule Coalescent model (GMYC). We could confirm the existence of species in all five darwinulid morphospecies and additional cryptic diversity in three morphospecies, namely in Penthesilenula brasiliensis, Darwinula stevensoni and in P. aotearoa. The number of cryptic species within one morphospecies varied between seven (P. brasiliensis), five to six (D. stevensoni) and two (P. aotearoa), respectively, depending on the method used. Cryptic species mainly followed continental distributions. We also found evidence for coexistence at the local scale for Brazilian cryptic species of P. brasiliensis and P. aotearoa. Our ITS2 data confirmed that species exist in darwinulids but detected far less EG species, namely two to three cryptic species in P. brasiliensis and no cryptic species at all in the other darwinulid morphospecies.Conclusions/SignificanceOur results clearly demonstrate that both species and cryptic diversity can be recognized in putative ancient asexual ostracods using the EG species concept, and that COI data are more suitable than ITS2 for this purpose. The discovery of up to eight cryptic species within a single morphospecies will significantly increase estimates of biodiversity in this asexual ostracod group. Which factors, other than long-term geographic isolation, are important for speciation processes in these ancient asexuals remains to be investigated.

Lichen myco- and photobiont diversity and their relationships at the edge of life (McMurdo Dry Valleys, Antarctica)

Lichen-forming fungi are among the most diverse group of organisms in Antarctica. Being poikilohydric, lichens are able to cope with harsh environmental conditions that exclude other organisms like vascular plants. The McMurdo Dry Valleys (Victoria Land, Continental Antarctica) are a hyperarid cold desert where macroscopic life is reduced to a few lichens and bryophyte species. We investigated the diversity of lichen-forming fungi and their associated photobionts in three valleys (Garwood, Marshall, and Miers). Correct identification of lichen-forming fungi from extreme ecosystems is complicated by the presence of numerous sterile and extremely modified thalli. To overcome this problem, we used a combined approach for the identification of the species present in the area, the first involving identification by means of standard characters and the second, two DNA-based (ITS region) species delimitation methods (General Mixed Yule-Coalescent model and genetic distances). In addition, we also used ITS sequences for the identification of the photobionts associated with the mycobionts. We studied the relationships between both bionts and assessed the degree of selectivity and specificity found in those associations. We also looked for landscape level spatial patterns in these associations. The two DNA-based methods performed quite differently, but 27 species of lichen-forming fungi and five putative species of photobionts were found in the studied area. Although there was a general trend for low selectivity in the relationships, high specificity was found in some associations and differential selectivity was observed in some lichen-forming fungi. No spatial structure was detected in the distribution of photobionts in the studied area.

Hiding deep in the trees: discovery of divergent mitochondrial lineages in Malagasy chameleons of theCalumma nasutumgroup

We conducted a comprehensive molecular phylogenetic study for a group of chameleons from Madagascar (Chamaeleonidae: Calumma nasutum group, comprising seven nominal species) to examine the genetic and species diversity in this widespread genus. Based on DNA sequences of the mitochondrial gene (ND2) from 215 specimens, we reconstructed the phylogeny using a Bayesian approach. Our results show deep divergences among several unnamed mitochondrial lineages that are difficult to identify morphologically. We evaluated lineage diversification using a number of statistical phylogenetic methods (general mixed Yule-coalescent model; SpeciesIdentifier; net p-distances) to objectively delimit lineages that we here consider as operational taxonomic units (OTUs), and for which the taxonomic status remains largely unknown. In addition, we compared molecular and morphological differentiation in detail for one particularly diverse clade (the C. boettgeri complex) from northern Madagascar. To assess the species boundaries within this group we used an integrative taxonomic approach, combining evidence from two independent molecular markers (ND2 and CMOS), together with genital and other external morphological characters, and conclude that some of the newly discovered OTUs are separate species (confirmed candidate species, CCS), while others should best be considered as deep conspecific lineages (DCLs). Our analysis supports a total of 33 OTUs, of which seven correspond to described species, suggesting that the taxonomy of the C. nasutum group is in need of revision.

Sequence-based species delimitation in the Balkan <i>Bythinella</i> Moquin-Tandon, 1856 (Gastropoda: Rissooidea) with general mixed yule coalescent model

Sequence-based species delimitation in the Balkan <i>Bythinella</i> Moquin-Tandon, 1856 (Gastropoda: Rissooidea) with general mixed yule coalescent model

Barcoding against a paradox? Combined molecular species delineations reveal multiple cryptic lineages in elusive meiofaunal sea slugs.

BackgroundMany marine meiofaunal species are reported to have wide distributions, which creates a paradox considering their hypothesized low dispersal abilities. Correlated with this paradox is an especially high taxonomic deficit for meiofauna, partly related to a lower taxonomic effort and partly to a high number of putative cryptic species. Molecular-based species delineation and barcoding approaches have been advocated for meiofaunal biodiversity assessments to speed up description processes and uncover cryptic lineages. However, these approaches show sensitivity to sampling coverage (taxonomic and geographic) and the success rate has never been explored on mesopsammic Mollusca.ResultsWe collected the meiofaunal sea-slug Pontohedyle (Acochlidia, Heterobranchia) from 28 localities worldwide. With a traditional morphological approach, all specimens fall into two morphospecies. However, with a multi-marker genetic approach, we reveal multiple lineages that are reciprocally monophyletic on single and concatenated gene trees in phylogenetic analyses. These lineages are largely concordant with geographical and oceanographic parameters, leading to our primary species hypothesis (PSH). In parallel, we apply four independent methods of molecular based species delineation: General Mixed Yule Coalescent model (GMYC), statistical parsimony, Bayesian Species Delineation (BPP) and Automatic Barcode Gap Discovery (ABGD). The secondary species hypothesis (SSH) is gained by relying only on uncontradicted results of the different approaches (‘minimum consensus approach’), resulting in the discovery of a radiation of (at least) 12 mainly cryptic species, 9 of them new to science, some sympatric and some allopatric with respect to ocean boundaries. However, the meiofaunal paradox still persists in some Pontohedyle species identified here with wide coastal and trans-archipelago distributions.ConclusionsOur study confirms extensive, morphologically cryptic diversity among meiofauna and accentuates the taxonomic deficit that characterizes meiofauna research. We observe for Pontohedyle slugs a high degree of morphological simplicity and uniformity, which we expect might be a general rule for meiofauna. To tackle cryptic diversity in little explored and hard-to-sample invertebrate taxa, at present, a combined approach seems most promising, such as multi-marker-barcoding (i.e., molecular systematics using mitochondrial and nuclear markers and the criterion of reciprocal monophyly) combined with a minimum consensus approach across independent methods of molecular species delineation to define candidate species.

Phylogenetic estimation error can decrease the accuracy of species delimitation: a Bayesian implementation of the general mixed Yule-coalescent model

BackgroundSpecies 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.ResultsWe 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.ConclusionOur 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.

Cryptic variation in an ecological indicator organism: mitochondrial and nuclear DNA sequence data confirm distinct lineages of Baetis harrisoni Barnard (Ephemeroptera: Baetidae) in southern Africa.

BackgroundBaetis harrisoni Barnard is a mayfly frequently encountered in river studies across Africa, but the external morphological features used for identifying nymphs have been observed to vary subtly between different geographic locations. It has been associated with a wide range of ecological conditions, including pH extremes of pH 2.9–10.0 in polluted waters. We present a molecular study of the genetic variation within B. harrisoni across 21 rivers in its distribution range in southern Africa.ResultsFour gene regions were examined, two mitochondrial (cytochrome c oxidase subunit I [COI] and small subunit ribosomal 16S rDNA [16S]) and two nuclear (elongation factor 1 alpha [EF1α] and phosphoenolpyruvate carboxykinase [PEPCK]). Bayesian and parsimony approaches to phylogeny reconstruction resulted in five well-supported major lineages, which were confirmed using a general mixed Yule-coalescent (GMYC) model. Results from the EF1α gene were significantly incongruent with both mitochondrial and nuclear (PEPCK) results, possibly due to incomplete lineage sorting of the EF1α gene. Mean between-clade distance estimated using the COI and PEPCK data was found to be an order of magnitude greater than the within-clade distance and comparable to that previously reported for other recognised Baetis species. Analysis of the Isolation by Distance (IBD) between all samples showed a small but significant effect of IBD. Within each lineage the contribution of IBD was minimal. Tentative dating analyses using an uncorrelated log-normal relaxed clock and two published estimates of COI mutation rates suggest that diversification within the group occurred throughout the Pliocene and mid-Miocene (~2.4–11.5 mya).ConclusionsThe distinct lineages of B. harrisoni correspond to categorical environmental variation, with two lineages comprising samples from streams that flow through acidic Table Mountain Sandstone and three lineages with samples from neutral-to-alkaline streams found within eastern South Africa, Malawi and Zambia. The results of this study suggest that B. harrisoni as it is currently recognised is not a single species with a wide geographic range and pH-tolerance, but may comprise up to five species under the phylogenetic species concept, each with limited pH-tolerances, and that the B. harrisoni species group is thus in need of taxonomic review.

Species identification in the taxonomically neglected, highly diverse, neotropical parasitoid wasp genus Notiospathius (Braconidae: Doryctinae) based on an integrative molecular and morphological approach

Various DNA sequence-based methods for species delineation have recently been developed to assess the species-richness of highly diverse, neglected invertebrate taxa. These methods, however, need to be tested under a variety of conditions, including the use of different markers and parameters. Here, we explored the species diversity of a species-rich group of braconid parasitoid wasps, the Neotropical genus Notiospathius, including 233 specimens from 10 different countries. We examined sequences of two mitochondrial (mt) (COI, cyt b) and one nuclear (wg) gene fragments. We analysed them separately as well as concatenating the mt data with the general mixed Yule-coalescent (GMYC) model for species delineation using different tree-building methods and parameters for reconstructing ultrametric trees. We evaluated the performance of GMYC analyses by comparing their species delineations with our morphospecies identifications. Reconstructing ultrametric trees with a relaxed lognormal clock rate using the program BEAST gave the most congruent results with morphology for the two mt markers. A tree obtained with wg using the programs MrBayes+Pathd8 had the fewest cases of incongruence with morphology, though the performance of this nuclear marker was considerably lower than that of COI and cyt b. Species delimitation using the coalescent prior to obtain ultrametric trees was morphologically more congruent with COI, whereas the Yule prior was more congruent with cyt b. The analyses concatenating the mt datasets failed to recover some species supported both by morphology and the separate analyses of the mt markers. The highest morphological congruence was obtained with the GMYC analysis on an ultrametric tree reconstructed with cyt b using the relaxed lognormal clock rate and the Yule prior, thus supporting the importance of using alternative markers when the information of the barcoding locus (COI) is not concordant with morphological evidence. Seventy-one species were delimited based on the congruence found among COI, cyt b and morphology. Both mt markers also revealed the existence of seven potential cryptic species. This high species richness from a scattered geographical sampling indicates that there is a remarkable number of Notiospathius species that remains undiscovered.

Toward a DNA Taxonomy of Alpine Rhithrogena (Ephemeroptera: Heptageniidae) Using a Mixed Yule-Coalescent Analysis of Mitochondrial and Nuclear DNA

Aquatic larvae of many Rhithrogena mayflies (Ephemeroptera) inhabit sensitive Alpine environments. A number of species are on the IUCN Red List and many recognized species have restricted distributions and are of conservation interest. Despite their ecological and conservation importance, ambiguous morphological differences among closely related species suggest that the current taxonomy may not accurately reflect the evolutionary diversity of the group. Here we examined the species status of nearly 50% of European Rhithrogena diversity using a widespread sampling scheme of Alpine species that included 22 type localities, general mixed Yule-coalescent (GMYC) model analysis of one standard mtDNA marker and one newly developed nDNA marker, and morphological identification where possible. Using sequences from 533 individuals from 144 sampling localities, we observed significant clustering of the mitochondrial (cox1) marker into 31 GMYC species. Twenty-one of these could be identified based on the presence of topotypes (expertly identified specimens from the species' type locality) or unambiguous morphology. These results strongly suggest the presence of both cryptic diversity and taxonomic oversplitting in Rhithrogena. Significant clustering was not detected with protein-coding nuclear PEPCK, although nine GMYC species were congruent with well supported terminal clusters of nDNA. Lack of greater congruence in the two data sets may be the result of incomplete sorting of ancestral polymorphism. Bayesian phylogenetic analyses of both gene regions recovered four of the six recognized Rhithrogena species groups in our samples as monophyletic. Future development of more nuclear markers would facilitate multi-locus analysis of unresolved, closely related species pairs. The DNA taxonomy developed here lays the groundwork for a future revision of the important but cryptic Rhithrogena genus in Europe.

SPECIATION AND DISPERSAL ALONG CONTINENTAL COASTLINES AND ISLAND ARCS IN THE INDO-WEST PACIFIC TURBINID GASTROPOD GENUSLUNELLA

Species trees were produced for the Indo-West Pacific (IWP) gastropod genus Lunella using MrBayes, BEAST, and *BEAST with sequence data from four genes. Three fossil records were used to calibrate a molecular clock. Eight cryptic species were recognized using statistical methods for species delimitation in combination with morphological differences. However, our results suggest caution in interpreting ESUs defined solely by the general mixed Yule Coalescent model in genera like Lunella, with lower dispersal abilities. Four almost entirely allopatric species groups were recovered that differ in ecology and distribution. Three groups occur predominantly along continental coastlines and one occurs on island arrays. Sympatric species occur only in the torquata and coronata groups along coastlines, whereas species in the cinerea group, distributed in two-dimensional island arrays, occur in complete allopatry. Dispersal along island arcs has been important in the maintenance of species distributions and gene flow among populations in the cinerea group. The emergence of new islands and their eventual subsidence over geological time has had important consequences for the isolation of populations and the eventual rise of new species in Lunella.

Evolutionary criteria outperform operational approaches in producing ecologically relevant fungal species inventories

Analyses of the structure and function of microbial communities are highly constrained by the diversity of organisms present within most environmental samples. A common approach is to rely almost entirely on DNA sequence data for estimates of microbial diversity, but to date there is no objective method of clustering sequences into groups that is grounded in evolutionary theory of what constitutes a biological lineage. The general mixed Yule-coalescent (GMYC) model uses a likelihood-based approach to distinguish population-level processes within lineages from processes associated with speciation and extinction, thus identifying a distinct point where extant lineages became independent. Using two independent surveys of DNA sequences associated with a group of ubiquitous plant-symbiotic fungi, we compared estimates of species richness derived using the GMYC model to those based on operational taxonomic units (OTUs) defined by fixed levels of sequence similarity. The model predicted lower species richness in these surveys than did traditional methods of sequence similarity. Here, we show for the first time that groups delineated by the GMYC model better explained variation in the distribution of fungi in relation to putative niche-based variables associated with host species identity, edaphic factors, and aspects of how the sampled ecosystems were managed. Our results suggest the coalescent-based GMYC model successfully groups environmental sequences of fungi into clusters that are ecologically more meaningful than more arbitrary approaches for estimating species richness.

DNA barcoding a highly diverse group of parasitoid wasps (Braconidae: Doryctinae) from a Mexican nature reserve

Background and aims. The preliminary results of a DNA barcoding study of the doryctine fauna of parasitoid wasps from the Chamela–Cuixmala Biosphere Reserve in Mexico, a region dominated by tropical dry forest, are presented. So far, three field trips have been carried out to the reserve and 468 specimens have been collected, of which 407 cox1 sequences were obtained.Materials and methods. The general mixed Yule-coalescent model was applied to a phylogram to investigate the number of evolutionary units that can be detected from the DNA sequence data examined.Results. A total of 185 barcoding species assigned to 20 identified doryctine genera were discriminated using the above model, 115 of which belong to the speciose genus Heterospilus, pointing out the extraordinary species richness of this subfamily of insects in a Mexican tropical dry forest.Conclusion. On the basis of the DNA barcodes generated, Ptesimogastroides Braet & van Achterberg is proposed to be a junior synonym of Ptesimogaster Marsh syn. nov. Neoheterospilus was also found deeply nested within a large Heterospilus clade, suggesting the paraphyly of the latter genus.