Evaluating multilocus Bayesian species delimitation for discovery of cryptic mycorrhizal diversity

The increasing availability of DNA sequence data enables exciting new opportunities for fungal ecology. However, it amplifies the challenge of how to objectively classify the diversity of fungal sequences into meaningful units, often in the absence of morphological characters. Here, we test the utility of modern multilocus Bayesian coalescent-based methods for delimiting cryptic fungal diversity in the orchid mycorrhiza morphospecies Serendipita vermifera. We obtained 147 fungal isolates from Caladenia, a speciose clade of Australian orchids known to associate with Serendipita fungi. DNA sequence data for 7 nuclear and mtDNA loci were used to erect competing species hypotheses by clustering isolates based on: (a) ITS sequence divergence, (b) Bayesian admixture analysis, and (c) mtDNA variation. We implemented two coalescent-based Bayesian methods to determine which species hypothesis best fitted our data. Both methods found strong support for eight species of Serendipita among our isolates, supporting species boundaries reflected in ITS divergence. Patterns of host plant association showed evidence for both generalist and specialist associations within the host genus Caladenia. Our findings demonstrate the utility of Bayesian species delimitation methods and suggest that wider application of these techniques will readily uncover new species in other cryptic fungal lineages.     

Species tree inference in a recent radiation of orioles (Genus Icterus): multiple markers and methods reveal cytonuclear discordance in the northern oriole group

Recent computational advances provide novel opportunities to infer species trees based on multiple independent loci. Thus, single gene trees no longer need suffice as proxies for species phylogenies. Several methods have been developed to deal with the challenges posed by incomplete and stochastic lineage sorting. In this study, we employed four Bayesian methods to infer the phylogeny of a clade of 11 recently diverged oriole species within the genus Icterus. We obtained well-resolved and mostly congruent phylogenies using a set of seven unlinked nuclear intron loci and sampling multiple individuals per species. Most notably, Bayesian concordance analysis generally agreed well with concatenation; the two methods agreed fully on eight of nine nodes. The coalescent-based method ^∗BEAST further supported six of these eight nodes. The fourth method used, BEST, failed to converge despite exhaustive efforts to optimize the tree search. Overall, the results obtained by new species tree methods and concatenation generally corroborate our findings from previous analyses and data sets. However, we found striking disagreement between mitochondrial and nuclear DNA involving relationships within the northern oriole group. Our results highlight the danger of reliance on mtDNA alone for phylogenetic inference. We demonstrate that in spite of low variability and incomplete lineage sorting, multiple nuclear loci can produce largely congruent phylogenies based on multiple species tree methods, even for very closely-related species.     

Data Concatenation,Bayesian Concordance and Coalescent-Based Analyses of the Species Tree for the Rapid Radiation of Triturus Newts

The phylogenetic relationships for rapid species radiations are difficult to disentangle. Here we study one such case, namely the genus Triturus, which is composed of the marbled and crested newts. We analyze data for 38 genetic markers, positioned in 3-prime untranslated regions of protein-coding genes, obtained with 454 sequencing. Our dataset includes twenty Triturus newts and represents all nine species. Bayesian analysis of population structure allocates all individuals to their respective species. The branching patterns obtained by data concatenation, Bayesian concordance analysis and coalescent-based estimations of the species tree differ from one another. The data concatenation based species tree shows high branch support but branching order is considerably affected by allele choice in the case of heterozygotes in the concatenation process. Bayesian concordance analysis expresses the conflict between individual gene trees for part of the Triturus species tree as low concordance factors. The coalescent-based species tree is relatively similar to a previously published species tree based upon morphology and full mtDNA and any conflicting internal branches are not highly supported. Our findings reflect high gene tree discordance due to incomplete lineage sorting (possibly aggravated by hybridization) in combination with low information content of the markers employed (as can be expected for relatively recent species radiations). This case study highlights the complexity of resolving rapid radiations and we acknowledge that to convincingly resolve the Triturus species tree even more genes will have to be consulted.     

Evolutionary History of the Morocco lizard-Fingered Geckos of the <Emphasis Type="Italic">Saurodactylus brosseti</Emphasis> Complex

Studies of biodiversity in the Maghreb have revealed high genetic diversity and divergent genetic lineages among many taxa including squamates. Geographic barriers such as the Atlas Mountains are one of the key factors promoting genetic differentiation and the high levels of endemism. The lizard-fingered gecko Saurodactylus brosseti is endemic to Morocco. Its range includes both sides of the Atlas Mountains, and although high diversity was previously identified within the species, much of the range was unsampled. To understand the evolutionary and biogeographical history of this species, we used mitochondrial and nuclear DNA sequence data from 64 populations sampled across the entire species range. We employed phylogenetic methods based on gene trees and species trees as well as a time calibrated Bayesian genealogy and coalescent species delimitation approaches. We uncovered four highly divergent and allopatric mitochondrial lineages that did not share haplotypes at variable nuclear loci, suggesting the four groups have been evolving independently since the Miocene, according to our molecular dating estimates. These results coupled with the geographic pattern of genetic diversity suggest a possible role of the Atlas Mountains for the divergence observed between the four lineages of S. brosseti, while each lineage probably later underwent several episodes of fragmentation followed by (re-) expansion during Pleistocene climatic oscillations. Bayesian species delimitation analysis indicates that the four lineages may well be distinct species but we suggest that detailed morphological analyses are needed prior to taxonomic changes. The four lineages represent ancient independent evolutionary units, and deserve conservation management as distinct entities.     

Using multilocus approach to uncover cryptic diversity within Pseudotrapelus lizards from Saudi Arabia

The agamid Pseudotrapelus lizards inhabit the mountainous areas of the Arabian Peninsula and eastern North Africa. Currently six Pseudotrapelus species are recognised, though diagnostic morphological characters are still lacking, creating great difficulty in describing new species. Recently, two specimens of Pseudotrapelus were collected from the vicinity of Riyadh in central Saudi Arabia, an area that was not sampled in previous phylogenetic studies. In here we used both mitochondrial and nuclear data to investigate the phylogenetic position of the new samples, and assess their phylogenetic relationships with the other recognised species of Pseudotrapelus from across the distribution range of the genus. We used a multilocus approach of haplotype networks, concatenated datasets and species trees, performed mitochondrial and nuclear species delimitation analyses, and estimated divergence times. In general, our results support previous molecular studies and uncover the presence of cryptic diversity within Pseudotrapelus. The phylogenetic structure of the genus is of two major clades and within them seven distinct, delimited phylogenetic groups belonging to the six recognised species and the seventh to the individuals from Riyadh. The Riyadh specimens were distinct in all analyses performed. We suggest that the new specimens from the Riyadh area are a distinct lineage, forming a clade with their phylogenetic relatives, P. sinaitus and P. chlodnickii. The clade formed by these three species diverged during the Late Miocene around 6.4 Ma, with cladogenesis possibly facilitated by vicariance and isolation caused due to climatic fluctuations and the progression of sandy areas. Our results suggest further morphological research is necessary to revise the taxonomic status of this lineage and of the entire genus.     

Phylogeny and species reassessment of Hyalopterus (Aphididae,Aphidinae)

The broadly distributed genus Hyalopterus currently comprises three formally recognized species that are highly similar morphologically and hence difficult to be identified with certainty. This group has undergone multiple revisions in the past century, but none of these has assessed species from Asia, which has hampered our understanding of the species diversity within this genus. Based on a comprehensive data set from morphological data and host-associated data, and by coalescent-based delimitation approaches, the Hyalopterus species boundaries, distribution and diversity were clarified here to further reveal the composition of the species. Two single-locus (ML-GMYC and mPTP) and two multilocus (BPP and STACEY) delimitation methods were conducted based on extensive sampling. Then, the phylogenetic relationships and morphological divergence were assessed. Our data strongly supported that the number of recognized species in Hyalopterus had likely been underestimated. The phylogenetic analyses recovered four major clades, which corresponded to distinct host-plant preferences. Also, the morphological analyses showed significant differentiation for only one of the newly recognized candidate species uncovered by the delimitation approaches, suggesting the existence of at least two independent evolutionary lineages within Hyalopterus arundiniformis, which showed different patterns of host association. Moreover, based on our data, the taxonomic misidentification of H. arundiniformis in China was corrected here. This study lays the groundwork for the thorough taxonomic revision of Hyalopterus and for future evolutionary studies and underlines the importance of an integrated framework for species determination.     

Species delimitation and phylogeography of Aphonopelma hentzi (Araneae, Mygalomorphae, Theraphosidae): cryptic diversity in North American tarantulas

Background

The primary objective of this study is to reconstruct the phylogeny of the hentzi species group and sister species in the North American tarantula genus, Aphonopelma, using a set of mitochondrial DNA markers that include the animal “barcoding gene”. An mtDNA genealogy is used to consider questions regarding species boundary delimitation and to evaluate timing of divergence to infer historical biogeographic events that played a role in shaping the present-day diversity and distribution. We aimed to identify potential refugial locations, directionality of range expansion, and test whether A. hentzi post-glacial expansion fit a predicted time frame.

Methods and Findings

A Bayesian phylogenetic approach was used to analyze a 2051 base pair (bp) mtDNA data matrix comprising aligned fragments of the gene regions CO1 (1165 bp) and ND1-16S (886 bp). Multiple species delimitation techniques (DNA tree-based methods, a “barcode gap” using percent of pairwise sequence divergence (uncorrected p-distances), and the GMYC method) consistently recognized a number of divergent and genealogically exclusive groups.

Conclusions

The use of numerous species delimitation methods, in concert, provide an effective approach to dissecting species boundaries in this spider group; as well they seem to provide strong evidence for a number of nominal, previously undiscovered, and cryptic species. Our data also indicate that Pleistocene habitat fragmentation and subsequent range expansion events may have shaped contemporary phylogeographic patterns of Aphonopelma diversity in the southwestern United States, particularly for the A. hentzi species group. These findings indicate that future species delimitation approaches need to be analyzed in context of a number of factors, such as the sampling distribution, loci used, biogeographic history, breadth of morphological variation, ecological factors, and behavioral data, to make truly integrative decisions about what constitutes an evolutionary lineage recognized as a “species”.     

Multi-locus species tree of the chub genus <Emphasis Type="Italic">Squalius</Emphasis> (Leuciscinae: Cyprinidae) from western Iberia: new insights into its evolutionary history

The phylogenetic relationships of the genus Squalius are believed to be well established based on the mitochondrial cytochrome b gene. Here, we inferred the phylogenetic relationships of all species inhabiting most of the western Iberia river systems using a nuclear multi-locus approach and different species tree methods: concatenation and coalescent-based methods (BEST and minimize-deep-coalescence). The dataset comprised sequences of seven coding and three non-coding regions belonging to seven nuclear genes, which were chosen to cover multiple biological functions: amh, bmp4, ef1a, egr2, irbp, rh and rpl8. We provide evidence for a conflicting topology between the nuDNA species tree and the widely reported mtDNA gene tree. S. pyrenaicus is rendered paraphyletic in all nuDNA species trees, with populations of the Tagus/Colares clustering with S. carolitertii, while populations from the Guadiana, Sado and Almargem form a separate clade. Although a larger sampling size encompassing the full spectrum of Squalius populations in western Iberia is still needed to fully elucidate the phylogeography and species delimitation of this genus, our results suggest that the two S. pyrenaicus clades may represent different species.     

Molecular phylogenetics and historical biogeography of Rhinolophus bats

The phylogenetic relationships within the horseshoe bats (genus Rhinolophus) are poorly resolved, particularly at deeper levels within the tree. We present a better-resolved phylogenetic hypothesis for 30 rhinolophid species based on parsimony and Bayesian analyses of the mitochondrial cytochrome b gene and three nuclear introns (TG, THY and PRKC1). Strong support was found for the existence of two geographic clades within the monophyletic Rhinolophidae: an African group and an Oriental assemblage. The relaxed Bayesian clock method indicated that the two rhinolophid clades diverged approximately 35 million years ago and results from Dispersal Vicariance (DIVA) analysis suggest that the horseshoe bats arose in Asia and subsequently dispersed into Europe and Africa.     

Species trees for the tree swallows (Genus Tachycineta): An alternative phylogenetic hypothesis to the mitochondrial gene tree

The New World swallow genus Tachycineta comprises nine species that collectively have a wide geographic distribution and remarkable variation both within- and among-species in ecologically important traits. Existing phylogenetic hypotheses for Tachycineta are based on mitochondrial DNA sequences, thus they provide estimates of a single gene tree. In this study we sequenced multiple individuals from each species at 16 nuclear intron loci. We used gene concatenated approaches (Bayesian and maximum likelihood) as well as coalescent-based species tree inference to reconstruct phylogenetic relationships of the genus. We examined the concordance and conflict between the nuclear and mitochondrial trees and between concatenated and coalescent-based inferences. Our results provide an alternative phylogenetic hypothesis to the existing mitochondrial DNA estimate of phylogeny. This new hypothesis provides a more accurate framework in which to explore trait evolution and examine the evolution of the mitochondrial genome in this group.     

Revisiting species delimitation within the genus Oxystele using DNA barcoding approach

The genus Oxystele, a member of the highly diverse marine gastropod superfamily Trochoidea, is endemic to southern Africa. Members of the genus include some of the most abundant molluscs on southern African shores and are important components of littoral biodiversity in rocky intertidal habitats. Species delimitation within the genus is still controversial, especially regarding the complex O. impervia / O. variegata. Here, we assessed species boundaries within the genus using DNA barcoding and phylogenetic tree reconstruction. We analysed 56 specimens using the mitochondrial gene COI. Our analysis delimits five molecular operational taxonomic units (MOTUs), and distinguishes O. impervia from O. variegata. However, we reveal important discrepancies between MOTUs and morphology-based species identification and discuss alternative hypotheses that can account for this. Finally, we indicate the need for future study that includes additional genes, and the combination of both morphology and genetic techniques (e.g. AFLP or microsatellites) to get deeper insight into species delimitation within the genus.     

Molecular phylogeny of Pamphagidae (Acridoidea,Orthoptera) from China based on mitochondrial cytochrome oxidase II sequences

Abstract Phylogenetic relationships of Pamphagidae were examined using cytochrome oxidase subunit II (COII) mtDNA sequences (684 bp). Twenty‐seven species of Acridoidea from 20 genera were sequenced to obtain mtDNA data, along with four species from the GenBank nucleotide database. The purpose of this study was analyzing the phylogenetic relationships among subfamilies within Pamphagidae and interpreting the phylogenetic position of this family within the Acridoidea superfamily. Phylogenetic trees were reconstructed using neighbor‐joining (NJ), maximum parsimony (MP) and Bayesian inference (BI) methods. The 684 bp analyzed fragment included 126 parsimony informative sites. Sequences diverged 1.0%–11.1% between genera within subfamilies, and 8.8%–12.3% between subfamilies. Amino acid sequence diverged 0–6.1% between genera within subfamilies, and 0.4%–7.5% between subfamilies. Our phylogenetic trees revealed the monophyly of Pamphagidae and three distinct major groups within this family. Moreover, several well supported and stable clades were found in Pamphagidae. The global clustering results were similar to that obtained through classical morphological classification: Prionotropisinae, Thrinchinae and Pamphaginae were monophyletic groups. However, the current genus Filchnerella (Prionotropisinae) was not a monophyletic group and the genus Asiotmethis (Prionotropisinae) was a sister group of the genus Thrinchus (Thrinchinae). Further molecular and morphological studies are required to clarify the phylogenetic relationships of the genera Filchnerella and Asiotmethis.     

Multilocus phylogeny and systematics of Iberian endemic Squalius (Actinopterygii,Leuciscidae)

Inferring the evolutionary history of a group of species can be challenging given the many factors involved. In recent years, the increased availability of sequences of multiple genes per species has spurred the development of new methodologies to analyse multilocus data sets. Two approaches that analyse such data are concatenated supermatrix and coalescent-based species-tree analyses. In this study, we used both of these methods to infer the phylogenetic relationships of Iberian species of the genus Squalius from one mitochondrial and six nuclear genes. We found mitonuclear discordance in the phylogenetic relationships of the group. According to the mitochondrial gene analysis, all species were recovered as monophyletic except S. pyrenaicus; besides, in the concatenated supermatrix analysis of the nuclear markers, this species resolved as polyphyletic with three divergent evolutionary lineages. The coalescent-based nuclear species-tree analysis rendered a well-resolved phylogeny compared with the supermatrix analysis, which was unable to discern between S. carolitertii, S. castellanus and one of the evolutionary lineages of S. pyrenaicus. This result is likely due to the better integration of population uncertainty in the coalescent approach. Furthermore, Bayesian multilocus species delimitation analyses based on a BPP approach strongly supported the distinct nuclear lineages as different species. Nevertheless, the supermatrix analysis was able to obtain well-supported relationships in the divergent lineages with low numbers of individuals. Our study highlights the usefulness of different analytical methodologies to obtain a more complete picture of the evolutionary history of taxa, especially when discordant patterns among genes are found.     

Molecular identification of selected bees from the Indian Himalaya: A preliminary effort

DNA barcoding has largely been tested for a wide range of taxa and evidenced as a reliable and rapid molecular tool for species-level identification. The present study lends to generate 156 DNA barcodes, of which 141 belonged to 30 morphologically identified bees from the Indian Himalayan Regions (IHRs). The generated barcode data along with 84 sequences of global database distinctly discriminated all the studied species with sufficient genetic distances and cohesive monophyletic clustering in Bayesian analysis (BA) phylogeny. The species delimitation methods, Automatic Barcode Gap Discovery (ABGD), Bayesian Poisson-Tree-Processes (bPTP), and General Mixed Yule-coalescent (GMYC) yielded 68, 70, and 71 molecular operational taxonomic units (MOTUs) respectively. The present DNA barcode-based examination detected the possible cryptic diversity in two Apis species (A. cerana and A. dorsata), Bombus hypnorum, Lepidotrigona arcifera, and Ceratina sutepensis. The present study also evidenced the species complexes within Bombus albopleuralis and Bombus trifasciatus in the IHRs. The species delimitation methods also detected an additional seven putative species from the IHRs, which were identified up to the genus level. In conclusion, this preliminary effort helps to develop a reliable barcode database of bees from the Indian IHRs to facilitate the future systematics study. These molecular data can be utilized to evaluate the population structures and assist to formulate the effective plans for bee conservation.     

Phylogeny, diversity, and species delimitation of the North American Round-Nosed Minnows (Teleostei: Dionda), as inferred from mitochondrial and nuclear DNA sequences

Accurate delimitation of species is a critical first step in protecting biodiversity. Detection of distinct species is especially important for groups of organisms that inhabit sensitive environments subject to recent degradation, such as creeks, springs, and rivers in arid or semi-desert regions. The genus Dionda currently includes six recognized and described species of minnows that live in clear springs and spring-fed creeks of Texas, New Mexico (USA), and northern Mexico, but the boundaries, delimitation, and characterization of species in this genus have not been examined rigorously. The habitats of some of the species in this genus are rapidly deteriorating, and many local populations of Dionda have been extirpated. Considering the increasing concerns over degradation of their habitat, and pending a more detailed morphological revision of the genus, we undertook a molecular survey based on four DNA regions to examine variation over the range of the genus, test species boundaries, and infer phylogenetic relationships within Dionda. Based on analyses of two mitochondrial (cytb and D-loop) and two nuclear (Rag1 and S7) DNA regions from specimens collected throughout the range of Dionda, we identified 12 distinct species in the genus. Formerly synonymized names are available for two of these species, and four other species remain undescribed. We also redefine the known range of six species. The limited distribution of several of the species, coupled with widespread habitat degradation, suggests that many of the species in this genus should be targets for conservation and recovery efforts.     

How to Handle Speciose Clades? Mass Taxon-Sampling as a Strategy towards Illuminating the Natural History of Campanula (Campanuloideae)

Background

Speciose clades usually harbor species with a broad spectrum of adaptive strategies and complex distribution patterns, and thus constitute ideal systems to disentangle biotic and abiotic causes underlying species diversification. The delimitation of such study systems to test evolutionary hypotheses is difficult because they often rely on artificial genus concepts as starting points. One of the most prominent examples is the bellflower genus Campanula with some 420 species, but up to 600 species when including all lineages to which Campanula is paraphyletic. We generated a large alignment of petD group II intron sequences to include more than 70% of described species as a reference. By comparison with partial data sets we could then assess the impact of selective taxon sampling strategies on phylogenetic reconstruction and subsequent evolutionary conclusions.

Methodology/Principal Findings

Phylogenetic analyses based on maximum parsimony (PAUP, PRAP), Bayesian inference (MrBayes), and maximum likelihood (RAxML) were first carried out on the large reference data set (D680). Parameters including tree topology, branch support, and age estimates, were then compared to those obtained from smaller data sets resulting from “classification-guided” (D088) and “phylogeny-guided sampling” (D101). Analyses of D088 failed to fully recover the phylogenetic diversity in Campanula, whereas D101 inferred significantly different branch support and age estimates.

Conclusions/Significance

A short genomic region with high phylogenetic utility allowed us to easily generate a comprehensive phylogenetic framework for the speciose Campanula clade. Our approach recovered 17 well-supported and circumscribed sub-lineages. Knowing these will be instrumental for developing more specific evolutionary hypotheses and guide future research, we highlight the predictive value of a mass taxon-sampling strategy as a first essential step towards illuminating the detailed evolutionary history of diverse clades.     

Reconciling gene trees of eastern North American minnows

Most eastern North American cyprinid fishes belong to a clade known as the "open posterior myodome" (OPM) minnows, but phylogenetic relationships within this clade have been difficult to ascertain. Previous attempts to resolve relationships among the generally benthic "chubs" and the more pelagic "shiners", that constitute the majority of OPM minnows, have led to highly discordant phylogenetic hypotheses. To further examine relationships among the OPM minnows, we utilized both a concatenated Bayesian approach and a coalescent-based species tree method to analyze data from six protein coding nuclear loci (Enc1, Ptr, Ryr3, Sh3px3, Tbr1, and Zic1), as well as the mitochondrial locus (Cytb). We focused our analyses on the chub-like genus Phenacobius, a group that has drifted topologically between other benthic chubs and the more pelagic shiners, and also included exemplar taxa from 11 other OPM lineages. Individual gene trees were highly discordant regarding relationships within Phenacobius and across the OPM clade. The concatenated Bayesian analysis and coalescent-based species tree reconstruction recovered slightly different phylogenetic topologies. Additionally, the posterior support values for clades using the coalescent-based approach were consistently lower than the concatenated analysis. However, Phenacobius was resolved as monophyletic and as the sister lineage to Erimystax regardless of the combined data approach taken. Furthermore, Phenacobius+Erimystax was recovered as more closely related to the shiners we examined than to other chubs. Relationships within Phenacobius varied depending on the combined phylogenetic method utilized. Our results highlight the importance of multi-locus, coalescent-based approaches for resolving the phylogeny of diverse clades like the eastern North American OPM minnows.