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.     

Cryptic Genetic Diversity Is Paramount in Small-Bodied Amphibians of the Genus Euparkerella (Anura: Craugastoridae) Endemic to the Brazilian Atlantic Forest

Morphological similarity associated to restricted distributions and low dispersal abilities make the direct developing “Terrarana” frogs of the genus Euparkerella a good model for examining diversification processes. We here infer phylogenetic relationships within the genus Euparkerella, using DNA sequence data from one mitochondrial and four nuclear genes coupled with traditional Bayesian phylogenetic reconstruction approaches and more recent coalescent methods of species tree inference. We also used Bayesian clustering analysis and a recent Bayesian coalescent-based approach specifically to infer species delimitation. The analysis of 39 individuals from the four known Euparkerella species uncovered high levels of genetic diversity, especially within the two previously morphologically-defined E. cochranae and E. brasiliensis. Within these species, the gene trees at five independent loci and trees from combined data (concatenated dataset and the species tree) uncovered six deeply diverged and geographically coherent evolutionary units, which may have diverged between the Miocene and the Pleistocene. These six units were also uncovered in the Bayesian clustering analysis, and supported by the Bayesian coalescent-based species delimitation (BPP), and Genealogical Sorting Index (GSI), providing thus strong evidence for underestimation of the current levels of diversity within Euparkerella. The cryptic diversity now uncovered opens new opportunities to examine the origins and maintenance of microendemism in the context of spatial heterogeneity and/or human induced fragmentation of the highly threatened Brazilian Atlantic forest hotspot.     

Not as Ubiquitous as We Thought: Taxonomic Crypsis,Hidden Diversity and Cryptic Speciation in the Cosmopolitan Fungus Thelonectria discophora (Nectriaceae,Hypocreales, Ascomycota)

The distribution of microbial species, including fungi, has long been considered cosmopolitan. Recently, this perception has been challenged by molecular studies in historical biogeography, phylogeny and population genetics. Here we explore this issue using the fungal morphological species Thelonectria discophora, one of the most common species of fungi in the family Nectriaceae, encountered in almost all geographic regions and considered as a cosmopolitan taxon. In order to determine if T. discophora is a single cosmopolitan species or an assemblage of sibling species, we conducted various phylogenetic analyses, including standard gene concatenation, Bayesian concordance methods, and coalescent-based species tree reconstruction on isolates collected from a wide geographic range. Results show that diversity among isolates referred as T. discophora is greatly underestimated and that it represents a species complex. Within this complex, sixteen distinct highly supported lineages were recovered, each of which has a restricted geographic distribution and ecology. The taxonomic status of isolates regarded as T. discophora is reconsidered, and the assumed cosmopolitan distribution of this species is rejected. We discuss how assumptions about geographically widespread species have implications regarding their taxonomy, true diversity, biological diversity conservation, and ecological functions.     

Phylogenomics and Coalescent Analyses Resolve Extant Seed Plant Relationships

The extant seed plants include more than 260,000 species that belong to five main lineages: angiosperms, conifers, cycads, Ginkgo, and gnetophytes. Despite tremendous effort using molecular data, phylogenetic relationships among these five lineages remain uncertain. Here, we provide the first broad coalescent-based species tree estimation of seed plants using genome-scale nuclear and plastid data By incorporating 305 nuclear genes and 47 plastid genes from 14 species, we identify that i) extant gymnosperms (i.e., conifers, cycads, Ginkgo, and gnetophytes) are monophyletic, ii) gnetophytes exhibit discordant placements within conifers between their nuclear and plastid genomes, and iii) cycads plus Ginkgo form a clade that is sister to all remaining extant gymnosperms. We additionally observe that the placement of Ginkgo inferred from coalescent analyses is congruent across different nucleotide rate partitions. In contrast, the standard concatenation method produces strongly supported, but incongruent placements of Ginkgo between slow- and fast-evolving sites. Specifically, fast-evolving sites yield relationships in conflict with coalescent analyses. We hypothesize that this incongruence may be related to the way in which concatenation methods treat sites with elevated nucleotide substitution rates. More empirical and simulation investigations are needed to understand this potential weakness of concatenation methods.     

Species tree estimation and the historical biogeography of heroine cichlids

Heroine cichlids are major components of the fish faunas in both Central America and the Caribbean. To examine the evolutionary patterns of how cichlids colonized both of these regions, we reconstructed the phylogenetic relationships among 23 cichlid lineages. We used three phylogenetically novel nuclear markers (Dystropin b, Myomesin1, and Wnt7b) in combination with sequence data from seven other gene regions (Nd2, Rag1, Enc1, Sreb2, Ptr, Plagl2, and Zic1) to elucidate the species tree of these cichlids. The species examined represent major heroine lineages in South America, Central America, and the Greater Antilles. The individual gene trees of these groups were topologically quite discordant. Therefore, we combined the genetic partitions and inferred the species tree using both concatenation and a coalescent-based Bayesian method. The two resulting phylogenetic topologies were largely concordant but differed in two fundamental ways. First, more nodes in the concatenated tree were supported with substantial or 100% Bayesian posterior support than in the coalescent-based tree. Second, there was a minor, but biogeographically critical, topological difference between the concatenated and coalescent-based trees. Nevertheless, both analyses recovered topologies consistent with the Greater Antillean heroines being phylogenetically nested within the largely Central American heroine radiation. This study suggests that reconstructions of cichlid phylogeny and historical biogeography should account for the vagaries of individual gene histories.     

Species trees for spiny lizards (Genus Sceloporus): Identifying points of concordance and conflict between nuclear and mitochondrial data

Spiny lizards (genus Sceloporus) represent one of the most diverse and species rich clades of squamate reptiles in continental North America. Sceloporus contains 90+ species, which are partitioned into 21 species groups containing anywhere from one to 15 species. Despite substantial progress towards elucidating the phylogeographic patterns for many species of Sceloporus, efforts to resolve the phylogenetic relationships among the major species groups remain limited. In this study, the phylogenetic relationships of 53 species of Sceloporus, representing all 21 species groups, are estimated based on four nuclear genes (BDNF, PNN, R35, RAG-1; >3.3 kb) and contrasted with a new mitochondrial DNA genealogy based on six genes (12S, ND1, ND4, and the histidine, serine, and leucine tRNA genes; >2.5 kb). Species trees estimated from the nuclear loci using data concatenation or a coalescent-based inference method result in concordant topologies, but the coalescent approach provides lower resolution and support. When comparing nuclear versus mtDNA-based topologies for Sceloporus species groups, conflicting relationships outnumber concordant relationships. Incongruence is not restricted to weak or unresolved nodes as might be expected under a scenario of rapid diversification, but extends to conflicts involving strongly support clades. The points of concordance and conflict between the nuclear and mtDNA data are discussed, and arguments for preferring the species trees estimated from the multilocus nuclear data are presented.     

Phylogeny estimation of the radiation of western North American chipmunks (Tamias) in the face of introgression using reproductive protein genes

The causes and consequences of rapid radiations are major unresolved issues in evolutionary biology. This is in part because phylogeny estimation is confounded by processes such as stochastic lineage sorting and hybridization. Because these processes are expected to be heterogeneous across the genome, comparison among marker classes may provide a means of disentangling these elements. Here we use introns from nuclear-encoded reproductive protein genes expected to be resistant to introgression to estimate the phylogeny of the western chipmunks (Tamias: subgenus: Neotamias), a rapid radiation that has experienced introgressive hybridization of mitochondrial DNA (mtDNA). We analyze the nuclear loci using coalescent-based species-tree estimation methods and concatenation to estimate a species tree and we use parametric bootstraps and coalescent simulations to differentiate between phylogenetic error, coalescent stochasticity and introgressive hybridization. Results indicate that the mtDNA gene tree reflects several introgression events that have occurred between taxa of varying levels of divergence and at different time points in the tree. T. panamintinus and T. speciosus appear to be fixed for ancient mitochondrial introgressions from T. minimus. A southern Rocky Mountains clade appears well sorted (i.e., species are largely monophyletic) at multiple nuclear loci, while five of six taxa are nonmonophyletic based on cytochrome b. Our simulations reject phylogenetic error and coalescent stochasticity as causes. The results represent an advance in our understanding of the processes at work during the radiation of Tamias and suggest that sampling reproductive-protein genes may be a viable strategy for phylogeny estimation of rapid radiations in which reproductive isolation is incomplete. However, a genome-scale survey that can statistically compare heterogeneity of genealogical process at many more loci will be necessary to test this conclusion.     

Multilocus phylogenetics of a rapid radiation in the genus Thomomys (Rodentia: Geomyidae)

Species complexes undergoing rapid radiation present a challenge in molecular systematics because of the possibility that ancestral polymorphism is retained in component gene trees. Coalescent theory has demonstrated that gene trees often fail to match lineage trees when taxon divergence times are less than the ancestral effective population sizes. Suggestions to increase the number of loci and the number of individuals per taxon have been proposed; however, phylogenetic methods to adequately analyze these data in a coalescent framework are scarce. We compare two approaches to estimating lineage (species) trees using multiple individuals and multiple loci: the commonly used partitioned Bayesian analysis of concatenated sequences and a modification of a newly developed hierarchical Bayesian method (BEST) that simultaneously estimates gene trees and species trees from multilocus data. We test these approaches on a phylogeny of rapidly radiating species wherein divergence times are likely to be smaller than effective population sizes, and incomplete lineage sorting is known, in the rodent genus, Thomomys. We use seven independent noncoding nuclear sequence loci (total approximately 4300 bp) and between 1 and 12 individuals per taxon to construct a phylogenetic hypothesis for eight Thomomys species. The majority-rule consensus tree from the partitioned concatenated analysis included 14 strongly supported bipartitions, corroborating monophyletic species status of five of the eight named species. The BEST tree strongly supported only the split between the two subgenera and showed very low support for any other clade. Comparison of both lineage trees to individual gene trees revealed that the concatenation method appears to ignore conflicting signals among gene trees, whereas the BEST tree considers conflicting signals and downweights support for those nodes. Bayes factor analysis of posterior tree distributions from both analyses strongly favor the model underlying the BEST analysis. This comparison underscores the risks of overreliance on results from concatenation, and ignoring the properties of coalescence, especially in cases of recent, rapid radiations.     

Cryptic diversity in the Mexican highlands: Thousands of UCE loci help illuminate phylogenetic relationships,species limits and divergence times of montane rattlesnakes (Viperidae: Crotalus)

With the continued adoption of genome‐scale data in evolutionary biology comes the challenge of adequately harnessing the information to make accurate phylogenetic inferences. Coalescent‐based methods of species tree inference have become common, and concatenation has been shown in simulation to perform well, particularly when levels of incomplete lineage sorting are low. However, simulation conditions are often overly simplistic, leaving empiricists with uncertainty regarding analytical tools. We use a large ultraconserved element data set (>3,000 loci) from rattlesnakes of the Crotalus triseriatus group to delimit lineages and estimate species trees using concatenation and several coalescent‐based methods. Unpartitioned and partitioned maximum likelihood and Bayesian analysis of the concatenated matrix yield a topology identical to coalescent analysis of a subset of the data in bpp . ASTRAL analysis on a subset of the more variable loci also results in a tree consistent with concatenation and bpp , whereas the SVDquartets phylogeny differs at additional nodes. The size of the concatenated matrix has a strong effect on species tree inference using SVDquartets , warranting additional investigation on optimal data characteristics for this method. Species delimitation analyses suggest up to 16 unique lineages may be present within the C. triseriatus group, with divergences occurring during the Neogene and Quaternary. Network analyses suggest hybridization within the group is relatively rare. Altogether, our results reaffirm the Mexican highlands as a biodiversity hotspot and suggest that coalescent‐based species tree inference on data subsets can provide a strongly supported species tree consistent with concatenation of all loci with a large amount of missing data.     

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.     

Evaluating evolutionary history in the face of high gene tree discordance in Australian Gehyra (Reptilia: Gekkonidae)

Species tree methods have provided improvements for estimating species relationships and the timing of diversification in recent radiations by allowing for gene tree discordance. Although gene tree discordance is often observed, most discordance is attributed to incomplete lineage sorting rather than other biological phenomena, and the causes of discordance are rarely investigated. We use species trees from multi-locus data to estimate the species relationships, evolutionary history and timing of diversification among Australian Gehyra—a group renowned for taxonomic uncertainty and showing a large degree of gene tree discordance. We find support for a recent Asian origin and two major clades: a tropically adapted clade and an arid adapted clade, with some exceptions, but no support for allopatric speciation driven by chromosomal rearrangement in the group. Bayesian concordance analysis revealed high gene tree discordance and comparisons of Robinson–Foulds distances showed that discordance between gene trees was significantly higher than that generated by topological uncertainty within each gene. Analysis of gene tree discordance and incomplete taxon sampling revealed that gene tree discordance was high whether terminal taxon or gene sampling was maximized, indicating discordance is due to biological processes, which may be important in contributing to gene tree discordance in many recently diversified organisms.     

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.     

Phylogeny Reconstruction and Hybrid Analysis of Populus (Salicaceae) Based on Nucleotide Sequences of Multiple Single-Copy Nuclear Genes and Plastid Fragments

Populus (Salicaceae) is one of the most economically and ecologically important genera of forest trees. The complex reticulate evolution and lack of highly variable orthologous single-copy DNA markers have posed difficulties in resolving the phylogeny of this genus. Based on a large data set of nuclear and plastid DNA sequences, we reconstructed robust phylogeny of Populus using parsimony, maximum likelihood and Bayesian inference methods. The resulting phylogenetic trees showed better resolution at both inter- and intra-sectional level than previous studies. The results revealed that (1) the plastid-based phylogenetic tree resulted in two main clades, suggesting an early divergence of the maternal progenitors of Populus; (2) three advanced sections (Populus, Aigeiros and Tacamahaca) are of hybrid origin; (3) species of the section Tacamahaca could be divided into two major groups based on plastid and nuclear DNA data, suggesting a polyphyletic nature of the section; and (4) many species proved to be of hybrid origin based on the incongruence between plastid and nuclear DNA trees. Reticulate evolution may have played a significant role in the evolution history of Populus by facilitating rapid adaptive radiations into different environments.     

Morphing of the phylogeographic lineages of the Balkan alpine newts (Ichthyosaura alpestris, Caudata, Salamandridae): in situ morphological diversification

Numerous alpine newt (Ichthyosaura alpestris) populations from the Balkans, representing all the previously established phylogeographic lineages, were studied for variations in various morphological characteristics (body size and shape, skull qualitative traits and number of trunk vertebrae). Here, we present a decoupling of morphological and mtDNA phylogeographic substructuring in the alpine newt on the Balkan Peninsula. In sharp contrast to other European newts (Triturus spp., Lissotriton spp.), the vast majority of morphological variation in the alpine newt is concentrated at the population level indicating an in situ morphological diversification. We found that the rate of morphological change is similar to the rate of mtDNA change. We hypothesize that the alpine newts are characterized by non-adaptive morphological evolution.     

Limited genetic divergence among Australian alpine Poa tussock grasses coupled with regional structuring points to ongoing gene flow and taxonomic challenges

Background and Aims

While molecular approaches can often accurately reconstruct species relationships, taxa that are incompletely differentiated pose a challenge even with extensive data. Such taxa are functionally differentiated, but may be genetically differentiated only at small and/or patchy regions of the genome. This issue is considered here in Poa tussock grass species that dominate grassland and herbfields in the Australian alpine zone.

Methods

Previously reported tetraploidy was confirmed in all species by sequencing seven nuclear regions and five microsatellite markers. A Bayesian approach was used to co-estimate nuclear and chloroplast gene trees with an overall dated species tree. The resulting species tree was used to examine species structure and recent hybridization, and intertaxon fertility was tested by experimental crosses.

Key Results

Species tree estimation revealed Poa gunnii, a Tasmanian endemic species, as sister to the rest of the Australian alpine Poa. The taxa have radiated in the last 0·5–1·2 million years and the non-gunnii taxa are not supported as genetically distinct. Recent hybridization following past species divergence was also not supported. Ongoing gene flow is suggested, with some broad-scale geographic structure within the group.

Conclusions

The Australian alpine Poa species are not genetically distinct despite being distinguishable phenotypically, suggesting recent adaptive divergence with ongoing intertaxon gene flow. This highlights challenges in using conventional molecular taxonomy to infer species relationships in recent, rapid radiations.     

Concatenation and concordance in the reconstruction of mouse lemur phylogeny: an empirical demonstration of the effect of allele sampling in phylogenetics

The systematics and speciation literature is rich with discussion relating to the potential for gene tree/species tree discordance. Numerous mechanisms have been proposed to generate discordance, including differential selection, long-branch attraction, gene duplication, genetic introgression, and/or incomplete lineage sorting. For speciose clades in which divergence has occurred recently and rapidly, recovering the true species tree can be particularly problematic due to incomplete lineage sorting. Unfortunately, the availability of multilocus or "phylogenomic" data sets does not simply solve the problem, particularly when the data are analyzed with standard concatenation techniques. In our study, we conduct a phylogenetic study for a nearly complete species sample of the dwarf and mouse lemur clade, Cheirogaleidae. Mouse lemurs (genus, Microcebus) have been intensively studied over the past decade for reasons relating to their high level of cryptic species diversity, and although there has been emerging consensus regarding the evolutionary diversity contained within the genus, there is no agreement as to the inter-specific relationships within the group. We attempt to resolve cheirogaleid phylogeny, focusing especially on the mouse lemurs, by employing a large multilocus data set. We compare the results of Bayesian concordance methods with those of standard gene concatenation, finding that though concatenation yields the strongest results as measured by statistical support, these results are found to be highly misleading. By employing an approach where individual alleles are treated as operational taxonomic units, we show that phylogenetic results are substantially influenced by the selection of alleles in the concatenation process.     

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.