Molecular phylogenetic relationships and phenotypic diversity in miniaturized toadlets, genus Brachycephalus (Amphibia: Anura: Brachycephalidae)

Toadlets of the genus Brachycephalus are endemic to the Atlantic rainforests of southeastern and southern Brazil. The 14 species currently described have snout-vent lengths less than 18 mm and are thought to have evolved through miniaturization: an evolutionary process leading to an extremely small adult body size. Here, we present the first comprehensive phylogenetic analysis for Brachycephalus, using a multilocus approach based on two nuclear (Rag-1 and Tyr) and three mitochondrial (Cyt b, 12S, and 16S rRNA) gene regions. Phylogenetic relationships were inferred using a partitioned Bayesian analysis of concatenated sequences and the hierarchical Bayesian method (BEST) that estimates species trees based on the multispecies coalescent model. Individual gene trees showed conflict and also varied in resolution. With the exception of the mitochondrial gene tree, no gene tree was completely resolved. The concatenated gene tree was completely resolved and is identical in topology and degree of statistical support to the individual mtDNA gene tree. On the other hand, the BEST species tree showed reduced significant node support relative to the concatenate tree and recovered a basal trichotomy, although some bipartitions were significantly supported at the tips of the species tree. Comparison of the log likelihoods for the concatenated and BEST trees suggests that the method implemented in BEST explains the multilocus data for Brachycephalus better than the Bayesian analysis of concatenated data. Landmark-based geometric morphometrics revealed marked variation in cranial shape between the species of Brachycephalus. In addition, a statistically significant association was demonstrated between variation in cranial shape and genetic distances estimated from the mtDNA and nuclear loci. Notably, B. ephippium and B. garbeana that are predicted to be sister-species in the individual and concatenated gene trees and the BEST species tree share an evolutionary novelty, the hyperossified dorsal plate.     

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.     

Is homoplasy or lineage sorting the source of incongruent mtdna and nuclear gene trees in the stiff-tailed ducks (Nomonyx-Oxyura)?

We evaluated the potential effects of homoplasy, ancestral polymorphism, and hybridization as obstacles to resolving phylogenetic relationships within Nomonyx-Oxyura stiff-tailed ducks (Oxyurinae; subtribe Oxyurina). Mitochondrial DNA (mtDNA) control region sequences from 94 individuals supported monophyly of mtDNA haplotypes for each of the six species and provided no evidence of extant incomplete lineage sorting or inter-specific hybridization. The ruddy ducks (O. j. jamaicensis,O. j. andina, O. j. ferruginea) are each others' closest relatives, but the lack of shared haplotypes between O. j. jamaicensis and O. j. ferruginea suggests long-standing historical isolation. In contrast, O. j. andina shares haplotypes with O. j. jamaicensis and O. j. ferruginea, which supports Todd's (1979) and Fjelds?'s (1986) hypothesis that O. j. andina is an intergrade or hybrid subspecies of O. j. jamaicensis and O. j. ferruginea. Control region data and a much larger data set composed of approximately 8800 base pairs of mitochondrial and nuclear sequence for each species indicate that the two New World species, O. vittata and O. jamaicensis, branch basally within Oxyura. A clade of three Old World species (O. australis, O. maccoa, O. leucocephala) is well supported, but different loci and also different characters within the mtDNA data support three different resolutions of the Old World clade, yielding an essentially unresolved trichotomy. Fundamentally different factors limited the resolution of the mtDNA and nuclear gene trees. Gene trees for most nuclear loci were unresolved due to slow rates of mutation and a lack of informative variation, whereas uncertain resolution of the mtDNA gene tree was due to homoplasy. Within the mtDNA, approximately equal numbers of characters supported each of three possible resolutions. Parametric and nonparametric bootstrap analyses suggest that resolution of the mtDNA tree based on ~4300 bp per taxon is uncertain but that complete mtDNA sequences would yield a fully resolved gene tree. A short internode separating O. leucocephala from (O. australis, O. maccoa) in the best mtDNA tree combined with long terminal branches and substantial rate variation among nucleotide sites allowed the small number of changes occurring on the internode to be obscured by homoplasy in a significant portion of simulated data sets. Although most nuclear loci were uninformative, two loci supported a resolution of the Old World clade (O. maccoa, O. leucocephala) that is incongruent with the best mtDNA tree. Thus, incongruence between nuclear and mtDNA trees may be due to random sorting of ancestral lineages during the short internode, homoplasy in the mtDNA data, or both. The Oxyura trichotomy represents a difficult though likely common problem in molecular systematics. Given a short internode, the mtDNA tree has a greater chance of being congruent with the history of speciation because its effective population size (N(e)) is one-quarter that of any nuclear locus, but its resolution is more likely to be obscured by homoplasy. In contrast, gene trees for more slowly evolving nuclear loci will be difficult to resolve due to a lack of substitutions during the internode, and when resolved are more likely to be incongruent with the species history due to the stochastic effects of lineage sorting. We suggest that researchers consider first whether independent gene trees are adequately resolved and then whether those trees are congruent with the species history. In the case of Oxyura, the answer to both questions may be no. Complete mtDNA sequences combined with data from a very large number of nuclear loci may be the only way to resolve such trichotomies.     

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.     

A species tree for the Australo-Papuan Fairy-wrens and allies (Aves: Maluridae)

We explored the efficacy of species tree methods at the family level in birds, using the Australo-Papuan Fairy-wrens (Passeriformes: Maluridae) as a model system. Fairy-wrens of the genus Malurus are known for high intensities of sexual selection, resulting in some cases in rapid speciation. This history suggests that incomplete lineage sorting (ILS) of neutrally evolving loci could be substantial, a situation that could compromise traditional methods of combining loci in phylogenetic analysis. Using 18 molecular markers (5 anonymous loci, 7 exons, 5 introns, and 1 mitochondrial DNA locus), we show that gene tree monophyly across species could be rejected for 16 of 18 loci, suggesting substantial ILS at the family level in these birds. Using the software Concaterpillar, we also detect three statistically distinct clusters of gene trees among the 18 loci. Despite substantial variation in gene trees, species trees constructed using four different species tree estimation methods (BEST, BUCKy, and STAR) were generally well supported and similar to each other and to the concatenation tree, with a few mild discordances at nodes that could be explained by rapid and recent speciation events. By contrast, minimizing deep coalescences produced a species tree that was topologically more divergent from those of the other methods as measured by multidimensional scaling of trees. Additionally, gene and species trees were topologically more similar in the BEST analysis, presumably because of the species tree prior employed in BEST which appropriately assumes that gene trees are correlated with each other and with the species tree. Among the 18 loci, we also discovered 102 independent indel markers, which also proved phylogenetically informative, primarily among genera, and displayed a ～4-fold bias towards deletions. As suggested in earlier work, the grasswrens (Amytornis) are sister to the rest of the family and the emu-wrens (Stipiturus) are sister to fairy-wrens (Malurus, Clytomyias). Our study shows that ILS is common at the family level in birds yet, despite this, species tree methods converge on broadly similar results for this family.     

Estimating species phylogeny from gene-tree probabilities despite incomplete lineage sorting: an example from Melanoplus grasshoppers

Estimating phylogenetic relationships among closely related species can be extremely difficult when there is incongruence among gene trees and between the gene trees and the species tree. Here we show that incorporating a model of the stochastic loss of gene lineages by genetic drift into the phylogenetic estimation procedure can provide a robust estimate of species relationships, despite widespread incomplete sorting of ancestral polymorphism. This approach is applied to a group of montane Melanoplus grasshoppers for which genealogical discordance among loci and incomplete lineage sorting obscures any obvious phylogenetic relationships among species. Unlike traditional treatments where gene trees estimated using standard phylogenetic methods are implicitly equated with the species tree, with the coalescent-based approach the species tree is modeled probabilistically from the estimated gene trees. The estimated species phylogeny (the ESP) is calculated for the grasshoppers from multiple gene trees reconstructed for nuclear loci and a mitochondrial gene. This empirical application is coupled with a simulation study to explore the performance of the coalescent-based approach. Specifically, we test the accuracy of the ESP given the data based on analyses of simulated data matching the multilocus data collected in Melanoplus (i.e., data were simulated for each locus with the same number of base pairs and locus-specific mutational models). The results of the study show that ESPs can be computed using the coalescent-based approach long before reciprocal monophyly has been achieved, and that these statistical estimates are accurate. This contrasts with analyses of the empirical data collected in Melanoplus and simulated data based on concatenation of multiple loci, for which the incomplete lineage sorting of recently diverged species posed significant problems. The strengths and potential challenges associated with incorporating an explicit model of gene-lineage coalescence into the phylogenetic procedure to obtain an ESP, as illustrated by application to Melanoplus, versus concatenation and consensus approaches are discussed. This study represents a fundamental shift in how species relationships are estimated - the relationship between the gene trees and the species phylogeny is modeled probabilistically rather than equating gene trees with a species tree.     

An improved phylogeny of the Andean tit-tyrants (Aves, Tyrannidae): more characters trump sophisticated analyses

The phylogeny of the flycatcher genus Anairetes was previously inferred using short fragments of mitochondrial DNA and parsimony and distance-based methods. The resulting topology spurred taxonomic revision and influenced understanding of Andean biogeography. More than a decade later, we revisit the phylogeny of Anairetes tit-tyrants using more mtDNA characters, seven unlinked loci (three mitochondrial genes, six nuclear loci), more closely related outgroup taxa, partitioned Bayesian analyses, and two coalescent species-tree approaches (Bayesian estimation of species trees, BEST; Bayesian evolutionary analysis by sampling trees, (*)BEAST). Of these improvements in data and analyses, the fourfold increase in mtDNA characters was both necessary and sufficient to incur a major shift in the topology and near-complete resolution. The species-tree analyses, while theoretically preferable to concatenation or single gene approaches, yielded topologies that were compatible with mtDNA but with weaker statistical resolution at nodes. The previous results that had led to taxonomic and biogeographic reappraisal were refuted, and the current results support the resurrection of the genus Uromyias as the sister clade to Anairetes. The sister relationship between these two genera corresponds to an ecological dichotomy between a depauperate humid cloud forest clade and a diverse dry-tolerant clade that has diversified along the latitudinal axis of the Andes. The species-tree results and the concatenation results each reaffirm the primacy of mtDNA to provide phylogenetic signal for avian phylogenies at the species and subspecies level. This is due in part to the abundance of informative characters in mtDNA, and in part to its lower effective population size that causes it to more faithfully track the species tree.     

Eight independent nuclear genes support monophyly of the plovers: The role of mutational variance in gene trees

Molecular phylogenies of Charadriiformes based on mtDNA genes and one to three nuclear loci do not support the traditional placement of Pluvialis in the plovers (Charadriidae), assigning it instead to oystercatchers, stilts, and avocets (Haematopodidae and Recurvirostridae). To investigate this hypothesis of plover paraphyly, the relationships among Pluvialis and closely related families were revisited by sequencing two individuals of all taxa except Peltohyas for eight independent single copy nuclear protein-coding loci selected for their informativeness at this phylogenetic depth. The species tree estimated jointly with the gene trees in the coalescent programme (*)BEAST strongly supported plover monophyly, as did Bayesian analysis of the concatenated matrix. The data sets that supported plover paraphyly in Baker et al. (2007) and Fain and Houde (2007) reflect two to four independent gene histories, and thus discordance with the plover monophyly species tree might have arisen by chance through stochastic mutational variance. For the plovers we conclude there is no conclusive evidence of coalescent variance from ancient incomplete lineage sorting across the interior branch leading to Pluvialis in the species tree. Rather, earlier studies seem have been misled by faster evolving mtDNA genes with high mutational variance, and a few nuclear genes that had low resolving power at the Pluvialis sister group level. These findings are of general relevance in avian phylogenetics, as they show that careful attention needs to be paid to the number and the phylogenetic informativeness of genes required to obtain accurate estimates of the species tree, especially where there is mutational heterogeneity in gene trees.     

THE INTERFACE BETWEEN PHYLOGENETICS AND POPULATION GENETICS: INVESTIGATING GENE TREES,SPECIES TREES,AND POPULATION DYNAMICS IN THE PHYLLOPHAGA FRATERNA SPECIES GROUP

This study uses traditional and contemporary phylogenetic and population genetic analyses to assess the causes of discordance (i.e., lineage sorting and introgression) among mitochondrial and nuclear gene trees for a clade of eastern North American scarab beetles (fraterna species group, genus Phyllophaga). I estimated gene trees using individual and combined analysis of one mitochondrial and two nuclear loci in MrBayes , and inferred a species tree using a hierarchical coalescent approach based on all loci in the program Best . Because hybridization violates the assumptions of Best , I tested for introgression by comparing species monophyly between the mitochondrial and nuclear gene trees based on the prediction that cytoplasmic genomes introgress more readily than nuclear genomes. Haplotype exclusivity was identified using Bayesian tests of monophyly and the genealogical sorting index. I used the results of the phylogenetic analyses and monophyly tests to develop an explicit hypothesis of introgression that could be tested in the program IMa. Results from these analyses provided evidence for introgression across clades within the fraterna group. The tiered analytical approach used in this study demonstrated how the use of multiple methods can identify when assumptions are violated and methods are prone to yield misleading results.     

TESTING MITOCHONDRIAL CAPTURE AND DEEP COALESCENCE IN AMAZONIAN CICHLID FISHES (CICHLIDAE: CICHLA)

Hybridization and introgression have important consequences in evolution, such as increasing the genetic diversity and adaptive potential of a species. One of their most conspicuous footprints is discordance among gene trees or between genes and phenotypes. However, most studies that report introgression fail to disprove the null hypothesis that genetic incongruence may result from stochastic sorting of ancestral allelic polymorphisms. In the case of ancient introgression, these two processes may be especially difficult to distinguish topologically, but they make different predictions about the patterns of coalescence among loci. Here we apply three methods, molecular dating, multispecies coalescent models, and gene tree simulation under coalescence, to compare these two hypotheses that explain the polyphyletic mtDNA of the butterfly peacock bass, Cichla orinocensis. In comparison with a species tree based on 20 unlinked nuclear loci, we determined that mtDNA divergences were too recent to be explained by ancestral polymorphism. Similarly, coalescent species tree branches were significantly shorter when putative introgressed mtDNA was incorporated, and simulations showed the mtDNA topology to be unlikely under lineage sorting only. We conclude that introgression approximately 1.5 million years ago resulted in capture by C. orinocensis of an mtDNA lineage ancestral to the modern subspecies C. oc. monoculus.     

Comparing two Bayesian methods for gene tree/species tree reconstruction: simulations with incomplete lineage sorting and horizontal gene transfer

With the increasing interest in recognizing the discordance between gene genealogies, various gene tree/species tree reconciliation methods have been developed. We present here the first attempt to assess and compare two such Bayesian methods, Bayesian estimation of species trees (BEST) and BUCKy (Bayesian untangling of concordance knots), in the presence of several known processes of gene tree discordance. DNA alignments were simulated under the influence of incomplete lineage sorting (ILS) and of horizontal gene transfer (HGT). BEST and BUCKy both account for uncertainty in gene tree estimation but differ substantially in their assumptions of what caused gene tree discordance. BEST estimates a species tree using the coalescent model, assuming that all gene tree discordance is due to ILS. BUCKy does not assume any specific biological process of gene tree discordance through the use of a nonparametric clustering of concordant genes. BUCKy estimates the concordance factor (CF) of a clade, which is defined as the proportion of genes that truly have the clade in their trees. The estimated concordance tree is then built from clades with the highest estimated CFs. Because of their different assumptions, it was expected that BEST would perform better in the presence of ILS and that BUCKy would perform better in the presence of HGT. As expected, the species tree was more accurately reconstructed by BUCKy in the presence of HGT, when the HGT events were unevenly placed across the species tree. BUCKy and BEST performed similarly in most other cases, including in the presence of strong ILS and of HGT events that were evenly placed across the tree. However, BUCKy was shown to underestimate the uncertainty in CF estimation, with short credibility intervals. Despite this, the discordance pattern estimated by BUCKy could be compared with the signature of ILS. The resulting test for the adequacy of the coalescent model proved to have low Type I error. It was powerful when HGT was the major source of discordance and when HGT events were unevenly placed across the species tree.     

Evolution and phylogenetic utility of the PHOT gene duplicates in the Verbena complex (Verbenaceae): dramatic intron size variation and footprint of ancestral recombination

A well-resolved species level phylogeny is critically important in studying organismal evolution (e.g., hybridization, polyploidization, adaptive speciation). Lack of appropriate molecular markers that give sufficient resolution to gene trees is one of the major impediments to inferring species level phylogenies. In addition, sampling multiple independent loci is essential to overcome the lineage sorting problem. The availability of nuclear loci has often been a limiting factor in plant species-level phylogenetic studies. Here the two PHOT loci were developed as new sources of nuclear gene trees. The PHOT1 and PHOT2 gene trees of the Verbena complex (Verbenaceae) are well resolved and have good clade support. These gene trees are consistent with each other and previously generated chloroplast and nuclear waxy gene trees in most of the phylogenetic backbone as well as some terminal relationships, but are incongruent in some other relationships. Locus-specific primers were optimized for amplifying and sequencing these two loci in all Lamiales. Comparing intron size in the context of the gene trees shows dramatic variation within the Verbena complex, particularly at the PHOT1 locus. These variations are largely caused by invasions of short transposable elements and frequent long deletions and insertions of unknown causes. In addition, inspection of DNA sequences and phylogenetic analyses unmask a clear footprint of ancestral recombination in one species.     

Nuclear loci and coalescent methods support ancient hybridization as cause of mitochondrial paraphyly between gadwall and falcated duck (Anas spp.)

Many species have mitochondrial DNA lineages that are phylogenetically intermixed with other species, but studies have rarely tested the cause of such paraphyly. In this study, we tested two hypotheses that could explain mitochondrial paraphyly of Holarctic gadwalls (Anas strepera) with respect to Asian falcated ducks (A. falcata). First, hybridization could have resulted in falcated duck mitochondrial DNA (mtDNA) introgressing into the gadwall gene pool. Second, gadwalls and falcated ducks could have diverged so recently that mtDNA lineages have not sorted to reciprocal monophyly. We used coalescent analyses of three independent loci to distinguish between these two hypotheses. Two lines of evidence support introgression. First, analyses of the three loci combined show that some introgression is necessary to explain current genetic diversity in gadwalls. Second, we generated alternative predictions regarding time since divergence estimated from mtDNA: falcated ducks and gadwalls would have diverged between 65,000 and 700,000 years before present (ybp) under the introgression hypothesis and between 11,000 and 76,000 ybp under the incomplete lineage sorting hypothesis. The two independent nuclear introns indicated that these species diverged between 210,000 and 5,200,000 ybp, which did not overlap the predicted time for incomplete lineage sorting. These analyses also suggested that ancient introgression ( approximately 14,000 ybp) has resulted in the widespread distribution and high frequency of falcated-like mtDNA (5.5% of haplotypes) in North America. This is the first study to use a rigorous quantitative framework to reject incomplete lineage sorting as the cause of mitochondrial paraphyly.     

Multilocus and morphological analysis of south-eastern Iberian Wall lizards (Squamata,Podarcis)

The phylogenetic relationships among the wall lizards of the Podarcis hispanicus complex that inhabit the south-east (SE) of the Iberian Peninsula and other lineages of the complex remain unclear. In this study, four mitochondrial and two nuclear markers were used to study genetic relationships within this complex. The phylogenetic analyses based on mtDNA gene trees constructed with ML and BI, and a species tree using *BEAST support three divergent clades in this region: the Valencia, Galera and Albacete/Murcia lineages. These three lineages were also corroborated in species delimitation analyses based on mtDNA using bPTP, mPTP, GMYC, ABGD and BAPS. Bayesian inference species delimitation method (BPP) based on both nuclear data and a combined data set (mtDNA + nuclear) showed high posterior probabilities for these three SE lineages (≥0.94) and another Bayesian analysis (STACEY) based on combined data set recovered the same three groups in this region. Divergence time dating of the species tree provided an estimated divergence of the Galera lineage from the other SE group (Podarcis vaucheri, (Albacete/Murcia, Valencia)) at 12.48 Ma. During this period, the Betic–Rifian arc was isolated, which could have caused the isolation of the Galera form distributed to the south of the Betic Corridor. Although lizards from the Albacete/Murcia and Galera lineage are morphologically similar, they clearly represent distinct genetic lineages. The noteworthy separation of the Galera lineage enables us to conclude that this lineage must be considered as a new full species.     

Phylogenetic relationships of phrynosomatid lizards based on nuclear and mitochondrial data,and a revised phylogeny for Sceloporus

Phrynosomatid lizards are among the most common and diverse groups of reptiles in western North America, Mexico, and Central America. Phrynosomatidae includes 136 species in 10 genera. Phrynosomatids are used as model systems in many research programs in evolution and ecology, and much of this research has been undertaken in a comparative phylogenetic framework. However, relationships among many phrynosomatid genera are poorly supported and in conflict between recent studies. Further, previous studies based on mitochondrial DNA sequences suggested that the most species-rich genus (Sceloporus) is possibly paraphyletic with respect to as many as four other genera (Petrosaurus, Sator, Urosaurus, and Uta). Here, we collect new sequence data from five nuclear genes and combine them with published data from one additional nuclear gene and five mitochondrial gene regions. We compare trees from nuclear and mitochondrial data from 37 phrynosomatid taxa, including a “species tree” (from BEST) for the nuclear data. We also present a phylogeny for 122 phrynosomatid species based on maximum likelihood analysis of the combined data, which provides a strongly-supported hypothesis for relationships among most phrynosomatid genera and includes most phrynosomatid species. Our results strongly support the monophyly of Sceloporus (including Sator) and many of the relationships within it. We present a new classification for phrynosomatid lizards and the genus Sceloporus, and offer a new tree with branch lengths for use in comparative studies.     

Multilocus analyses of an Antarctic fish species flock (Teleostei, Notothenioidei, Trematominae): phylogenetic approach and test of the early-radiation event

Clades that have undergone episodes of rapid cladogenesis are challenging from a phylogenetic point of view. They are generally characterised by short or missing internal branches in phylogenetic trees and by conflicting topologies among individual gene trees. This may be the case of the subfamily Trematominae, a group of marine teleosts of coastal Antarctic waters, which is considered to have passed through a period of rapid diversification. Despite much phylogenetic attention, the relationships among Trematominae species remain unclear. In contrast to previous studies that were mostly based on concatenated datasets of mitochondrial and/or single nuclear loci, we applied various single-locus and multilocus phylogenetic approaches to sequences from 11 loci (eight nuclear) and we also used several methods to assess the hypothesis of a radiation event in Trematominae evolution. Diversification rate analyses support the hypothesis of a period of rapid diversification during Trematominae history and only a few nodes in the hypothetical species tree were consistently resolved with various phylogenetic methods. We detected significant discrepancies among trees from individual genes of these species, most probably resulting from incomplete lineage sorting, suggesting that concatenation of loci is not the most appropriate way to investigate Trematominae species interrelationships. These data also provide information about the possible effects of historic climate changes on the diversification rate of this group of fish.     

THE INFLUENCE OF SAMPLING DESIGN ON SPECIES TREE INFERENCE: A NEW RELATIONSHIP FOR THE NEW WORLD CHICKADEES (AVES: POECILE)

In this study, we explore the long‐standing issue of how many loci are needed to infer accurate phylogenetic relationships, and whether loci with particular attributes (e.g., parsimony informativeness, variability, gene tree resolution) outperform others. To do so, we use an empirical data set consisting of the seven species of chickadees (Aves: Paridae), an analytically tractable, recently diverged group, and well‐studied ecologically but lacking a nuclear phylogeny. We estimate relationships using 40 nuclear loci and mitochondrial DNA using four coalescent‐based species tree inference methods (BEST, *BEAST, STEM, STELLS). Collectively, our analyses contrast with previous studies and support a sister relationship between the Black‐capped and Carolina Chickadee, two superficially similar species that hybridize along a long zone of contact. Gene flow is a potential source of conflict between nuclear and mitochondrial gene trees, yet we find a significant, albeit low, signal of gene flow. Our results suggest that relatively few loci with high information content may be sufficient for estimating an accurate species tree, but that substantially more loci are necessary for accurate parameter estimation. We provide an empirical reference point for researchers designing sampling protocols with the purpose of inferring phylogenies and population parameters of closely related taxa.     

INFERRING PHYLOGENIES FROM mtDNA VARIATION: MITOCHONDRIAL-GENE TREES VERSUS NUCLEAR-GENE TREES

An accurately resolved gene tree may not be congruent with the species tree because of lineage sorting of ancestral polymorphisms. DNA sequences from the mitochondrially encoded genes (mtDNA) are attractive sources of characters for estimating the phylogenies of recently evolved taxa because mtDNA evolves rapidly, but its utility is limited because the mitochondrial genes are inherited as a single linkage group (haplotype) and provide only one independent estimate of the species tree. In contrast, a set of nuclear genes can be selected from distinct chromosomes, such that each gene tree provides an independent estimate of the species tree. Another aspect of the gene-tree versus species-tree problem, however, favors the use of mtDNA for inferring species trees. For a three-species segment of a phylogeny, the branching order of a gene tree will correspond to that of the species tree if coalescence of the alleles or haplotypes occurred in the internode between the first and second bifurcation. From neutral theory, it is apparent that the probability of coalescence increases as effective population size decreases. Because the mitochondrial genome is maternally inherited and effectively haploid, its effective population size is one-fourth that of a nuclear-autosomal gene. Thus, the mitochondrial-haplotype tree has a substantially higher probability of accurately tracking a short internode than does a nuclear-autosomal-gene tree. When an internode is sufficiently long that the probability that the mitochondrial-haplotype tree will be congruent with the species tree is 0.95, the probability that a nuclear-autosomalgene tree will be congruent is only 0.62. If each of k independently sampled nuclear-gene trees has a probability of congruence with the species tree of 0.62, then a sample of 16 such trees would be required to be as confident of the inference based on the mitochondrial-haplotype tree. A survey of mtDNA-haplotype diversity in 34 species of birds indicates that coalescence is generally very recent, which suggests that coalescence times are typically much shorter than internodal branch lengths of the species tree, and that sorting of mtDNA lineages is not likely to confound the species tree. Hybridization resulting in transfer of mtDNA haplotypes among branches could also result in a haplotype tree that is incongruent with the species tree; if undetected, this could confound the species tree. However, hybridization is usually easy to detect and should be incorporated in the historical narrative of the group, because reticulation, as well as cladistic events, contributed to the evolution of the group.     

Nuclear gene sequences resolve species phylogeny and mitochondrial introgression in Leptocarabus beetles showing trans-species polymorphisms

We studied the phylogenetic relationships among Japanese Leptocarabus ground beetles, which show extensive trans-species polymorphisms in mitochondrial gene genealogies. Simultaneous analysis of combined nuclear data with partial sequences from the long-wavelength rhodopsin, wingless, phosphoenolpyruvate carboxykinase, and 28S rRNA genes resolved the relationships among the five species, although separate analyses of these genes provided topologies with low resolution. For both the nuclear gene tree resulting from the combined data from four genes and a mitochondrial cytochrome oxidase subunit I (COI) gene tree, we applied a Bayesian divergence time estimation using a common calibration method to identify mitochondrial introgression events that occurred after speciation. Three mitochondrial lineages shared by two or three species were likely subject to introgression due to interspecific hybridization because the coalescent times for these lineages were much shorter than the corresponding speciation times estimated from nuclear gene sequences. We demonstrated that when species phylogeny is fully resolved with nuclear gene sequence data, comparative analysis of nuclear and mitochondrial gene trees can be used to infer introgressive hybridization events that might cause trans-species polymorphisms in mitochondrial gene trees.     

Differentiating between hypotheses of lineage sorting and introgression in New Zealand alpine cicadas (Maoricicada Dugdale)

Lineage sorting and introgression can lead to incongruence among gene phylogenies, complicating the inference of species trees for large groups of taxa that have recently and rapidly radiated. In addition, it can be difficult to determine which of these processes is responsible for this incongruence. We explore these issues with the radiation of New Zealand alpine cicadas of the genus Maoricicada Dugdale. Gene trees were estimated from four putative independent loci: mitochondrial DNA (2274 nucleotides), elongation factor 1-alpha (1275 nucleotides), period (1709 nucleotides), and calmodulin (678 nucleotides). We reconstructed phylogenies using maximum likelihood and Bayesian methods from 44 individuals representing the 19 species and subspecies of Maoricicada and two outgroups. Species-level relationships were reconstructed using a novel extension of gene tree parsimony, whereby gene trees were weighted by their Bayesian posterior probabilities. The inferred gene trees show marked incongruence in the placement of some taxa, especially the enigmatic forest and scrub dwelling species, M. iolanthe. Using the species tree estimated by gene tree parsimony, we simulated coalescent gene trees in order to test the null hypothesis that the nonrandom placement of M. iolanthe among gene trees has arisen by chance. Under the assumptions of constant population size, known generation time, and panmixia, we were able to reject this null hypothesis. Furthermore, because the two alternative placements of M. iolanthe are in each case with species that share a similar song structure, we conclude that it is more likely that an ancient introgression event rather than lineage sorting has caused this incongruence.