Why Do Phylogenomic Data Sets Yield Conflicting Trees? Data Type Influences the Avian Tree of Life more than Taxon Sampling.

Phylogenomics, the use of large-scale data matrices in phylogenetic analyses, has been viewed as the ultimate solution to the problem of resolving difficult nodes in the tree of life. However, it has become clear that analyses of these large genomic data sets can also result in conflicting estimates of phylogeny. Here, we use the early divergences in Neoaves, the largest clade of extant birds, as a "model system" to understand the basis for incongruence among phylogenomic trees. We were motivated by the observation that trees from two recent avian phylogenomic studies exhibit conflicts. Those studies used different strategies: 1) collecting many characters [$\sim$ 42 mega base pairs (Mbp) of sequence data] from 48 birds, sometimes including only one taxon for each major clade; and 2) collecting fewer characters ($\sim$ 0.4 Mbp) from 198 birds, selected to subdivide long branches. However, the studies also used different data types: the taxon-poor data matrix comprised 68% non-coding sequences whereas coding exons dominated the taxon-rich data matrix. This difference raises the question of whether the primary reason for incongruence is the number of sites, the number of taxa, or the data type. To test among these alternative hypotheses we assembled a novel, large-scale data matrix comprising 90% non-coding sequences from 235 bird species. Although increased taxon sampling appeared to have a positive impact on phylogenetic analyses the most important variable was data type. Indeed, by analyzing different subsets of the taxa in our data matrix we found that increased taxon sampling actually resulted in increased congruence with the tree from the previous taxon-poor study (which had a majority of non-coding data) instead of the taxon-rich study (which largely used coding data). We suggest that the observed differences in the estimates of topology for these studies reflect data-type effects due to violations of the models used in phylogenetic analyses, some of which may be difficult to detect. If incongruence among trees estimated using phylogenomic methods largely reflects problems with model fit developing more "biologically-realistic" models is likely to be critical for efforts to reconstruct the tree of life. [Birds; coding exons; GTR model; model fit; Neoaves; non-coding DNA; phylogenomics; taxon sampling.].

Concurrent Infection of Batrachochytrium dendrobatidis and Ranavirus among Native Amphibians from Northeastern Oklahoma, USA.

Global amphibian decline continues to be a great concern despite our increased understanding of the causes behind the observed patterns of the decline, such as habitat modification and infectious diseases. Although there is a large body of literature on the topic of amphibian infectious diseases, pathogen prevalence and distribution among entire communities of species in many regions remain poorly understood. In addition to these geographic gaps in our understanding, past work has focused largely on individual pathogens, either Batrachochytrium dendrobatidis (Bd) or ranavirus (RV), rather than dual infection rates among host species. We sampled for prevalence and infection load of both pathogens in 514 amphibians across 16 total sites in northeastern Oklahoma. Amphibians were caught by hand, net, or seine; they were swabbed to screen for Bd; and liver tissue samples were collected to screen for RV. Overall results of quantitative PCR assays showed that 7% of screened individuals were infected with RV only, 37% were infected with Bd only, and 9% were infected with both pathogens simultaneously. We also documented disease presence in several rare amphibian species that are currently being monitored as species of concern due to their small population sizes in Oklahoma. This study synthesizes a growing body of research regarding infectious diseases among amphibian communities in the central United States.

Homoplastic microinversions and the avian tree of life.

BACKGROUND: Microinversions are cytologically undetectable inversions of DNA sequences that accumulate slowly in genomes. Like many other rare genomic changes (RGCs), microinversions are thought to be virtually homoplasy-free evolutionary characters, suggesting that they may be very useful for difficult phylogenetic problems such as the avian tree of life. However, few detailed surveys of these genomic rearrangements have been conducted, making it difficult to assess this hypothesis or understand the impact of microinversions upon genome evolution. RESULTS: We surveyed non-coding sequence data from a recent avian phylogenetic study and found substantially more microinversions than expected based upon prior information about vertebrate inversion rates, although this is likely due to underestimation of these rates in previous studies. Most microinversions were lineage-specific or united well-accepted groups. However, some homoplastic microinversions were evident among the informative characters. Hemiplasy, which reflects differences between gene trees and the species tree, did not explain the observed homoplasy. Two specific loci were microinversion hotspots, with high numbers of inversions that included both the homoplastic as well as some overlapping microinversions. Neither stem-loop structures nor detectable sequence motifs were associated with microinversions in the hotspots. CONCLUSIONS: Microinversions can provide valuable phylogenetic information, although power analysis indicates that large amounts of sequence data will be necessary to identify enough inversions (and similar RGCs) to resolve short branches in the tree of life. Moreover, microinversions are not perfect characters and should be interpreted with caution, just as with any other character type. Independent of their use for phylogenetic analyses, microinversions are important because they have the potential to complicate alignment of non-coding sequences. Despite their low rate of accumulation, they have clearly contributed to genome evolution, suggesting that active identification of microinversions will prove useful in future phylogenomic studies.

Phylogenomic evidence for multiple losses of flight in ratite birds.

Ratites (ostriches, emus, rheas, cassowaries, and kiwis) are large, flightless birds that have long fascinated biologists. Their current distribution on isolated southern land masses is believed to reflect the breakup of the paleocontinent of Gondwana. The prevailing view is that ratites are monophyletic, with the flighted tinamous as their sister group, suggesting a single loss of flight in the common ancestry of ratites. However, phylogenetic analyses of 20 unlinked nuclear genes reveal a genome-wide signal that unequivocally places tinamous within ratites, making ratites polyphyletic and suggesting multiple losses of flight. Phenomena that can mislead phylogenetic analyses, including long branch attraction, base compositional bias, discordance between gene trees and species trees, and sequence alignment errors, have been eliminated as explanations for this result. The most plausible hypothesis requires at least three losses of flight and explains the many morphological and behavioral similarities among ratites by parallel or convergent evolution. Finally, this phylogeny demands fundamental reconsideration of proposals that relate ratite evolution to continental drift.

Duplication of accelerated evolution and growth hormone gene in passerine birds.

We report the discovery of a duplication of the growth hormone (GH) gene in a major group of birds, the passerines (Aves: Passeriformes). Phylogenetic analysis of 1.3-kb partial DNA sequences of GH genes for 24 species of passerines and numerous outgroups indicates that the duplication occurred in the ancestral lineage of extant passerines. Both duplicates and their open-reading frames are preserved throughout the passerine clade, and both duplicates are expressed in the zebra finch brain, suggesting that both are likely to be functional. The estimated rates of amino acid evolution are more than 10-fold higher in passerine GH genes than in those of their closest nonpasserine relatives. In addition, although the 84 codons sequenced are generally highly conserved for both passerines and nonpasserines, comparisons of the nonsynonymous/synonymous substitution ratios and the rate of predicted amino acid changes indicate that the 2 gene duplicates are evolving under different selective pressures and may be functionally divergent. The evidence of differential selection, coupled with the preservation of both gene copies in all major lineages since the origin of passerines, suggests that the duplication may be of adaptive significance, with possible implications for the explosive diversification of the passerine clade.

The effect of marker choice on estimated levels of introgression across an avian (Pipridae: Manacus) hybrid zone.

Hybrid zones are often characterized by narrow, coincident clines for diverse traits, suggesting that little introgression occurs across them. However, this pattern may result from a bias in focussing on traits that are diagnostic of parental populations. Such choice of highly differentiated traits may cause us to overlook differential introgression in nondiagnostic traits and to distort our perception of hybrid zones. We tested this hypothesis in an avian hybrid zone by comparing cline structure in two sets of molecular markers: isozyme and restriction fragment length polymorphism markers chosen for differentiation between parental forms, and microsatellite markers chosen for polymorphism. Two cline-fitting methods showed that cline centre positions of microsatellite alleles were more variable than those of isozyme and restriction fragment length polymorphism markers, and several were significantly shifted from those of the diagnostic markers. Cline widths of microsatellite alleles were also variable and two- to eightfold wider than those of the diagnostic markers. These patterns are consistent with the idea that markers chosen for differentiation are more likely to be under purifying selection, and studies focussed on these markers will underestimate overall introgression across hybrid zones. Our results suggest that neutral and positively selected alleles may introgress freely across many hybrid zones without altering perceived boundaries between hybridizing forms.

Parsimony and model-based analyses of indels in avian nuclear genes reveal congruent and incongruent phylogenetic signals.

Insertion/deletion (indel) mutations, which are represented by gaps in multiple sequence alignments, have been used to examine phylogenetic hypotheses for some time. However, most analyses combine gap data with the nucleotide sequences in which they are embedded, probably because most phylogenetic datasets include few gap characters. Here, we report analyses of 12,030 gap characters from an alignment of avian nuclear genes using maximum parsimony (MP) and a simple maximum likelihood (ML) framework. Both trees were similar, and they exhibited almost all of the strongly supported relationships in the nucleotide tree, although neither gap tree supported many relationships that have proven difficult to recover in previous studies. Moreover, independent lines of evidence typically corroborated the nucleotide topology instead of the gap topology when they disagreed, although the number of conflicting nodes with high bootstrap support was limited. Filtering to remove short indels did not substantially reduce homoplasy or reduce conflict. Combined analyses of nucleotides and gaps resulted in the nucleotide topology, but with increased support, suggesting that gap data may prove most useful when analyzed in combination with nucleotide substitutions.

A phylogenomic study of birds reveals their evolutionary history.

Deep avian evolutionary relationships have been difficult to resolve as a result of a putative explosive radiation. Our study examined approximately 32 kilobases of aligned nuclear DNA sequences from 19 independent loci for 169 species, representing all major extant groups, and recovered a robust phylogeny from a genome-wide signal supported by multiple analytical methods. We documented well-supported, previously unrecognized interordinal relationships (such as a sister relationship between passerines and parrots) and corroborated previously contentious groupings (such as flamingos and grebes). Our conclusions challenge current classifications and alter our understanding of trait evolution; for example, some diurnal birds evolved from nocturnal ancestors. Our results provide a valuable resource for phylogenetic and comparative studies in birds.

Molecular phylogenetic analysis of Fringillidae, "New World nine-primaried oscines" (Aves: Passeriformes).

Systematic studies of Fringillidae have long been problematic due to their apparent recent and explosive diversification. We present phylogenetic hypotheses of 44 fringillids that represent the overall diversity of the family, based on 3.2 kb of mitochondrial DNA sequences, and phylogenetic analyses for a subset of fringillids based on new and published mitochondrial cytochrome b sequences. Monophyly of Fringillidae and its two constituent subfamilies, Fringillinae and Emberizinae, was consistently supported with the exceptions of Peucedramus being placed outside of Fringillinae and Euphonia being placed within Fringillinae instead of within Emberizinae. Within Emberizinae, Thraupini (tanagers), Cardinalini (cardinals and grosbeaks), and Emberizini (New World sparrows) did not form separate monophyletic groups. Our results indicate that Emberizinae consists of three clades, each with a different overall geographical distribution. Several taxa traditionally considered members of Thraupini fall outside of the thraupine clade, including the only North American genus, Piranga. Consequently, the thraupine clade includes only Neotropical species. Increasing evidence suggests that Fringillidae, often called "New World nine-primaried oscines," does not in fact have a New World origin.

TESTING HYPOTHESES OF NEURAL EVOLUTION IN GYMNOTIFORM ELECTRIC FISHES USING PHYLOGENETIC CHARACTER DATA.

In this paper, we propose a method to test alternative hypotheses of phenotypic evolution. The method compares patterns observed in phylogenetic character data with patterns expected by explicit models of evolutionary process. Observed patterns of character-state diversity are assessed from four properties of character-state change derived from a phylogenetic analysis: the sequence and correlation of transformations on a cladogram and the spatial and functional localization of these transformations to parts of an organism. Patterns expressed in terms of the localization of transformations are compared with the expectations of null models that the number of transformations is proportional to measures of size or complexity. Deviations from the values expected by the null models are then compared with qualitative expectations of the models. The method is applied to characters in the nervous system of gymnotiform electric fishes. Patterns in the diversity of 63 reconstructed character-state changes are compared with the expectations of 10 published models of neural evolution. A total of 63 expectations are reviewed, of which 33 (52%) are found to be consistent with the gymnotiform neural data. In general, the models reviewed are not successful at making global predictions, in part because they have been cast in excessively general terms. The data support the conclusion that evolution in the nervous system of gymnotiforms has involved a mosaic of processes, each operating differentially on functional and developmental systems and at different spatial and temporal scales. The results also indicate that more refined models are required, each making more explicit predictions.