Complexity of avian evolution revealed by family-level genomes.

Despite tremendous efforts in the past decades, relationships among main avian lineages remain heavily debated without a clear resolution. Discrepancies have been attributed to diversity of species sampled, phylogenetic method and the choice of genomic regions1-3. Here we address these issues by analysing the genomes of 363 bird species4 (218 taxonomic families, 92% of total). Using intergenic regions and coalescent methods, we present a well-supported tree but also a marked degree of discordance. The tree confirms that Neoaves experienced rapid radiation at or near the Cretaceous-Palaeogene boundary. Sufficient loci rather than extensive taxon sampling were more effective in resolving difficult nodes. Remaining recalcitrant nodes involve species that are a challenge to model due to either extreme DNA composition, variable substitution rates, incomplete lineage sorting or complex evolutionary events such as ancient hybridization. Assessment of the effects of different genomic partitions showed high heterogeneity across the genome. We discovered sharp increases in effective population size, substitution rates and relative brain size following the Cretaceous-Palaeogene extinction event, supporting the hypothesis that emerging ecological opportunities catalysed the diversification of modern birds. The resulting phylogenetic estimate offers fresh insights into the rapid radiation of modern birds and provides a taxon-rich backbone tree for future comparative studies.

A region of suppressed recombination misleads neoavian phylogenomics.

Genomes are typically mosaics of regions with different evolutionary histories. When speciation events are closely spaced in time, recombination makes the regions sharing the same history small, and the evolutionary history changes rapidly as we move along the genome. When examining rapid radiations such as the early diversification of Neoaves 66 Mya, typically no consistent history is observed across segments exceeding kilobases of the genome. Here, we report an exception. We found that a 21-Mb region in avian genomes, mapped to chicken chromosome 4, shows an extremely strong and discordance-free signal for a history different from that of the inferred species tree. Such a strong discordance-free signal, indicative of suppressed recombination across many millions of base pairs, is not observed elsewhere in the genome for any deep avian relationships. Although long regions with suppressed recombination have been documented in recently diverged species, our results pertain to relationships dating circa 65 Mya. We provide evidence that this strong signal may be due to an ancient rearrangement that blocked recombination and remained polymorphic for several million years prior to fixation. We show that the presence of this region has misled previous phylogenomic efforts with lower taxon sampling, showing the interplay between taxon and locus sampling. We predict that similar ancient rearrangements may confound phylogenetic analyses in other clades, pointing to a need for new analytical models that incorporate the possibility of such events.

Exploring Conflicts in Whole Genome Phylogenetics: A Case Study Within Manakins (Aves: Pipridae).

Some phylogenetic problems remain unresolved even when large amounts of sequence data are analyzed and methods that accommodate processes such as incomplete lineage sorting are employed. In addition to investigating biological sources of phylogenetic incongruence, it is also important to reduce noise in the phylogenomic dataset by using appropriate filtering approach that addresses gene tree estimation errors. We present the results of a case study in manakins, focusing on the very difficult clade comprising the genera Antilophia and Chiroxiphia. Previous studies suggest that Antilophia is nested within Chiroxiphia, though relationships among Antilophia+Chiroxiphia species have been highly unstable. We extracted more than 11,000 loci (ultra-conserved elements and introns) from whole genomes and conducted analyses using concatenation and multispecies coalescent methods. Topologies resulting from analyses using all loci differed depending on the data type and analytical method, with 2 clades (Antilophia+Chiroxiphia and Manacus+Pipra+Machaeopterus) in the manakin tree showing incongruent results. We hypothesized that gene trees that conflicted with a long coalescent branch (e.g., the branch uniting Antilophia+Chiroxiphia) might be enriched for cases of gene tree estimation error, so we conducted analyses that either constrained those gene trees to include monophyly of Antilophia+Chiroxiphia or excluded these loci. While constraining trees reduced some incongruence, excluding the trees led to completely congruent species trees, regardless of the data type or model of sequence evolution used. We found that a suite of gene metrics (most importantly the number of informative sites and likelihood of intralocus recombination) collectively explained the loci that resulted in non-monophyly of Antilophia+Chiroxiphia. We also found evidence for introgression that may have contributed to the discordant topologies we observe in Antilophia+Chiroxiphia and led to deviations from expectations given the multispecies coalescent model. Our study highlights the importance of identifying factors that can obscure phylogenetic signal when dealing with recalcitrant phylogenetic problems, such as gene tree estimation error, incomplete lineage sorting, and reticulation events. [Birds; c-gene; data type; gene estimation error; model fit; multispecies coalescent; phylogenomics; reticulation].

Systems biology as a framework to understand the physiological and endocrine bases of behavior and its evolution-From concepts to a case study in birds.

Organismal behavior, with its tremendous complexity and diversity, is generated by numerous physiological systems acting in coordination. Understanding how these systems evolve to support differences in behavior within and among species is a longstanding goal in biology that has captured the imagination of researchers who work on a multitude of taxa, including humans. Of particular importance are the physiological determinants of behavioral evolution, which are sometimes overlooked because we lack a robust conceptual framework to study mechanisms underlying adaptation and diversification of behavior. Here, we discuss a framework for such an analysis that applies a "systems view" to our understanding of behavioral control. This approach involves linking separate models that consider behavior and physiology as their own networks into a singular vertically integrated behavioral control system. In doing so, hormones commonly stand out as the links, or edges, among nodes within this system. To ground our discussion, we focus on studies of manakins (Pipridae), a family of Neotropical birds. These species have numerous physiological and endocrine specializations that support their elaborate reproductive displays. As a result, manakins provide a useful example to help imagine and visualize the way systems concepts can inform our appreciation of behavioral evolution. In particular, manakins help clarify how connectedness among physiological systems-which is maintained through endocrine signaling-potentiate and/or constrain the evolution of complex behavior to yield behavioral differences across taxa. Ultimately, we hope this review will continue to stimulate thought, discussion, and the emergence of research focused on integrated phenotypes in behavioral ecology and endocrinology.

Categorical edge-based analyses of phylogenomic data reveal conflicting signals for difficult relationships in the avian tree.

Phylogenetic analyses fail to yield a satisfactory resolution of some relationships in the tree of life even with genome-scale datasets, so the failure is unlikely to reflect limitations in the amount of data. Gene tree conflicts are particularly notable in studies focused on these contentious nodes, and taxon sampling, different analytical methods, and/or data type effects can further confound analyses. Although many efforts have been made to incorporate biological conflicts, few studies have curated individual genes for their efficiency in phylogenomic studies. Here, we conduct an edge-based analysis of Neoavian evolution, examining the phylogenetic efficacy of two recent phylogenomic bird datasets and three datatypes (ultraconserved elements [UCEs], introns, and coding regions). We assess the potential causes for biases in signal-resolution for three difficult nodes: the earliest divergence of Neoaves, the position of the enigmatic Hoatzin (Opisthocomus hoazin), and the position of owls (Strigiformes). We observed extensive conflict among genes for all data types and datasets even after meticulous curation. Edge-based analyses (EBA) increased congruence and provided information about the impact of data type, GC content variation (GCCV), and outlier genes on each of nodes we examined. First, outlier gene signals appeared to drive different patterns of support for the relationships among the earliest diverging Neoaves. Second, the placement of Hoatzin was highly variable, although our EBA did reveal a previously unappreciated data type effect with an impact on its position. It also revealed that the resolution with the most support here was Hoatzin + shorebirds. Finally, GCCV, rather than data type (i.e., coding vs non-coding) per se, was correlated with a signal that supports monophyly of owls + Accipitriformes (hawks, eagles, and vultures). Eliminating high GCCV loci increased the signal for owls + mousebirds. Categorical EBA was able to reveal the nature of each edge and provide a way to highlight especially problematic branches that warrant a further examination. The current study increases our understanding about the contentious parts of the avian tree, which show even greater conflicts than appreciated previously.

Historical specimens and the limits of subspecies phylogenomics in the New World quails (Odontophoridae).

As phylogenomics focuses on comprehensive taxon sampling at the species and population/subspecies levels, incorporating genomic data from historical specimens has become increasingly common. While historical samples can fill critical gaps in our understanding of the evolutionary history of diverse groups, they also introduce additional sources of phylogenomic uncertainty, making it difficult to discern novel evolutionary relationships from artifacts caused by sample quality issues. These problems highlight the need for improved strategies to disentangle artifactual patterns from true biological signal as historical specimens become more prevalent in phylogenomic datasets. Here, we tested the limits of historical specimen-driven phylogenomics to resolve subspecies-level relationships within a highly polytypic family, the New World quails (Odontophoridae), using thousands of ultraconserved elements (UCEs). We found that relationships at and above the species-level were well-resolved and highly supported across all analyses, with the exception of discordant relationships within the two most polytypic genera which included many historical specimens. We examined the causes of discordance and found that inferring phylogenies from subsets of taxa resolved the disagreements, suggesting that analyzing subclades can help remove artifactual causes of discordance in datasets that include historical samples. At the subspecies-level, we found well-resolved geographic structure within the two most polytypic genera, including the most polytypic species in this family, Northern Bobwhites (Colinus virginianus), demonstrating that variable sites within UCEs are capable of resolving phylogenetic structure below the species level. Our results highlight the importance of complete taxonomic sampling for resolving relationships among polytypic species, often through the inclusion of historical specimens, and we propose an integrative strategy for understanding and addressing the uncertainty that historical samples sometimes introduce to phylogenetic analyses.

Earth history and the passerine superradiation.

Avian diversification has been influenced by global climate change, plate tectonic movements, and mass extinction events. However, the impact of these factors on the diversification of the hyperdiverse perching birds (passerines) is unclear because family level relationships are unresolved and the timing of splitting events among lineages is uncertain. We analyzed DNA data from 4,060 nuclear loci and 137 passerine families using concatenation and coalescent approaches to infer a comprehensive phylogenetic hypothesis that clarifies relationships among all passerine families. Then, we calibrated this phylogeny using 13 fossils to examine the effects of different events in Earth history on the timing and rate of passerine diversification. Our analyses reconcile passerine diversification with the fossil and geological records; suggest that passerines originated on the Australian landmass ∼47 Ma; and show that subsequent dispersal and diversification of passerines was affected by a number of climatological and geological events, such as Oligocene glaciation and inundation of the New Zealand landmass. Although passerine diversification rates fluctuated throughout the Cenozoic, we find no link between the rate of passerine diversification and Cenozoic global temperature, and our analyses show that the increases in passerine diversification rate we observe are disconnected from the colonization of new continents. Taken together, these results suggest more complex mechanisms than temperature change or ecological opportunity have controlled macroscale patterns of passerine speciation.

A phylogenomic supermatrix of Galliformes (Landfowl) reveals biased branch lengths.

Building taxon-rich phylogenies is foundational for macroevolutionary studies. One approach to improve taxon sampling beyond individual studies is to build supermatricies of publicly available data, incorporating taxa sampled across different studies and utilizing different loci. Most existing supermatrix studies have focused on loci commonly sequenced with Sanger technology ("legacy" markers, such as mitochondrial data and small numbers of nuclear loci). However, incorporating phylogenomic studies into supermatrices allows problem nodes to be targeted and resolved with considerable amounts of data, while improving taxon sampling with legacy data. Here we estimate phylogeny from a galliform supermatrix which includes well-known model and agricultural species such as the chicken and turkey. We assembled a supermatrix comprising 4500 ultra-conserved elements (UCEs) collected as part of recent phylogenomic studies in this group and legacy mitochondrial and nuclear (intron and exon) sequences. Our resulting phylogeny included 88% of extant species and recovered well-accepted relationships with strong support. However, branch lengths, which are particularly important in down-stream macroevolutionary studies, appeared vastly skewed. Taxa represented only by rapidly evolving mitochondrial data had high proportions of missing data and exhibited long terminal branches. Conversely, taxa sampled for slowly evolving UCEs with low proportions of missing data exhibited substantially shorter terminal branches. We explored several branch length re-estimation methods with particular attention to terminal branches and conclude that re-estimation using well-sampled mitochondrial sequences may be a pragmatic approach to obtain trees suitable for macroevolutionary analysis.

Phylogenomics of manakins (Aves: Pipridae) using alternative locus filtering strategies based on informativeness.

Target capture sequencing effectively generates molecular marker arrays useful for molecular systematics. These extensive data sets are advantageous where previous studies using a few loci have failed to resolve relationships confidently. Moreover, target capture is well-suited to fragmented source DNA, allowing data collection from species that lack fresh tissues. Herein we use target capture to generate data for a phylogeny of the avian family Pipridae (manakins), a group that has been the subject of many behavioral and ecological studies. Most manakin species feature lek mating systems, where males exhibit complex behavioral displays including mechanical and vocal sounds, coordinated movements of multiple males, and high speed movements. We analyzed thousands of ultraconserved element (UCE) loci along with a smaller number of coding exons and their flanking regions from all but one species of Pipridae. We examined three different methods of phylogenetic estimation (concatenation and two multispecies coalescent methods). Phylogenetic inferences using UCE data yielded strongly supported estimates of phylogeny regardless of analytical method. Exon probes had limited capability to capture sequence data and resulted in phylogeny estimates with reduced support and modest topological differences relative to the UCE trees, although these conflicts had limited support. Two genera were paraphyletic among all analyses and data sets, with Antilophia nested within Chiroxiphia and Tyranneutes nested within Neopelma. The Chiroxiphia-Antilophia clade was an exception to the generally high support we observed; the topology of this clade differed among analyses, even those based on UCE data. To further explore relationships within this group, we employed two filtering strategies to remove low-information loci. Those analyses resulted in distinct topologies, suggesting that the relationships we identified within Chiroxiphia-Antilophia should be interpreted with caution. Despite the existence of a few continuing uncertainties, our analyses resulted in a robust phylogenetic hypothesis of the family Pipridae that provides a comparative framework for future ecomorphological and behavioral studies.

Comparative Genomics Reveals a Burst of Homoplasy-Free Numt Insertions.

Mitochondrial DNA sequences are frequently transferred into the nuclear genome, giving rise to numts (nuclear mitochondrial DNA segments). In the absence of whole genomes, avian numts have been suggested to be rare and relatively short. We examined 64 bird genomes to test hypotheses regarding numt frequency, distribution among taxa, and likelihood of homoplasy. We discovered 100-fold variation in numt number across species. Two songbirds, Geospiza fortis (Darwin's finch) and Zonotrichia albicollis (white-throated sparrow) had the largest number of numts. Ancestral state reconstruction of 957 numt insertions in these two species and their close relatives indicated a remarkable acceleration of numt insertion in the ancestor of Geospiza and Zonotrichia followed by slower, continued accumulation in each lineage. These numts appear to result primarily from de novo insertion with the duplication of existing numts representing a secondary pathway. Insertion events were essentially homoplasy-free and numts appear to represent perfect rare genomic changes.

An evolutionary model motivated by physicochemical properties of amino acids reveals variation among proteins.

Motivation: The relative rates of amino acid interchanges over evolutionary time are likely to vary among proteins. Variation in those rates has the potential to reveal information about constraints on proteins. However, the most straightforward model that could be used to estimate relative rates of amino acid substitution is parameter-rich and it is therefore impractical to use for this purpose. Results: A six-parameter model of amino acid substitution that incorporates information about the physicochemical properties of amino acids was developed. It showed that amino acid side chain volume, polarity and aromaticity have major impacts on protein evolution. It also revealed variation among proteins in the relative importance of those properties. The same general approach can be used to improve the fit of empirical models such as the commonly used PAM and LG models. Availability and implementation: Perl code and test data are available from https://github.com/ebraun68/sixparam. Supplementary information: Supplementary data are available at Bioinformatics online.

What are the roles of taxon sampling and model fit in tests of cyto-nuclear discordance using avian mitogenomic data?

Conflicts between nuclear and mitochondrial phylogenies have led to uncertainty for some relationships within the tree of life. These conflicts have led some to question the value of mitochondrial DNA in phylogenetics now that genome-scale nuclear data can be readily obtained. However, since mitochondrial DNA is maternally inherited and does not recombine, its phylogeny should be closer to the species tree. Additionally, its rapid evolutionary rate may drive accumulation of mutations along short internodes where relevant information from nuclear loci may be limited. In this study, we examine the mitochondrial phylogeny of Cavitaves to elucidate its congruence with recently published nuclear phylogenies of this group of birds. Cavitaves includes the orders Trogoniformes (trogons), Bucerotiformes (hornbills), Coraciiformes (kingfishers and allies), and Piciformes (woodpeckers and allies). We hypothesized that sparse taxon sampling in previously published mitochondrial trees was responsible for apparent cyto-nuclear discordance. To test this hypothesis, we assembled 27 additional Cavitaves mitogenomes and estimated phylogenies using seven different taxon sampling schemes ranging from five to 42 ingroup species. We also tested the role that partitioning and model choice played in the observed discordance. Our analyses demonstrated that improved taxon sampling could resolve many of the disagreements. Similarly, partitioning was valuable in improving congruence with the topology from nuclear phylogenies, though the model used to generate the mitochondrial phylogenies had less influence. Overall, our results suggest that the mitochondrial tree is trustworthy when partitioning is used with suitable taxon sampling.

A simple strategy for recovering ultraconserved elements, exons, and introns from low coverage shotgun sequencing of museum specimens: Placement of the partridge genus Tropicoperdix within the galliformes.

Next-generation DNA sequencing (NGS) offers a promising way to obtain massive numbers of orthologous loci to understand phylogenetic relationships among organisms. Of particular interest are old museum specimens and other samples with degraded DNA, where traditional sequencing methods have proven to be challenging. Low coverage shotgun sequencing and sequence capture are two widely used NGS approaches for degraded DNA. Sequence capture can yield sequence data for large numbers of orthologous loci, but it can only be used to sequence genomic regions near conserved sequences that can be used as probes. Low coverage shotgun sequencing has the potential to yield different data types throughout the genome. However, many studies using this method have often generated mitochondrial sequences, and few nuclear sequences, suggesting orthologous nuclear sequences are likely harder to recover. To determine the phylogenetic position of the galliform genus Tropicoperdix, whose phylogenetic position is currently uncertain, we explored two strategies to maximize data extraction from low coverage shotgun sequencing from approximately 100-year-old museum specimens from two species of Tropicoperdix. One approach, a simple read mapping strategy, outperformed the other (a reduced complexity assembly approach), and allowed us to obtain a large number of ultraconserved element (UCE) loci, relatively conserved exons, more variable introns, as well as mitochondrial genomes. Additionally, we demonstrated some simple approaches to explore possible artifacts that may result from the use of degraded DNA. Our data placed Tropicoperdix within a clade that includes many taxa characterized with ornamental eyespots (peafowl, argus pheasants, and peacock pheasants), and established relationships among species within the genus. Therefore, our study demonstrated that low coverage shotgun sequencing can easily be leveraged to yield substantial amounts and varying types of data, which opens the door for many research questions that might require information from different data types from museum specimens.

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.].

Avoiding Missing Data Biases in Phylogenomic Inference: An Empirical Study in the Landfowl (Aves: Galliformes).

Production of massive DNA sequence data sets is transforming phylogenetic inference, but best practices for analyzing such data sets are not well established. One uncertainty is robustness to missing data, particularly in coalescent frameworks. To understand the effects of increasing matrix size and loci at the cost of increasing missing data, we produced a 90 taxon, 2.2 megabase, 4,800 locus sequence matrix of landfowl using target capture of ultraconserved elements. We then compared phylogenies estimated with concatenated maximum likelihood, quartet-based methods executed on concatenated matrices and gene tree reconciliation methods, across five thresholds of missing data. Results of maximum likelihood and quartet analyses were similar, well resolved, and demonstrated increasing support with increasing matrix size and sparseness. Conversely, gene tree reconciliation produced unexpected relationships when we included all informative loci, with certain taxa placed toward the root compared with other approaches. Inspection of these taxa identified a prevalence of short average contigs, which potentially biased gene tree inference and caused erroneous results in gene tree reconciliation. This suggests that the more problematic missing data in gene tree-based analyses are partial sequences rather than entire missing sequences from locus alignments. Limiting gene tree reconciliation to the most informative loci solved this problem, producing well-supported topologies congruent with concatenation and quartet methods. Collectively, our analyses provide a well-resolved phylogeny of landfowl, including strong support for previously problematic relationships such as those among junglefowl (Gallus), and clarify the position of two enigmatic galliform genera (Lerwa, Melanoperdix) not sampled in previous molecular phylogenetic studies.

How do seemingly non-vagile clades accomplish trans-marine dispersal? Trait and dispersal evolution in the landfowl (Aves: Galliformes).

Dispersal ability is a key factor in determining insular distributions and island community composition, yet non-vagile terrestrial organisms widely occur on oceanic islands. The landfowl (pheasants, partridges, grouse, turkeys, quails and relatives) are generally poor dispersers, but the Old World quail (Coturnix) are a notable exception. These birds evolved small body sizes and high-aspect-ratio wing shapes, and hence are capable of trans-continental migrations and trans-oceanic colonization. Two monotypic partridge genera, Margaroperdix of Madagascar and Anurophasis of alpine New Guinea, may represent additional examples of trans-marine dispersal in landfowl, but their body size and wing shape are typical of poorly dispersive continental species. Here, we estimate historical relationships of quail and their relatives using phylogenomics, and infer body size and wing shape evolution in relation to trans-marine dispersal events. Our results show that Margaroperdix and Anurophasis are nested within the Coturnix quail, and are each 'island giants' that independently evolved from dispersive, Coturnix-like ancestral populations that colonized and were subsequently isolated on Madagascar and New Guinea. This evolutionary cycle of gain and loss of dispersal ability, coupled with extinction of dispersive taxa, can result in the false appearance that non-vagile taxa somehow underwent rare oceanic dispersal.

Historical relationships of three enigmatic phasianid genera (Aves: Galliformes) inferred using phylogenomic and mitogenomic data.

The phylogeny of the Phasianidae (pheasants, partridges, and allies) has been studied extensively. However, these studies have largely ignored three enigmatic genera because of scarce DNA source material and limited overlapping phylogenetic data: blood pheasants (Ithaginis), snow partridges (Lerwa), and long-billed partridges (Rhizothera). Thus, phylogenetic positions of these three genera remain uncertain in what is otherwise a well-resolved phylogeny. Previous studies using different data types place Lerwa and Ithaginis in similar positions, but the absence of overlapping data means the relationship between them could not be inferred. Rhizothera was originally described in the genus Perdix (true partridges), although a partial cytochrome b (CYB) sequence suggests it is sister to Pucrasia (koklass pheasant). To identify robust relationships among Ithaginis, Lerwa, Rhizothera, and their phasianid relatives, we used 3692 ultra-conserved element (UCE) loci and complete mitogenomes from 19 species including previously hypothesized relatives of the three focal genera and representatives from all major phasianid clades. We used DNA extracted from historical specimen toepads for species that lacked fresh tissue in museum collections. Maximum likelihood and multispecies coalescent UCE analyses strongly supported Lerwa sister to a large clade which included Ithaginis at its base, and also including turkey, grouse, typical pheasants, tragopans, Pucrasia, and Perdix. Rhizothera was also in this clade, sister to a diverse group comprising Perdix, typical pheasants, Pucrasia, turkey and grouse. Mitogenomic genealogies differed from UCEs topologies, supporting a sister relationship between Ithaginis and Lerwa rather than a grade. The position of Rhizothera using mitogenomes depended on analytical choices. Unpartitioned and codon-based analyses placed Rhizothera sister to a tragopan clade, whereas a partitioned DNA model of the mitogenome was congruent with UCE results. In all mitogenome analyses, Pucrasia was sister to a clade including Perdix and the typical pheasants with high support, in contrast to UCEs and published nuclear intron data. Due to the strong support and consistent topology provided by all UCE analyses, we have identified phylogenetic relationships of these three enigmatic, poorly-studied, phasianid taxa.

Phylogenomic analysis supports multiple instances of polyphyly in the oomycete peronosporalean lineage.

The study of biological diversification of oomycetes has been a difficult task for more than a century. Pioneer researchers used morphological characters to describe this heterogeneous group, and physiological and genetic tools expanded knowledge of these microorganisms. However, research on oomycete diversification is limited by conflicting phylogenies. Using whole genomic data from 17 oomycete taxa, we obtained a dataset of 277 core orthologous genes shared among these genomes. Analyses of this dataset resulted in highly congruent and strongly supported estimates of oomycete phylogeny when we used concatenated maximum likelihood and coalescent-based methods; the one important exception was the position of Albugo. Our results supported the position of Phytopythium vexans (formerly in Pythium clade K) as a sister clade to the Phytophthora-Hyaloperonospora clade. The remaining clades comprising Pythium sensu lato formed two monophyletic groups. One group was composed of three taxa that correspond to Pythium clades A, B and C, and the other group contained taxa representing clades F, G and I, in agreement with previous Pythium phylogenies. However, the group containing Pythium clades F, G and I was placed as sister to the Phytophthora-Hyaloperonospora-Phytopythium clade, thus confirming the lack of monophyly of Pythium sensu lato. Multispecies coalescent methods revealed that the white blister rust, Albugo laibachii, could not be placed with a high degree of confidence. Our analyses show that genomic data can resolve the oomycete phylogeny and provide a phylogenetic framework to study the evolution of oomycete lifestyles.

Analysis of a Rapid Evolutionary Radiation Using Ultraconserved Elements: Evidence for a Bias in Some Multispecies Coalescent Methods.

Rapid evolutionary radiations are expected to require large amounts of sequence data to resolve. To resolve these types of relationships many systematists believe that it will be necessary to collect data by next-generation sequencing (NGS) and use multispecies coalescent ("species tree") methods. Ultraconserved element (UCE) sequence capture is becoming a popular method to leverage the high throughput of NGS to address problems in vertebrate phylogenetics. Here we examine the performance of UCE data for gallopheasants (true pheasants and allies), a clade that underwent a rapid radiation 10-15 Ma. Relationships among gallopheasant genera have been difficult to establish. We used this rapid radiation to assess the performance of species tree methods, using ∼600 kilobases of DNA sequence data from ∼1500 UCEs. We also integrated information from traditional markers (nuclear intron data from 15 loci and three mitochondrial gene regions). Species tree methods exhibited troubling behavior. Two methods [Maximum Pseudolikelihood for Estimating Species Trees (MP-EST) and Accurate Species TRee ALgorithm (ASTRAL)] appeared to perform optimally when the set of input gene trees was limited to the most variable UCEs, though ASTRAL appeared to be more robust than MP-EST to input trees generated using less variable UCEs. In contrast, the rooted triplet consensus method implemented in Triplec performed better when the largest set of input gene trees was used. We also found that all three species tree methods exhibited a surprising degree of dependence on the program used to estimate input gene trees, suggesting that the details of likelihood calculations (e.g., numerical optimization) are important for loci with limited phylogenetic information. As an alternative to summary species tree methods we explored the performance of SuperMatrix Rooted Triple - Maximum Likelihood (SMRT-ML), a concatenation method that is consistent even when gene trees exhibit topological differences due to the multispecies coalescent. We found that SMRT-ML performed well for UCE data. Our results suggest that UCE data have excellent prospects for the resolution of difficult evolutionary radiations, though specific attention may need to be given to the details of the methods used to estimate species trees.

Early mesozoic coexistence of amniotes and hepadnaviridae.

Hepadnaviridae are double-stranded DNA viruses that infect some species of birds and mammals. This includes humans, where hepatitis B viruses (HBVs) are prevalent pathogens in considerable parts of the global population. Recently, endogenized sequences of HBVs (eHBVs) have been discovered in bird genomes where they constitute direct evidence for the coexistence of these viruses and their hosts from the late Mesozoic until present. Nevertheless, virtually nothing is known about the ancient host range of this virus family in other animals. Here we report the first eHBVs from crocodilian, snake, and turtle genomes, including a turtle eHBV that endogenized >207 million years ago. This genomic "fossil" is >125 million years older than the oldest avian eHBV and provides the first direct evidence that Hepadnaviridae already existed during the Early Mesozoic. This implies that the Mesozoic fossil record of HBV infection spans three of the five major groups of land vertebrates, namely birds, crocodilians, and turtles. We show that the deep phylogenetic relationships of HBVs are largely congruent with the deep phylogeny of their amniote hosts, which suggests an ancient amniote-HBV coexistence and codivergence, at least since the Early Mesozoic. Notably, the organization of overlapping genes as well as the structure of elements involved in viral replication has remained highly conserved among HBVs along that time span, except for the presence of the X gene. We provide multiple lines of evidence that the tumor-promoting X protein of mammalian HBVs lacks a homolog in all other hepadnaviruses and propose a novel scenario for the emergence of X via segmental duplication and overprinting of pre-existing reading frames in the ancestor of mammalian HBVs. Our study reveals an unforeseen host range of prehistoric HBVs and provides novel insights into the genome evolution of hepadnaviruses throughout their long-lasting association with amniote hosts.