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Rivers frequently delimit the geographic ranges of species in the Amazon Basin. These rivers also define the boundaries between genetic clusters within many species, yet river boundaries have been documented to break down in headwater regions where rivers are narrower. To explore the evolutionary implications of headwater contact zones in Amazonia, we examined genetic variation in the Blue-capped Manakin (Lepidothrix coronata), a species previously shown to contain several genetically and phenotypically distinct populations across the western Amazon Basin. We collected restriction site-associated DNA sequence data (RADcap) for 706 individuals and found that spatial patterns of genetic structure indicate several rivers, particularly the Amazon and Ucayali, are dispersal barriers for L. coronata. We also found evidence that genetic connectivity is elevated across several headwater regions, highlighting the importance of headwater gene flow for models of Amazonian diversification. The headwater region of the Ucayali River provided a notable exception to findings of headwater gene flow by harboring non-admixed populations of L. coronata on opposite sides of a < 1-km-wide river channel with a known dynamic history, suggesting that additional prezygotic barriers may be limiting gene flow in this region., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Society for the Study of Evolution (SSE). All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The clapper rail (Rallus crepitans), of the family Rallidae, is a secretive marsh bird species that is adapted for high salinity habitats. They are very similar in appearance to the closely related king rail (R. elegans), but while king rails are limited primarily to freshwater marshes, clapper rails are highly adapted to tolerate salt marshes. Both species can be found in brackish marshes where they freely hybridize, but the distribution of their respective habitats precludes the formation of a continuous hybrid zone and secondary contact can occur repeatedly. This system, thus, provides unique opportunities to investigate the underlying mechanisms driving their differential salinity tolerance as well as the maintenance of the species boundary between the 2 species. To facilitate these studies, we assembled a de novo reference genome assembly for a female clapper rail. Chicago and HiC libraries were prepared as input for the Dovetail HiRise pipeline to scaffold the genome. The pipeline, however, did not recover the Z chromosome so a custom script was used to assemble the Z chromosome. We generated a near chromosome level assembly with a total length of 994.8 Mb comprising 13,226 scaffolds. The assembly had a scaffold N50 was 82.7 Mb, L50 of four, and had a BUSCO completeness score of 92%. This assembly is among the most contiguous genomes among the species in the family Rallidae. It will serve as an important tool in future studies on avian salinity tolerance, interspecific hybridization, and speciation., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

The Order Pelagibacterales (SAR11) is the most abundant group of heterotrophic bacterioplankton in global oceans and comprises multiple subclades with unique spatiotemporal distributions. Subclade IIIa is the primary SAR11 group in brackish waters and shares a common ancestor with the dominant freshwater IIIb (LD12) subclade. Despite its dominance in brackish environments, subclade IIIa lacks systematic genomic or ecological studies. Here, we combine closed genomes from new IIIa isolates, new IIIa MAGS from San Francisco Bay (SFB), and 460 highly complete publicly available SAR11 genomes for the most comprehensive pangenomic study of subclade IIIa to date. Subclade IIIa represents a taxonomic family containing three genera (denoted as subgroups IIIa.1, IIIa.2, and IIIa.3) that had distinct ecological distributions related to salinity. The expansion of taxon selection within subclade IIIa also established previously noted metabolic differentiation in subclade IIIa compared to other SAR11 subclades such as glycine/serine prototrophy, mosaic glyoxylate shunt presence, and polyhydroxyalkanoate synthesis potential. Our analysis further shows metabolic flexibility among subgroups within IIIa. Additionally, we find that subclade IIIa.3 bridges the marine and freshwater clades based on its potential for compatible solute transport, iron utilization, and bicarbonate management potential. Pure culture experimentation validated differential salinity ranges in IIIa.1 and IIIa.3 and provided detailed IIIa cell size and volume data. This study is an important step forward for understanding the genomic, ecological, and physiological differentiation of subclade IIIa and the overall evolutionary history of SAR11., (© 2023. The Author(s).)

North American sunfishes (Family Centrarchidae) are among the most popular sportfish throughout the United States and Canada. Despite the popularity of sunfishes, their ecological importance, and their extensive stocking and aquacultural history, few molecular studies have examined the evolutionary relationships and species boundaries among members of this group, many of which are known to hybridize. Here, we describe a chromosome-scale genome assembly representing Bluegill (Lepomis macrochirus), one of the most widespread centrarchid species. By combining long-read, Oxford Nanopore sequencing data with short-insert, whole-genome and HiC sequence reads, we produced an assembly (Lm&#95;LA&#95;1.1) having a total length of 889 Mb including 1,841 scaffolds and having a scaffold N50 of 36 Mb, L50 of 12, N90 of 29 Mb, and L90 of 22. We detected 99% (eukaryota&#95;odb10) and 98% (actinopterygii&#95;odb10) universal single-copy orthologs (BUSCOs), and ab initio gene prediction performed using this new assembly identified a set of 17,233 genes that were supported by external (OrthoDB v10) data. This new assembly provides an important addition to the growing set of assemblies already available for spiny-rayed fishes (Acanthomorpha), and it will serve as a resource for future studies that focus on the complex evolutionary history of centrarchids., Competing Interests: Conflicts of interest None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Genetics Society of America.)

Secondary contact between species often results in the formation of a hybrid zone, with the eventual fates of the hybridizing species dependent on evolutionary and ecological forces. We examine this process in the Amazon Basin by conducting the first genomic and phenotypic characterization of the hybrid zone formed after secondary contact between two obligate army-ant-followers: the White-breasted Antbird (Rhegmatorhina hoffmannsi) and the Harlequin Antbird (Rhegmatorhina berlepschi). We found a major geographic displacement (∼120 km) between the mitochondrial and nuclear clines, and we explore potential hypotheses for the displacement, including sampling error, genetic drift, and asymmetric cytonuclear incompatibilities. We cannot exclude roles for sampling error and genetic drift in contributing to the discordance; however, the data suggest expansion and unidirectional introgression of hoffmannsi into the distribution of berlepschi., (© 2021 The Authors. Evolution © 2021 The Society for the Study of Evolution.)

The Neotropics harbor the most species-rich freshwater fish fauna on the planet, but the timing of that exceptional diversification remains unclear. Did the Neotropics accumulate species steadily throughout their long history, or attain their remarkable diversity recently? Biologists have long debated the relative support for these museum and cradle hypotheses, but few phylogenies of megadiverse tropical clades have included sufficient taxa to distinguish between them. We used 1288 ultraconserved element loci spanning 293 species, 211 genera, and 21 families of characoid fishes to reconstruct a new, fossil-calibrated phylogeny and infer the most likely diversification scenario for a clade that includes a third of Neotropical fish diversity. This phylogeny implies paraphyly of the traditional delimitation of Characiformes because it resolves the largely Neotropical Characoidei as the sister lineage of Siluriformes (catfishes), rather than the African Citharinodei. Time-calibrated phylogenies indicate an ancient origin of major characoid lineages and reveal a much more recent emergence of most characoid species. Diversification rate analyses infer increased speciation and decreased extinction rates during the Oligocene at around 30 Ma during a period of mega-wetland formation in the proto-Orinoco-Amazonas. Three species-rich and ecomorphologically diverse lineages (Anostomidae, Serrasalmidae, and Characidae) that originated more than 60 Ma in the Paleocene experienced particularly notable bursts of Oligocene diversification and now account collectively for 68% of the approximately 2150 species of Characoidei. In addition to paleogeographic changes, we discuss potential accelerants of diversification in these three lineages. While the Neotropics accumulated a museum of ecomorphologically diverse characoid lineages long ago, this geologically dynamic region also cradled a much more recent birth of remarkable species-level diversity. [Biodiversity; Characiformes; macroevolution; Neotropics; phylogenomics; ultraconserved elements.]., (© The Author(s) 2021. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Black-throated Flowerpiercers (Diglossa brunneiventris) are one species representing a phenotypically specialized group of tanagers (Thraupidae) that have hooked bills which allow them to feed by stealing nectar from the base of flowers. Members of the genus are widely distributed in montane regions from Mexico to northern Argentina, and previous studies of Diglossa have focused on their systematics, phylogenetics, and interesting natural history. Despite numerous studies of species within the genus, no genome assembly exists to represent these nectivorous tanagers. We described the assembly of a genome sequence representing a museum-vouchered, wild, female D. brunneiventris collected in Peru. By combining Pacific Biosciences Sequel long-read technology with 10× linked-read and reference-based scaffolding, we produced a 1.08 Gbp pseudochromosomal assembly including 600 scaffolds with a scaffold N50 of 67.3 Mbp, a scaffold L50 of 6, and a BUSCO completeness score of 95%. This new assembly improves representation of the diverse species that comprise the tanagers, improves on scaffold lengths and contiguity when compared to existing genomic resources for tanagers, and provides another avenue of research into the genetic basis of adaptations common to a nectivorous lifestyle among vertebrates., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

The lack of genomic resources for tropical canopy trees is impeding several research avenues in tropical forest biology. We present genome assemblies for two Neotropical hardwood species, Jacaranda copaia and Handroanthus (formerly Tabebuia) guayacan, that are model systems for research on tropical tree demography and flowering phenology. For each species, we combined Illumina short-read data with in vitro proximity-ligation (Chicago) libraries to generate an assembly. For Jacaranda copaia, we obtained 104X physical coverage and produced an assembly with N50/N90 scaffold lengths of 1.020/0.277 Mbp. For H. guayacan, we obtained 129X coverage and produced an assembly with N50/N90 scaffold lengths of 0.795/0.165 Mbp. J. copaia and H. guayacan assemblies contained 95.8% and 87.9% of benchmarking orthologs, although they constituted only 77.1% and 66.7% of the estimated genome sizes of 799 and 512 Mbp, respectively. These differences were potentially due to high repetitive sequence content (>59.31% and 45.59%) and high heterozygosity (0.5% and 0.8%) in each species. Finally, we compared each new assembly to a previously sequenced genome for Handroanthus impetiginosus using whole-genome alignment. This analysis indicated extensive gene duplication in H. impetiginosus since its divergence from H. guayacan., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

Northern bobwhites ( Colinus virginianus ) are small quails in the New World Quail family (Odontophoridae) and are one of the most phenotypically diverse avian species. Despite extensive research on bobwhite ecology, genomic studies investigating the evolution of phenotypic diversity in this species are lacking. Here, we present a new, highly contiguous assembly for bobwhites using tissue samples from a vouchered, wild, female bird collected in Louisiana. By performing a de novo assembly and scaffolding the assembly with Dovetail Chicago and HiC libraries and the HiRise pipeline, we produced an 866.8 Mb assembly including 1,512 scaffolds with a scaffold N50 of 66.8 Mb, a scaffold L90 of 17, and a BUSCO completeness score of 90.8%. This new assembly represents approximately 96% of the non-repetitive and 84% of the entire bobwhite genome size, greatly improves scaffold lengths and contiguity compared to an existing draft bobwhite genome, and provides an important tool for future studies of evolutionary and functional genomics in bobwhites., (Copyright © 2019 Salter et al.)

Resolving patterns of ancient and rapid diversifications is one of the most challenging tasks in evolutionary biology. These difficulties arise from confusing phylogenetic signals that are associated with the interplay of incomplete lineage sorting (ILS) and homoplasy. Phylogenomic analyses of hundreds, or even thousands, of loci offer the potential to resolve such contentious relationships. Yet, how much useful phylogenetic information these large data sets contain remains uncertain and often goes untested. Here, we assess the utility of different data filtering approaches to maximize phylogenetic information and minimize noise when reconstructing an ancient radiation of Neotropical electric knifefishes (Order Gymnotiformes) using ultraconserved elements. We found two contrasting hypotheses of gymnotiform evolutionary relationships depending on whether phylogenetic inferences were based on concatenation or coalescent methods. In the first case, all analyses inferred a previously-and commonly-proposed hypothesis, where the family Apteronotidae was found as the sister group to all other gymnotiform families. In contrast, coalescent-based analyses suggested a novel hypothesis where families producing pulse-type (viz., Gymnotidae, Hypopomidae, and Rhamphichthyidae) and wave-type electric signals (viz., Apteronotidae, Sternopygidae) were reciprocally monophyletic. Nodal support for this second hypothesis increased when analyzing loci with the highest phylogenetic information content and further increased when data were pruned using targeted filtering methods that maximized phylogenetic informativeness at the deepest nodes of the Gymnotiformes. Bayesian concordance analyses and topology tests of individual gene genealogies demonstrated that the difficulty of resolving this radiation was likely due to high gene-tree incongruences that resulted from ILS. We show that data filtering reduces gene-tree heterogeneity and increases nodal support and consistency of species trees using coalescent methods; however, we failed to observe the same effect when using concatenation methods. Furthermore, the targeted filtering strategies applied here support the use of "gene data interrogation" rather than "gene genealogy interrogation" approaches in phylogenomic analyses, to extract phylogenetic signal from intractable portions of the Tree of Life., (© The Author(s) 2018. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Advances in high-throughput sequencing techniques now allow relatively easy and affordable sequencing of large portions of the genome, even for nonmodel organisms. Many phylogenetic studies reduce costs by focusing their sequencing efforts on a selected set of targeted loci, commonly enriched using sequence capture. The advantage of this approach is that it recovers a consistent set of loci, each with high sequencing depth, which leads to more confidence in the assembly of target sequences. High sequencing depth can also be used to identify phylogenetically informative allelic variation within sequenced individuals, but allele sequences are infrequently assembled in phylogenetic studies. Instead, many scientists perform their phylogenetic analyses using contig sequences which result from the de novo assembly of sequencing reads into contigs containing only canonical nucleobases, and this may reduce both statistical power and phylogenetic accuracy. Here, we develop an easy-to-use pipeline to recover allele sequences from sequence capture data, and we use simulated and empirical data to demonstrate the utility of integrating these allele sequences to analyses performed under the multispecies coalescent model. Our empirical analyses of ultraconserved element locus data collected from the South American hummingbird genus Topaza demonstrate that phased allele sequences carry sufficient phylogenetic information to infer the genetic structure, lineage divergence, and biogeographic history of a genus that diversified during the last 3 myr. The phylogenetic results support the recognition of two species and suggest a high rate of gene flow across large distances of rainforest habitats but rare admixture across the Amazon River. Our simulations provide evidence that analyzing allele sequences leads to more accurate estimates of tree topology and divergence times than the more common approach of using contig sequences.

Evolutionary transitions between fresh and salt water happen infrequently among bacterioplankton. Within the ubiquitous and highly abundant heterotrophic Alphaproteobacteria order Pelagibacterales (SAR11), most members live in marine habitats, but the LD12 subclade has evolved as a unique freshwater lineage. LD12 cells occur as some of the most dominant freshwater bacterioplankton, yet this group has remained elusive to cultivation, hampering a more thorough understanding of its biology. Here, we report the first successful isolation of an LD12 representative, strain LSUCC0530, using high-throughput dilution-to-extinction cultivation methods, and its complete genome sequence. Growth experiments corroborate ecological data suggesting active populations of LD12 in brackish water up to salinities of ~5. LSUCC0530 has the smallest closed genome thus far reported for a SAR11 strain (1.16 Mbp). The genome affirms many previous metabolic predictions from cultivation-independent analyses, like a complete Embden-Meyerhof-Parnas glycolysis pathway, but also provides novel insights, such as the first isocitrate dehydrogenase in LD12, a likely homologous recombination of malate synthase from outside of the SAR11 clade, and analogous substitutions of ion transporters with others that occur throughout the rest of the SAR11 clade. Growth data support metagenomic recruitment results suggesting temperature-based ecotype diversification within LD12. Key gene losses for osmolyte uptake provide a succinct hypothesis for the evolutionary transition of LD12 from salt to freshwater. For strain LSUCC0530, we propose the provisional nomenclature Candidatus fonsibacter ubiquis.

The rapid diversification of Myotis bats into more than 100 species is one of the most extensive mammalian radiations available for study. Efforts to understand relationships within Myotis have primarily utilized mitochondrial markers and trees inferred from nuclear markers lacked resolution. Our current understanding of relationships within Myotis is therefore biased towards a set of phylogenetic markers that may not reflect the history of the nuclear genome. To resolve this, we sequenced the full mitochondrial genomes of 37 representative Myotis, primarily from the New World, in conjunction with targeted sequencing of 3648 ultraconserved elements (UCEs). We inferred the phylogeny and explored the effects of concatenation and summary phylogenetic methods, as well as combinations of markers based on informativeness or levels of missing data, on our results. Of the 294 phylogenies generated from the nuclear UCE data, all are significantly different from phylogenies inferred using mitochondrial genomes. Even within the nuclear data, quartet frequencies indicate that around half of all UCE loci conflict with the estimated species tree. Several factors can drive such conflict, including incomplete lineage sorting, introgressive hybridization, or even phylogenetic error. Despite the degree of discordance between nuclear UCE loci and the mitochondrial genome and among UCE loci themselves, the most common nuclear topology is recovered in one quarter of all analyses with strong nodal support. Based on these results, we re-examine the evolutionary history of Myotis to better understand the phenomena driving their unique nuclear, mitochondrial, and biogeographic histories.

Ostariophysi is a superorder of bony fishes including more than 10,300 species in 1100 genera and 70 families. This superorder is traditionally divided into five major groups (orders): Gonorynchiformes (milkfishes and sandfishes), Cypriniformes (carps and minnows), Characiformes (tetras and their allies), Siluriformes (catfishes), and Gymnotiformes (electric knifefishes). Unambiguous resolution of the relationships among these lineages remains elusive, with previous molecular and morphological analyses failing to produce a consensus phylogeny. In this study, we use over 350 ultraconserved element (UCEs) loci comprising 5 million base pairs collected across 35 representative ostariophysan species to compile one of the most data-rich phylogenies of fishes to date. We use these data to infer higher level (interordinal) relationships among ostariophysan fishes, focusing on the monophyly of the Characiformes-one of the most contentiously debated groups in fish systematics. As with most previous molecular studies, we recover a non-monophyletic Characiformes with the two monophyletic suborders, Citharinoidei and Characoidei, more closely related to other ostariophysan clades than to each other. We also explore incongruence between results from different UCE data sets, issues of orthology, and the use of morphological characters in combination with our molecular data. [Conserved sequence; ichthyology; massively parallel sequencing; morphology; next-generation sequencing; UCEs.]., (© The Author(s) 2017. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The phylogeny of eutherian mammals contains some of the most recalcitrant nodes in the tetrapod tree of life. We combined comprehensive taxon and character sampling to explore three of the most debated interordinal relationships among placental mammals. We performed in silico extraction of ultraconserved element loci from 72 published genomes and invitro enrichment and sequencing of ultraconserved elements from 28 additional mammals, resulting in alignments of 3,787 loci. We analyzed these data using concatenated and multispecies coalescent phylogenetic approaches, topological tests, and exploration of support among individual loci to identify the root of Eutheria and the sister groups of tree shrews (Scandentia) and horses (Perissodactyla). Individual loci provided weak, but often consistent support for topological hypotheses. Although many gene trees lacked accepted species-tree relationships, summary coalescent topologies were largely consistent with inferences from concatenation. At the root of Eutheria, we identified consistent support for a sister relationship between Xenarthra and Afrotheria (i.e., Atlantogenata). At the other nodes of interest, support was less consistent. We suggest Scandentia is the sister of Primatomorpha (Euarchonta), but we failed to reject a sister relationship between Scandentia and Glires. Similarly, we suggest Perissodactyla is sister to Cetartiodactyla (Euungulata), but a sister relationship between Perissodactyla and Chiroptera remains plausible., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Sequence capture and restriction site associated DNA sequencing (RAD-Seq) are two genomic enrichment strategies for applying next-generation sequencing technologies to systematics studies. At shallow timescales, such as within species, RAD-Seq has been widely adopted among researchers, although there has been little discussion of the potential limitations and benefits of RAD-Seq and sequence capture. We discuss a series of issues that may impact the utility of sequence capture and RAD-Seq data for shallow systematics in non-model species. We review prior studies that used both methods, and investigate differences between the methods by re-analyzing existing RAD-Seq and sequence capture data sets from a Neotropical bird (Xenops minutus). We suggest that the strengths of RAD-Seq data sets for shallow systematics are the wide dispersion of markers across the genome, the relative ease and cost of laboratory work, the deep coverage and read overlap at recovered loci, and the high overall information that results. Sequence capture's benefits include flexibility and repeatability in the genomic regions targeted, success using low-quality samples, more straightforward read orthology assessment, and higher per-locus information content. The utility of a method in systematics, however, rests not only on its performance within a study, but on the comparability of data sets and inferences with those of prior work. In RAD-Seq data sets, comparability is compromised by low overlap of orthologous markers across species and the sensitivity of genetic diversity in a data set to an interaction between the level of natural heterozygosity in the samples examined and the parameters used for orthology assessment. In contrast, sequence capture of conserved genomic regions permits interrogation of the same loci across divergent species, which is preferable for maintaining comparability among data sets and studies for the purpose of drawing general conclusions about the impact of historical processes across biotas. We argue that sequence capture should be given greater attention as a method of obtaining data for studies in shallow systematics and comparative phylogeography., (© The Author(s) 2016. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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., (© The Author(s) 2016. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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., (© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Unlabelled: Targeted enrichment of conserved and ultraconserved genomic elements allows universal collection of phylogenomic data from hundreds of species at multiple time scales (<5 Ma to > 300 Ma). Prior to downstream inference, data from these types of targeted enrichment studies must undergo preprocessing to assemble contigs from sequence data; identify targeted, enriched loci from the off-target background data; align enriched contigs representing conserved loci to one another; and prepare and manipulate these alignments for subsequent phylogenomic inference. PHYLUCE is an efficient and easy-to-install software package that accomplishes these tasks across hundreds of taxa and thousands of enriched loci., Availability and Implementation: PHYLUCE is written for Python 2.7. PHYLUCE is supported on OSX and Linux (RedHat/CentOS) operating systems. PHYLUCE source code is distributed under a BSD-style license from https://www.github.com/faircloth-lab/phyluce/ PHYLUCE is also available as a package (https://binstar.org/faircloth-lab/phyluce) for the Anaconda Python distribution that installs all dependencies, and users can request a PHYLUCE instance on iPlant Atmosphere (tag: phyluce). The software manual and a tutorial are available from http://phyluce.readthedocs.org/en/latest/ and test data are available from doi: 10.6084/m9.figshare.1284521., Contact: brant@faircloth-lab.org, Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Determining the evolutionary relationships among the major lineages of extant birds has been one of the biggest challenges in systematic biology. To address this challenge, we assembled or collected the genomes of 48 avian species spanning most orders of birds, including all Neognathae and two of the five Palaeognathae orders. We used these genomes to construct a genome-scale avian phylogenetic tree and perform comparative genomic analyses., Findings: Here we present the datasets associated with the phylogenomic analyses, which include sequence alignment files consisting of nucleotides, amino acids, indels, and transposable elements, as well as tree files containing gene trees and species trees. Inferring an accurate phylogeny required generating: 1) A well annotated data set across species based on genome synteny; 2) Alignments with unaligned or incorrectly overaligned sequences filtered out; and 3) Diverse data sets, including genes and their inferred trees, indels, and transposable elements. Our total evidence nucleotide tree (TENT) data set (consisting of exons, introns, and UCEs) gave what we consider our most reliable species tree when using the concatenation-based ExaML algorithm or when using statistical binning with the coalescence-based MP-EST algorithm (which we refer to as MP-EST*). Other data sets, such as the coding sequence of some exons, revealed other properties of genome evolution, namely convergence., Conclusions: The Avian Phylogenomics Project is the largest vertebrate phylogenomics project to date that we are aware of. The sequence, alignment, and tree data are expected to accelerate analyses in phylogenomics and other related areas.

Comparative genetic studies of non-model organisms are transforming rapidly due to major advances in sequencing technology. A limiting factor in these studies has been the identification and screening of orthologous loci across an evolutionarily distant set of taxa. Here, we evaluate the efficacy of genomic markers targeting ultraconserved DNA elements (UCEs) for analyses at shallow evolutionary timescales. Using sequence capture and massively parallel sequencing to generate UCE data for five co-distributed Neotropical rainforest bird species, we recovered 776-1516 UCE loci across the five species. Across species, 53-77% of the loci were polymorphic, containing between 2.0 and 3.2 variable sites per polymorphic locus, on average. We performed species tree construction, coalescent modeling, and species delimitation, and we found that the five co-distributed species exhibited discordant phylogeographic histories. We also found that species trees and divergence times estimated from UCEs were similar to the parameters obtained from mtDNA. The species that inhabit the understory had older divergence times across barriers, contained a higher number of cryptic species, and exhibited larger effective population sizes relative to the species inhabiting the canopy. Because orthologous UCEs can be obtained from a wide array of taxa, are polymorphic at shallow evolutionary timescales, and can be generated rapidly at low cost, they are an effective genetic marker for studies investigating evolutionary patterns and processes at shallow timescales.

Although massively parallel sequencing has facilitated large-scale DNA sequencing, comparisons among distantly related species rely upon small portions of the genome that are easily aligned. Methods are needed to efficiently obtain comparable DNA fragments prior to massively parallel sequencing, particularly for biologists working with non-model organisms. We introduce a new class of molecular marker, anchored by ultraconserved genomic elements (UCEs), that universally enable target enrichment and sequencing of thousands of orthologous loci across species separated by hundreds of millions of years of evolution. Our analyses here focus on use of UCE markers in Amniota because UCEs and phylogenetic relationships are well-known in some amniotes. We perform an in silico experiment to demonstrate that sequence flanking 2030 UCEs contains information sufficient to enable unambiguous recovery of the established primate phylogeny. We extend this experiment by performing an in vitro enrichment of 2386 UCE-anchored loci from nine, non-model avian species. We then use alignments of 854 of these loci to unambiguously recover the established evolutionary relationships within and among three ancient bird lineages. Because many organismal lineages have UCEs, this type of genetic marker and the analytical framework we outline can be applied across the tree of life, potentially reshaping our understanding of phylogeny at many taxonomic levels.

Polymerase chain reaction (PCR) is a basic molecular biology technique with a multiplicity of uses, including deoxyribonucleic acid cloning and sequencing, functional analysis of genes, diagnosis of diseases, genotyping and discovery of genetic variants. Reliable primer design is crucial for successful PCR, and for over a decade, the open-source Primer3 software has been widely used for primer design, often in high-throughput genomics applications. It has also been incorporated into numerous publicly available software packages and web services. During this period, we have greatly expanded Primer3's functionality. In this article, we describe Primer3's current capabilities, emphasizing recent improvements. The most notable enhancements incorporate more accurate thermodynamic models in the primer design process, both to improve melting temperature prediction and to reduce the likelihood that primers will form hairpins or dimers. Additional enhancements include more precise control of primer placement-a change motivated partly by opportunities to use whole-genome sequences to improve primer specificity. We also added features to increase ease of use, including the ability to save and re-use parameter settings and the ability to require that individual primers not be used in more than one primer pair. We have made the core code more modular and provided cleaner programming interfaces to further ease integration with other software. These improvements position Primer3 for continued use with genome-scale data in the decade ahead.