Robb T. Brumfield

Whole-genome analyses resolve early branches in the tree of life of modern birds

To better determine the history of modern birds, we performed a genome-scale phylogenetic analysis of 48 species representing all orders of Neoaves using phylogenomic methods created to handle genome-scale data. We recovered a highly resolved tree that confirms previously controversial sister or close relationships. We identified the first divergence in Neoaves, two groups we named Passerea and Columbea, representing independent lineages of diverse and convergently evolved land and water bird species. Among Passerea, we infer the common ancestor of core landbirds to have been an apex predator and confirm independent gains of vocal learning. Among Columbea, we identify pigeons and flamingoes as belonging to sister clades. Even with whole genomes, some of the earliest branches in Neoaves proved challenging to resolve, which was best explained by massive protein-coding sequence convergence and high levels of incomplete lineage sorting that occurred during a rapid radiation after the Cretaceous-Paleogene mass extinction event about 66 million years ago.

The utility of single nucleotide polymorphisms in inferences of population history

Single nucleotide polymorphisms (SNPs) represent the most widespread type of sequence variation in genomes, yet they have only emerged recently as valuable genetic markers for revealing the evolutionary history of populations. Their occurrence throughout the genome also makes them ideal for analyses of speciation and historical demography, especially in light of recent theory suggesting that many unlinked nuclear loci are needed to estimate population genetic parameters with statistical confidence. In spite of having lower variation compared with microsatellites, SNPs should make the comparison of genomic diversities and histories of different species (the core goal of comparative biogeography) more straightforward than has been possible with microsatellites. The most pervasive, but correctable, complication to SNP analysis is a bias towards analyzing only the most variable loci, an artifact that is usually introduced by the limited number of individuals used to screen initially for polymorphisms. Although the use of SNPs as markers in population studies is still new, innovative methods for SNP identification, automated screening, haplotype inference and statistical analysis might quickly make SNPs the marker of choice. Traditionally, phylogeography has used gene trees of nonrecombining, uniparentally inherited LOCI (see Glossary), such as mitochondrial DNA or the vertebrate Y chromosome, to study the geographical distribution of genetic variation within species [1]. As evolutionary biologists have started to examine variation in recombining, biparentally inherited loci, a natural outgrowth of phylogeography is a shift from gene trees to analyses, based on COALESCENT THEORY, of multi-locus, recombining histories. This new discipline, dubbed historical demography [2,3] or statistical phylogeography [4], is concerned less with gene trees than with estimating population parameters such as genetic diversities, divergence times, growth rates and gene flow between populations. The shift in focus is, in part, a result of recent advances in population genetics, which suggest that, from a statistical standpoint, the ability of single-locus phylogeography to determine the timing of speciation events and the historical demography of populations has been overestimated [3‐7]. The errors surrounding estimates of divergence times, rates of gene flow and population-size changes during speciation are all reduced substantially when information from multiple unlinked loci is combined [8,9]. With the move to analyses of multiple loci, phylogeographers must re-learn an old lesson: that the number of loci required to estimate the preceding parameters with statistical confidence can be soberingly large because of the high stochasticity of the gene tree of any single locus [10]. What is required is a suite of unlinked nuclear genetic markers that can capture a genome-wide picture of the population history [3,11‐14]. The polymerase chain reaction (PCR) as well as fluorescent sequencing and fragment analysis technologies have catalyzed a revolution in the development of genetic markers for the analysis of natural populations. Emphasizing discoveries in nonmodel species, we discuss one emerging marker of great relevance to historical demography: single nucleotide polymorphisms (SNPs).

Ultraconserved Elements Anchor Thousands of Genetic Markers Spanning Multiple Evolutionary 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.

Applications of next-generation sequencing to phylogeography and phylogenetics

This is a time of unprecedented transition in DNA sequencing technologies. Next-generation sequencing (NGS) clearly holds promise for fast and cost-effective generation of multilocus sequence data for phylogeography and phylogenetics. However, the focus on non-model organisms, in addition to uncertainty about which sample preparation methods and analyses are appropriate for different research questions and evolutionary timescales, have contributed to a lag in the application of NGS to these fields. Here, we outline some of the major obstacles specific to the application of NGS to phylogeography and phylogenetics, including the focus on non-model organisms, the necessity of obtaining orthologous loci in a cost-effective manner, and the predominate use of gene trees in these fields. We describe the most promising methods of sample preparation that address these challenges. Methods that reduce the genome by restriction digest and manual size selection are most appropriate for studies at the intraspecific level, whereas methods that target specific genomic regions (i.e., target enrichment or sequence capture) have wider applicability from the population level to deep-level phylogenomics. Additionally, we give an overview of how to analyze NGS data to arrive at data sets applicable to the standard toolkit of phylogeography and phylogenetics, including initial data processing to alignment and genotype calling (both SNPs and loci involving many SNPs). Even though whole-genome sequencing is likely to become affordable rather soon, because phylogeography and phylogenetics rely on analysis of hundreds of individuals in many cases, methods that reduce the genome to a subset of loci should remain more cost-effective for some time to come.

More than 1000 ultraconserved elements provide evidence that turtles are the sister group of archosaurs

We present the first genomic-scale analysis addressing the phylogenetic position of turtles, using over 1000 loci from representatives of all major reptile lineages including tuatara. Previously, studies of morphological traits positioned turtles either at the base of the reptile tree or with lizards, snakes and tuatara (lepidosaurs), whereas molecular analyses typically allied turtles with crocodiles and birds (archosaurs). A recent analysis of shared microRNA families found that turtles are more closely related to lepidosaurs. To test this hypothesis with data from many single-copy nuclear loci dispersed throughout the genome, we used sequence capture, high-throughput sequencing and published genomes to obtain sequences from 1145 ultraconserved elements (UCEs) and their variable flanking DNA. The resulting phylogeny provides overwhelming support for the hypothesis that turtles evolved from a common ancestor of birds and crocodilians, rejecting the hypothesized relationship between turtles and lepidosaurs.

ESTIMATING SPECIES TREES USING MULTIPLE‐ALLELE DNA SEQUENCE DATA

Abstract Several techniques, such as concatenation and consensus methods, are available for combining data from multiple loci to produce a single statement of phylogenetic relationships. However, when multiple alleles are sampled from individual species, it becomes more challenging to estimate relationships at the level of species, either because concatenation becomes inappropriate due to conflicts among individual gene trees, or because the species from which multiple alleles have been sampled may not form monophyletic groups in the estimated tree. We propose a Bayesian hierarchical model to reconstruct species trees from multipleallele, multilocus sequence data, building on a recently proposed method for estimating species trees from single allele multilocus data. A two-step Markov Chain Monte Carlo (MCMC) algorithm is adopted to estimate the posterior distribution of the species tree. The model is applied to estimate the posterior distribution of species trees for two multiple-allele datasets—yeast (Saccharomyces) and birds (Manacus—manakins). The estimates of the species trees using our method are consistent with those inferred from other methods and genetic markers, but in contrast to other species tree methods, it provides credible regions for the species tree. The Bayesian approach described here provides a powerful framework for statistical testing and integration of population genetics and phylogenetics.

A Phylogeny of Birds Based on Over 1,500 Loci Collected by Target Enrichment and High-Throughput Sequencing

Evolutionary relationships among birds in Neoaves, the clade comprising the vast majority of avian diversity, have vexed systematists due to the ancient, rapid radiation of numerous lineages. We applied a new phylogenomic approach to resolve relationships in Neoaves using target enrichment (sequence capture) and high-throughput sequencing of ultraconserved elements (UCEs) in avian genomes. We collected sequence data from UCE loci for 32 members of Neoaves and one outgroup (chicken) and analyzed data sets that differed in their amount of missing data. An alignment of 1,541 loci that allowed missing data was 87% complete and resulted in a highly resolved phylogeny with broad agreement between the Bayesian and maximum-likelihood (ML) trees. Although results from the 100% complete matrix of 416 UCE loci were similar, the Bayesian and ML trees differed to a greater extent in this analysis, suggesting that increasing from 416 to 1,541 loci led to increased stability and resolution of the tree. Novel results of our study include surprisingly close relationships between phenotypically divergent bird families, such as tropicbirds (Phaethontidae) and the sunbittern (Eurypygidae) as well as between bustards (Otididae) and turacos (Musophagidae). This phylogeny bolsters support for monophyletic waterbird and landbird clades and also strongly supports controversial results from previous studies, including the sister relationship between passerines and parrots and the non-monophyly of raptorial birds in the hawk and falcon families. Although significant challenges remain to fully resolving some of the deep relationships in Neoaves, especially among lineages outside the waterbirds and landbirds, this study suggests that increased data will yield an increasingly resolved avian phylogeny.

The drivers of tropical speciation

Since the recognition that allopatric speciation can be induced by large-scale reconfigurations of the landscape that isolate formerly continuous populations, such as the separation of continents by plate tectonics, the uplift of mountains or the formation of large rivers, landscape change has been viewed as a primary driver of biological diversification. This process is referred to in biogeography as vicariance. In the most species-rich region of the world, the Neotropics, the sundering of populations associated with the Andean uplift is ascribed this principal role in speciation. An alternative model posits that rather than being directly linked to landscape change, allopatric speciation is initiated to a greater extent by dispersal events, with the principal drivers of speciation being organism-specific abilities to persist and disperse in the landscape. Landscape change is not a necessity for speciation in this model. Here we show that spatial and temporal patterns of genetic differentiation in Neotropical birds are highly discordant across lineages and are not reconcilable with a model linking speciation solely to landscape change. Instead, the strongest predictors of speciation are the amount of time a lineage has persisted in the landscape and the ability of birds to move through the landscape matrix. These results, augmented by the observation that most species-level diversity originated after episodes of major Andean uplift in the Neogene period, suggest that dispersal and differentiation on a matrix previously shaped by large-scale landscape events was a major driver of avian speciation in lowland Neotropical rainforests.

Ecology Predicts Levels of Genetic Differentiation in Neotropical Birds

Despite the theoretical link between the ecology and the population genetics of species, little empirical evidence is available that corroborates the association. Here, we examined genetic variation in 40 codistributed species of lowland Neotropical rain forest birds that have populations isolated on either side of the Andes, the Amazon River, and the Madeira River. We found widely varying levels of genetic divergence among these taxa across the same biogeographic barriers. Our investigation of the extent to which ecological traits predicted the amount of cross‐barrier divergence revealed a strongly significant relationship between the forest stratum at which a species forages and the level of cross‐barrier genetic differentiation. Canopy species had statistically lower genetic divergence values across the Andes and the two Amazonian rivers than did understory birds. We hypothesize that the association reflects an effect of dispersal propensity, which is greater in canopy birds, on the movement of alleles among demes (i.e., migration) and, consequently, on the interdemic proportion of the genetic variance. Differences in dispersal propensity may also explain the observation that understory species contain a significantly greater number of subspecies than do canopy species. This result indicates that higher rates of diversification may occur in lineages with lower dispersal propensity.

LINEAGE DIVERSIFICATION AND MORPHOLOGICAL EVOLUTION IN A LARGE-SCALE CONTINENTAL RADIATION: THE NEOTROPICAL OVENBIRDS AND WOODCREEPERS (AVES: FURNARIIDAE)

Patterns of diversification in species‐rich clades provide insight into the processes that generate biological diversity. We tested different models of lineage and phenotypic diversification in an exceptional continental radiation, the ovenbird family Furnariidae, using the most complete species‐level phylogenetic hypothesis produced to date for a major avian clade (97% of 293 species). We found that the Furnariidae exhibit nearly constant rates of lineage accumulation but show evidence of constrained morphological evolution. This pattern of sustained high rates of speciation despite limitations on phenotypic evolution contrasts with the results of most previous studies of evolutionary radiations, which have found a pattern of decelerating diversity‐dependent lineage accumulation coupled with decelerating or constrained phenotypic evolution. Our results suggest that lineage accumulation in tropical continental radiations may not be as limited by ecological opportunities as in temperate or island radiations. More studies examining patterns of both lineage and phenotypic diversification are needed to understand the often complex tempo and mode of evolutionary radiations on continents.