Extreme elevational migration spurred cryptic speciation in giant hummingbirds.

The ecoevolutionary drivers of species niche expansion or contraction are critical for biodiversity but challenging to infer. Niche expansion may be promoted by local adaptation or constrained by physiological performance trade-offs. For birds, evolutionary shifts in migratory behavior permit the broadening of the climatic niche by expansion into varied, seasonal environments. Broader niches can be short-lived if diversifying selection and geography promote speciation and niche subdivision across climatic gradients. To illuminate niche breadth dynamics, we can ask how "outlier" species defy constraints. Of the 363 hummingbird species, the giant hummingbird (Patagona gigas) has the broadest climatic niche by a large margin. To test the roles of migratory behavior, performance trade-offs, and genetic structure in maintaining its exceptional niche breadth, we studied its movements, respiratory traits, and population genomics. Satellite and light-level geolocator tracks revealed an >8,300-km loop migration over the Central Andean Plateau. This migration included a 3-wk, ~4,100-m ascent punctuated by upward bursts and pauses, resembling the acclimatization routines of human mountain climbers, and accompanied by surging blood-hemoglobin concentrations. Extreme migration was accompanied by deep genomic divergence from high-elevation resident populations, with decisive postzygotic barriers to gene flow. The two forms occur side-by-side but differ almost imperceptibly in size, plumage, and respiratory traits. The high-elevation resident taxon is the world's largest hummingbird, a previously undiscovered species that we describe and name here. The giant hummingbirds demonstrate evolutionary limits on niche breadth: when the ancestral niche expanded due to evolution (or loss) of an extreme migratory behavior, speciation followed.

Forebrain nuclei linked to woodpecker territorial drum displays mirror those that enable vocal learning in songbirds.

Vocal learning is thought to have evolved in 3 orders of birds (songbirds, parrots, and hummingbirds), with each showing similar brain regions that have comparable gene expression specializations relative to the surrounding forebrain motor circuitry. Here, we searched for signatures of these same gene expression specializations in previously uncharacterized brains of 7 assumed vocal non-learning bird lineages across the early branches of the avian family tree. Our findings using a conserved marker for the song system found little evidence of specializations in these taxa, except for woodpeckers. Instead, woodpeckers possessed forebrain regions that were anatomically similar to the pallial song nuclei of vocal learning birds. Field studies of free-living downy woodpeckers revealed that these brain nuclei showed increased expression of immediate early genes (IEGs) when males produce their iconic drum displays, the elaborate bill-hammering behavior that individuals use to compete for territories, much like birdsong. However, these specialized areas did not show increased IEG expression with vocalization or flight. We further confirmed that other woodpecker species contain these brain nuclei, suggesting that these brain regions are a common feature of the woodpecker brain. We therefore hypothesize that ancient forebrain nuclei for refined motor control may have given rise to not only the song control systems of vocal learning birds, but also the drumming system of woodpeckers.

Extraordinary levels of per- and polyfluoroalkyl substances (PFAS) in vertebrate animals at a New Mexico desert oasis: Multiple pathways for wildlife and human exposure.

Per- and polyfluoroalkyl substances (PFAS) in the environment pose persistent and complex threats to human and wildlife health. Around the world, PFAS point sources such as military bases expose thousands of populations of wildlife and game species, with potentially far-reaching implications for population and ecosystem health. But few studies shed light on the extent to which PFAS permeate food webs, particularly ecologically and taxonomically diverse communities of primary and secondary consumers. Here we conducted >2000 assays to measure tissue-concentrations of 17 PFAS in 23 species of mammals and migratory birds at Holloman Air Force Base (AFB), New Mexico, USA, where wastewater catchment lakes form biodiverse oases. PFAS concentrations were among the highest reported in animal tissues, and high levels have persisted for at least three decades. Twenty of 23 species sampled at Holloman AFB were heavily contaminated, representing middle trophic levels and wetland to desert microhabitats, implicating pathways for PFAS uptake: ingestion of surface water, sediments, and soil; foraging on aquatic invertebrates and plants; and preying upon birds or mammals. The hazardous long carbon-chain form, perfluorooctanosulfonic acid (PFOS), was most abundant, with liver concentrations averaging >10,000 ng/g wet weight (ww) in birds and mammals, respectively, and reaching as high 97,000 ng/g ww in a 1994 specimen. Perfluorohexanesulfonic acid (PFHxS) averaged thousands of ng/g ww in the livers of aquatic birds and littoral-zone house mice, but one order of magnitude lower in the livers of upland desert rodent species. Piscivores and upland desert songbirds were relatively uncontaminated. At control sites, PFAS levels were strikingly lower on average and different in composition. In sum, legacy PFAS at this desert oasis have permeated local aquatic and terrestrial food webs across decades, severely contaminating populations of resident and migrant animals, and exposing people via game meat consumption and outdoor recreation.

Contrasting drivers of diversity in hosts and parasites across the tropical Andes.

Geographic turnover in community composition is created and maintained by eco-evolutionary forces that limit the ranges of species. One such force may be antagonistic interactions among hosts and parasites, but its general importance is unknown. Understanding the processes that underpin turnover requires distinguishing the contributions of key abiotic and biotic drivers over a range of spatial and temporal scales. Here, we address these challenges using flexible, nonlinear models to identify the factors that underlie richness (alpha diversity) and turnover (beta diversity) patterns of interacting host and parasite communities in a global biodiversity hot spot. We sampled 18 communities in the Peruvian Andes, encompassing ∼1,350 bird species and ∼400 hemosporidian parasite lineages, and spanning broad ranges of elevation, climate, primary productivity, and species richness. Turnover in both parasite and host communities was most strongly predicted by variation in precipitation, but secondary predictors differed between parasites and hosts, and between contemporary and phylogenetic timescales. Host communities shaped parasite diversity patterns, but there was little evidence for reciprocal effects. The results for parasite communities contradicted the prevailing view that biotic interactions filter communities at local scales while environmental filtering and dispersal barriers shape regional communities. Rather, subtle differences in precipitation had strong, fine-scale effects on parasite turnover while host-community effects only manifested at broad scales. We used these models to map bird and parasite turnover onto the ecological gradients of the Andean landscape, illustrating beta-diversity hot spots and their mechanistic underpinnings.

Hummingbird blood traits track oxygen availability across space and time.

Predictable trait variation across environments suggests shared adaptive responses via repeated genetic evolution, phenotypic plasticity or both. Matching of trait-environment associations at phylogenetic and individual scales implies consistency between these processes. Alternatively, mismatch implies that evolutionary divergence has changed the rules of trait-environment covariation. Here we tested whether species adaptation alters elevational variation in blood traits. We measured blood for 1217 Andean hummingbirds of 77 species across a 4600-m elevational gradient. Unexpectedly, elevational variation in haemoglobin concentration ([Hb]) was scale independent, suggesting that physics of gas exchange, rather than species differences, determines responses to changing oxygen pressure. However, mechanisms of [Hb] adjustment did show signals of species adaptation: Species at either low or high elevations adjusted cell size, whereas species at mid-elevations adjusted cell number. This elevational variation in red blood cell number versus size suggests that genetic adaptation to high altitude has changed how these traits respond to shifts in oxygen availability.

Blood Variation Implicates Respiratory Limits on Elevational Ranges of Andean Birds.

AbstractThe extent to which species ranges reflect intrinsic physiological tolerances is a major question in evolutionary ecology. To date, consensus has been hindered by the limited tractability of experimental approaches across most of the tree of life. Here, we apply a macrophysiological approach to understand how hematological traits related to oxygen transport shape elevational ranges in a tropical biodiversity hot spot. Along Andean elevational gradients, we measured traits that affect blood oxygen-carrying capacity-total and cellular hemoglobin concentration and hematocrit, the volume percentage of red blood cells-for 2,355 individuals of 136 bird species. We used these data to evaluate the influence of hematological traits on elevational ranges. First, we asked whether the sensitivity of hematological traits to changes in elevation is predictive of elevational range breadth. Second, we asked whether variance in hematological traits changed as a function of distance to the nearest elevational range limit. We found that birds showing greater hematological sensitivity had broader elevational ranges, consistent with the idea that a greater acclimatization capacity facilitates elevational range expansion. We further found reduced variation in hematological traits in birds sampled near their elevational range limits and at high absolute elevations, patterns consistent with intensified natural selection, reduced effective population size, or compensatory changes in other cardiorespiratory traits. Our findings suggest that constraints on hematological sensitivity and local genetic adaptation to oxygen availability promote the evolution of the narrow elevational ranges that underpin tropical montane biodiversity.

Comparing divergence landscapes from reduced-representation and whole genome resequencing in the yellow-rumped warbler (Setophaga coronata) species complex.

Researchers seeking to generate genomic data for non-model organisms are faced with a number of trade-offs when deciding which method to use. The selection of reduced representation approaches versus whole genome resequencing will ultimately affect the marker density, sequencing depth, and the number of individuals that can multiplexed. These factors can affect researchers' ability to accurately characterize certain genomic features, such as landscapes of divergence-how FST varies across the genomes. To provide insight into the effect of sequencing method on the estimation of divergence landscapes, we applied an identical bioinformatic pipeline to three generations of sequencing data (GBS, ddRAD, and WGS) produced for the same system, the yellow-rumped warbler species complex. We compare divergence landscapes generated using each method for the myrtle warbler (Setophaga coronata coronata) and the Audubon's warbler (S. c. auduboni), and for Audubon's warblers with deeply divergent mtDNA resulting from mitochondrial introgression. We found that most high-FST peaks were not detected in the ddRAD data set, and that while both GBS and WGS were able to identify the presence of large peaks, WGS was superior at a finer scale. Comparing Audubon's warblers with divergent mitochondrial haplotypes, only WGS allowed us to identify small (10-20 kb) regions of elevated differentiation, one of which contained the nuclear-encoded mitochondrial gene NDUFAF3. We calculated the cost per base pair for each method and found it was comparable between GBS and WGS, but significantly higher for ddRAD. These comparisons highlight the advantages of WGS over reduced representation methods when characterizing landscapes of divergence.

Detecting turnover among complex communities using null models: a case study with sky-island haemosporidian parasites.

Turnover in species composition between sites, or beta diversity, is a critical component of species diversity that is typically influenced by geography, environment, and biotic interactions. Quantifying turnover is particularly challenging, however, in multi-host, multi-parasite assemblages where undersampling is unavoidable, resulting in inflated estimates of turnover and uncertainty about its spatial scale. We developed and implemented a framework using null models to test for community turnover in avian haemosporidian communities of three sky islands in the southwestern United States. We screened 776 birds for haemosporidian parasites from three genera (Parahaemoproteus, Plasmodium, and Leucocytozoon) by amplifying and sequencing a mitochondrial DNA barcode. We detected infections in 280 birds (36.1%), sequenced 357 infections, and found a total of 99 parasite haplotypes. When compared to communities simulated from a regional pool, we observed more unique, single-mountain haplotypes and fewer haplotypes shared among three mountain ranges than expected, indicating that haemosporidian communities differ to some degree among adjacent mountain ranges. These results were robust even after pruning datasets to include only identical sets of host species, and they were consistent for two of the three haemosporidian genera. The two more distant mountain ranges were more similar to each other than the one located centrally, suggesting that the differences we detected were due to stochastic colonization-extirpation dynamics. These results demonstrate that avian haemosporidian communities of temperate-zone forests differ on relatively fine spatial scales between adjacent sky islands. Null models are essential tools for testing the spatial scale of turnover in complex, undersampled, and poorly known systems.

Pervasive Genomic Signatures of Local Adaptation to Altitude Across Highland Specialist Andean Hummingbird Populations.

Populations along steep environmental gradients are subject to differentiating selection that can result in local adaptation, despite countervailing gene flow, and genetic drift. In montane systems, where species are often restricted to narrow ranges of elevation, it is unclear whether the selection is strong enough to influence functional differentiation of subpopulations differing by a few hundred meters in elevation. We used targeted capture of 12 501 exons from across the genome, including 271 genes previously implicated in altitude adaptation, to test for adaptation to local elevations for 2 highland hummingbird species, Coeligena violifer (n = 62) and Colibri coruscans (n = 101). For each species, we described population genetic structure across the complex geography of the Peruvian Andes and, while accounting for this structure, we tested whether elevational allele frequency clines in single nucleotide polymorphisms (SNPs) showed evidence for local adaptation to elevation. Although the 2 species exhibited contrasting population genetic structures, we found signatures of clinal genetic variation with shifts in elevation in both. The genes with SNP-elevation associations included candidate genes previously discovered for high-elevation adaptation as well as others not previously identified, with cellular functions related to hypoxia response, energy metabolism, and immune function, among others. Despite the homogenizing effects of gene flow and genetic drift, natural selection on parts of the genome evidently optimizes elevation-specific cellular function even within elevation range-restricted montane populations. Consequently, our results suggest local adaptation occurring in narrow elevation bands in tropical mountains, such as the Andes, may effectively make them "taller" biogeographic barriers.

Evolution between forest macrorefugia is linked to discordance between genetic and morphological variation in Neotropical passerines.

The central Andean rainforests and the Atlantic Forest are two similar biomes that are fully isolated by xerophytic and open-vegetation regions (the Chaco and Cerrado, respectively). Even though there is evidence suggesting that these rainforests have been connected in the past, their dynamics of connection, the geographic areas that bridged these regions, and the biological processes that have promoted diversification between them remain to be studied. In this research, we used three passerine species (Poecilotriccus plumbeiceps, Phylloscartes ventralis and Cacicus chrysopterus) as models to address whether the Andean and the Atlantic forests have acted as a refugia system (macrorefugia), and to evaluate biogeographic hypotheses of diversification and connection between them. In order to achieve these goals, we performed traditional phylogeographic analyses and compared alternative biogeographic scenarios by using Approximate Bayesian Computation. Additionally, we performed morphological analyses to evaluate phenotypic divergence between these regions. Our findings support that both rainforest regions acted as refugia, but that the impact of their isolation was stronger on the genetic than on the morphologic characters. Our results provided evidence that both geographic isolation as well as ecological factors have modeled the external traits of forest organisms in the region. Regarding the connection routes between the Andes and the Atlantic Forest, the genetic data rejected the hypothesis of a Chaco connection in the tested species, providing evidence for a connection through the Cerrado or through the transition between the Cerrado and Chaco, in a process that could have started as early as the Late Miocene.

Extreme and variable torpor among high-elevation Andean hummingbird species.

Torpor is thought to be particularly important for small endotherms occupying cold environments and with limited fat reserves to fuel metabolism, yet among birds deep torpor is both rare and variable in extent. We investigated torpor in hummingbirds at approximately 3800 m.a.s.l. in the tropical Andes by monitoring body temperature (Tb) in 26 individuals of six species held captive overnight and experiencing natural air temperature (Ta) patterns. All species used pronounced torpor, with one Metallura phoebe reaching a minimum Tb of 3.26°C, the lowest yet reported for any bird or non-hibernating mammal. The extent and duration of torpor varied among species, with overnight body mass (Mb) loss negatively correlated with both minimum Tb and bout duration. We found a significant phylogenetic signal for minimum Tb and overnight Mb loss, consistent with evolutionarily conserved thermoregulatory traits. Our findings suggest deep torpor is routine for high Andean hummingbirds, but evolved species differences affect its depth.

High-altitude adaptations mitigate risk for hypertension and diabetes-associated anemia.

BACKGROUND: Human populations native to high altitude exhibit numerous genetic adaptations to hypobaric hypoxia. Among Tibetan plateau peoples, these include increased vasodilation and uncoupling of erythropoiesis from hypoxia. OBJECTIVE/METHODS: We tested the hypothesis that these high-altitude adaptations reduce risk for hypertension and diabetes-associated anemia among the Mosuo, a Tibetan-descended population in the mountains of Southwest China that is experiencing rapid economic change and increased chronic disease risk. RESULTS: Hypertension was substantially less common among Mosuo than low-altitude Han populations, and models fit to the Han predicted higher probability of hypertension than models fit to the Mosuo. Diabetes was positively associated with anemia among the Han, but not the Mosuo. CONCLUSION: The Mosuo have lower risk for hypertension and diabetes-associated anemia than the Han, supporting the hypothesis that high-altitude adaptations affecting blood and circulation intersect with chronic disease processes to lower risk for these outcomes. As chronic diseases continue to grow as global health concerns, it is important to investigate how they may be affected by local genetic adaptations.

Seasonal and elevational variation in glucose and glycogen in two songbird species.

Birds naturally maintain high glucose concentrations in the blood and tissues, even when relying on fat to meet the metabolic demands of flight or thermogenesis. One possibility is that high glucose levels might be needed to deal with these metabolic demands. Thus, we hypothesized that birds chronically exposed to colder temperatures and higher elevations have higher circulating glucose and tissue free glucose and glycogen compared to conspecifics living at warmer temperatures and lower elevations. Adult House Sparrows (Passer domesticus) and House Finches (Haemorhous mexicanus) were captured from Phoenix, AZ (340 m elevation), and Albuquerque, NM (1600 m elevation), during the summer and winter months. We measured plasma glucose, as well as free glucose and glycogen from multiple tissues. In general, high elevation and colder temperatures were associated with higher tissue glycogen and higher free glucose concentrations in the brain. These findings indicate that glucose and glycogen are subject to seasonal phenotypic flexibility as well as geographic variations that may relate to local food availability and abundance.

Deeply conserved susceptibility in a multi-host, multi-parasite system.

Variation in susceptibility is ubiquitous in multi-host, multi-parasite assemblages, and can have profound implications for ecology and evolution in these systems. The extent to which susceptibility to parasites is phylogenetically conserved among hosts can be revealed by analysing diverse regional communities. We screened for haemosporidian parasites in 3983 birds representing 40 families and 523 species, spanning ~ 4500 m elevation in the tropical Andes. To quantify the influence of host phylogeny on infection status, we applied Bayesian phylogenetic multilevel models that included a suite of environmental, spatial, temporal, life history and ecological predictors. We found evidence of deeply conserved susceptibility across the avian tree; host phylogeny explained substantial variation in infection status, and results were robust to phylogenetic uncertainty. Our study suggests that susceptibility is governed, in part, by conserved, latent aspects of anti-parasite defence. This demonstrates the importance of deep phylogeny for understanding present-day ecological interactions.

Predictable evolution toward flightlessness in volant island birds.

Birds are prolific colonists of islands, where they readily evolve distinct forms. Identifying predictable, directional patterns of evolutionary change in island birds, however, has proved challenging. The "island rule" predicts that island species evolve toward intermediate sizes, but its general applicability to birds is questionable. However, convergent evolution has clearly occurred in the island bird lineages that have undergone transitions to secondary flightlessness, a process involving drastic reduction of the flight muscles and enlargement of the hindlimbs. Here, we investigated whether volant island bird populations tend to change shape in a way that converges subtly on the flightless form. We found that island bird species have evolved smaller flight muscles than their continental relatives. Furthermore, in 366 populations of Caribbean and Pacific birds, smaller flight muscles and longer legs evolved in response to increasing insularity and, strikingly, the scarcity of avian and mammalian predators. On smaller islands with fewer predators, birds exhibited shifts in investment from forelimbs to hindlimbs that were qualitatively similar to anatomical rearrangements observed in flightless birds. These findings suggest that island bird populations tend to evolve on a trajectory toward flightlessness, even if most remain volant. This pattern was consistent across nine families and four orders that vary in lifestyle, foraging behavior, flight style, and body size. These predictable shifts in avian morphology may reduce the physical capacity for escape via flight and diminish the potential for small-island taxa to diversify via dispersal.

Stability-Mediated Epistasis Restricts Accessible Mutational Pathways in the Functional Evolution of Avian Hemoglobin.

If the fitness effects of amino acid mutations are conditional on genetic background, then mutations can have different effects depending on the sequential order in which they occur during evolutionary transitions in protein function. A key question concerns the fraction of possible mutational pathways connecting alternative functional states that involve transient reductions in fitness. Here we examine the functional effects of multiple amino acid substitutions that contributed to an evolutionary transition in the oxygenation properties of avian hemoglobin (Hb). The set of causative changes included mutations at intradimer interfaces of the Hb tetramer. Replacements at such sites may be especially likely to have epistatic effects on Hb function since residues at intersubunit interfaces are enmeshed in networks of salt bridges and hydrogen bonds between like and unlike subunits; mutational reconfigurations of these atomic contacts can affect allosteric transitions in quaternary structure and the propensity for tetramer-dimer dissociation. We used ancestral protein resurrection in conjunction with a combinatorial protein engineering approach to synthesize genotypes representing the complete set of mutational intermediates in all possible forward pathways that connect functionally distinct ancestral and descendent genotypes. The experiments revealed that 1/2 of all possible forward pathways included mutational intermediates with aberrant functional properties because particular combinations of mutations promoted tetramer-dimer dissociation. The subset of mutational pathways with unstable intermediates may be selectively inaccessible, representing evolutionary roads not taken. The experimental results also demonstrate how epistasis for particular functional properties of proteins may be mediated indirectly by mutational effects on quaternary structural stability.

Detecting introgression despite phylogenetic uncertainty: The case of the South American siskins.

Genetic introgression among closely related species is a widespread phenomenon across the Tree of Life and could be an important source of adaptive variation during early stages of diversification. In particular, genomic studies have revealed that many rapidly radiating clades tend to have complex, reticulate evolutionary histories. Although rapid radiations appear to be susceptible to introgression, they present special challenges for its detection because formal tests require accurate phylogenies, and paradoxically, introgression itself may obscure evolutionary relationships. To address this methodological challenge, we assessed introgression in a recent, rapid avian radiation in the Andes, the South American siskins (Spinus). Using ~45,000 SNPs, we estimated the Spinus phylogeny using multiple analytical approaches and recovered four strongly conflicting topologies. We performed a series of complimentary introgression tests that included valid tests for each of the likely species trees. From the consilience of test results, we inferred multiple introgression events among Andean Spinus in a way that was robust to phylogenetic uncertainty in the species tree. Positive tests for introgression were corroborated by independent population structure and ancestral assignment analyses, as well as a striking geographic pattern of mitochondrial haplotype sharing among species. The methodological approach we describe could be applied using any genomewide data, including SNP data, for clades without fully resolvable species trees. Our discovery of multiple introgression events within the Andean radiation of Spinus siskins is consistent with an emerging paradigm, that introgression tends to accompany the early stages of diversification.

Forest corridors between the central Andes and the southern Atlantic Forest enabled dispersal and peripatric diversification without niche divergence in a passerine.

The central Andean rainforests and the Atlantic Forest are separated by the Chaco and the Cerrado domains. Despite this isolation, diverse evidence suggests that these rainforests have been connected in the past. However, little is known about the timing and geographic positions of these connections, as well as their effects on diversification of species. In this study, we used the Black-goggled Tanager (Trichothraupis melanops, Thraupidae) as a model to study whether the Andean and the Atlantic forests have acted as a refugia system, and to evaluate biogeographic hypotheses of diversification and connection between these rainforests. We compared alternative biogeographic scenarios by using Approximate Bayesian Computation (ABC), modeled range shifts across time, and assessed niche divergence between regions. The results indicated that the major phylogeographic gap within T. melanops is located between these rainforests. The ABC analysis supported peripatric diversification, with initial dispersal from the Atlantic Forest to the Andes during the Mid-Pleistocene. Also, the results supported an Andean-Atlantic forests connection through the current Cerrado-Chaco transition, linking the southern Atlantic Forest with the central Andes. Our findings, taken together with other studies, support that the connection between these biomes has been recurrent, and that has occurred mostly through the Cerrado and/or the Cerrado-Chaco transition. The data also support that the connection dynamic has played an important role in the biological diversification, by promoting peripatric divergence in some forest taxa restricted to both biomes.

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.].

Why are tropical mountain passes "low" for some species? Genetic and stable-isotope tests for differentiation, migration and expansion in elevational generalist songbirds.

Most tropical bird species have narrow elevational ranges, likely reflecting climatic specialization. This is consistent with Janzen's Rule, the tendency for mountain passes to be effectively "higher" in the tropics. Hence, those few tropical species that occur across broad elevational gradients (elevational generalists) represent a contradiction to Janzen's Rule. Here, we aim to address the following questions. Are elevational generalists being sundered by diversifying selection along the gradient? Does elevational movement cause these species to resist diversification or specialization? Have they recently expanded, suggesting that elevational generalism is short-lived in geological time? To answer these questions, we tested for differentiation, movement and expansion in four elevational generalist songbird species on the Andean west slope. We used morphology and mtDNA to test for genetic differentiation between high- and low-elevation populations. To test for elevational movements, we measured hydrogen isotope (δ2 H) values of metabolically inert feathers and metabolically active liver. Morphology differed for House Wren (Troglodytes aedon) and Hooded Siskin (Spinus magellanicus), but not for Cinereous Conebill (Conirostrum cinereum) and Rufous-collared Sparrow (Zonotrichia capensis) respectively. mtDNA was structured by elevation only in Z. capensis. δ2 H data indicated elevational movements by two tree- and shrub-foraging species with moderate-to-high vagility (C. cinereum and S. magellanicus), and sedentary behaviour by two terrestrial-foraging species with low-to-moderate vagility (T. aedon and Z. capensis). In S. magellanicus, elevational movements and lack of mtDNA structure contrast with striking morphological divergence, suggesting strong diversifying selection on body proportions across the c. 50 km gradient. All species except C. cinereum exhibited mtDNA haplotype variation consistent with recent population expansion across the elevational gradient, potentially concurrent with Holocene anthropogenic habitat conversion for agriculture. In different ways, each species defies the tendency for tropical birds to have long-term stable distributions and sedentary habits. We conclude that tropical elevational generalism is rare due to evolutionary instability.