Complexity of avian evolution revealed by family-level genomes.

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

Gene expression plasticity followed by genetic change during colonization in a high-elevation environment.

Phenotypic plasticity facilitates organismal invasion of novel environments, and the resultant phenotypic change may later be modified by genetic change, so called 'plasticity first.' Herein, we quantify gene expression plasticity and regulatory adaptation in a wild bird (Eurasian Tree Sparrow) from its original lowland (ancestral stage), experimentally implemented hypoxia acclimation (plastic stage), and colonized highland (colonized stage). Using a group of co-expressed genes from the cardiac and flight muscles, respectively, we demonstrate that gene expression plasticity to hypoxia tolerance is more often reversed than reinforced at the colonized stage. By correlating gene expression change with muscle phenotypes, we show that colonized tree sparrows reduce maladaptive plasticity that largely associated with decreased hypoxia tolerance. Conversely, adaptive plasticity that is congruent with increased hypoxia tolerance is often reinforced in the colonized tree sparrows. Genes displaying large levels of reinforcement or reversion plasticity (i.e. 200% of original level) show greater genetic divergence between ancestral and colonized populations. Overall, our work demonstrates that gene expression plasticity at the initial stage of high-elevation colonization can be reversed or reinforced through selection-driven adaptive modification.

Metabolic and microbial changes in light-vented bulbul during recent northward range expansion.

Endotherms recently expanding to cold environments generally exhibit strong physiological acclimation to sustain high body temperature. During this process, gut microbes likely play a considerable role in host physiological functions, including digestion and thermogenesis. The light-vented bulbul Pycnonotus sinensis represents one such species. It used to be restricted to the Oriental realm but expanded its distribution range north to the Palearctic areas during the past few decades. Here, we explored the seasonal dynamics of the resting metabolic rate (RMR) and microbiota for local and newly colonized populations of the species. Our results showed that the mass-adjusted RMR and body mass were positively correlated with latitude variations in both seasons. Consistently, the gut microbiota showed a corresponding variation to the northern cold environments. In the two northern populations, the alpha diversity decreased compared with those of the two southern populations. Significant differences were detected in dominant phyla, such as Firmicutes, Bacteroidetes, Proteobacteria, and Desulfobacterota in both seasons. The core microbiota showed geographic differences in the winter, including the elevated relative abundance of 5 species in northern populations. Finally, to explore the link between microbial communities and host metabolic thermogenesis, we conducted a correlation analysis between microbiota and mass-adjusted RMR. We found that more genera were significantly correlated with mass-adjusted RMR in the wintering season compared to the breeding season (71 vs. 23). These results suggest that microbiota of the lighted-vented bulbul linked with thermogenesis in diversity and abundance under northward expansion.

Gene flow and an anomaly zone complicate phylogenomic inference in a rapidly radiated avian family (Prunellidae).

BACKGROUND: Resolving the phylogeny of rapidly radiating lineages presents a challenge when building the Tree of Life. An Old World avian family Prunellidae (Accentors) comprises twelve species that rapidly diversified at the Pliocene-Pleistocene boundary. RESULTS: Here we investigate the phylogenetic relationships of all species of Prunellidae using a chromosome-level de novo assembly of Prunella strophiata and 36 high-coverage resequenced genomes. We use homologous alignments of thousands of exonic and intronic loci to build the coalescent and concatenated phylogenies and recover four different species trees. Topology tests show a large degree of gene tree-species tree discordance but only 40-54% of intronic gene trees and 36-75% of exonic genic trees can be explained by incomplete lineage sorting and gene tree estimation errors. Estimated branch lengths for three successive internal branches in the inferred species trees suggest the existence of an empirical anomaly zone. The most common topology recovered for species in this anomaly zone was not similar to any coalescent or concatenated inference phylogenies, suggesting presence of anomalous gene trees. However, this interpretation is complicated by the presence of gene flow because extensive introgression was detected among these species. When exploring tree topology distributions, introgression, and regional variation in recombination rate, we find that many autosomal regions contain signatures of introgression and thus may mislead phylogenetic inference. Conversely, the phylogenetic signal is concentrated to regions with low-recombination rate, such as the Z chromosome, which are also more resistant to interspecific introgression. CONCLUSIONS: Collectively, our results suggest that phylogenomic inference should consider the underlying genomic architecture to maximize the consistency of phylogenomic signal.

Landscape Heterogeneity Explains the Genetic Differentiation of a Forest Bird across the Sino-Himalayan Mountains.

Mountains are the world's most important centers of biodiversity. The Sino-Himalayan Mountains are global biodiversity hotspot due to their extremely high species richness and endemicity. Ample research investigated the impact of the Qinghai-Tibet Plateau uplift and Quaternary glaciations in driving species diversification in plants and animals across the Sino-Himalayan Mountains. However, little is known about the role of landscape heterogeneity and other environmental features in driving diversification in this region. We utilized whole genomes and phenotypic data in combination with landscape genetic approaches to investigate population structure, demography, and genetic diversity in a forest songbird species native to the Sino-Himalayan Mountains, the red-billed leiothrix (Leiothrix lutea). We identified 5 phylogeographic clades, including 1 in the East of China, 1 in Yunnan, and 3 in Tibet, roughly consistent with differences in song and plumage coloration but incongruent with traditional subspecies boundaries. Isolation-by-resistance model best explained population differentiation within L. lutea, with extensive secondary contact after allopatric isolation leading to admixture among clades. Ecological niche modeling indicated relative stability in the extent of suitable distribution areas of the species across Quaternary glacial cycles. Our results underscore the importance of mountains in the diversification of this species, given that most of the distinct genetic clades are concentrated in a relatively small area in the Sino-Himalayan Mountain region, while a single shallow clade populates vast lower-lying areas to the east. This study highlights the crucial role of landscape heterogeneity in promoting differentiation and provides a deep genomic perspective on the mechanisms through which diversity hotspots form.

When colors mislead: Genomics and bioacoustics prompt re-classification of Asian flycatcher radiation (Aves: Niltavinae).

Traditional classification of many animals, including birds, has been highly dependent on external morphological characters like plumage coloration. However, both bioacoustics and genetic or genomic data have revolutionized our understanding of the relationships of certain lineages and led to sweeping taxonomic re-organizations. In this study, we present a case of erroneous delimitation of genus boundaries in the species-rich flycatcher subfamily Niltavinae. Genera within this subfamily have historically been delineated based on blue versus brown male body plumage until recent studies based on a few mitochondrial and nuclear loci unearthed several cases of generic misclassification. Here we use extensive bioacoustic data from 43 species and genomic data from 28 species for a fundamental reclassification of species in the Niltavinae. Our study reveals that song is an important trait to classify these birds even at the genus level, whereas plumage traits exhibit ample convergence and have led to numerous historic misattributions. Our taxonomic re-organization leads to new biogeographic limits of major genera, such that the genus Cyornis now only extends as far east as the islands of Sulawesi, Sula, and Banggai, whereas Eumyias is redefined to extend far beyond Wallace's Line to the islands of Seram and Timor. Our conclusions advise against an over-reliance on morphological traits and underscore the importance of integrative datasets.

Phylogenetic conflict between species tree and maternally inherited gene trees in a clade of Emberiza buntings (Aves: Emberizidae).

Different genomic regions may reflect conflicting phylogenetic topologies primarily due to incomplete lineage sorting and/or gene flow. Genomic data are necessary to reconstruct the true species tree and explore potential causes of phylogenetic conflict. Here, we investigate the phylogenetic relationships of four Emberiza species (Aves: Emberizidae) and discuss the potential causes of the observed mitochondrial non-monophyly of Emberiza godlewskii (Godlewski's bunting) using phylogenomic analyses based on whole genome resequencing data from 41 birds. Analyses based on both the whole mitochondrial genome and ~39 kilobases from the non-recombining W chromosome reveal sister relationships between each the northern and southern populations of E. godlewskii with E. cioides and E. cia, respectively. In contrast, the monophyly of E. godlewskii is reflected by the phylogenetic signal of autosomal and Z chromosomal sequence data as well as demographic inference analyses, which - in combination - support the following tree topology: (((E. godlewskii, E. cia), E. cioides), E. jankowskii). Using D-statistics, we detected multiple gene flow events among different lineages, indicating pervasive introgressive hybridization within this clade. Introgression from an unsampled lineage that is sister to E. cioides or introgression from an unsampled mitochondrial + W chromosomal lineage of E. cioides into northern E. godlewskii may explain the phylogenetic conflict between the species tree estimated from genome-wide data versus mtDNA/W tree topologies. These results underscore the importance of using genomic data for phylogenetic reconstruction and species delimitation.

Introgression underlies phylogenetic uncertainty but not parallel plumage evolution in a recent songbird radiation.

Instances of parallel phenotypic evolution offer great opportunities to understand the evolutionary processes underlying phenotypic changes. However, confirming parallel phenotypic evolution and studying its causes requires a robust phylogenetic framework. One such example is the "black-and-white wagtails", a group of five species in the songbird genus Motacilla: one species, Motacilla alba, shows wide intra-specific plumage variation, while the four others form two pairs of very similar-looking species (M. aguimp + M. samveasnae and M. grandis + M. maderaspatensis, respectively). However, the two species in each of these pairs were not recovered as sisters in previous phylogenetic inferences. Their relationships varied depending on the markers used, suggesting that gene tree heterogeneity might have hampered accurate phylogenetic inference. Here, we use whole genome resequencing data to explore the phylogenetic relationships within this group, with a special emphasis on characterizing the extent of gene tree heterogeneity and its underlying causes. We first used multispecies coalescent methods to generate a "complete evidence" phylogenetic hypothesis based on genome-wide variants, while accounting for incomplete lineage sorting (ILS) and introgression. We then investigated the variation in phylogenetic signal across the genome, to quantify the extent of discordance across genomic regions, and test its underlying causes. We found that wagtail genomes are mosaics of regions supporting variable genealogies, because of ILS and inter-specific introgression. The most common topology across the genome, supporting M. alba and M. aguimp as sister species, appears to be influenced by ancient introgression. Additionally, we inferred another ancient introgression event, between M. alba and M. grandis. By combining results from multiple analyses, we propose a phylogenetic network for the black-and-white wagtails that confirms that similar phenotypes evolved in non-sister lineages, supporting parallel plumage evolution. Furthermore, the inferred reticulations do not connect species with similar plumage coloration, suggesting that introgression does not underlie parallel plumage evolution in this group. Our results demonstrate the importance of investigation of genome-wide patterns of gene tree heterogeneity to help understanding the mechanisms underlying phenotypic evolution.

Recurrent selection and reduction in recombination shape the genomic landscape of divergence across multiple population pairs of Green-backed Tit.

Speciation is fundamental for building and maintaining biodiversity. The formation of the highly differentiated genomic regions between diverging taxa has been interpreted as a result of divergence with gene flow, linked selection, and reduction in recombination. It is challenging to unravel these nonexclusive processes in shaping genomic divergence. Here, we investigate the relative roles of these processes in shaping genomic differentiation in a montane bird, the Green-backed Tit (Parus monticolus). Our genetic structure and demographic analyses identify that four genetic lineages diverge between 838 and 113 thousand years ago and there is evidence of secondary gene flow. The highly divergent genomic regions do not increase with the divergence time, as we found that the old lineages show relatively fewer numbers and smaller sizes of highly differentiated regions than the young divergent lineages (numbers, 118-138 vs. 156-289; sizes, 5.9-6.9 vs. 7.8-14.5 megabase). Across the genome, the outlier windows show a reduction in nucleotide diversity, absolute genetic divergence, and recombination rate, suggesting recurrent selection in regions with low recombination being the major driver of genomic divergence. Finally, we show that secondary gene flow tends to affect the highly differentiated genomic regions if these regions are less likely to be the targets of selection. Altogether, our study shows how common ancestry, recurrent selection, low recombination rate, and gene flow have contributed to the emergence of genomic islands at different stages of speciation.

Species-specific traits mediate avian demographic responses under past climate change.

Anticipating species' responses to environmental change is a pressing mission in biodiversity conservation. Despite decades of research investigating how climate change may affect population sizes, historical context is lacking, and the traits that mediate demographic sensitivity to changing climate remain elusive. We use whole-genome sequence data to reconstruct the demographic histories of 263 bird species over the past million years and identify networks of interacting morphological and life history traits associated with changes in effective population size (Ne) in response to climate warming and cooling. Our results identify direct and indirect effects of key traits representing dispersal, reproduction and survival on long-term demographic responses to climate change, thereby highlighting traits most likely to influence population responses to ongoing climate warming.

Limited Song Mixing Without Genomic Gene Flow in a Contact Zone Between Two Songbird Species.

Song is considered to play an important role in the maintenance of prezygotic reproductive isolation between closely related songbird species. Therefore, song mixing in a contact zone between closely related species is often considered as evidence of hybridization. The Sichuan Leaf Warbler Phylloscopus forresti and the Gansu Leaf Warbler Phylloscopus kansuensis, which diverged 2 million years ago, have formed a contact zone in the south of the Gansu Province of China, where mixed songs have been observed. In this study, we investigated the potential causes and consequences of song mixing by integrating bioacoustic, morphological, mitochondrial, and genomic data with field ecological observations. We found that the two species display no apparent morphological differences, whereas their songs differ dramatically. We demonstrated that ∼11% of the males in the contact zone sang mixed songs. Two males singing mixed song were genotyped, and both were found to be P. kansuensis. Despite the presence of mixed singers, population genomic analyses detected no signs of recent gene flow between the two species, although two possible cases of mitochondrial introgression were identified. We conclude that the rather limited song mixing does not lead to, or result from, hybridization, and hence does not result in the breakdown of reproductive barriers between these cryptic species.

Divergent contributions of coding and noncoding sequences to initial high-altitude adaptation in passerine birds endemic to the Qinghai-Tibet Plateau.

Early events in the evolution of an ancestral lineage can shape the adaptive patterns of descendant species, but the evolutionary mechanisms driving initial adaptation from an ancestor remain largely unexplored. High-altitude adaptations have been extensively explored from the viewpoint of protein-coding genes; however, the contribution of noncoding regions remains relatively neglected. Here, we integrate genomic and transcriptomic data to investigate adaptive evolution in the ancestor of three high-altitude snowfinch species endemic to the Qinghai-Tibet Plateau. Our genome-wide scan for adaptation in the snowfinch ancestor identifies strong adaptation signals in functions of development and metabolism for the coding genes, but in functions of the nervous system development for noncoding regions. This pattern is exclusive to the snowfinch ancestor compared to a control ancestral lineage subject to weak selection. Changes in noncoding regions in the snowfinch ancestor, especially those nearest to coding genes, may be disproportionately associated with the differential expression of genes in the brain tissue compared to other tissues. Extensive gene expression in the brain tissue can be further altered via genetic regulatory networks of transcription factors harbouring potential accelerated regulatory regions (e.g., the development-related transcription factor YEATS4). Altogether, our study provides new evidence concerning how coding and noncoding sequences work through decoupled pathways in initial adaptation to the selective pressure of high-altitude environments. The analysis highlights the idea that noncoding sequences may be promising elements in facilitating the rapid evolution and adaptation to high altitudes.

Changes in the functional diversity of modern bird species over the last million years.

Despite evidence of declining biosphere integrity, we currently lack understanding of how the functional diversity associated with changes in abundance among ecological communities has varied over time and before widespread human disturbances. We combine morphological, ecological, and life-history trait data for >260 extant bird species with genomic-based estimates of changing effective population size (Ne) to quantify demographic-based shifts in avian functional diversity over the past million years and under pre-anthropogenic climate warming. We show that functional diversity was relatively stable over this period, but underwent significant changes in some key areas of trait space due to changing species abundances. Our results suggest that patterns of population decline over the Pleistocene have been concentrated in particular regions of trait space associated with extreme reproductive strategies and low dispersal ability, consistent with an overall erosion of functional diversity. Further, species most sensitive to climate warming occupied a relatively narrow region of functional space, indicating that the largest potential population increases and decreases under climate change will occur among species with relatively similar trait sets. Overall, our results identify fluctuations in functional space of extant species over evolutionary timescales and represent the demographic-based vulnerability of different regions of functional space among these taxa. The integration of paleodemographic dynamics with functional trait data enhances our ability to quantify losses of biosphere integrity before anthropogenic disturbances and attribute contemporary biodiversity loss to different drivers over time.

Genomic and phenotypic changes associated with alterations of migratory behaviour in a songbird.

The seasonal migration of birds is a fascinating natural wonder. Avian migratory behaviour changes are common and are probably a polygenic process as avian migration is governed by multiple correlated components with a variable genetic basis. However, the genetic and phenotypic changes involving migration changes are poorly studied. Using one annotated near-chromosomal level de novo genome assembly, 50 resequenced genomes, hundreds of morphometric data and species distribution information, we investigated population structure and genomic and phenotypic differences associated with differences in migratory behaviour in a songbird species, Yellow-throated Bunting Emberiza elegans (Aves: Emberizidae). Population genomic analyses reveal extensive gene flow between the southern resident and the northern migratory populations of this species. The hand-wing index is significantly lower in the resident populations than in the migratory populations, indicating reduced flight efficiency of the resident populations. Here, we discuss the possibility that nonmigratory populations may have originated from migratory populations though migration loss. We further infer that the alterations of genes related to energy metabolism, nervous system and circadian rhythm may have played major roles in regulating migration change. Our study sheds light on phenotypic and polygenic changes involving migration change.

A de novo assembled genome of the Tibetan Partridge (Perdix hodgsoniae) and its high-altitude adaptation.

The Tibetan Partridge (Perdix hodgsoniae) is an endemic species distributed in high-altitude areas of 3600-5600 m on the Qinghai-Tibet Plateau. To explore how the species is adapted to the high elevation environment, we assembled a draft genome based on both the Illumina and PacBio sequencing platforms with its population genetics and genomics analysis. In total, 134.74 Gb short reads and 30.81 Gb long reads raw data were generated. The 1.05-Gb assembled genome had a contig N50 of 4.56 Mb, with 91.94% complete BUSCOs. The 17 457 genes were annotated, and 11.35% of the genome was composed of repeat sequences. The phylogenetic tree showed that P. hodgsoniae was located at the basal position of the clade, including Golden Pheasant (Chrysolophus pictus), Common Pheasant (Phasianus colchicus), and Mikado Pheasant (Syrmaticus mikado). We found that 1014, 2595, and 2732 of the 6641 one-to-one orthologous genes were under positive selection in P. hodgsoniae, detected using PAML, BUSTED, and aBSREL programs, respectively, of which 965 genes were common under positive selection with 3 different programs. Several positively selected genes and immunity pathways relevant to high-altitude adaptation were detected. Gene family evolution showed that 99 gene families experienced significant expansion events, while 6 gene families were under contraction. The total number of olfactory receptor genes was relatively low in P. hodgsoniae. Genomic data provide an important resource for a further study on the evolutionary history of P. hodgsoniae, which provides a new insight into its high-altitude adaptation mechanisms.

Genetic mechanism of adaptive evolution: the example of adaptation to high altitudes.

Since Darwin's time, elucidating the mechanism of adaptive evolution has been one of the most important scientific issues in evolutionary biology and ecology. Adaptive evolution usually means that species evolve special phenotypic traits to increase fitness under selective pressures. Phenotypic adaptation can be observed at different hierarchical levels of morphology, physiology, biochemistry, histology, and behavior. With the breakthroughs of molecular biology and next-generation sequencing technologies, mounting evidence has uncovered the genetic architecture driving adaptive complex phenotypes. Studying the molecular genetic mechanisms of evolutionary adaption will enable us to understand the forces shaping biodiversity and set up genotype-phenotype-environment interactions. Genetic bases of adaptive evolution have been explained by multiple hypotheses, including major-effect genes, supergenes, polygenicity, noncoding regions, repeated regions, and introgression. The strong selection pressure exerted by high-altitude extreme environments greatly promotes the occurrence of phenotypic and genetic adaptation in species. Studies on multi-omics data provide new insights into adaptive evolution. In this review, we systematically summarize the genetic mechanism of adaptive evolution, research progress in adaptation to high-altitude environmental conditions, and existing challenges and discuss the future perspectives, thereby providing guidance for researchers in this field.

Eating More and Fighting Less: Social Foraging Is a Potential Advantage for Successful Expansion of Bird Source Populations.

Animals can expand distributions in response to climatic and environmental changes, but the potential expansive ability of a source population is rarely evaluated using designed experiments. Group foraging can increase survival in new environments, but it also increases intraspecific competition. The trade-off between benefit and conflict needs to be determined. The expanding Light-vented Bulbul Pycnonotus sinensis was used as a model to test mechanisms promoting successful expansion. Social foraging and its advantages were evaluated using lab-designed feeding trials. Consuming novel foods was compared between bulbuls and a sympatric, nonexpansive relative species, the finchbill Spizixos semitorques, from native areas at both solitary and social levels. Bulbuls increased their eating times when transferred from solitary to group, whereas social context did not affect finchbills. Bulbuls were significantly more likely to eat with their companions than finchbills when in a group. Thus, exploring food resources in a bulbul source population was facilitated by social context, indicating that social foraging is an important means by which birds successfully expand and respond to environmental changes. This research increases understanding of successful expansion mechanisms and will consequently help predict invasive potentials of alien species.

Orthologous microsatellites, transposable elements, and DNA deletions correlate with generation time and body mass in neoavian birds.

The rate of mutation accumulation in germline cells can be affected by cell replication and/or DNA damage, which are further related to life history traits such as generation time and body mass. Leveraging the existing datasets of 233 neoavian bird species, here, we investigated whether generation time and body mass contribute to the interspecific variation of orthologous microsatellite length, transposable element (TE) length, and deletion length and how these genomic attributes affect genome sizes. In nonpasserines, we found that generation time is correlated to both orthologous microsatellite length and TE length, and body mass is negatively correlated to DNA deletions. These patterns are less pronounced in passerines. In all species, we found that DNA deletions relate to genome size similarly as TE length, suggesting a role of body mass dynamics in genome evolution. Our results indicate that generation time and body mass shape the evolution of genomic attributes in neoavian birds.

The combination of genomic offset and niche modelling provides insights into climate change-driven vulnerability.

Global warming is increasingly exacerbating biodiversity loss. Populations locally adapted to spatially heterogeneous environments may respond differentially to climate change, but this intraspecific variation has only recently been considered when modelling vulnerability under climate change. Here, we incorporate intraspecific variation in genomic offset and ecological niche modelling to estimate climate change-driven vulnerability in two bird species in the Sino-Himalayan Mountains. We found that the cold-tolerant populations show higher genomic offset but risk less challenge for niche suitability decline under future climate than the warm-tolerant populations. Based on a genome-niche index estimated by combining genomic offset and niche suitability change, we identified the populations with the least genome-niche interruption as potential donors for evolutionary rescue, i.e., the populations tolerant to climate change. We evaluated potential rescue routes via a landscape genetic analysis. Overall, we demonstrate that the integration of genomic offset, niche suitability modelling, and landscape connectivity can improve climate change-driven vulnerability assessments and facilitate effective conservation management.