Museum genomics approach to study the taxonomy and evolution of Woolly-necked storks using historic specimens.

The accessibility of genomic tools in evolutionary biology has allowed for a thorough exploration of various evolutionary processes associated with adaptation and speciation. However, genomic studies in natural systems present numerous challenges, reflecting the inherent complexities of studying organisms in their native habitats. The utilization of museum specimens for genomics research has received increased attention in recent times, facilitated by advancements in ancient DNA techniques. In this study, we have utilized a museum genomics approach to analyze historic specimens of Woolly-necked storks (Ciconia spp.) and examine their genetic composition and taxonomic status and explore the evolutionary and adaptive trajectories of populations over the years. The Woolly-necked storks are distributed in Asia and Africa with a taxonomic classification that has been a matter of ambiguity. Asian and African Woollynecks were recently recognized as different species based on their morphological differences; however, their genomic validation was lacking. In this study, we have used ∼70-year-old museum samples for whole-genome population-scale sequencing. Our study has revealed that Asian and African Woollynecks are genetically distinct, consistent with the current taxonomic classification based on morphological features. However, we also found a high genetic divergence between the Asian subspecies Ciconia episcopus neglecta and Ciconia episcopus episcopus, suggesting this classification requires a detailed examination to explore processes of ongoing speciation. Because taxonomic classification directly impacts conservation efforts, and there is evidence of declining populations of Asian Woollynecks in Southeast Asia, our results highlight that population-scale studies are urgent to determine the genetic, ecological, and phylogenetic diversity of these birds.

Low Mutation Load in a Supergene Underpinning Alternative Male Mating Strategies in Ruff (Calidris pugnax).

A paradox in evolutionary biology is how supergenes can maintain high fitness despite reduced effective population size, the suppression of recombination, and the expected accumulation of mutational load. The ruff supergene involves 2 rare inversion haplotypes (satellite and faeder). These are recessive lethals but with dominant effects on male mating strategies, plumage, and body size. Sequence divergence to the wild-type (independent) haplotype indicates that the inversion could be as old as 4 million years. Here, we have constructed a highly contiguous genome assembly of the inversion region for both the independent and satellite haplotypes. Based on the new data, we estimate that the recombination event(s) creating the satellite haplotype occurred only about 70,000 yr ago. Contrary to expectations for supergenes, we find no substantial expansion of repeats and only a modest mutation load on the satellite and faeder haplotypes despite high sequence divergence to the non-inverted haplotype (1.46%). The essential centromere protein N (CENPN) gene is disrupted by the inversion and is as well conserved on the inversion haplotypes as on the noninversion haplotype. These results suggest that the inversion may be much younger than previously thought. The low mutation load, despite recessive lethality, may be explained by the introgression of the inversion from a now extinct lineage.

Genomic Variation, Population History, and Long-Term Genetic Adaptation to High Altitudes in Tibetan Partridge (Perdix hodgsoniae).

Species residing across elevational gradients display adaptations in response to environmental changes such as oxygen availability, ultraviolet radiation, and temperature. Here, we study genomic variation, gene expression, and long-term adaptation in Tibetan Partridge (Perdix hodgsoniae) populations residing across the elevational gradient of the Tibetan Plateau. We generated a high-quality draft genome and used it to carry out downstream population genomic and transcriptomic analysis. The P. hodgsoniae populations residing across various elevations were genetically distinct, and their phylogenetic clustering was consistent with their geographic distribution. We identified possible evidence of gene flow between populations residing in <3,000 and >4,200 m elevation that is consistent with known habitat expansion of high-altitude populations of P. hodgsoniae to a lower elevation. We identified a 60 kb haplotype encompassing the Estrogen Receptor 1 (ESR1) gene, showing strong genetic divergence between populations of P. hodgsoniae. We identified six single nucleotide polymorphisms within the ESR1 gene fixed for derived alleles in high-altitude populations that are strongly conserved across vertebrates. We also compared blood transcriptome profiles and identified differentially expressed genes (such as GAPDH, LDHA, and ALDOC) that correlated with differences in altitude among populations of P. hodgsoniae. These candidate genes from population genomics and transcriptomics analysis were enriched for neutrophil degranulation and glycolysis pathways, which are known to respond to hypoxia and hence may contribute to long-term adaptation to high altitudes in P. hodgsoniae. Our results highlight Tibetan Partridges as a useful model to study molecular mechanisms underlying long-term adaptation to high altitudes.

Identification of major histocompatibility complex genotypes associated with resistance to an amphibian emerging infectious disease.

Amphibian chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd), emerged from Asia and spread globally. By comparing functional MHC IIß1 alleles from an Asian Bd-resistant anuran species (Bufo gargarizans) with those of an Australasian Bd-susceptible species (Litoria caerulea), we identified MHC genotypes associated with Bd resistance. These alleles encode a glycine deletion (G90ß1) and adjacent motifs in the deepest pathogen-derived peptide-binding groove. Every Bd-resistant individual, but no susceptible individuals, possessed at least one allele encoding the variant. We detected trans-species polymorphism at the end of the MHC IIß1 sequences. The G90ß1 deletion was encoded by different alleles in the two species, suggesting it may have evolved independently in each species rather than having been derived from a common ancestor. These results are consistent with a scenario by which MHC adaptations that confer resistance to the pathogen have evolved by convergent evolution. Immunogenetic studies such as this are critical to ongoing conservation efforts.

Gene flow drives genomic diversity in Asian Pikas distributed along the core and range-edge habitats in the Himalayas.

Studying the genetic variation among different species distributed across their core and range-edge habitats can provide valuable insights into how genetic variation changes across the species' distribution range. This information can be important for understanding local adaptation, as well as for conservation and management efforts. In this study, we have carried out genomic characterization of six species of Asian Pikas distributed along their core and range-edge habitats in the Himalayas. We utilized a population genomics approach using ~28,000 genome-wide SNP markers obtained from restriction-site associated DNA sequencing. We identified low nucleotide diversity and high inbreeding coefficients in all six species across their core and range-edge habitats. We also identified evidence of gene flow among genetically diverse species. Our results provide evidence of reduced genetic diversity in Asian pikas distributed across the Himalayas and the neighboring regions and indicate that recurrent gene flow is possibly a key mechanism for maintaining genetic diversity and adaptive potential in these pikas. However, full-scale genomics studies that utilize whole-genome sequencing approaches will be needed to quantify the direction and timing of gene flow and functional changes associated with introgressed regions in the genome. Our results represent an important step toward understanding the patterns and consequences of gene flow in species, sampled at the least studied, yet climatically vulnerable part of their habitat that can be further used to inform conservation strategies that promote connectivity and gene flow between populations.

Rapid adaptive radiation of Darwin's finches depends on ancestral genetic modules.

Recent adaptive radiations are models for investigating mechanisms contributing to the evolution of biodiversity. An unresolved question is the relative importance of new mutations, ancestral variants, and introgressive hybridization for phenotypic evolution and speciation. Here, we address this issue using Darwin's finches and investigate the genomic architecture underlying their phenotypic diversity. Admixture mapping for beak and body size in the small, medium, and large ground finches revealed 28 loci showing strong genetic differentiation. These loci represent ancestral haplotype blocks with origins predating speciation events during the Darwin's finch radiation. Genes expressed in the developing beak are overrepresented in these genomic regions. Ancestral haplotypes constitute genetic modules for selection and act as key determinants of the unusual phenotypic diversity of Darwin's finches. Such ancestral haplotype blocks can be critical for how species adapt to environmental variability and change.

The genome sequence of the avian vampire fly (Philornis downsi), an invasive nest parasite of Darwin's finches in Galápagos.

The invasive avian vampire fly (Philornis downsi, Diptera: Muscidae) is considered one of the greatest threats to the endemic avifauna of the Galápagos Islands. The fly larvae parasitize nearly every passerine species, including Darwin's finches. Most P. downsi research to date has focused on the effects of the fly on avian host fitness and mitigation methods. A lag in research related to the genetics of this invasion demonstrates, in part, the need to develop full-scale genomic resources with which to address further questions within this system. In this study, an adult female P. downsi was sequenced to generate a high-quality genome assembly. We examined various features of the genome (e.g., coding regions and noncoding transposable elements) and carried out comparative genomics analysis against other dipteran genomes. We identified lists of gene families that are significantly expanding or contracting in P. downsi that are related to insecticide resistance, detoxification, and counter defense against host immune responses. The P. downsi genome assembly provides an important resource for studying the molecular basis of successful invasion in the Galápagos and the dynamics of its population across multiple islands. The findings of significantly changing gene families associated with insecticide resistance and immune responses highlight the need for further investigations into the role of different gene families in aiding the fly's successful invasion. Furthermore, this genomic resource provides a necessary tool to better inform future research studies and mitigation strategies aimed at minimizing the fly's impact on Galápagos birds.

Exploring potentialities of avian genomic research in Nepalese Himalayas.

Nepal, a small landlocked country in South Asia, holds about 800 km of Himalayan Mountain range including the Earth's highest mountain. Within such a mountain range in the north and plain lowlands in the south, Nepal provides a habitat for about 9% of global avian fauna. However, this diversity is underrated because of the lack of enough studies, especially using molecular tools to quantify and understand the distribution patterns of diversity. In this study, we reviewed the studies in the last two decades (2000‒2019) that used molecular methods to study the biodiversity in Nepal to examine the ongoing research trend and focus. Although Nepalese Himalaya has many opportunities for cutting-edge molecular research, our results indicated that the rate of genetic/genomic studies is much slower compared to the regional trends. We found that genetic research in Nepal heavily relies on resources from international institutes and that too is mostly limited to research on species monitoring, distribution, and taxonomic validations. Local infrastructures to carry out cutting-edge genomic research in Nepal are still in their infancy and there is a strong need for support from national/international scientists, universities, and governmental agencies to expand such genomic infrastructures in Nepal. We particularly highlight avian fauna as a potential future study system in this region that can be an excellent resource to explore key biological questions such as understanding eco-physiology and molecular basis of organismal persistence to changing environment, evolutionary processes underlying divergence and speciation, or mechanisms of endemism and restrictive distribution of species. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40657-021-00290-5.

A bird-like genome from a frog: Mechanisms of genome size reduction in the ornate burrowing frog, Platyplectrum ornatum.

The diversity of genome sizes across the tree of life is of key interest in evolutionary biology. Various correlates of variation in genome size, such as accumulation of transposable elements (TEs) or rate of DNA gain and loss, are well known, but the underlying molecular mechanisms driving or constraining genome size are poorly understood. Here, we study one of the smallest genomes among frogs characterized thus far, that of the ornate burrowing frog (Platyplectrum ornatum) from Australia, and compare it to other published frog and vertebrate genomes to examine the forces driving reduction in genome size. At ∼1.06 gigabases (Gb), the P. ornatum genome is like that of birds, revealing four major mechanisms underlying TE dynamics: reduced abundance of all major classes of TEs; increased net deletion bias in TEs; drastic reduction in intron lengths; and expansion via gene duplication of the repertoire of TE-suppressing Piwi genes, accompanied by increased expression of Piwi-interacting RNA (piRNA)-based TE-silencing pathway genes in germline cells. Transcriptomes from multiple tissues in both sexes corroborate these results and provide insight into sex-differentiation pathways in Platyplectrum Genome skimming of two closely related frog species (Lechriodus fletcheri and Limnodynastes fletcheri) confirms a reduction in TEs as a major driver of genome reduction in Platyplectrum and supports a macroevolutionary scenario of small genome size in frogs driven by convergence in life history, especially rapid tadpole development and tadpole diet. The P. ornatum genome offers a model for future comparative studies on mechanisms of genome size reduction in amphibians and vertebrates generally.

Female-biased gene flow between two species of Darwin's finches.

The mosaic nature of hybrid genomes is well recognized, but little is known of how they are shaped initially by patterns of breeding, selection, recombination and differential incompatibilities. On the small Galápagos island of Daphne Major, two species of Darwin's finches, Geospiza fortis and G. scandens, hybridize rarely and back-cross bidirectionally with little or no loss of fitness under conditions of plentiful food. We used whole-genome sequences to compare genomes from periods before and after successful interbreeding followed by back-crossing. We inferred extensive introgression from G. fortis to G. scandens on autosomes and mitochondria but not on the Z chromosome. The unique combination of long-term field observations and genomic data shows that the reduction of gene flow for Z-linked loci primarily reflects female-biased gene flow, arising from a hybrid-male disadvantage in competition for high-quality territories and mates, rather than from genetic incompatibilities at Z-linked loci.

Mutations Upstream of the TBX5 and PITX1 Transcription Factor Genes Are Associated with Feathered Legs in the Domestic Chicken.

Feathered leg is a trait in domestic chickens that has undergone intense selection by fancy breeders. Previous studies have shown that two major loci controlling feathered leg are located on chromosomes 13 and 15. Here, we present genetic evidence for the identification of candidate causal mutations at these loci. This was accomplished by combining classical linkage mapping using an experimental cross segregating for feathered leg and high-resolution identical-by-descent mapping using whole-genome sequence data from 167 samples of chicken with or without feathered legs. The first predicted causal mutation is a single-base change located 25 kb upstream of the gene for the forelimb-specific transcription factor TBX5 on chromosome 15. The second is a 17.7-kb deletion located ∼200 kb upstream of the gene for the hindlimb-specific transcription factor PITX1 on chromosome 13. These mutations are predicted to activate TBX5 and repress PITX1 expression, respectively. The study reveals a remarkable convergence in the evolution of the feathered-leg phenotype in domestic chickens and domestic pigeons, as this phenotype is caused by noncoding mutations upstream of the same two genes. Furthermore, the PITX1 causal variants are large overlapping deletions, 17.7 kb in chicken and 44 kb in pigeons. The results of the present study are consistent with the previously proposed model for pigeon that feathered leg is caused by reduced PITX1 expression and ectopic expression of TBX5 in hindlimb buds resulting in a shift of limb identity from hindlimb to more forelimb-like identity.

Integrating natural history collections and comparative genomics to study the genetic architecture of convergent evolution.

Evolutionary convergence has been long considered primary evidence of adaptation driven by natural selection and provides opportunities to explore evolutionary repeatability and predictability. In recent years, there has been increased interest in exploring the genetic mechanisms underlying convergent evolution, in part, owing to the advent of genomic techniques. However, the current 'genomics gold rush' in studies of convergence has overshadowed the reality that most trait classifications are quite broadly defined, resulting in incomplete or potentially biased interpretations of results. Genomic studies of convergence would be greatly improved by integrating deep 'vertical', natural history knowledge with 'horizontal' knowledge focusing on the breadth of taxonomic diversity. Natural history collections have and continue to be best positioned for increasing our comprehensive understanding of phenotypic diversity, with modern practices of digitization and databasing of morphological traits providing exciting improvements in our ability to evaluate the degree of morphological convergence. Combining more detailed phenotypic data with the well-established field of genomics will enable scientists to make progress on an important goal in biology: to understand the degree to which genetic or molecular convergence is associated with phenotypic convergence. Although the fields of comparative biology or comparative genomics alone can separately reveal important insights into convergent evolution, here we suggest that the synergistic and complementary roles of natural history collection-derived phenomic data and comparative genomics methods can be particularly powerful in together elucidating the genomic basis of convergent evolution among higher taxa. This article is part of the theme issue 'Convergent evolution in the genomics era: new insights and directions'.

A comparison of the association between large haplotype blocks under selection and the presence/absence of inversions.

Inversions may contribute to ecological adaptation and phenotypic diversity, and with the advent of "second" and "third" generation sequencing technologies, the ability to detect inversion polymorphisms has been enhanced dramatically. A key molecular consequence of an inversion is the suppression of recombination allowing independent accumulation of genetic changes between alleles over time. This may lead to the development of divergent haplotype blocks maintained by balancing selection. Thus, divergent haplotype blocks are often considered as indicating the presence of an inversion. In this paper, we first review the features of a 7.7 Mb inversion causing the Rose-comb phenotype in chicken, as a model for how inversions evolve and directly affect phenotypes. Second, we compare the genetic basis for alternative mating strategies in ruff and timing of reproduction in Atlantic herring, both associated with divergent haplotype blocks. Alternative male mating strategies in ruff are associated with a 4.5 Mb inversion that occurred about 4 million years ago. In fact, the ruff inversion shares some striking features with the Rose-comb inversion such as disruption of a gene at one of the inversion breakpoints and generation of a new allele by recombination between the inverted and noninverted alleles. In contrast, inversions do not appear to be a major reason for the fairly large haplotype blocks (range 10-200 kb) associated with ecological adaptation in the herring. Thus, it is important to note that divergent haplotypes may also be maintained by natural selection in the absence of structural variation.

Embracing heterogeneity: coalescing the Tree of Life and the future of phylogenomics.

Building the Tree of Life (ToL) is a major challenge of modern biology, requiring advances in cyberinfrastructure, data collection, theory, and more. Here, we argue that phylogenomics stands to benefit by embracing the many heterogeneous genomic signals emerging from the first decade of large-scale phylogenetic analysis spawned by high-throughput sequencing (HTS). Such signals include those most commonly encountered in phylogenomic datasets, such as incomplete lineage sorting, but also those reticulate processes emerging with greater frequency, such as recombination and introgression. Here we focus specifically on how phylogenetic methods can accommodate the heterogeneity incurred by such population genetic processes; we do not discuss phylogenetic methods that ignore such processes, such as concatenation or supermatrix approaches or supertrees. We suggest that methods of data acquisition and the types of markers used in phylogenomics will remain restricted until a posteriori methods of marker choice are made possible with routine whole-genome sequencing of taxa of interest. We discuss limitations and potential extensions of a model supporting innovation in phylogenomics today, the multispecies coalescent model (MSC). Macroevolutionary models that use phylogenies, such as character mapping, often ignore the heterogeneity on which building phylogenies increasingly rely and suggest that assimilating such heterogeneity is an important goal moving forward. Finally, we argue that an integrative cyberinfrastructure linking all steps of the process of building the ToL, from specimen acquisition in the field to publication and tracking of phylogenomic data, as well as a culture that values contributors at each step, are essential for progress.

Rapid hybrid speciation in Darwin's finches.

Homoploid hybrid speciation in animals has been inferred frequently from patterns of variation, but few examples have withstood critical scrutiny. Here we report a directly documented example, from its origin to reproductive isolation. An immigrant Darwin's finch to Daphne Major in the Galápagos archipelago initiated a new genetic lineage by breeding with a resident finch (Geospiza fortis). Genome sequencing of the immigrant identified it as a G. conirostris male that originated on Española >100 kilometers from Daphne Major. From the second generation onward, the lineage bred endogamously and, despite intense inbreeding, was ecologically successful and showed transgressive segregation of bill morphology. This example shows that reproductive isolation, which typically develops over hundreds of generations, can be established in only three.

Moderate nucleotide diversity in the Atlantic herring is associated with a low mutation rate.

The Atlantic herring is one of the most abundant vertebrates on earth but its nucleotide diversity is moderate (π = 0.3%), only three-fold higher than in human. Here, we present a pedigree-based estimation of the mutation rate in this species. Based on whole-genome sequencing of four parents and 12 offspring, the estimated mutation rate is 2.0 × 10-9 per base per generation. We observed a high degree of parental mosaicism indicating that a large fraction of these de novo mutations occurred during early germ cell development. The estimated mutation rate - the lowest among vertebrates analyzed to date - partially explains the discrepancy between the rather low nucleotide diversity in herring and its huge census population size. But a species like the herring will never reach its expected nucleotide diversity because of fluctuations in population size over the millions of years it takes to build up high nucleotide diversity.

Parallel adaptive evolution of geographically distant herring populations on both sides of the North Atlantic Ocean.

Atlantic herring is an excellent species for studying the genetic basis of adaptation in geographically distant populations because of its characteristically large population sizes and low genetic drift. In this study we compared whole-genome resequencing data of Atlantic herring populations from both sides of the Atlantic Ocean. An important finding was the very low degree of genetic differentiation among geographically distant populations (fixation index = 0.026), suggesting lack of reproductive isolation across the ocean. This feature of the Atlantic herring facilitates the detection of genetic factors affecting adaptation because of the sharp contrast between loci showing genetic differentiation resulting from natural selection and the low background noise resulting from genetic drift. We show that genetic factors associated with timing of reproduction are shared between genetically distinct and geographically distant populations. The genes for thyroid-stimulating hormone receptor (TSHR), the SOX11 transcription factor (SOX11), calmodulin (CALM), and estrogen receptor 2 (ESR2A), all with a significant role in reproductive biology, were among the loci that showed the most consistent association with spawning time throughout the species range. In fact, the same two SNPs located at the 5' end of TSHR showed the most significant association with spawning time in both the east and west Atlantic. We also identified unexpected haplotype sharing between spring-spawning oceanic herring and autumn-spawning populations across the Atlantic Ocean and the Baltic Sea. The genomic regions showing this pattern are unlikely to control spawning time but may be involved in adaptation to ecological factor(s) shared among these populations.

Gene flow, ancient polymorphism, and ecological adaptation shape the genomic landscape of divergence among Darwin's finches.

Genomic comparisons of closely related species have identified "islands" of locally elevated sequence divergence. Genomic islands may contain functional variants involved in local adaptation or reproductive isolation and may therefore play an important role in the speciation process. However, genomic islands can also arise through evolutionary processes unrelated to speciation, and examination of their properties can illuminate how new species evolve. Here, we performed scans for regions of high relative divergence (FST) in 12 species pairs of Darwin's finches at different genetic distances. In each pair, we identify genomic islands that are, on average, elevated in both relative divergence (FST) and absolute divergence (dXY). This signal indicates that haplotypes within these genomic regions became isolated from each other earlier than the rest of the genome. Interestingly, similar numbers of genomic islands of elevated dXY are observed in sympatric and allopatric species pairs, suggesting that recent gene flow is not a major factor in their formation. We find that two of the most pronounced genomic islands contain the ALX1 and HMGA2 loci, which are associated with variation in beak shape and size, respectively, suggesting that they are involved in ecological adaptation. A subset of genomic island regions, including these loci, appears to represent anciently diverged haplotypes that evolved early during the radiation of Darwin's finches. Comparative genomics data indicate that these loci, and genomic islands in general, have exceptionally low recombination rates, which may play a role in their establishment.

The genetic basis for ecological adaptation of the Atlantic herring revealed by genome sequencing.

Ecological adaptation is of major relevance to speciation and sustainable population management, but the underlying genetic factors are typically hard to study in natural populations due to genetic differentiation caused by natural selection being confounded with genetic drift in subdivided populations. Here, we use whole genome population sequencing of Atlantic and Baltic herring to reveal the underlying genetic architecture at an unprecedented detailed resolution for both adaptation to a new niche environment and timing of reproduction. We identify almost 500 independent loci associated with a recent niche expansion from marine (Atlantic Ocean) to brackish waters (Baltic Sea), and more than 100 independent loci showing genetic differentiation between spring- and autumn-spawning populations irrespective of geographic origin. Our results show that both coding and non-coding changes contribute to adaptation. Haplotype blocks, often spanning multiple genes and maintained by selection, are associated with genetic differentiation.