A nuclear genome assembly of an extinct flightless bird, the little bush moa.

We present a draft genome of the little bush moa (Anomalopteryx didiformis)-one of approximately nine species of extinct flightless birds from Aotearoa, New Zealand-using ancient DNA recovered from a fossil bone from the South Island. We recover a complete mitochondrial genome at 249.9× depth of coverage and almost 900 megabases of a male moa nuclear genome at ~4 to 5× coverage, with sequence contiguity sufficient to identify more than 85% of avian universal single-copy orthologs. We describe a diverse landscape of transposable elements and satellite repeats, estimate a long-term effective population size of ~240,000, identify a diverse suite of olfactory receptor genes and an opsin repertoire with sensitivity in the ultraviolet range, show that the wingless moa phenotype is likely not attributable to gene loss or pseudogenization, and identify potential function-altering coding sequence variants in moa that could be synthesized for future functional assays. This genomic resource should support further studies of avian evolution and morphological divergence.

Diversity and evolution of the vertebrate chemoreceptor gene repertoire.

Chemoreception - the ability to smell and taste - is an essential sensory modality of most animals. The number and type of chemical stimuli that animals can perceive depends primarily on the diversity of chemoreceptors they possess and express. In vertebrates, six families of G protein-coupled receptors form the core of their chemosensory system, the olfactory/pheromone receptor gene families OR, TAAR, V1R and V2R, and the taste receptors T1R and T2R. Here, we study the vertebrate chemoreceptor gene repertoire and its evolutionary history. Through the examination of 1,527 vertebrate genomes, we uncover substantial differences in the number and composition of chemoreceptors across vertebrates. We show that the chemoreceptor gene families are co-evolving, highly dynamic, and characterized by lineage-specific expansions (for example, OR in tetrapods; TAAR, T1R in teleosts; V1R in mammals; V2R, T2R in amphibians) and losses. Overall, amphibians, followed by mammals, are the vertebrate clades with the largest chemoreceptor repertoires. While marine tetrapods feature a convergent reduction of chemoreceptor numbers, the number of OR genes correlates with habitat in mammals and birds and with migratory behavior in birds, and the taste receptor repertoire correlates with diet in mammals and with aquatic environment in fish.

Convergence in hearing-related genes between echolocating birds and mammals.

Echolocation, the detection of objects by means of sound waves, has evolved independently in diverse animals. Echolocators include not only mammals such as toothed whales and yangochiropteran and rhinolophoid bats but also Rousettus fruit bats, as well as two bird lineages, oilbirds and swiftlets. In whales and yangochiropteran and rhinolophoid bats, positive selection and molecular convergence has been documented in key hearing-related genes, such as prestin (SLC26A5), but few studies have examined these loci in other echolocators. Here, we examine patterns of selection and convergence in echolocation-related genes in echolocating birds and Rousettus bats. Fewer of these loci were under selection in Rousettus or birds compared with classically recognized echolocators, and elevated convergence (compared to outgroups) was not evident across this gene set. In certain genes, however, we detected convergent substitutions with potential functional relevance, including convergence between Rousettus and classic echolocators in prestin at a site known to affect hair cell electromotility. We also detected convergence between Yangochiroptera, Rhinolophidea, and oilbirds in TMC1, an important mechanosensory transduction channel in vertebrate hair cells, and observed an amino acid change at the same site within the pore domain. Our results suggest that although most proteins implicated in echolocation in specialized mammals may not have been recruited in birds or Rousettus fruit bats, certain hearing-related loci may have undergone convergent functional changes. Investigating adaptations in diverse echolocators will deepen our understanding of this unusual sensory modality.

How do honeyeaters drink nectar?

We investigated the kinematics and biomechanics of nectar feeding in five species of honeyeater (Phylidonyris novaehollandiae, Acanthagenys rufogularis, Ptilotula penicillata, Certhionyx variegatus, Manorina flavigula). There is abundant information on honeyeater foraging behaviors and ecological relationships with plants, but there has never been an examination of their nectar-feeding from kinematic and biomechanical perspectives. We analyzed high-speed video of feeding in captive individuals to describe the kinematics of their nectar feeding, with specific focus on describing tongue movements and bill-tongue coordination, and to characterize the mechanism of nectar uptake in the tongue. We found clear interspecific variation in kinematics and tongue filling mechanics. Species varied in lick frequency, tongue velocity, and protrusion and retraction duration, which, in some cases, are relevant for differences in tongue filling mechanisms. We found support for the use of capillary filling in Certhionyx variegatus only. By contrast, Phylidonyris novaehollandiae, Acanthagenys rufogularis, Ptilotula penicillata, and Manorina flavigula employed a modified version of the expansive filling mechanism seen in hummingbirds, as there was dorsoventral expansion of the tongue body, even the portions that remain outside the nectar, once the tongue tip entered the nectar. All species use fluid trapping in the distal fimbriated portion of the tongue, which supports previous hypotheses describing the honeyeater tongue as a "paintbrush."

Spatial and temporal resource partitioning in a mixed-species colony of avian echolocators.

Resource partitioning may facilitate the coexistence of sympatric species with similar ecological requirements. Here, we study a colony of unusual echolocating birds called swiftlets, which nest underground on an island off the coast of Singapore. The colony comprises two congeneric swiftlet species, black-nest swiftlets (Aerodramus maximus) and edible-nest swiftlets (A. fuciphagus), nesting at high densities and in close proximity. Bioacoustic recordings and monitoring of nesting biology at the site across multiple seasons revealed significant differences in echolocation calls as well as survival rates between the species, with the larger black-nest swiftlet nesting at locations with the highest fledging rates. We also observe an additional off-season breeding peak by the smaller species, the edible-nest swiftlet. Unexpectedly, off-season egg-hatching rates were significantly higher compared with the rates during the shared breeding season (mean difference = 14%). Our study on the breeding biology of these echolocating cave-dwelling birds provides an example of spatial and temporal strategies that animals employ to partition resources within a confined habitat.

Historic samples reveal loss of wild genotype through domestic chicken introgression during the Anthropocene.

Human activities have precipitated a rise in the levels of introgressive gene flow among animals. The investigation of conspecific populations at different time points may shed light on the magnitude of human-mediated introgression. We used the red junglefowl Gallus gallus, the wild ancestral form of the chicken, as our study system. As wild junglefowl and domestic chickens readily admix, conservationists fear that domestic introgression into junglefowl may compromise their wild genotype. By contrasting the whole genomes of 51 chickens with 63 junglefowl from across their natural range, we found evidence of a loss of the wild genotype across the Anthropocene. When comparing against the genomes of junglefowl from approximately a century ago using rigorous ancient-DNA protocols, we discovered that levels of domestic introgression are not equal among and within modern wild populations, with the percentage of domestic ancestry around 20-50%. We identified a number of domestication markers in which chickens are deeply differentiated from historic junglefowl regardless of breed and/or geographic provenance, with eight genes under selection. The latter are involved in pathways dealing with development, reproduction and vision. The wild genotype is an allelic reservoir that holds most of the genetic diversity of G. gallus, a species which is immensely important to human society. Our study provides fundamental genomic infrastructure to assist in efforts to prevent a further loss of the wild genotype through introgression of domestic alleles.

Loss of a gluconeogenic muscle enzyme contributed to adaptive metabolic traits in hummingbirds.

Hummingbirds possess distinct metabolic adaptations to fuel their energy-demanding hovering flight, but the underlying genomic changes are largely unknown. Here, we generated a chromosome-level genome assembly of the long-tailed hermit and screened for genes that have been specifically inactivated in the ancestral hummingbird lineage. We discovered that FBP2 (fructose-bisphosphatase 2), which encodes a gluconeogenic muscle enzyme, was lost during a time period when hovering flight evolved. We show that FBP2 knockdown in an avian muscle cell line up-regulates glycolysis and enhances mitochondrial respiration, coincident with an increased mitochondria number. Furthermore, genes involved in mitochondrial respiration and organization have up-regulated expression in hummingbird flight muscle. Together, these results suggest that FBP2 loss was likely a key step in the evolution of metabolic muscle adaptations required for true hovering flight.

T1R2-mediated sweet sensing in a lizard.

Sugars are an important class of nutrients found in the flowers and fruits of angiosperms (flowering plants). Although T1R2-T1R3 has been identified as the mammalian sweet receptor, some birds rely on a repurposed T1R1-T1R3 savory receptor to sense sugars. Moreover, as the radiation of flowering plants occurred later than the last common ancestor of amniotes, sugar may not have been an important diet item for amniotes early in evolution, raising the question of whether T1R2-T1R3 is a universal sugar sensor or only a mammalian innovation. Here, using brief-access behavioral tests and functional characterization of taste receptors, we demonstrate that the nectar-taking Madagascar giant day gecko (Phelsuma grandis) can sense sugars through the T1R2-T1R3 receptor. These results reveal the existence of T1R2-based sweet taste in a non-avian reptile, which has important implications for our understanding of the evolutionary history of sugar detection in amniotes.

A single residue confers selective loss of sugar sensing in wrynecks.

Sensory receptors evolve, and changes to their response profiles can directly impact sensory perception and affect diverse behaviors, from mate choice to foraging decisions.1-3 Although receptor sensitivities can be highly contingent on changes occurring early in a lineage's evolutionary history,4 subsequent shifts in a species' behavior and ecology may exert selective pressure to modify and even reverse sensory receptor capabilities.5-7 Neither the extent to which sensory reversion occurs nor the mechanisms underlying such shifts is well understood. Using receptor profiling and behavioral tests, we uncover both an early gain and an unexpected subsequent loss of sugar sensing in woodpeckers, a primarily insectivorous family of landbirds.8,9 Our analyses show that, similar to hummingbirds10 and songbirds,4 the ancestors of woodpeckers repurposed their T1R1-T1R3 savory receptor to detect sugars. Importantly, whereas woodpeckers seem to have broadly retained this ability, our experiments demonstrate that wrynecks (an enigmatic ant-eating group sister to all other woodpeckers) selectively lost sugar sensing through a novel mechanism involving a single amino acid change in the T1R3 transmembrane domain. The identification of this molecular microswitch responsible for a sensory shift in taste receptors provides an example of the molecular basis of a sensory reversion in vertebrates and offers novel insights into structure-function relationships during sensory receptor evolution.

Synergism, Bifunctionality, and the Evolution of a Gradual Sensory Trade-off in Hummingbird Taste Receptors.

Sensory receptor evolution can imply trade-offs between ligands, but the extent to which such trade-offs occur and the underlying processes shaping their evolution is not well understood. For example, hummingbirds have repurposed their ancestral savory receptor (T1R1-T1R3) to detect sugars, but the impact of this sensory shift on amino acid perception is unclear. Here, we use functional and behavioral approaches to show that the hummingbird T1R1-T1R3 acts as a bifunctional receptor responsive to both sugars and amino acids. Our comparative analyses reveal substantial functional diversity across the hummingbird radiation and suggest an evolutionary timeline for T1R1-T1R3 retuning. Finally, we identify a novel form of synergism between sugars and amino acids in vertebrate taste receptors. This work uncovers an unexplored axis of sensory diversity, suggesting new ways in which nectar chemistry and pollinator preferences can coevolve.

Evolution of Brain-Expressed Biogenic Amine Receptors into Olfactory Trace Amine-Associated Receptors.

The family of trace amine-associated receptors (TAARs) is distantly related to G protein-coupled biogenic aminergic receptors. TAARs are found in the brain as well as in the olfactory epithelium where they detect biogenic amines. However, the functional relationship of receptors from distinct TAAR subfamilies and in different species is still uncertain. Here, we perform a thorough phylogenetic analysis of 702 TAAR-like (TARL) and TAAR sequences from 48 species. We show that a clade of Tarl genes has greatly expanded in lampreys, whereas the other Tarl clade consists of only one or two orthologs in jawed vertebrates and is lost in amniotes. We also identify two small clades of Taar genes in sharks related to the remaining Taar genes in bony vertebrates, which are divided into four major clades. We further identify ligands for 61 orphan TARLs and TAARs from sea lamprey, shark, ray-finned fishes, and mammals, as well as novel ligands for two 5-hydroxytryptamine receptor 4 orthologs, a serotonin receptor subtype closely related to TAARs. Our results reveal a pattern of functional convergence and segregation: TARLs from sea lamprey and bony vertebrate olfactory TAARs underwent independent expansions to function as chemosensory receptors, whereas TARLs from jawed vertebrates retain ancestral response profiles and may have similar functions to TAAR1 in the brain. Overall, our data provide a comprehensive understanding of the evolution and ligand recognition profiles of TAARs and TARLs.

Evolution of sensory systems.

Sensory systems evolve and enable organisms to perceive their sensory Umwelt, the unique set of cues relevant for their survival. The multiple components that comprise sensory systems - the receptors, cells, organs, and dedicated high-order circuits - can vary greatly across species. Sensory receptor gene families can expand and contract across lineages, resulting in enormous sensory diversity. Comparative studies of sensory receptor function have uncovered the molecular basis of receptor properties and identified novel sensory receptor classes and noncanonical sensory strategies. Phylogenetically informed comparisons of sensory systems across multiple species can pinpoint when sensory changes evolve and highlight the role of contingency in sensory system evolution.

Early origin of sweet perception in the songbird radiation.

Early events in the evolutionary history of a clade can shape the sensory systems of descendant lineages. Although the avian ancestor may not have had a sweet receptor, the widespread incidence of nectar-feeding birds suggests multiple acquisitions of sugar detection. In this study, we identify a single early sensory shift of the umami receptor (the T1R1-T1R3 heterodimer) that conferred sweet-sensing abilities in songbirds, a large evolutionary radiation containing nearly half of all living birds. We demonstrate sugar responses across species with diverse diets, uncover critical sites underlying carbohydrate detection, and identify the molecular basis of sensory convergence between songbirds and nectar-specialist hummingbirds. This early shift shaped the sensory biology of an entire radiation, emphasizing the role of contingency and providing an example of the genetic basis of convergence in avian evolution.

Functional evolution of vertebrate sensory receptors.

Sensory receptors enable animals to perceive their external world, and functional properties of receptors evolve to detect the specific cues relevant for an organism's survival. Changes in sensory receptor function or tuning can directly impact an organism's behavior. Functional tests of receptors from multiple species and the generation of chimeric receptors between orthologs with different properties allow for the dissection of the molecular basis of receptor function and identification of the key residues that impart functional changes in different species. Knowledge of these functionally important sites facilitates investigation into questions regarding the role of epistasis and the extent of convergence, as well as the timing of sensory shifts relative to other phenotypic changes. However, as receptors can also play roles in non-sensory tissues, and receptor responses can be modulated by numerous other factors including varying expression levels, alternative splicing, and morphological features of the sensory cell, behavioral validation can be instrumental in confirming that responses observed in heterologous systems play a sensory role. Expression profiling of sensory cells and comparative genomics approaches can shed light on cell-type specific modifications and identify other proteins that may affect receptor function and can provide insight into the correlated evolution of complex suites of traits. Here we review the evolutionary history and diversity of functional responses of the major classes of sensory receptors in vertebrates, including opsins, chemosensory receptors, and ion channels involved in temperature-sensing, mechanosensation and electroreception.

Duplications and Functional Convergence of Intestinal Carbohydrate-Digesting Enzymes.

Vertebrate diets and digestive physiologies vary tremendously. Although the contribution of ecological and behavioral features to such diversity is well documented, the roles and identities of individual intestinal enzymes shaping digestive traits remain largely unexplored. Here, we show that the sucrase-isomaltase (SI)/maltase-glucoamylase (MGAM) dual enzyme system long assumed to be the conserved disaccharide and starch digestion framework in all vertebrates is absent in many lineages. Our analyses indicate that independent duplications of an ancestral SI gave rise to the mammalian-specific MGAM, as well as to other duplicates in fish and birds. Strikingly, the duplicated avian enzyme exhibits similar activities to MGAM, revealing an unexpected case of functional convergence. Our results highlight digestive enzyme variation as a key uncharacterized component of dietary diversity in vertebrates.

Non-classical amine recognition evolved in a large clade of olfactory receptors.

Biogenic amines are important signaling molecules, and the structural basis for their recognition by G Protein-Coupled Receptors (GPCRs) is well understood. Amines are also potent odors, with some activating olfactory trace amine-associated receptors (TAARs). Here, we report that teleost TAARs evolved a new way to recognize amines in a non-classical orientation. Chemical screens de-orphaned eleven zebrafish TAARs, with agonists including serotonin, histamine, tryptamine, 2-phenylethylamine, putrescine, and agmatine. Receptors from different clades contact ligands through aspartates on transmembrane α-helices III (canonical Asp(3.32)) or V (non-canonical Asp(5.42)), and diamine receptors contain both aspartates. Non-classical monoamine recognition evolved in two steps: an ancestral TAAR acquired Asp(5.42), gaining diamine sensitivity, and subsequently lost Asp(3.32). Through this transformation, the fish olfactory system dramatically expanded its capacity to detect amines, ecologically significant aquatic odors. The evolution of a second, alternative solution for amine detection by olfactory receptors highlights the tremendous structural versatility intrinsic to GPCRs.

Sensory biology. Evolution of sweet taste perception in hummingbirds by transformation of the ancestral umami receptor.

Sensory systems define an animal's capacity for perception and can evolve to promote survival in new environmental niches. We have uncovered a noncanonical mechanism for sweet taste perception that evolved in hummingbirds since their divergence from insectivorous swifts, their closest relatives. We observed the widespread absence in birds of an essential subunit (T1R2) of the only known vertebrate sweet receptor, raising questions about how specialized nectar feeders such as hummingbirds sense sugars. Receptor expression studies revealed that the ancestral umami receptor (the T1R1-T1R3 heterodimer) was repurposed in hummingbirds to function as a carbohydrate receptor. Furthermore, the molecular recognition properties of T1R1-T1R3 guided taste behavior in captive and wild hummingbirds. We propose that changing taste receptor function enabled hummingbirds to perceive and use nectar, facilitating the massive radiation of hummingbird species.

The hummingbird's tongue: a self-assembling capillary syphon.

We present the results of a combined experimental and theoretical investigation of the dynamics of drinking in ruby-throated hummingbirds. In vivo observations reveal elastocapillary deformation of the hummingbird's tongue and capillary suction along its length. By developing a theoretical model for the hummingbird's drinking process, we investigate how the elastocapillarity affects the energy intake rate of the bird and how its open tongue geometry reduces resistance to nectar uptake. We note that the tongue flexibility is beneficial for accessing, transporting and unloading the nectar. We demonstrate that the hummingbird can attain the fastest nectar uptake when its tongue is roughly semicircular. Finally, we assess the relative importance of capillary suction and a recently proposed fluid trapping mechanism, and conclude that the former is important in many natural settings.

Agonists for 13 trace amine-associated receptors provide insight into the molecular basis of odor selectivity.

Trace amine-associated receptors (TAARs) are vertebrate olfactory receptors. However, ligand recognition properties of TAARs remain poorly understood, as most are "orphan receptors" without known agonists. Here, we identify the first ligands for many rodent TAARs and classify these receptors into two subfamilies based on the phylogeny and binding preference for primary or tertiary amines. Some mouse and rat orthologs have similar response profiles, although independent Taar7 gene expansions led to highly related receptors with altered ligand specificities. Using chimeric TAAR7 receptors, we identified an odor contact site in transmembrane helix III that functions as a selectivity filter. Homology models based on the ß(2) adrenergic receptor structure indicate spatial proximity of this site to the ligand. Gain-of-function mutations at this site created olfactory receptors with radically altered odor recognition properties. These studies provide new TAAR ligands, valuable tools for studying receptor function, and general insights into the molecular pharmacology of G protein-coupled receptors.