Two centuries of biodiversity discovery and loss in Singapore.

There is an urgent need for reliable data on the impacts of deforestation on tropical biodiversity. The city-state of Singapore has one of the most detailed biodiversity records in the tropics, dating back to the turn of the 19th century. In 1819, Singapore was almost entirely covered in primary forest, but this has since been largely cleared. We compiled more than 200 y of records for 10 major taxonomic groups in Singapore (>50,000 individual records; >3,000 species), and we estimated extinction rates using recently developed and novel statistical models that account for "dark extinctions," i.e., extinctions of undiscovered species. The estimated overall extinction rate was 37% (95% CI [31 to 42%]). Extrapolating our Singapore observations to a future business-as-usual deforestation scenario for Southeast Asia suggests that 18% (95% CI [16 to 22%]) of species will be lost regionally by 2100. Our extinction estimates for Singapore and Southeast Asia are a factor of two lower than previous estimates that also attempted to account for dark extinctions. However, we caution that particular groups such as large mammals, forest-dependent birds, orchids, and butterflies are disproportionately vulnerable.

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.

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.

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.

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.

Genomes From Historic DNA Unveil Massive Hidden Extinction and Terminal Endangerment in a Tropical Asian Songbird Radiation.

Quantifying the magnitude of the global extinction crisis is important but remains challenging, as many extinction events pass unnoticed owing to our limited taxonomic knowledge of the world's organisms. The increasing rarity of many taxa renders comprehensive sampling difficult, further compounding the problem. Vertebrate lineages such as birds, which are thought to be taxonomically well understood, are therefore used as indicator groups for mapping and quantifying global extinction. To test whether extinction patterns are adequately gauged in well-studied groups, we implemented ancient-DNA protocols and retrieved whole genomes from the historic DNA of museum specimens in a widely known songbird radiation of shamas (genus Copsychus) that is assumed to be of least conservation concern. We uncovered cryptic diversity and an unexpected degree of hidden extinction and terminal endangerment. Our analyses reveal that >40% of the phylogenetic diversity of this radiation is already either extinct in the wild or nearly so, including the two genomically most distinct members of this group (omissus and nigricauda), which have so far flown under the conservation radar as they have previously been considered subspecies. Comparing the genomes of modern samples with those from roughly a century ago, we also found a significant decrease in genetic diversity and a concomitant increase in homozygosity affecting various taxa, including small-island endemics that are extinct in the wild as well as subspecies that remain widespread across the continental scale. Our application of modern genomic approaches demonstrates elevated levels of allelic and taxonomic diversity loss in a songbird clade that has not been listed as globally threatened, highlighting the importance of ongoing reassessments of extinction incidence even across well-studied animal groups. Key words: extinction, introgression, white-rumped shama, conservation.

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.

Using historical genome-wide DNA to unravel the confused taxonomy in a songbird lineage that is extinct in the wild.

Urgent conservation action for terminally endangered species is sometimes hampered by taxonomic uncertainty, especially in illegally traded animals that are often cross-bred in captivity. To overcome these problems, we used a genomic approach to analyze historical DNA from museum samples across the Asian Pied Starling (Gracupica contra) complex in tropical Asia, a popular victim of the ongoing songbird crisis whose distinct Javan population ("Javan Pied Starling") is extinct in the wild and subject to admixture in captivity. Comparing genomic profiles across the entire distribution, we detected three deeply diverged lineages at the species level characterized by a lack of genomic intermediacy near areas of contact. Our study demonstrates that the use of historical DNA can be instrumental in delimiting species in situations of taxonomic uncertainty, especially when modern admixture may obfuscate species boundaries. Results of our research will enable conservationists to commence a dedicated ex situ breeding program for the Javan Pied Starling, and serve as a blueprint for similar conservation problems involving terminally endangered species subject to allelic infiltration from close congeners.

Phylogenomics of white-eyes, a 'great speciator', reveals Indonesian archipelago as the center of lineage diversity.

Archipelagoes serve as important 'natural laboratories' which facilitate the study of island radiations and contribute to the understanding of evolutionary processes. The white-eye genus Zosterops is a classical example of a 'great speciator', comprising c. 100 species from across the Old World, most of them insular. We achieved an extensive geographic DNA sampling of Zosterops by using historical specimens and recently collected samples. Using over 700 genome-wide loci in conjunction with coalescent species tree methods and gene flow detection approaches, we untangled the reticulated evolutionary history of Zosterops, which comprises three main clades centered in Indo-Africa, Asia, and Australasia, respectively. Genetic introgression between species permeates the Zosterops phylogeny, regardless of how distantly related species are. Crucially, we identified the Indonesian archipelago, and specifically Borneo, as the major center of diversity and the only area where all three main clades overlap, attesting to the evolutionary importance of this region.

The conservation value of admixed phenotypes in a critically endangered species complex.

In today's environmental crisis, conservationists are increasingly confronted with terminally endangered species whose last few surviving populations may be affected by allelic introgression from closely related species. Yet there is a worrying lack of evidence-based recommendations and solutions for this emerging problem. We analyzed genome-wide DNA markers and plumage variability in a critically endangered insular songbird, the Black-winged Myna (BWM, Acridotheres melanopterus). This species is highly threatened by the illegal wildlife trade, with its wild population numbering in the low hundreds, and its continued survival urgently depending on ex-situ breeding. Its three subspecies occur along a geographic gradient of melanism and are variably interpreted as three species. However, our integrative approach revealed that melanism poorly reflects the pattern of limited genomic differentiation across BWM subspecies. We also uncovered allelic introgression into the most melanistic subspecies, tertius, from the all-black congeneric Javan Myna (A. javanicus), which is native to the same islands. Based on our results, we recommend the establishment of three separate breeding programs to maintain subspecific traits that may confer local adaptation, but with the option of occasional cross-breeding between insurance populations in order to boost genetic diversity and increase overall viability prospects of each breeding program. Our results underscore the importance of evidence-based integrative approaches when determining appropriate conservation units. Given the rapid increase of terminally endangered organisms in need of ex-situ conservation, this study provides an important blueprint for similar programs dealing with phenotypically variable species.

Gene Flow in the Müllerian Mimicry Ring of a Poisonous Papuan Songbird Clade (Pitohui; Aves).

Müllerian mimicry rings are remarkable symbiotic species assemblages in which multiple members share a similar phenotype. However, their evolutionary origin remains poorly understood. Although gene flow among species has been shown to generate mimetic patterns in some Heliconius butterflies, mimicry is believed to be due to true convergence without gene flow in many other cases. We investigated the evolutionary history of multiple members of a passerine mimicry ring in the poisonous Papuan pitohuis. Previous phylogenetic evidence indicates that the aposematic coloration shared by many, but not all, members of this genus is ancestral and has only been retained by members of the mimicry ring. Using a newly assembled genome and thousands of genomic DNA markers, we demonstrate gene flow from the hooded pitohui (Pitohui dichrous) into the southern variable pitohui (Pitohui uropygialis), consistent with shared patterns of aposematic coloration. The vicinity of putatively introgressed loci is significantly enriched for genes that are important in melanin pigment expression and toxin resistance, suggesting that gene flow may have been instrumental in the sharing of plumage patterns and toxicity. These results indicate that interspecies gene flow may be a more general mechanism in generating mimicry rings than hitherto appreciated.

Population divergence and gene flow in two East Asian shorebirds on the verge of speciation.

Genetic isolation of populations over evolutionary time leads to the formation of independent species. We examined a pair of shorebirds - the Kentish Plover Charadrius alexandrinus and the enigmatic White-faced Plover C. dealbatus - which display strong plumage differentiation, yet show minimal genetic divergence based on previous mitochondrial and microsatellite work. Two scenarios may lead to this situation: (1) they represent clinal or poorly diverged populations with limited genomic differentiation despite substantial plumage variation, or (2) they are diverging taxa at the cusp of speciation, with ongoing limited gene flow obliterating signals of differentiation in traditional genetic markers. We compared the genotypes of 98 plovers (59 Kentish Plovers, 35 White-faced Plovers and 4 genomic hybrids) sampled in eastern Asia and Europe using ddRADSeq to harvest over 8000 genome-wide SNPs. In contrast to previous studies, our analyses revealed two well defined genomic clusters, with limited hybridization and a narrow contact zone. We also uncovered significant differences in bill length and further sex-specific differences in size, which may signal differences in mate choice between Kentish and White-faced Plovers. Our results support the hypothesis that this shorebird duo is on the verge of speciation.

Speciation in Indo-Pacific swiftlets (Aves: Apodidae): integrating molecular and phenotypic data for a new provisional taxonomy of the Collocalia esculenta complex.

White-bellied swiftlets of the Collocalia esculenta complex constitute a radiation of colony-breeding swifts distributed throughout the tropical Indo-Pacific region. Resolution of their taxonomy is challenging due to their morphological uniformity. To analyze the evolutionary history of this complex, we combine new biometric measurements and results from plumage assessment of museum specimens with novel as well as previously published molecular data. Together, this body of information constitutes the largest systematic dataset for white-bellied swiftlets yet compiled, drawn from 809 individuals belonging to 32 taxa for which new molecular, biometric, and/or plumage data are presented. We propose changing the classification of white-bellied swiftlets, for which two species are currently recognized, to elevate eight regional forms to species level, and we also describe two new subspecies. The ten taxa we recommend recognizing at the species level are: Collocalia linchi (Java to Lombok, Sumatran hills), C. dodgei (montane Borneo), C. natalis (Christmas Island), C. affinis (Greater Sundas, including the Thai-Malay Peninsula and Andaman-Nicobar Islands), C. marginata (Philippines), C. isonota (Philippines), C. sumbawae (west Lesser Sundas), C. neglecta (east Lesser Sundas), C. esculenta (Sulawesi, Moluccas, New Guinea, Bismarck Archipelago, Solomon Islands), and C. uropygialis (Vanuatu, New Caledonia). Future molecular and morphological work is needed to resolve questions of speciation and population affinities in the Philippines, Christmas Island, Wallacea and central Melanesia, and to shed light on historic diversification and patterns of gene flow in the complex.

A robust nonparametric method for quantifying undetected extinctions.

How many species have gone extinct in modern times before being described by science? To answer this question, and thereby get a full assessment of humanity's impact on biodiversity, statistical methods that quantify undetected extinctions are required. Such methods have been developed recently, but they are limited by their reliance on parametric assumptions; specifically, they assume the pools of extant and undetected species decay exponentially, whereas real detection rates vary temporally with survey effort and real extinction rates vary with the waxing and waning of threatening processes. We devised a new, nonparametric method for estimating undetected extinctions. As inputs, the method requires only the first and last date at which each species in an ensemble was recorded. As outputs, the method provides estimates of the proportion of species that have gone extinct, detected, or undetected and, in the special case where the number of undetected extant species in the present day is assumed close to zero, of the absolute number of undetected extinct species. The main assumption of the method is that the per-species extinction rate is independent of whether a species has been detected or not. We applied the method to the resident native bird fauna of Singapore. Of 195 recorded species, 58 (29.7%) have gone extinct in the last 200 years. Our method projected that an additional 9.6 species (95% CI 3.4, 19.8) have gone extinct without first being recorded, implying a true extinction rate of 33.0% (95% CI 31.0%, 36.2%). We provide R code for implementing our method. Because our method does not depend on strong assumptions, we expect it to be broadly useful for quantifying undetected extinctions.

Ancient horizontal transfers of retrotransposons between birds and ancestors of human pathogenic nematodes.

Parasite host switches may trigger disease emergence, but prehistoric host ranges are often unknowable. Lymphatic filariasis and loiasis are major human diseases caused by the insect-borne filarial nematodes Brugia, Wuchereria and Loa. Here we show that the genomes of these nematodes and seven tropical bird lineages exclusively share a novel retrotransposon, AviRTE, resulting from horizontal transfer (HT). AviRTE subfamilies exhibit 83-99% nucleotide identity between genomes, and their phylogenetic distribution, paleobiogeography and invasion times suggest that HTs involved filarial nematodes. The HTs between bird and nematode genomes took place in two pantropical waves, >25-22 million years ago (Myr ago) involving the Brugia/Wuchereria lineage and >20-17 Myr ago involving the Loa lineage. Contrary to the expectation from the mammal-dominated host range of filarial nematodes, we hypothesize that these major human pathogens may have independently evolved from bird endoparasites that formerly infected the global breadth of avian biodiversity.

Population genetics of the olive-winged bulbul (Pycnonotus plumosus) in a tropical urban-fragmented landscape.

With increasing urbanization, urban-fragmented landscapes are becoming more and more prevalent worldwide. Such fragmentation may lead to small, isolated populations that face great threats from genetic factors that affect even avian species with high dispersal propensities. Yet few studies have investigated the population genetics of species living within urban-fragmented landscapes in the Old World tropics, in spite of the high levels of deforestation and fragmentation within this region. We investigated the evolutionary history and population genetics of the olive-winged bulbul (Pycnonotus plumosus) in Singapore, a highly urbanized island which retains <5% of its original forest cover in fragments. Combining our own collected and sequenced samples with those from the literature, we conducted phylogenetic and population genetic analyses. We revealed high genetic diversity, evidence for population expansion, and potential presence of pronounced gene flow across the population in Singapore. This suggests increased chances of long-term persistence for the olive-winged bulbul and the ecosystem services it provides within this landscape.