Hybrid de novo genome assembly of the sexually dimorphic Lady Amherst's pheasant.

Pheasants are an important group of birds, valued for their economic benefit as poultry birds, game birds, and as ornamental species for their plumage. Lady Amherst's pheasant Chrysolophus amherstiae is an ornamental species, valued for its elaborate and beautiful plumage. In this study, we present a high-quality de novo hybrid genome assembly of C. amherstiae. Previous attempts to sequence the genome of this species resulted in draft-level assemblies, which are not available in the public domain. Using a combination of Illumina short reads and Oxford Nanopore's long-reads, we assembled a high-quality genome of N50 ~3.9 Mb and near complete BUSCO assessment. We observed a correlation between effective population size and past climatic conditions, with an increase in population size during the warm interglacial periods. We further observed significant fluctuations in genes involved with the immune system and visual perception. C. amherstiae is a highly dimorphic species, and significant fluctuations in gene families involved in immune response, visual perception, among others, suggesting a role of mate choice and sexual selection in the evolution and maintenance of exaggerated traits in the males.

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.

Megafaunal extinctions, not climate change, may explain Holocene genetic diversity declines in Numenius shorebirds.

Understanding the relative contributions of historical and anthropogenic factors to declines in genetic diversity is important for informing conservation action. Using genome-wide DNA of fresh and historic specimens, including that of two species widely thought to be extinct, we investigated fluctuations in genetic diversity and present the first complete phylogenomic tree for all nine species of the threatened shorebird genus Numenius, known as whimbrels and curlews. Most species faced sharp declines in effective population size, a proxy for genetic diversity, soon after the Last Glacial Maximum (around 20,000 years ago). These declines occurred prior to the Anthropocene and in spite of an increase in the breeding area predicted by environmental niche modeling, suggesting that they were not caused by climatic or recent anthropogenic factors. Crucially, these genetic diversity declines coincide with mass extinctions of mammalian megafauna in the Northern Hemisphere. Among other factors, the demise of ecosystem-engineering megafauna which maintained open habitats may have been detrimental for grassland and tundra-breeding Numenius shorebirds. Our work suggests that the impact of historical factors such as megafaunal extinction may have had wider repercussions on present-day population dynamics of open habitat biota than previously appreciated.

About 20,000 years ago, the Earth was a much colder world roamed by giant mastodons, gigantic elks, woolly mammoths and sabre-tooth tigers. Yet these imposing creatures were living on borrowed time: by the start of the Holocene, around 10,000 years later, many animals over 45kg had vanished across the Northern Hemisphere, closing the book on what is known as the Quaternary extinction event. As large grazers disappeared, the landscape likely changed too. Where open tundra and grasslands may have once dominated, woodlands and shrubs probably took over, creating ripple effects for surviving species. These extinction events took place in a changing world, with glaciers starting to retreat about 20,000 years ago and human populations colonizing an increasing share of this planet's land area. In fact, since the end of this last glacial maximum, ecosystems have been reshaped by a succession and a combination of climatic, historical and human-driven forces. This makes it difficult for scientists to disentangle the relative contribution of these factors on the lives of animals. Tan et al. decided to explore this question by reconstructing how effective population sizes changed over the past 20,000 years for nine species of curlews and whimbrels. These shorebirds, which together comprise the genus Numenius, breed slowly and nest in open environments such as moorlands or tundra. Many are currently under threat. Fluctuations in the numbers of breeding individuals affect the genetic diversity of a species, and these events leave tell-tale genetic signatures that can be uncovered through DNA analyses. Tan et al. had enough fresh and museum samples to infer these changes for five Numenius species, revealing that genetic diversity brutally dropped soon after the last glacial period ended. At the time, humans were yet to make significant changes on their environment and a warming world should have supported population growth. Tan et al. suggest that, instead, this sharp decline is linked to the late Quaternary extinctions of large mammals: with the demise of grazing animals which could keep woodlands at bay, the shorebirds lost their open nesting grounds. This event has left its mark in the genome of existing species, with these birds still exhibiting a low level of genetic diversity that may put them at further risk for extinction.

Genomic Insights Into the Evolution and Demographic History of the SARS-CoV-2 Omicron Variant: Population Genomics Approach.

Background: A thorough understanding of the patterns of genetic subdivision in a pathogen can provide crucial information that is necessary to prevent disease spread. For SARS-CoV-2, the availability of millions of genomes makes this task analytically challenging, and traditional methods for understanding genetic subdivision often fail. Objective: The aim of our study was to use population genomics methods to identify the subtle subdivisions and demographic history of the Omicron variant, in addition to those captured by the Pango lineage. Methods: We used a combination of an evolutionary network approach and multivariate statistical protocols to understand the subdivision and spread of the Omicron variant. We identified subdivisions within the BA.1 and BA.2 lineages and further identified the mutations associated with each cluster. We further characterized the overall genomic diversity of the Omicron variant and assessed the selection pressure for each of the genetic clusters identified. Results: We observed concordant results, using two different methods to understand genetic subdivision. The overall pattern of subdivision in the Omicron variant was in broad agreement with the Pango lineage definition. Further, 1 cluster of the BA.1 lineage and 3 clusters of the BA.2 lineage revealed statistically significant signatures of selection or demographic expansion (Tajima's D<-2), suggesting the role of microevolutionary processes in the spread of the virus. Conclusions: We provide an easy framework for assessing the genetic structure and demographic history of SARS-CoV-2, which can be particularly useful for understanding the local history of the virus. We identified important mutations that are advantageous to some lineages of Omicron and aid in the transmission of the virus. This is crucial information for policy makers, as preventive measures can be designed to mitigate further spread based on a holistic understanding of the variability of the virus and the evolutionary processes aiding its spread.

Next Generation Sequencing Revolutionizes Organismal Biology Research in Bats.

The advent of next generation sequencing technologies (NGS) has greatly accelerated our understanding of critical aspects of organismal biology from non-model organisms. Bats form a particularly interesting group in this regard, as genomic data have helped unearth a vast spectrum of idiosyncrasies in bat genomes associated with bat biology, physiology, and evolution. Bats are important bioindicators and are keystone species to many eco-systems. They often live in proximity to humans and are frequently associated with emerging infectious diseases, including the COVID-19 pandemic. Nearly four dozen bat genomes have been published to date, ranging from drafts to chromosomal level assemblies. Genomic investigations in bats have also become critical towards our understanding of disease biology and host-pathogen coevolution. In addition to whole genome sequencing, low coverage genomic data like reduced representation libraries, resequencing data, etc. have contributed significantly towards our understanding of the evolution of natural populations, and their responses to climatic and anthropogenic perturbations. In this review, we discuss how genomic data have enhanced our understanding of physiological adaptations in bats (particularly related to ageing, immunity, diet, etc.), pathogen discovery, and host pathogen co-evolution. In comparison, the application of NGS towards population genomics, conservation, biodiversity assessment, and functional genomics has been appreciably slower. We reviewed the current areas of focus, identifying emerging topical research directions and providing a roadmap for future genomic studies in bats.

Using Environmental Sampling to Enable Zoonotic Pandemic Preparedness.

The current pandemic caused by the SARS CoV-2, tracing back its origin possibly to a coronavirus associated with bats, has ignited renewed interest in understanding zoonotic spillovers across the globe. While research is more directed towards solving the problem at hand by finding therapeutic strategies and novel vaccine techniques, it is important to address the environmental drivers of pathogen spillover and the complex biotic and abiotic drivers of zoonoses. The availability of cutting-edge genomic technologies has contributed enormously to preempt viral emergence from wildlife. However, there is still a dearth of studies from species-rich South Asian countries, especially from India. In this review, we outline the importance of studying disease dynamics through environmental sampling from wildlife in India and how ecological parameters of both the virus and the host community may play a role in mediating cross-species spillovers. Non-invasive sampling using feces, urine, shed hair, saliva, shed skin, and feathers has been instrumental in providing genetic information for both the host and their associated pathogens. Here, we discuss the advances made in environmental sampling protocols and strategies to generate genetic data from such samples towards the surveillance and characterization of potentially zoonotic pathogens. We primarily focus on bat-borne or small mammal-borne zoonoses and propose a conceptual framework for non-invasive strategies to tackle the threat of emerging zoonotic infections.

Island Biogeography Revisited: Museomics Reveals Affinities of Shelf Island Birds Determined by Bathymetry and Paleo-Rivers, Not by Distance to Mainland.

Island biogeography is one of the most powerful subdisciplines of ecology: its mathematical predictions that island size and distance to mainland determine diversity have withstood the test of time. A key question is whether these predictions follow at a population-genomic level. Using rigorous ancient-DNA protocols, we retrieved approximately 1,000 genomic markers from approximately 100 historic specimens of two Southeast Asian songbird complexes from across the Sunda Shelf archipelago collected 1893-1957. We show that the genetic affinities of populations on small shelf islands defy the predictions of geographic distance and appear governed by Earth-historic factors including the position of terrestrial barriers (paleo-rivers) and persistence of corridors (Quaternary land bridges). Our analyses suggest that classic island-biogeographic predictors may not hold well for population-genomic dynamics on the thousands of shelf islands across the globe, which are exposed to dynamic changes in land distribution during Quaternary climate change.

Novel genome reveals susceptibility of popular gamebird, the red-legged partridge (Alectoris rufa, Phasianidae), to climate change.

We produced a high-quality de novo genome assembly of the red-legged partridge A. rufa, the first reference genome of its genus, by utilising novel 10× Chromium technology. The estimated genome size was 1.19 Gb with an overall genome heterozygosity of 0.0022; no runs of homozygosity were observed. In total, 21,589 protein coding genes were identified and assigned to 16,772 orthologs. Of these, 201 emerged as unique to Alectoris and were enriched for positive regulation of epithelial cell migration, viral genome integration and maturation. Using PSMC analysis, we inferred a major demographic decline commencing ~140,000 years ago, consistent with forest expansion and reduction of open habitats during the Eemian interglacial. Present-day populations exhibit the historically lowest genetic diversity. Besides implications for management and conservation, this genome also promises key insights into the physiology of these birds with a view to improving poultry husbandry practices.

Gene Flow in Volant Vertebrates: Species Biology, Ecology and Climate Change.

Gene flow, the exchange of genetic material between populations is an important biological process, which shapes and maintains biodiversity. The successful movement of individuals between populations depends on multiple factors determined by species biology and the environment. One of the most important factors regulating gene flow is the ability to move, and flight allows individuals to easily move across geographical barriers. Volant vertebrates are found on some of the remotest islands and contribute significantly to the biodiversity and ecosystem. The availability of next-generation sequencing data for non-model animals has substantially improved our understanding of gene flow and its consequences, allowing us to look at fine-scale patterns. However, most of our understanding regarding gene flow comes from the temperate regions and the Neotropics. The lack of studies from species-rich Asia is striking. In this review, we outline the importance of gene flow and the factors affecting gene flow, especially for volant vertebrates. We especially discuss research studies from tropical biomes of South and Southeast Asia, highlight the lacuna in literature and provide an outline for future studies in this species-rich region.

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.

Last Glacial Maximum led to community-wide population expansion in a montane songbird radiation in highland Papua New Guinea.

BACKGROUND: Quaternary climate fluctuations are an engine of biotic diversification. Global cooling cycles, such as the Last Glacial Maximum (LGM), are known to have fragmented the ranges of higher-latitude fauna and flora into smaller refugia, dramatically reducing species ranges. However, relatively less is known about the effects of cooling cycles on tropical biota. RESULTS: We analyzed thousands of genome-wide DNA markers across an assemblage of three closely related understorey-inhabiting scrubwrens (Sericornis and Aethomyias; Aves) from montane forest along an elevational gradient on Mt. Wilhelm, the highest mountain of Papua New Guinea. Despite species-specific differences in elevational preference, we found limited differentiation within each scrubwren species, but detected a strong genomic signature of simultaneous population expansions at 27-29 ka, coinciding with the onset of the LGM. CONCLUSION: The remarkable synchronous timing of population expansions of all three species demonstrates the importance of global cooling cycles in expanding highland habitat. Global cooling cycles have likely had strongly different impacts on tropical montane areas versus boreal and temperate latitudes, leading to population expansions in the former and serious fragmentation in the latter.

Quaternary land bridges have not been universal conduits of gene flow.

Quaternary climate oscillations are a well-known driver of animal diversification, but their effects are most well studied in areas where glaciations lead to habitat fragmentation. In large areas of the planet, however, glaciations have had the opposite effect, but here their impacts are much less well understood. This is especially true in Southeast Asia, where cyclical changes in land distribution have generated enormous land expansions during glacial periods. In this study, we selected a panel of five songbird species complexes covering a range of ecological specificities to investigate the effects Quaternary land bridges have had on the connectivity of Southeast Asian forest biota. Specifically, we combined morphological and bioacoustic analysis with an arsenal of population genomic and modelling approaches applied to thousands of genome-wide DNA markers across a total of more than 100 individuals. Our analyses show that species dependent on forest understorey exhibit deep differentiation between Borneo and western Sundaland, with no evidence of gene flow during the land bridges accompanying the last 1-2 ice ages. In contrast, dispersive canopy species and habitat generalists have experienced more recent gene flow. Our results argue that there remains much cryptic species-level diversity to be discovered in Southeast Asia even in well-known animal groups such as birds, especially in nondispersive forest understorey inhabitants. We also demonstrate that Quaternary land bridges have not been equally suitable conduits of gene flow for all species complexes and that life history is a major factor in predicting relative population divergence time across Quaternary climate fluctuations.

Chromosome-level assembly of the horseshoe crab genome provides insights into its genome evolution.

The evolutionary history of horseshoe crabs, spanning approximately 500 million years, is characterized by remarkable morphological stasis and a low species diversity with only four extant species. Here we report a chromosome-level genome assembly for the mangrove horseshoe crab (Carcinoscorpius rotundicauda) using PacBio reads and Hi-C data. The assembly spans 1.67 Gb with contig N50 of 7.8 Mb and 98% of the genome assigned to 16 chromosomes. The genome contains five Hox clusters with 34 Hox genes, the highest number reported in any invertebrate. Detailed analysis of the genome provides evidence that suggests three rounds of whole-genome duplication (WGD), raising questions about the relationship between WGD and species radiation. Several gene families, particularly those involved in innate immunity, have undergone extensive tandem duplication. These expanded gene families may be important components of the innate immune system of horseshoe crabs, whose amebocyte lysate is a sensitive agent for detecting endotoxin contamination.

Novel de Novo Genome of Cynopterus brachyotis Reveals Evolutionarily Abrupt Shifts in Gene Family Composition across Fruit Bats.

Major novel physiological or phenotypic adaptations often require accompanying modifications at the genic level. Conversely, the detection of considerable contractions and/or expansions of gene families can be an indicator of fundamental but unrecognized physiological change. We sequenced a novel fruit bat genome (Cynopterus brachyotis) and adopted a comparative approach to reconstruct the evolution of fruit bats, mapping contractions and expansions of gene families along their evolutionary history. Despite a radical change in life history as compared with other bats (e.g., loss of echolocation, large size, and frugivory), fruit bats have undergone surprisingly limited change in their genic composition, perhaps apart from a potentially novel gene family expansion relating to telomere protection and longevity. In sharp contrast, within fruit bats, the new Cynopterus genome bears the signal of unusual gene loss and gene family contraction, despite its similar morphology and lifestyle to two other major fruit bat lineages. Most missing genes are regulatory, immune-related, and olfactory in nature, illustrating the diversity of genomic strategies employed by bats to contend with responses to viral infection and olfactory requirements. Our results underscore that significant fluctuations in gene family composition are not always associated with obvious examples of novel physiological and phenotypic adaptations but may often relate to less-obvious shifts in immune strategies.

Historic reveals Anthropocene threat to a tropical urban fruit bat.

Anthropogenic activities have propelled the Earth into a crisis characterized by unprecedented levels of environmental degradation and habitat loss, generating changes in global climatic regimes and initiating the planet's Sixth Extinction Catastrophe [1]. Loss of population genetic diversity is known to be a harbinger of local and global extinction events [2]. However, there is a lack of direct empirical evidence of historic losses of genetic diversity through periods of anthropogenically linked environmental degradation. We present genomic DNA information from a population of Sunda fruit bats (Cynopterus brachyotis) from Singapore, an exceptionally well-studied tropical rainforest island that has undergone substantial environmental degradation and fragmentation through the Anthropocene of the 1930-1950s [3]. As an effective pollinator and seed disperser, C. brachyotis represents an important keystone species in Singapore's ecosystem [4]. Here we show that comparison of historic DNA from individuals collected in 1931 with modern specimens reveals a nearly 30-fold reduction in effective population size and corresponding levels of decline in genetic diversity estimates. Coalescent population models indicate that Singapore's C. brachyotis bats underwent a continuous decline in genetic diversity followed by a stark bottleneck in approximately the 1940s, consistent with the estimated onset of the Anthropocene [5]. C. brachyotis continues to be considered common across Singapore [4], yet our results reveal large-scale impacts of the Anthropocene on biotic communities, even in those species thought to be tolerant to the effects of environmental degradation.

Fluctuating fortunes: genomes and habitat reconstructions reveal global climate-mediated changes in bats' genetic diversity.

Over the last approximately 2.6 Myr, Earth's climate has been dominated by cyclical ice ages that have profoundly affected species' population sizes, but the impact of impending anthropogenic climate change on species' extinction potential remains a worrying problem. We investigated 11 bat species from different taxonomic, ecological and geographical backgrounds using combined information from palaeoclimatic habitat reconstructions and genomes to analyse biotic impacts of historic climate change. We discover tightly correlated fluctuations between species' historic distribution and effective population size, identify frugivores as particularly susceptible to global warming, pinpoint large insectivores as having overall low effective population size and flag the onset of the Holocene (approx. 10-12 000 years ago) as the period with the generally lowest effective population sizes across the last approximately 1 Myr. Our study shows that combining genomic and palaeoclimatological approaches reveals effects of climatic shifts on genetic diversity and may help predict impacts of future climate change.

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.

Conservation genomics in the fight to help the recovery of the critically endangered Siamese crocodile Crocodylus siamensis.

Endangered species are often characterized by low genetic diversity and it is imperative for conservation efforts to incorporate the knowledge obtained from genetic studies for effective management. However, despite the promise of technological advances in sequencing, application of genome-wide data to endangered populations remains uncommon. In the present study we pursued a holistic conservation-genomic approach to inform a field-based management programme of a Critically Endangered species, the Siamese crocodile Crocodylus siamensis. Using thousands of single nucleotide polymorphisms from throughout the genome, we revealed signals of introgression from two other crocodile species within our sample of both wild and captive-bred Siamese crocodiles from Cambodia. Our genetic screening of the Siamese crocodiles resulted in the subsequent re-introduction of 12 individuals into the wild as well as the selection of four individuals for captive breeding programmes. Comparison of intraspecific genetic diversity revealed an alarmingly low contemporary effective population size in the wild (<50) with evidence of a recent bottleneck around Tonle Sap Lake. We also projected a probable future extinction in the wild (within fewer than five generations) in this population in the absence of re-introduction efforts. However, an increase in the number of potential breeders through re-introductions, including the one resulting from this project, could counter this trend. Our results have been implemented in ongoing re-introduction and captive breeding programmes, with major implications for the conservation management of Siamese crocodiles, and provide a blueprint for the rescue effort of other "terminally ill" populations of critically endangered species.

Novel genome and genome-wide SNPs reveal early fragmentation effects in an edge-tolerant songbird population across an urbanized tropical metropolis.

Although edge-tolerant species are known to benefit from habitat fragmentation, less is known about the population genetic impacts fragmentation may exert on edge-tolerant species. We examined the landscape genomic structure of an edge-tolerant forest-dependent bird species, the Striped Tit-Babbler Mixornis gularis, in the heavily urbanized island of Singapore to determine if two centuries of fragmentation have led to signs of isolation and loss of population-genetic diversity in different parts of the island. We obtained a high-quality complete reference genome with 78x coverage. Using almost 4000 SNPs from double-digest RAD-Sequencing across 46 individuals, we found that the population has likely experienced a recent contraction in effective population size and presently exhibits low population genetic diversity. Using empirical and simulation-based landscape genomic analyses, we also found that the subtle population genetic structure observed in the Striped Tit-Babbler population in Singapore is likely driven by isolation by distance resulting from limited dispersal. Our results demonstrate that population genetic impoverishment and subdivision can accumulate at relatively rapid rates in edge-tolerant bird species such as the Striped Tit-Babbler as a result of fragmentation, and that subtle spatial genetic structure can be detected over fine spatial and temporal scales using relatively few multilocus genomic SNPs.