Bats live longer than similar-sized mammals, but the number of lineages that have independently evolved extreme longevity has not previously been determined. Here we reconstruct the evolution of size-corrected longevity on a recent molecular phylogeny and find that at least four lineages of bats have lifespans more than fourfold those of similar-sized placental mammals, with the ancestral bat projected to live 2.6 times as long. We then evaluate a series of phylogenetic generalized least-squares models containing up to nine variables hypothesized to influence extrinsic mortality. These analyses reveal that body mass and hibernation predict longevity. Among hibernators, longevity is predicted by the absolute value of the median latitude of the species range and cave use, while cave use and lack of sexual dimorphism predict longevity among non-hibernators. The importance of torpor in extending lifespan is further supported by the one lineage with extreme longevity that does not hibernate but exhibits flexible thermoregulation, the common vampire bat. We propose several potential mechanisms that may enable bats to live so long, and suggest that the ability to tolerate a wide range of body temperatures could be important for surviving viral or other pathogen infections.

Throughout their evolutionary history, genomes acquire new genetic material that facilitates phenotypic innovation and diversification. Developmental processes associated with reproduction are particularly likely to involve novel genes. Abundant gene creation impacts the evolution of chromosomal gene content and general regulatory mechanisms such as dosage compensation. Numerous studies in model organisms have found complex and, at times contradictory, relationships among these genomic attributes highlighting the need to examine these patterns in other systems characterized by abundant sexual selection. Therefore, we examined the association among novel gene creation, tissue-specific gene expression, and chromosomal gene content within stalk-eyed flies. Flies in this family are characterized by strong sexual selection and the presence of a newly evolved X chromosome. We generated RNA-seq transcriptome data from the testes for three species within the family and from seven additional tissues in the highly dimorphic species,Teleopsis dalmanni Analysis of dipteran gene orthology reveals dramatic testes-specific gene creation in stalk-eyed flies, involving numerous gene families that are highly conserved in other insect groups. Identification of X-linked genes for the three species indicates that the X chromosome arose prior to the diversification of the family. The most striking feature of this X chromosome is that it is highly masculinized, containing nearly twice as many testes-specific genes as expected based on its size. All the major processes that may drive differential sex chromosome gene content-creation of genes with male-specific expression, development of male-specific expression from pre-existing genes, and movement of genes with male-specific expression-are elevated on the X chromosome ofT. dalmanni This masculinization occurs despite evidence that testes expressed genes do not achieve the same levels of gene expression on the X chromosome as they do on the autosomes.

Many bird species produce temporally coordinated duets and choruses, requiring the rapid integration of auditory perception and motor production. While males and females of some species are known to participate in these displays for sex-specific purposes, few studies have identified perceptual features that trigger sex-specific contributions of coordinated song. Especially little is known about perception and production in duetting suboscine passerines, which are thought to have innate songs and largely static, rather than dynamic, vocal behavior. Here, we used synthetic stimuli in a playback experiment on chestnut-backed antbirds (Myrmeciza exsul) to (1) test whether differences in song frequency (Hz) can trigger sex-specific vocal behavior in a suboscine passerine (2) test for the functions of duetting in males and females of this species, and (3) determine whether these suboscines can dynamically adjust the temporal and spectral features of their songs. We found sex-specific responses to synthetic playback manipulated in song frequency (Hz), providing evidence that in this context males sing in duets for general territory defense and females join in for mate guarding purposes. In addition, we found that the birds altered the frequency, duration, and timing of their songs depending on the frequency of the playback songs. Thus, we show that these birds integrate spectral and temporal information about conspecific songs and actively modulate their responses in sex-specific ways.

Comparative analyses of bats indicate that hibernation is associated with increased longevity among species. However, it is not yet known if hibernation affects biological ageing of individuals. Here, we use DNA methylation (DNAm) as an epigenetic biomarker of ageing to determine the effect of hibernation on the big brown bat, Eptesicus fuscus. First, we compare epigenetic age, as predicted by a multi-species epigenetic clock, between hibernating and non-hibernating animals and find that hibernation is associated with epigenetic age. Second, we identify genomic sites that exhibit hibernation-associated change in DNAm, independent of age, by comparing samples taken from the same individual in hibernating and active seasons. This paired comparison identified over 3000 differentially methylated positions (DMPs) in the genome. Genome-wide association comparisons to tissue-specific functional elements reveals that DMPs with elevated DNAm during winter occur at sites enriched for quiescent chromatin states, whereas DMPs with reduced DNAm during winter occur at sites enriched for transcription enhancers. Furthermore, genes nearest DMPs are involved in regulation of metabolic processes and innate immunity. Finally, significant overlap exists between genes nearest hibernation DMPs and genes nearest previously identified longevity DMPs. Taken together, these results are consistent with hibernation influencing ageing and longevity in bats.

Exceptionally long-lived species, including many bats, rarely show overt signs of aging, making it difficult to determine why species differ in lifespan. Here, we use DNA methylation (DNAm) profiles from 712 known-age bats, representing 26 species, to identify epigenetic changes associated with age and longevity. We demonstrate that DNAm accurately predicts chronological age. Across species, longevity is negatively associated with the rate of DNAm change at age-associated sites. Furthermore, analysis of several bat genomes reveals that hypermethylated age- and longevity-associated sites are disproportionately located in promoter regions of key transcription factors (TF) and enriched for histone and chromatin features associated with transcriptional regulation. Predicted TF binding site motifs and enrichment analyses indicate that age-related methylation change is influenced by developmental processes, while longevity-related DNAm change is associated with innate immunity or tumorigenesis genes, suggesting that bat longevity results from augmented immune response and cancer suppression.

Observations of animals feeding in aggregations are often interpreted as events of social foraging, but it can be difficult to determine whether the animals arrived at the foraging sites after collective search [1-4] or whether they found the sites by following a leader [5, 6] or even independently, aggregating as an artifact of food availability [7, 8]. Distinguishing between these explanations is important, because functionally, they might have very different consequences. In the first case, the animals could benefit from the presence of conspecifics, whereas in the second and third, they often suffer from increased competition [3, 9-13]. Using novel miniature sensors, we recorded GPS tracks and audio of five species of bats, monitoring their movement and interactions with conspecifics, which could be inferred from the audio recordings. We examined the hypothesis that food distribution plays a key role in determining social foraging patterns [14-16]. Specifically, this hypothesis predicts that searching for an ephemeral resource (whose distribution in time or space is hard to predict) is more likely to favor social foraging [10, 13-15] than searching for a predictable resource. The movement and social interactions differed between bats foraging on ephemeral versus predictable resources. Ephemeral species changed foraging sites and showed large temporal variation nightly. They aggregated with conspecifics as was supported by playback experiments and computer simulations. In contrast, predictable species were never observed near conspecifics and showed high spatial fidelity to the same foraging sites over multiple nights. Our results suggest that resource (un)predictability influences the costs and benefits of social foraging.

Hearing loss is a hallmark of aging, typically initially affecting the higher frequencies. In echolocating bats, the ability to discern high frequencies is essential. However, nothing is known about age-related hearing loss in bats, and they are often assumed to be immune to it. We tested the hearing of 47 wild Egyptian fruit bats by recording their auditory brainstem response and cochlear microphonics, and we also assessed the cochlear histology in four of these bats. We used the bats' DNA methylation profile to evaluate their age and found that bats exhibit age-related hearing loss, with more prominent deterioration at the higher frequencies. The rate of the deterioration was ∼1 dB per year, comparable to the hearing loss observed in humans. Assessing the noise in the fruit bat roost revealed that these bats are exposed to continuous immense noise-mostly of social vocalizations-supporting the assumption that bats might be partially resistant to loud noise. Thus, in contrast to previous assumptions, our results suggest that bats constitute a model animal for the study of age-related hearing loss.

Polymorphisms of single amino acid repeats (SARPs) are a potential source of genetic variation for rapidly evolving morphological traits. Here, we characterize variation in and test for an association between SARPs and head shape, a trait under strong sexual selection, in the stalk-eyed fly, Teleopsis dalmanni. Using an annotated expressed sequence tag database developed from eye-antennal imaginal disc tissues in T. dalmanni we identified 98 genes containing nine or more consecutive copies of a single amino acid. We then quantify variation in length and allelic diversity for 32 codon and 15 noncodon repeat regions in a large outbred population. We also assessed the frequency with which amino acid repeats are either gained or lost by identifying sequence similarities between T. dalmanni SARP loci and their orthologs in Drosophila melanogaster. Finally, to identify SARP containing genes that may influence head development we conducted a two-generation association study after assortatively mating for extreme relative eyespan.

In Drosophila, the Enhancer of split complex (E(spl)-C) comprises 11 bHLH and Bearded genes that function during Notch signaling to repress proneural identity in the developing peripheral nervous system. Comparison with other insects indicates that the basal state for Diptera is a single bHLH and Bearded homolog and that the expansion of the gene complex occurred in the lineage leading to Drosophila. However, comparative genomic data from other fly species that would elucidate the origin and sequence of gene duplication for the complex is lacking. Therefore, in order to examine the evolutionary history of the complex within Diptera, we reconstructed, using several fosmid clones, the entire E(spl)-complex in the stalk-eyed fly, Teleopsis dalmanni and collected additional homologs of E(spl)-C genes from searches of dipteran EST databases and the Glossina morsitans genome assembly.

Although sex chromosome meiotic drive has been observed in a variety of species for over 50 years, the genes causing drive are only known in a few cases, and none of these cases cause distorted sex-ratios in nature. In stalk-eyed flies (Teleopsis dalmanni), driving X chromosomes are commonly found at frequencies approaching 30% in the wild, but the genetic basis of drive has remained elusive due to reduced recombination between driving and non-driving X chromosomes. Here, we used RNAseq to identify transcripts that are differentially expressed between males carrying either a driving X (XSR) or a standard X chromosome (XST), and found hundreds of these, the majority of which are X-linked. Drive-associated transcripts show increased levels of sequence divergence (dN/dS) compared to a control set, and are predominantly expressed either in testes or in the gonads of both sexes. Finally, we confirmed that XSR and XST are highly divergent by estimating sequence differentiation between the RNAseq pools. We found that X-linked transcripts were often strongly differentiated (whereas most autosomal transcripts were not), supporting the presence of a relatively large region of recombination suppression on XSR presumably caused by one or more inversions. We have identified a group of genes that are good candidates for further study into the causes and consequences of sex-chromosome drive, and demonstrated that meiotic drive has had a profound effect on sequence evolution and gene expression of X-linked genes in this species.

Sex chromosome meiotic drive has been suggested as a cause of several evolutionary genetic phenomena, including genomic conflicts that give rise to reproductive isolation between new species. In this paper we present a population genetic analysis of X chromosome drive in the stalk-eyed fly, Teleopsis dalmanni, to determine how this natural polymorphism influences genetic diversity. We analyzed patterns of DNA sequence variation at two X-linked regions (comprising 1325 bp) approximately 50 cM apart and one autosomal region (comprising 921 bp) for 50 males, half of which were collected in the field from one of two allopatric locations and the other half were derived from lab-reared individuals with known brood sex ratios. These two populations are recently diverged but exhibit partial postzygotic reproductive isolation, i.e. crosses produce sterile hybrid males and fertile females. We find no nucleotide or microsatellite variation on the drive X chromosome, whereas the same individuals show levels of variation at autosomal regions that are similar to field-collected flies. Furthermore, one field-caught individual collected 10 years previously had a nearly identical X haplotype to the drive X, and is over 2% divergent from other haplotypes sampled from the field. These results are consistent with a selective sweep that has removed genetic variation from much of the drive X chromosome. We discuss how this finding may relate to the rapid evolution of postzygotic reproductive isolation that has been documented for these flies.

Many species of stalk-eyed flies (Diopsidae) possess highly-exaggerated, sexually dimorphic eye-stalks that play an important role in the mating system of these flies. Eye-stalks are increasingly being used as a model system for studying sexual selection, but little is known about the genetic mechanisms producing variation in these ornamental traits. Therefore, we constructed an EST database of genes expressed in the developing eye-antennal imaginal disc of the highly dimorphic species Teleopsis dalmanni. We used this set of genes to construct microarray slides and compare patterns of gene expression between lines of flies with divergent eyespan.

Stalk-eyed flies (family Diopsidae) are a model system for studying sexual selection due to the elongated and sexually dimorphic eye-stalks found in many species. These flies are of additional interest because their X chromosome is derived largely from an autosomal arm in other flies. To identify candidate genes required for development of dimorphic eyestalks and investigate how sex-biased expression arose on the novel X, we compared gene expression between males and females using oligonucleotide microarrays and RNA from developing eyestalk tissue or adult heads in the dimorphic diopsid, Teleopsis dalmanni. Microarray analysis revealed sex-biased expression for 26% of 3,748 genes expressed in eye-antennal imaginal discs and concordant sex-biased expression for 86 genes in adult heads. Overall, 415 female-biased and 482 male-biased genes were associated with dimorphic eyestalk development but not differential expression in the adult head. Functional analysis revealed that male-biased genes are disproportionately associated with growth and mitochondrial function while female-biased genes are associated with cell differentiation and patterning or are novel transcripts. With regard to chromosomal effects, dosage compensation occurs by elevated expression of X-linked genes in males. Genes with female-biased expression were more common on the X and less common on autosomes than expected, while male-biased genes exhibited no chromosomal pattern. Rates of protein evolution were lower for female-biased genes but higher for genes that moved on or off the novel X chromosome. These findings cannot be due to meiotic sex chromosome inactivation or by constraints associated with dosage compensation. Instead, they could be consistent with sexual conflict in which female-biased genes on the novel X act primarily to reduce eyespan in females while other genes increase eyespan in both sexes. Additional information on sex-biased gene expression in other tissues and related sexually monomorphic species could confirm this interpretation.

For echolocating bats, hearing is essential for survival. Specializations for detecting and processing high frequency sounds are apparent throughout their auditory systems. Recent studies on echolocating mammals have reported evidence of parallel evolution in some hearing-related genes in which distantly related groups of echolocating animals (bats and toothed whales), cluster together in gene trees due to apparent amino acid convergence. However, molecular adaptations can occur not only in coding sequences, but also in the regulation of gene expression. The aim of this study was to examine the expression of hearing-related genes in the inner ear of developing big brown bats, Eptesicus fuscus, during the period in which echolocation vocalizations increase dramatically in frequency. We found that seven genes were significantly upregulated in juveniles relative to adults, and that the expression of four genes through development correlated with estimated age. Compared to available data for mice, it appears that expression of some hearing genes is extended in juvenile bats. These results are consistent with a prolonged growth period required to develop larger cochlea relative to body size, a later maturation of high frequency hearing, and a greater dependence on high frequency hearing in echolocating bats.

Multiple methods have been developed to infer behavioral states from animal movement data, but rarely has their accuracy been assessed from independent evidence, especially for location data sampled with high temporal resolution. Here we evaluate the performance of behavioral segmentation methods using acoustic recordings that monitor prey capture attempts.

Stalk-eyed flies in the genus Teleopsis carry selfish genetic elements that induce sex ratio (SR) meiotic drive and impact the fitness of male and female carriers. Here, we assemble and describe a chromosome-level genome assembly of the stalk-eyed fly, Teleopsis dalmanni, to elucidate patterns of divergence associated with SR. The genome contains tens of thousands of transposable element (TE) insertions and hundreds of transcriptionally and insertionally active TE families. By resequencing pools of SR and ST males using short and long reads, we find widespread differentiation and divergence between XSR and XST associated with multiple nested inversions involving most of the SR haplotype. Examination of genomic coverage and gene expression data revealed seven X-linked genes with elevated expression and coverage in SR males. The most extreme and likely drive candidate involves an XSR-specific expansion of an array of partial copies of JASPer, a gene necessary for maintenance of euchromatin and associated with regulation of TE expression. In addition, we find evidence for rapid protein evolution between XSR and XST for testis expressed and novel genes, that is, either recent duplicates or lacking a Dipteran ortholog, including an X-linked duplicate of maelstrom, which is also involved in TE silencing. Overall, the evidence suggests that this ancient XSR polymorphism has had a variety of impacts on repetitive DNA and its regulation in this species.