Bats possess extraordinary adaptations, including flight, echolocation, extreme longevity and unique immunity. High-quality genomes are crucial for understanding the molecular basis and evolution of these traits. Here we incorporated long-read sequencing and state-of-the-art scaffolding protocols 1 to generate, to our knowledge, the first reference-quality genomes of six bat species (Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pipistrellus kuhlii and Molossus molossus). We integrated gene projections from our 'Tool to infer Orthologs from Genome Alignments' (TOGA) software with de novo and homology gene predictions as well as short-and long-read transcriptomics to generate highly complete gene annotations. To resolve the phylogenetic position of bats within Laurasiatheria, we applied several phylogenetic methods to comprehensive sets of orthologous protein-coding and noncoding regions of the genome, and identified a basal origin for bats within Scrotifera. Our genome-wide screens revealed positive selection on hearing-related genes in the ancestral branch of bats, which is indicative of laryngeal echolocation being an ancestral trait in this clade. We found selection and loss of immunity-related genes (including pro-inflammatory NF-κB regulators) and expansions of anti-viral APOBEC3 genes, which highlights molecular mechanisms that may contribute to the exceptional immunity of bats. Genomic integrations of diverse viruses provide a genomic record of historical tolerance to viral infection in bats. Finally, we found and experimentally validated bat-specific variation in microRNAs, which may regulate bat-specific gene-expression programs. Our reference-quality bat genomes provide the resources required to uncover and validate the genomic basis of adaptations of bats, and stimulate new avenues of research that are directly relevant to human health and disease 1. With more than 1,400 species identified to date 2 , bats (Chiroptera) account for about 20% of all extant mammal species. Bats are found around the world and successfully occupy diverse ecological niches 1. Their global success is attributed to an extraordinary suite of adaptations 1 including powered flight, laryngeal echolocation, vocal learning, exceptional longevity and a unique immune system that probably enables bats to better tolerate viruses that are lethal to other mammals (such as severe acute respiratory syndrome-related coronavirus, Middle East respiratory syndrome-related coronavirus and Ebola virus) 3. Bats therefore represent important model systems for the study of

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.

42 43 ^Joint first authors 44 *joint senior/corresponding authors emails 45 Michael Hiller: hiller@mpi-cbg.de 46 Sonja Vernes: sonja.vernes@mpi.nl 47 Eugene Myers: gene@mpi-cbg.de 48 Emma C. Teeling: emma.teeling@ucd.ie 49 2 Abstract: Bats account for ~20% of all extant mammal species and are considered exceptional 50given their extraordinary adaptations, including biosonar, true flight, extreme longevity, and 51 unparalleled immune systems. To understand these adaptations, we generated reference-quality 52 genomes of six species representing the key divergent lineages. We assembled these genomes 53 with a novel pipeline incorporating state-of-the-art long-read and long-range sequencing and 54 assembly techniques. The genomes were annotated using a maximal evidence approach, de 55 novo predictions, protein/mRNA alignments, Iso-seq long read and RNA-seq short read 56 transcripts, and gene projections from our new TOGA pipeline, retrieving virtually all (>99%) 57 mammalian BUSCO genes. Phylogenetic analyses of 12,931 protein coding-genes and 10,857 58 conserved non-coding elements identified across 48 mammalian genomes helped to resolve 59 bats' closest extant relatives within Laurasiatheria, supporting a basal position for bats within 60 Scrotifera. Genome-wide screens along the bat ancestral branch revealed (a) selection on 61 hearing-involved genes (e.g LRP2, SERPINB6, TJP2), which suggest that laryngeal 62 echolocation is a shared ancestral trait of bats; (b) selection (e.g INAVA, CXCL13, NPSR1) and 63 loss of immunity related proteins (e.g. LRRC70, IL36G), including pro-inflammatory NF-kB 64 signalling; and (c) expansion of the APOBEC family, associated with restricting viral infection, 65 transposon activity and interferon signalling. We also identified unique integrated viruses, 66 indicating that bats have a history of tolerating viral pathogens, lethal to other mammal species. 67Non-coding RNA analyses identified variant and novel microRNAs, revealing regulatory 68 relationships that may contribute to phenotypic diversity in bats. Together, our reference-69 quality genomes, high-quality annotations, genome-wide screens and in-vitro tests revealed 70 previously unknown genomic adaptations in bats that may explain their extraordinary traits. 71 72

Telomeres are used increasingly in ecology and evolution as biomarkers for ageing and environmental stress, and are typically measured from DNA extracted from nonlethally sampled blood. However, obtaining blood is not always possible in field conditions and only limited amounts can be taken from small mammals, such as bats, which moreover lack nucleated red blood cells and hence yield relatively low amounts of DNA. As telomere length can vary within species according to age and tissue, it is important to determine which tissues serve best as a representation of the organism as a whole. Here, we investigated whether wing tissue biopsies, a rapid and relatively noninvasive tissue collection method, could serve as a proxy for other tissues when measuring relative telomere length (rTL) in the Egyptian fruit bat (Rousettus aegyptiacus). Telomeres were measured from blood, brain, heart, kidney, liver lung, muscle and wing, and multiple wing biopsies were taken from the same individuals to determine intra‐individual repeatability of rTL measured by using qPCR. Wing rTL correlated with rTL estimates from most tissues apart from blood. Blood rTL was not significantly correlated with rTL from any other tissue. Blood and muscle rTLs were significantly longer compared with other tissues, while lung displayed the shortest rTLs. Individual repeatability of rTL measures from wing tissue was high (>70%). Here we show the relationships between tissue telomere dynamics for the first time in a bat, and our results provide support for the use of wing tissue for rTL measurements.

Bats hold considerable potential for understanding exceptional longevity because some species can live eight times longer than other mammals of similar size [1]. Estimating their age or longevity is difficult because they show few signs of aging. DNA methylation (DNAm) provides a potential solution given its utility for estimating age [2-4] and lifespan [5-7] in humans. Here, we profile DNAm from wing biopsies of nearly 700 individuals representing 26 bat species and demonstrate that DNAm can predict chronological age accurately. Furthermore, the rate DNAm changes at age-informative sites is negatively related to longevity. To identify longevity-informative sites, we compared DNAm rates between three long-lived and two short-lived species. Hypermethylated age and longevity sites are enriched for histone and chromatin features associated with transcriptional regulation and preferentially located in the promoter regions of helix-turn-helix transcription factors (TFs). 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, in part, from augmented immune response and cancer suppression.