Inhibition of METTL3 Results in a Cell-Intrinsic Interferon Response That Enhances Antitumor Immunity.

Therapies that enhance antitumor immunity have altered the natural history of many cancers. Consequently, leveraging nonoverlapping mechanisms to increase immunogenicity of cancer cells remains a priority. Using a novel enzymatic inhibitor of the RNA methyl-transferase METTL3, we demonstrate a global decrease in N6-methyladenosine (m6A) results in double-stranded RNA (dsRNA) formation and a profound cell-intrinsic interferon response. Through unbiased CRISPR screens, we establish dsRNA-sensing and interferon signaling are primary mediators that potentiate T-cell killing of cancer cells following METTL3 inhibition. We show in a range of immunocompetent mouse models that although METTL3 inhibition is equally efficacious to anti-PD-1 therapy, the combination has far greater preclinical activity. Using SPLINTR barcoding, we demonstrate that anti-PD-1 therapy and METTL3 inhibition target distinct malignant clones, and the combination of these therapies overcomes clones insensitive to the single agents. These data provide the mole-cular and preclinical rationale for employing METTL3 inhibitors to promote antitumor immunity in the clinic. SIGNIFICANCE: This work demonstrates that METTL3 inhibition stimulates a cell-intrinsic interferon response through dsRNA formation. This immunomodulatory mechanism is distinct from current immunotherapeutic agents and provides the molecular rationale for combination with anti-PD-1 immune-checkpoint blockade to augment antitumor immunity. This article is featured in Selected Articles from This Issue, p. 2109.

Downregulation of Dystrophin Expression Occurs across Diverse Tumors, Correlates with the Age of Onset, Staging and Reduced Survival of Patients.

Altered dystrophin expression was found in some tumors and recent studies identified a developmental onset of Duchenne muscular dystrophy (DMD). Given that embryogenesis and carcinogenesis share many mechanisms, we analyzed a broad spectrum of tumors to establish whether dystrophin alteration evokes related outcomes. Transcriptomic, proteomic, and mutation datasets from fifty tumor tissues and matching controls (10,894 samples) and 140 corresponding tumor cell lines were analyzed. Interestingly, dystrophin transcripts and protein expression were found widespread across healthy tissues and at housekeeping gene levels. In 80% of tumors, DMD expression was reduced due to transcriptional downregulation and not somatic mutations. The full-length transcript encoding Dp427 was decreased in 68% of tumors, while Dp71 variants showed variability of expression. Notably, low expression of dystrophins was associated with a more advanced stage, older age of onset, and reduced survival across different tumors. Hierarchical clustering analysis of DMD transcripts distinguished malignant from control tissues. Transcriptomes of primary tumors and tumor cell lines with low DMD expression showed enrichment of specific pathways in the differentially expressed genes. Pathways consistently identified: ECM-receptor interaction, calcium signaling, and PI3K-Akt are also altered in DMD muscle. Therefore, the importance of this largest known gene extends beyond its roles identified in DMD, and certainly into oncology.

Predicting genes associated with RNA methylation pathways using machine learning.

RNA methylation plays an important role in functional regulation of RNAs, and has thus attracted an increasing interest in biology and drug discovery. Here, we collected and collated transcriptomic, proteomic, structural and physical interaction data from the Harmonizome database, and applied supervised machine learning to predict novel genes associated with RNA methylation pathways in human. We selected five types of classifiers, which we trained and evaluated using cross-validation on multiple training sets. The best models reached 88% accuracy based on cross-validation, and an average 91% accuracy on the test set. Using protein-protein interaction data, we propose six molecular sub-networks linking model predictions to previously known RNA methylation genes, with roles in mRNA methylation, tRNA processing, rRNA processing, but also protein and chromatin modifications. Our study exemplifies how access to large omics datasets joined by machine learning methods can be used to predict gene function.

Nectar-feeding bats and birds show parallel molecular adaptations in sugar metabolism enzymes.

In most vertebrates, the demand for glucose as the primary substrate for cellular respiration is met by the breakdown of complex carbohydrates, or energy is obtained by protein and lipid catabolism. In contrast, a few bat and bird species have convergently evolved to subsist on nectar, a sugar-rich mixture of glucose, fructose, and sucrose.1-4 How these nectar-feeders have adapted to cope with life-long high sugar intake while avoiding the onset of metabolic syndrome and diabetes5-7 is not understood. We analyzed gene sequences obtained from 127 taxa, including 22 nectar-feeding bat and bird genera that collectively encompass four independent origins of nectarivory. We show these divergent taxa have undergone pervasive molecular adaptation in sugar catabolism pathways, including parallel selection in key glycolytic and fructolytic enzymes. We also uncover convergent amino acid substitutions in the otherwise evolutionarily conserved aldolase B (ALDOB), which catalyzes rate-limiting steps in fructolysis and glycolysis, and the mitochondrial gatekeeper pyruvate dehydrogenase (PDH), which links glycolysis and the tricarboxylic acid cycle. Metabolomic profile and enzyme functional assays are consistent with increased respiratory flux in nectar-feeding bats and help explain how these taxa can both sustain hovering flight and efficiently clear simple sugars. Taken together, our results indicate that nectar-feeding bats and birds have undergone metabolic adaptations that have enabled them to exploit a unique energy-rich dietary niche among vertebrates.

Dietary Diversification and Specialization in Neotropical Bats Facilitated by Early Molecular Evolution.

Dietary adaptation is a major feature of phenotypic and ecological diversification, yet the genetic basis of dietary shifts is poorly understood. Among mammals, Neotropical leaf-nosed bats (family Phyllostomidae) show unmatched diversity in diet; from a putative insectivorous ancestor, phyllostomids have radiated to specialize on diverse food sources including blood, nectar, and fruit. To assess whether dietary diversification in this group was accompanied by molecular adaptations for changing metabolic demands, we sequenced 89 transcriptomes across 58 species and combined these with published data to compare ∼13,000 protein coding genes across 66 species. We tested for positive selection on focal lineages, including those inferred to have undergone dietary shifts. Unexpectedly, we found a broad signature of positive selection in the ancestral phyllostomid branch, spanning genes implicated in the metabolism of all major macronutrients, yet few positively selected genes at the inferred switch to plantivory. Branches corresponding to blood- and nectar-based diets showed selection in loci underpinning nitrogenous waste excretion and glycolysis, respectively. Intriguingly, patterns of selection in metabolism genes were mirrored by those in loci implicated in craniofacial remodeling, a trait previously linked to phyllostomid dietary specialization. Finally, we show that the null model of the widely-used branch-site test is likely to be misspecified, with the implication that the test is too conservative and probably under-reports true cases of positive selection. Our findings point to a complex picture of adaptive radiation, in which the evolution of new dietary specializations has been facilitated by early adaptations combined with the generation of new genetic variation.

Small-molecule inhibition of METTL3 as a strategy against myeloid leukaemia.

N6-methyladenosine (m6A) is an abundant internal RNA modification1,2 that is catalysed predominantly by the METTL3-METTL14 methyltransferase complex3,4. The m6A methyltransferase METTL3 has been linked to the initiation and maintenance of acute myeloid leukaemia (AML), but the potential of therapeutic applications targeting this enzyme remains unknown5-7. Here we present the identification and characterization of STM2457, a highly potent and selective first-in-class catalytic inhibitor of METTL3, and a crystal structure of STM2457 in complex with METTL3-METTL14. Treatment of tumours with STM2457 leads to reduced AML growth and an increase in differentiation and apoptosis. These cellular effects are accompanied by selective reduction of m6A levels on known leukaemogenic mRNAs and a decrease in their expression consistent with a translational defect. We demonstrate that pharmacological inhibition of METTL3 in vivo leads to impaired engraftment and prolonged survival in various mouse models of AML, specifically targeting key stem cell subpopulations of AML. Collectively, these results reveal the inhibition of METTL3 as a potential therapeutic strategy against AML, and provide proof of concept that the targeting of RNA-modifying enzymes represents a promising avenue for anticancer therapy.

Interrogating Phylogenetic Discordance Resolves Deep Splits in the Rapid Radiation of Old World Fruit Bats (Chiroptera: Pteropodidae).

The family Pteropodidae (Old World fruit bats) comprises $>$200 species distributed across the Old World tropics and subtropics. Most pteropodids feed on fruit, suggesting an early origin of frugivory, although several lineages have shifted to nectar-based diets. Pteropodids are of exceptional conservation concern with $>$50% of species considered threatened, yet the systematics of this group has long been debated, with uncertainty surrounding early splits attributed to an ancient rapid diversification. Resolving the relationships among the main pteropodid lineages is essential if we are to fully understand their evolutionary distinctiveness, and the extent to which these bats have transitioned to nectar-feeding. Here we generated orthologous sequences for $>$1400 nuclear protein-coding genes (2.8 million base pairs) across 114 species from 43 genera of Old World fruit bats (57% and 96% of extant species- and genus-level diversity, respectively), and combined phylogenomic inference with filtering by information content to resolve systematic relationships among the major lineages. Concatenation and coalescent-based methods recovered three distinct backbone topologies that were not able to be reconciled by filtering via phylogenetic information content. Concordance analysis and gene genealogy interrogation show that one topology is consistently the best supported, and that observed phylogenetic conflicts arise from both gene tree error and deep incomplete lineage sorting. In addition to resolving long-standing inconsistencies in the reported relationships among major lineages, we show that Old World fruit bats have likely undergone at least seven independent dietary transitions from frugivory to nectarivory. Finally, we use this phylogeny to identify and describe one new genus. [Chiroptera; coalescence; concordance; incomplete lineage sorting; nectar feeder; species tree; target enrichment.].

Inflammatory Signals Induce AT2 Cell-Derived Damage-Associated Transient Progenitors that Mediate Alveolar Regeneration.

Tissue regeneration is a multi-step process mediated by diverse cellular hierarchies and states that are also implicated in tissue dysfunction and pathogenesis. Here we leveraged single-cell RNA sequencing in combination with in vivo lineage tracing and organoid models to finely map the trajectories of alveolar-lineage cells during injury repair and lung regeneration. We identified a distinct AT2-lineage population, damage-associated transient progenitors (DATPs), that arises during alveolar regeneration. We found that interstitial macrophage-derived IL-1ß primes a subset of AT2 cells expressing Il1r1 for conversion into DATPs via a HIF1α-mediated glycolysis pathway, which is required for mature AT1 cell differentiation. Importantly, chronic inflammation mediated by IL-1ß prevents AT1 differentiation, leading to aberrant accumulation of DATPs and impaired alveolar regeneration. Together, this stepwise mapping to cell fate transitions shows how an inflammatory niche controls alveolar regeneration by controlling stem cell fate and behavior.

Positive Selection and Inactivation in the Vision and Hearing Genes of Cetaceans.

The transition to an aquatic lifestyle in cetaceans (whales and dolphins) resulted in a radical transformation in their sensory systems. Toothed whales acquired specialized high-frequency hearing tied to the evolution of echolocation, whereas baleen whales evolved low-frequency hearing. More generally, all cetaceans show adaptations for hearing and seeing underwater. To determine the extent to which these phenotypic changes have been driven by molecular adaptation, we performed large-scale targeted sequence capture of 179 sensory genes across the Cetacea, incorporating up to 54 cetacean species from all major clades as well as their closest relatives, the hippopotamuses. We screened for positive selection in 167 loci related to vision and hearing and found that the diversification of cetaceans has been accompanied by pervasive molecular adaptations in both sets of genes, including several loci implicated in nonsyndromic hearing loss. Despite these findings, however, we found no direct evidence of positive selection at the base of odontocetes coinciding with the origin of echolocation, as found in studies examining fewer taxa. By using contingency tables incorporating taxon- and gene-based controls, we show that, although numbers of positively selected hearing and nonsyndromic hearing loss genes are disproportionately high in cetaceans, counts of vision genes do not differ significantly from expected values. Alongside these adaptive changes, we find increased evidence of pseudogenization of genes involved in cone-mediated vision in mysticetes and deep-diving odontocetes.

Phylogenomic Resolution of the Cetacean Tree of Life Using Target Sequence Capture.

The evolution of cetaceans, from their early transition to an aquatic lifestyle to their subsequent diversification, has been the subject of numerous studies. However, although the higher-level relationships among cetacean families have been largely settled, several aspects of the systematics within these groups remain unresolved. Problematic clades include the oceanic dolphins (37 spp.), which have experienced a recent rapid radiation, and the beaked whales (22 spp.), which have not been investigated in detail using nuclear loci. The combined application of high-throughput sequencing with techniques that target specific genomic sequences provide a powerful means of rapidly generating large volumes of orthologous sequence data for use in phylogenomic studies. To elucidate the phylogenetic relationships within the Cetacea, we combined sequence capture with Illumina sequencing to generate data for $\sim $3200 protein-coding genes for 68 cetacean species and their close relatives including the pygmy hippopotamus. By combining data from $>$38,000 exons with existing sequences from 11 cetaceans and seven outgroup taxa, we produced the first comprehensive comparative genomic data set for cetaceans, spanning 6,527,596 aligned base pairs (bp) and 89 taxa. Phylogenetic trees reconstructed with maximum likelihood and Bayesian inference of concatenated loci, as well as with coalescence analyses of individual gene trees, produced mostly concordant and well-supported trees. Our results completely resolve the relationships among beaked whales as well as the contentious relationships among oceanic dolphins, especially the problematic subfamily Delphinidae. We carried out Bayesian estimation of species divergence times using MCMCTree and compared our complete data set to a subset of clocklike genes. Analyses using the complete data set consistently showed less variance in divergence times than the reduced data set. In addition, integration of new fossils (e.g., Mystacodon selenensis) indicates that the diversification of Crown Cetacea began before the Late Eocene and the divergence of Crown Delphinidae as early as the Middle Miocene. [Cetaceans; phylogenomics; Delphinidae; Ziphiidae; dolphins; whales.].

Resolving evolutionary relationships among six closely related taxa of the horseshoe bats (Rhinolophus) with targeted resequencing data.

Recently diverged taxa are often characterised by high rates of introgressive hybridization and incomplete lineage sorting, both of which can complicate phylogenetic reconstructions of species histories. Here we use a sequence capture approach to obtain genome-wide data to resolve the evolutionary relationships, and infer the extent and timescale of hybridization and introgression events, among six recently diverged taxa of the horseshoe bat species complexes Rhinolophus sinicus and R. thomasi. We show that two different methods of species tree reconstruction applied to a set of ~1500 nuclear loci all recover species trees with similar topologies, differing from the previous phylogeny based on two nuclear loci. By comparing the tree topology obtained from the nuclear loci with that inferred from the mitochondrial genome, we observed at least three cases of conflict, each of which likely results from past introgression. Of these, the occurrence of a highly similar mitogenome sequence shared by individuals of two taxa in a sympatric region points to very recent mtDNA introgression. The other cases are characterised by greater divergence and strong phylogeographic structure among putative introgressed individuals and their source populations, and thus likely reflect more ancient hybridization events. These results also suggest that two of the subspecies (R. s. septentrionalis and the undescribed taxon R. s. ssp) are likely to represent full species, warranting full taxonomic descriptions. This work adds a growing number of studies showing the potential problems of relying solely on mitochondrial sequences, or a limited number of loci, to infer phylogenetic relationships among recently diverged taxa.

A phylogenomic framework and timescale for comparative studies of tunicates.

BACKGROUND: Tunicates are the closest relatives of vertebrates and are widely used as models to study the evolutionary developmental biology of chordates. Their phylogeny, however, remains poorly understood, and to date, only the 18S rRNA nuclear gene and mitogenomes have been used to delineate the major groups of tunicates. To resolve their evolutionary relationships and provide a first estimate of their divergence times, we used a transcriptomic approach to build a phylogenomic dataset including all major tunicate lineages, consisting of 258 evolutionarily conserved orthologous genes from representative species. RESULTS: Phylogenetic analyses using site-heterogeneous CAT mixture models of amino acid sequence evolution resulted in a strongly supported tree topology resolving the relationships among four major tunicate clades: (1) Appendicularia, (2) Thaliacea + Phlebobranchia + Aplousobranchia, (3) Molgulidae, and (4) Styelidae + Pyuridae. Notably, the morphologically derived Thaliacea are confirmed as the sister group of the clade uniting Phlebobranchia + Aplousobranchia within which the precise position of the model ascidian genus Ciona remains uncertain. Relaxed molecular clock analyses accommodating the accelerated evolutionary rate of tunicates reveal ancient diversification (~ 450-350 million years ago) among the major groups and allow one to compare their evolutionary age with respect to the major vertebrate model lineages. CONCLUSIONS: Our study represents the most comprehensive phylogenomic dataset for the main tunicate lineages. It offers a reference phylogenetic framework and first tentative timescale for tunicates, allowing a direct comparison with vertebrate model species in comparative genomics and evolutionary developmental biology studies.

Comparative genomics reveals contraction in olfactory receptor genes in bats.

Gene loss and gain during genome evolution are thought to play important roles in adaptive phenotypic diversification. Among mammals, bats possess the smallest genomes and have evolved the unique abilities of powered flight and laryngeal echolocation. To investigate whether gene family evolution has contributed to the genome downsizing and phenotypic diversification in this group, we performed comparative evolutionary analyses of complete proteome data for eight bat species, including echolocating and non-echolocating forms, together with the proteomes of 12 other laurasiatherian mammals. Our analyses revealed extensive gene loss in the most recent ancestor of bats, and also of carnivores (both >1,000 genes), although this gene contraction did not appear to correlate with the reduction in genome size in bats. Comparisons of highly dynamic families suggested that expansion and contraction affected genes with similar functions (immunity, response to stimulus) in all laurasiatherian lineages. However, the magnitude and direction of these changes varied greatly among groups. In particular, our results showed contraction of the Olfactory Receptor (OR) gene repertoire in the last common ancestor of all bats, as well as that of the echolocating species studied. In contrast, non-echolocating fruit bats showed evidence of expansion in ORs, supporting a "trade-off" between sensory modalities.

A phylogenomic analysis of the role and timing of molecular adaptation in the aquatic transition of cetartiodactyl mammals.

Recent studies have reported multiple cases of molecular adaptation in cetaceans related to their aquatic abilities. However, none of these has included the hippopotamus, precluding an understanding of whether molecular adaptations in cetaceans occurred before or after they split from their semi-aquatic sister taxa. Here, we obtained new transcriptomes from the hippopotamus and humpback whale, and analysed these together with available data from eight other cetaceans. We identified more than 11 000 orthologous genes and compiled a genome-wide dataset of 6845 coding DNA sequences among 23 mammals, to our knowledge the largest phylogenomic dataset to date for cetaceans. We found positive selection in nine genes on the branch leading to the common ancestor of hippopotamus and whales, and 461 genes in cetaceans compared to 64 in hippopotamus. Functional annotation revealed adaptations in diverse processes, including lipid metabolism, hypoxia, muscle and brain function. By combining these findings with data on protein-protein interactions, we found evidence suggesting clustering among gene products relating to nervous and muscular systems in cetaceans. We found little support for shared ancestral adaptations in the two taxa; most molecular adaptations in extant cetaceans occurred after their split with hippopotamids.

Family Wide Molecular Adaptations to Underground Life in African Mole-Rats Revealed by Phylogenomic Analysis.

During their evolutionary radiation, mammals have colonized diverse habitats. Arguably the subterranean niche is the most inhospitable of these, characterized by reduced oxygen, elevated carbon dioxide, absence of light, scarcity of food, and a substrate that is energetically costly to burrow through. Of all lineages to have transitioned to a subterranean niche, African mole-rats are one of the most successful. Much of their ecological success can be attributed to a diet of plant storage organs, which has allowed them to colonize climatically varied habitats across sub-Saharan Africa, and has probably contributed to the evolution of their diverse social systems. Yet despite their many remarkable phenotypic specializations, little is known about molecular adaptations underlying these traits. To address this, we sequenced the transcriptomes of seven mole-rat taxa, including three solitary species, and combined new sequences with existing genomic data sets. Alignments of more than 13,000 protein-coding genes encompassed, for the first time, all six genera and the full spectrum of ecological and social variation in the clade. We detected positive selection within the mole-rat clade and along ancestral branches in approximately 700 genes including loci associated with tumorigenesis, aging, morphological development, and sociality. By combining these results with gene ontology annotation and protein-protein networks, we identified several clusters of functionally related genes. This family wide analysis of molecular evolution in mole-rats has identified a suite of positively selected genes, deepening our understanding of the extreme phenotypic traits exhibited by this group.

Divergent evolutionary rates in vertebrate and mammalian specific conserved non-coding elements (CNEs) in echolocating mammals.

BACKGROUND: The majority of DNA contained within vertebrate genomes is non-coding, with a certain proportion of this thought to play regulatory roles during development. Conserved Non-coding Elements (CNEs) are an abundant group of putative regulatory sequences that are highly conserved across divergent groups and thus assumed to be under strong selective constraint. Many CNEs may contain regulatory factor binding sites, and their frequent spatial association with key developmental genes - such as those regulating sensory system development - suggests crucial roles in regulating gene expression and cellular patterning. Yet surprisingly little is known about the molecular evolution of CNEs across diverse mammalian taxa or their role in specific phenotypic adaptations. We examined 3,110 vertebrate-specific and ~82,000 mammalian-specific CNEs across 19 and 9 mammalian orders respectively, and tested for changes in the rate of evolution of CNEs located in the proximity of genes underlying the development or functioning of auditory systems. As we focused on CNEs putatively associated with genes underlying the development/functioning of auditory systems, we incorporated echolocating taxa in our dataset because of their highly specialised and derived auditory systems. RESULTS: Phylogenetic reconstructions of concatenated CNEs broadly recovered accepted mammal relationships despite high levels of sequence conservation. We found that CNE substitution rates were highest in rodents and lowest in primates, consistent with previous findings. Comparisons of CNE substitution rates from several genomic regions containing genes linked to auditory system development and hearing revealed differences between echolocating and non-echolocating taxa. Wider taxonomic sampling of four CNEs associated with the homeobox genes Hmx2 and Hmx3 - which are required for inner ear development - revealed family-wise variation across diverse bat species. Specifically within one family of echolocating bats that utilise frequency-modulated echolocation calls varying widely in frequency and intensity high levels of sequence divergence were found. CONCLUSIONS: Levels of selective constraint acting on CNEs differed both across genomic locations and taxa, with observed variation in substitution rates of CNEs among bat species. More work is needed to determine whether this variation can be linked to echolocation, and wider taxonomic sampling is necessary to fully document levels of conservation in CNEs across diverse taxa.

Molecular evolution of growth hormone and insulin-like growth factor 1 receptors in long-lived, small-bodied mammals.

Mammals typically display a robust positive relationship between lifespan and body size. Two groups that deviate markedly from this pattern are bats and African mole-rats, with members of both groups being extremely long-lived given their body size, with the maximum documented lifespan for many species exceeding 20 years. A recent genomics study of the exceptionally long-lived Brandt's bat, Myotis brandtii (41 years), suggested that its longevity and small body size may be at least partly attributed to key amino acid substitutions in the transmembrane domains of the receptors of growth hormone (GH) and insulin-like growth factor 1 (IGF1). However, whereas elevated longevity is likely to be common across all 19 bat families, the reported amino acid substitutions were only observed in two closely related bat families. To test the hypothesis that an altered GH/IGF1 axis relates to the longevity of African mole-rats and bats, we compared and analysed the homologous coding gene sequences in genomic and transcriptomic data from 26 bat species, five mole-rats and 38 outgroup species. Phylogenetic analyses of both genes recovered the majority of nodes in the currently accepted species tree with high support. Compared to other clades, such as primates and carnivores, the bats and rodents had longer branch lengths. The single 24 amino acid transmembrane domain of IGF1R was found to be more conserved across mammals compared to that of GHR. Within bats, considerable variation in the transmembrane domain of GHR was found, including a previously unreported deletion in Emballonuridae. The transmembrane domains of rodents were found to be more conserved, with mole-rats lacking uniquely conserved amino acid substitutions. Molecular evolutionary analyses showed that both genes were under purifying selection in bats and mole-rats. Our findings suggest that while the previously documented mutations may confer some additional lifespan to Myotis bats, other, as yet unknown, genetic differences are likely to account for the long lifespans observed in many bat and mole-rat species.

Phylogenomic analyses elucidate the evolutionary relationships of bats.

Molecular phylogenetics has rapidly established the evolutionary positions of most major mammal groups, yet analyses have repeatedly failed to agree on that of bats (order Chiroptera). Moreover, the relationship among the major bat lineages has proven equally contentious, with ongoing disagreements about whether echolocating bats are paraphyletic or a true group having profound implications for whether echolocation evolved once or possibly multiple times. By generating new bat genome data and applying model-based phylogenomic analyses designed to accommodate heterogeneous evolutionary processes, we show that-contrary to recent suggestions-bats are not closely related to odd-toed ungulates but instead have a more ancient origin as sister group to a large clade of carnivores, ungulates, and cetaceans. Additionally, we provide the first genome-scale support showing that laryngeal echolocating bats are not a true group and that this paraphyly is robust to their position within mammals. We suggest that earlier disagreements in the literature may reflect model misspecification, long-branch artifacts, poor taxonomic coverage, and differences in the phylogenetic markers used. These findings are a timely reminder of the relevance of experimental design and careful statistical analysis as we move into the phylogenomic era.

Genome-wide signatures of convergent evolution in echolocating mammals.

Evolution is typically thought to proceed through divergence of genes, proteins and ultimately phenotypes. However, similar traits might also evolve convergently in unrelated taxa owing to similar selection pressures. Adaptive phenotypic convergence is widespread in nature, and recent results from several genes have suggested that this phenomenon is powerful enough to also drive recurrent evolution at the sequence level. Where homoplasious substitutions do occur these have long been considered the result of neutral processes. However, recent studies have demonstrated that adaptive convergent sequence evolution can be detected in vertebrates using statistical methods that model parallel evolution, although the extent to which sequence convergence between genera occurs across genomes is unknown. Here we analyse genomic sequence data in mammals that have independently evolved echolocation and show that convergence is not a rare process restricted to several loci but is instead widespread, continuously distributed and commonly driven by natural selection acting on a small number of sites per locus. Systematic analyses of convergent sequence evolution in 805,053 amino acids within 2,326 orthologous coding gene sequences compared across 22 mammals (including four newly sequenced bat genomes) revealed signatures consistent with convergence in nearly 200 loci. Strong and significant support for convergence among bats and the bottlenose dolphin was seen in numerous genes linked to hearing or deafness, consistent with an involvement in echolocation. Unexpectedly, we also found convergence in many genes linked to vision: the convergent signal of many sensory genes was robustly correlated with the strength of natural selection. This first attempt to detect genome-wide convergent sequence evolution across divergent taxa reveals the phenomenon to be much more pervasive than previously recognized.