The genome sequence of Molossusnigricans (Chiroptera, Molossidae; Miller, 1902).

We present a genome assembly from an individual male Molossus nigricans (Chordata; Mammalia; Chiroptera; Molossidae). The genome sequence is 2.41 gigabases in span. The majority of the assembly is scaffolded into 24 chromosomal pseudomolecules, with the X sex chromosome assembled.

Using machine learning to decode animal communication.

New methods promise transformative insights and conservation benefits.

Chiropterans Are a Hotspot for Horizontal Transfer of DNA Transposons in Mammalia.

Horizontal transfer of transposable elements (TEs) is an important mechanism contributing to genetic diversity and innovation. Bats (order Chiroptera) have repeatedly been shown to experience horizontal transfer of TEs at what appears to be a high rate compared with other mammals. We investigated the occurrence of horizontally transferred (HT) DNA transposons involving bats. We found over 200 putative HT elements within bats; 16 transposons were shared across distantly related mammalian clades, and 2 other elements were shared with a fish and two lizard species. Our results indicate that bats are a hotspot for horizontal transfer of DNA transposons. These events broadly coincide with the diversification of several bat clades, supporting the hypothesis that DNA transposon invasions have contributed to genetic diversification of bats.

The genome sequence of the northern bat, Eptesicus nilssonii (Keyserling & Blasius, 1839).

We present a genome assembly from an individual Eptesicus nilssonii (the northern bat; Chordata; Mammalia; Chiroptera; Vespertilionidae), derived from the placental tissue of a pregnancy that resulted a male pup. The genome sequence is 2,064.1 megabases in span. Most of the assembly is scaffolded into 26 chromosomal pseudomolecules, including the X and Y sex chromosomes. The mitochondrial genome has also been assembled and is 17.04 kilobases in length.

The genome sequence of the Soprano Pipistrelle, Pipistrellus pygmaeus (Leach, 1825).

We present a genome assembly from an individual male Pipistrellus pygmaeus (the Soprano Pipistrelle; Chordata; Mammalia; Chiroptera; Vespertilionidae). The genome sequence is 1,895.1 megabases in span. Most of the assembly is scaffolded into 23 chromosomal pseudomolecules, including the X and Y sex chromosomes. The mitochondrial genome has also been assembled and is 17.18 kilobases in length.

The pale spear-nosed bat: A neuromolecular and transgenic model for vocal learning.

Vocal learning, the ability to produce modified vocalizations via learning from acoustic signals, is a key trait in the evolution of speech. While extensively studied in songbirds, mammalian models for vocal learning are rare. Bats present a promising study system given their gregarious natures, small size, and the ability of some species to be maintained in captive colonies. We utilize the pale spear-nosed bat (Phyllostomus discolor) and report advances in establishing this species as a tractable model for understanding vocal learning. We have taken an interdisciplinary approach, aiming to provide an integrated understanding across genomics (Part I), neurobiology (Part II), and transgenics (Part III). In Part I, we generated new, high-quality genome annotations of coding genes and noncoding microRNAs to facilitate functional and evolutionary studies. In Part II, we traced connections between auditory-related brain regions and reported neuroimaging to explore the structure of the brain and gene expression patterns to highlight brain regions. In Part III, we created the first successful transgenic bats by manipulating the expression of FoxP2, a speech-related gene. These interdisciplinary approaches are facilitating a mechanistic and evolutionary understanding of mammalian vocal learning and can also contribute to other areas of investigation that utilize P. discolor or bats as study species.

ARHGEF39, a Gene Implicated in Developmental Language Disorder, Activates RHOA and Is Involved in Cell De-Adhesion and Neural Progenitor Cell Proliferation.

ARHGEF39 was previously implicated in developmental language disorder (DLD) via a functional polymorphism that can disrupt post-transcriptional regulation by microRNAs. ARHGEF39 is part of the family of Rho guanine nucleotide exchange factors (RhoGEFs) that activate small Rho GTPases to regulate a wide variety of cellular processes. However, little is known about the function of ARHGEF39, or how its function might contribute to neurodevelopment or related disorders. Here, we explore the molecular function of ARHGEF39 and show that it activates the Rho GTPase RHOA and that high ARHGEF39 expression in cell cultures leads to an increase of detached cells. To explore its role in neurodevelopment, we analyse published single cell RNA-sequencing data and demonstrate that ARHGEF39 is a marker gene for proliferating neural progenitor cells and that it is co-expressed with genes involved in cell division. This suggests a role for ARHGEF39 in neurogenesis in the developing brain. The co-expression of ARHGEF39 with other RHOA-regulating genes supports RHOA as substrate of ARHGEF39 in neural cells, and the involvement of RHOA in neuropsychiatric disorders highlights a potential link between ARHGEF39 and neurodevelopment and disorder. Understanding the GTPase substrate, co-expression network, and processes downstream of ARHGEF39 provide new avenues for exploring the mechanisms by which altered expression levels of ARHGEF39 may contribute to neurodevelopment and associated disorders.

Contradictory Phylogenetic Signals in the Laurasiatheria Anomaly Zone.

Relationships among laurasiatherian clades represent one of the most highly disputed topics in mammalian phylogeny. In this study, we attempt to disentangle laurasiatherian interordinal relationships using two independent genome-level approaches: (1) quantifying retrotransposon presence/absence patterns, and (2) comparisons of exon datasets at the levels of nucleotides and amino acids. The two approaches revealed contradictory phylogenetic signals, possibly due to a high level of ancestral incomplete lineage sorting. The positions of Eulipotyphla and Chiroptera as the first and second earliest divergences were consistent across the approaches. However, the phylogenetic relationships of Perissodactyla, Cetartiodactyla, and Ferae, were contradictory. While retrotransposon insertion analyses suggest a clade with Cetartiodactyla and Ferae, the exon dataset favoured Cetartiodactyla and Perissodactyla. Future analyses of hitherto unsampled laurasiatherian lineages and synergistic analyses of retrotransposon insertions, exon and conserved intron/intergenic sequences might unravel the conflicting patterns of relationships in this major mammalian clade.

Hyperkinetic stereotyped movements in a boy with biallelic CNTNAP2 variants.

BACKGROUND: Heterozygous variants in CNTNAP2 have been implicated in a wide range of neurological phenotypes, including intellectual disability (ID), epilepsy, autistic spectrum disorder (ASD), and impaired language. However, heterozygous variants can also be found in unaffected individuals. Biallelic CNTNAP2 variants are rarer and cause a well-defined genetic syndrome known as CASPR2 deficiency disorder, a condition characterised by ID, early-onset refractory epilepsy, language impairment, and autistic features. CASE-REPORT: A 7-year-old boy presented with hyperkinetic stereotyped movements that started during early infancy and persisted over childhood. Abnormal movements consisted of rhythmic and repetitive shaking of the four limbs, with evident stereotypic features. Additional clinical features included ID, attention deficit-hyperactivity disorder (ADHD), ASD, and speech impairment, consistent with CASPR2 deficiency disorder. Whole-genome array comparative genomic hybridization detected a maternally inherited 0.402 Mb duplication, which involved intron 1, exon 2, and intron 2 of CNTNAP2 (c.97 +?_209-?dup). The affected region in intron 1 contains a binding site for the transcription factor FOXP2, potentially leading to abnormal CNTNAP2 expression regulation. Sanger sequencing of the coding region of CNTNAP2 also identified a paternally-inherited missense variant c.2752C > T, p.(Leu918Phe). CONCLUSION: This case expands the molecular and phenotypic spectrum of CASPR2 deficiency disorder, suggesting that Hyperkinetic stereotyped movements may be a rare, yet significant, clinical feature of this complex neurological disorder. Furthermore, the identification of an in-frame, largely non-coding duplication in CNTNAP2 points to a sophisticated underlying molecular mechanism, likely involving impaired FOXP2 binding.

The multi-dimensional nature of vocal learning.

How learning affects vocalizations is a key question in the study of animal communication and human language. Parallel efforts in birds and humans have taught us much about how vocal learning works on a behavioural and neurobiological level. Subsequent efforts have revealed a variety of cases among mammals in which experience also has a major influence on vocal repertoires. Janik and Slater (Anim. Behav.60, 1-11. (doi:10.1006/anbe.2000.1410)) introduced the distinction between vocal usage and production learning, providing a general framework to categorize how different types of learning influence vocalizations. This idea was built on by Petkov and Jarvis (Front. Evol. Neurosci.4, 12. (doi:10.3389/fnevo.2012.00012)) to emphasize a more continuous distribution between limited and more complex vocal production learners. Yet, with more studies providing empirical data, the limits of the initial frameworks become apparent. We build on these frameworks to refine the categorization of vocal learning in light of advances made since their publication and widespread agreement that vocal learning is not a binary trait. We propose a novel classification system, based on the definitions by Janik and Slater, that deconstructs vocal learning into key dimensions to aid in understanding the mechanisms involved in this complex behaviour. We consider how vocalizations can change without learning, and a usage learning framework that considers context specificity and timing. We identify dimensions of vocal production learning, including the copying of auditory models (convergence/divergence on model sounds, accuracy of copying), the degree of change (type and breadth of learning) and timing (when learning takes place, the length of time it takes and how long it is retained). We consider grey areas of classification and current mechanistic understanding of these behaviours. Our framework identifies research needs and will help to inform neurobiological and evolutionary studies endeavouring to uncover the multi-dimensional nature of vocal learning. This article is part of the theme issue 'Vocal learning in animals and humans'.

Neuroanatomy of the grey seal brain: bringing pinnipeds into the neurobiological study of vocal learning.

Comparative animal studies of complex behavioural traits, and their neurobiological underpinnings, can increase our understanding of their evolution, including in humans. Vocal learning, a potential precursor to human speech, is one such trait. Mammalian vocal learning is under-studied: most research has either focused on vocal learning in songbirds or its absence in non-human primates. Here, we focus on a highly promising model species for the neurobiology of vocal learning: grey seals (Halichoerus grypus). We provide a neuroanatomical atlas (based on dissected brain slices and magnetic resonance images), a labelled MRI template, a three-dimensional model with volumetric measurements of brain regions, and histological cortical stainings. Four main features of the grey seal brain stand out: (i) it is relatively big and highly convoluted; (ii) it hosts a relatively large temporal lobe and cerebellum; (iii) the cortex is similar to that of humans in thickness and shows the expected six-layered mammalian structure; (iv) there is expression of FoxP2 present in deeper layers of the cortex; FoxP2 is a gene involved in motor learning, vocal learning, and spoken language. Our results could facilitate future studies targeting the neural and genetic underpinnings of mammalian vocal learning, thus bridging the research gap from songbirds to humans and non-human primates. Our findings are relevant not only to vocal learning research but also to the study of mammalian neurobiology and cognition more in general. This article is part of the theme issue 'Vocal learning in animals and humans'.

The vocal development of the pale spear-nosed bat is dependent on auditory feedback.

Human vocal development and speech learning require acoustic feedback, and humans who are born deaf do not acquire a normal adult speech capacity. Most other mammals display a largely innate vocal repertoire. Like humans, bats are thought to be one of the few taxa capable of vocal learning as they can acquire new vocalizations by modifying vocalizations according to auditory experiences. We investigated the effect of acoustic deafening on the vocal development of the pale spear-nosed bat. Three juvenile pale spear-nosed bats were deafened, and their vocal development was studied in comparison with an age-matched, hearing control group. The results show that during development the deafened bats increased their vocal activity, and their vocalizations were substantially altered, being much shorter, higher in pitch, and more aperiodic than the vocalizations of the control animals. The pale spear-nosed bat relies on auditory feedback for vocal development and, in the absence of auditory input, species-atypical vocalizations are acquired. This work serves as a basis for further research using the pale spear-nosed bat as a mammalian model for vocal learning, and contributes to comparative studies on hearing impairment across species. This article is part of the theme issue 'Vocal learning in animals and humans'.

Large-scale genome sampling reveals unique immunity and metabolic adaptations in bats.

Comprising more than 1,400 species, bats possess adaptations unique among mammals including powered flight, unexpected longevity, and extraordinary immunity. Some of the molecular mechanisms underlying these unique adaptations includes DNA repair, metabolism and immunity. However, analyses have been limited to a few divergent lineages, reducing the scope of inferences on gene family evolution across the Order Chiroptera. We conducted an exhaustive comparative genomic study of 37 bat species, one generated in this study, encompassing a large number of lineages, with a particular emphasis on multi-gene family evolution across immune and metabolic genes. In agreement with previous analyses, we found lineage-specific expansions of the APOBEC3 and MHC-I gene families, and loss of the proinflammatory PYHIN gene family. We inferred more than 1,000 gene losses unique to bats, including genes involved in the regulation of inflammasome pathways such as epithelial defence receptors, the natural killer gene complex and the interferon-gamma induced pathway. Gene set enrichment analyses revealed genes lost in bats are involved in defence response against pathogen-associated molecular patterns and damage-associated molecular patterns. Gene family evolution and selection analyses indicate bats have evolved fundamental functional differences compared to other mammals in both innate and adaptive immune system, with the potential to enhance antiviral immune response while dampening inflammatory signalling. In addition, metabolic genes have experienced repeated expansions related to convergent shifts to plant-based diets. Our analyses support the hypothesis that, in tandem with flight, ancestral bats had evolved a unique set of immune adaptations whose functional implications remain to be explored.

Towards complete and error-free genome assemblies of all vertebrate species.

High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are available for only a few non-microbial species1-4. To address this issue, the international Genome 10K (G10K) consortium5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling highly accurate and nearly complete reference genomes. Here we present lessons learned from generating assemblies for 16 species that represent six major vertebrate lineages. We confirm that long-read sequencing technologies are essential for maximizing genome quality, and that unresolved complex repeats and haplotype heterozygosity are major sources of assembly error when not handled correctly. Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries. These include the identification of many false gene duplications, increases in gene sizes, chromosome rearrangements that are specific to lineages, a repeated independent chromosome breakpoint in bat genomes, and a canonical GC-rich pattern in protein-coding genes and their regulatory regions. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an international effort to generate high-quality, complete reference genomes for all of the roughly 70,000 extant vertebrate species and to help to enable a new era of discovery across the life sciences.

DNA methylation predicts age and provides insight into exceptional longevity of 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.

Hearing sensitivity and amplitude coding in bats are differentially shaped by echolocation calls and social calls.

Differences in auditory perception between species are influenced by phylogenetic origin and the perceptual challenges imposed by the natural environment, such as detecting prey- or predator-generated sounds and communication signals. Bats are well suited for comparative studies on auditory perception since they predominantly rely on echolocation to perceive the world, while their social calls and most environmental sounds have low frequencies. We tested if hearing sensitivity and stimulus level coding in bats differ between high and low-frequency ranges by measuring auditory brainstem responses (ABRs) of 86 bats belonging to 11 species. In most species, auditory sensitivity was equally good at both high- and low-frequency ranges, while amplitude was more finely coded for higher frequency ranges. Additionally, we conducted a phylogenetic comparative analysis by combining our ABR data with published data on 27 species. Species-specific peaks in hearing sensitivity correlated with peak frequencies of echolocation calls and pup isolation calls, suggesting that changes in hearing sensitivity evolved in response to frequency changes of echolocation and social calls. Overall, our study provides the most comprehensive comparative assessment of bat hearing capacities to date and highlights the evolutionary pressures acting on their sensory perception.

Six reference-quality genomes reveal evolution of bat adaptations.

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 protocols1 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 disease1.