Kalina T. J. Davies

Oculocutaneous albinism—characterized by absent or decreased melanin in the skin, eyes and hair—often co-occurs with sensory, skin and immunity disorders. The genetic basis of albinism in humans is complex, with many loci implicated in multiple forms of the disorder. Less is known about the underlying genetic causes of albinism and leucism in other species, and cross-species studies of the molecular correlates of hypopigmentation could highlight common and conserved pathways underlying mammalian pigmentation disorders. We characterize the putative causal loci of reduced pigmentation in an Egyptian fruit bat (Rousettus aegyptiacus) that displayed features indicative of albinism. Despite albino or leucistic individuals having been reported in >100 bat species, the associated genetic backgrounds have not previously been studied. We used a digital gene expression panel to quantify mRNA levels in wing membrane samples of four candidate pigmentation genes in the focal albino fruit bat and control individuals. Significantly reduced SLC45A2 mRNA expression was found in the albino compared to five bats with typical coloration. Additionally, intraspecific sequence analyses of the albino bat SLC45A2 coding sequence identified two missense mutations, E18A and Q298R, the former of which was private to the albino bat. Position 18 of SLC45A2 was otherwise found to be highly conserved across 60 bat species and has not been previously linked to human albinism. By identifying SLC45A2 as the likely contributing locus, our results also indicate further support for the necessity of genetic testing for the reliable categorization of hypopigmented animals as either albino or leucistic.

Sensory organs must develop alongside the skull within which they are largely encased, and this relationship can manifest as the skull constraining the organs, organs constraining the skull, or organs constraining one another in relative size. How this interplay between sensory organs and the developing skull plays out during the evolution of sensory diversity; however, remains unknown. Here, we examine the developmental sequence of the cochlea, the organ responsible for hearing and echolocation, in species with distinct diet and echolocation types within the ecologically diverse bat super‐family Noctilionoidea. We found the size and shape of the cochlea largely correlates with skull size, with exceptions of Pteronotus parnellii , whose high duty cycle echolocation (nearly constant emission of sound pulses during their echolocation process allowing for detailed information gathering, also called constant frequency echolocation) corresponds to a larger cochlear and basal turn, and Monophyllus redmani , a small‐bodied nectarivorous bat, for which interactions with other sensory organs restrict cochlea size. Our findings support the existence of developmental constraints, suggesting that both developmental and anatomical factors may act synergistically during the development of sensory systems in noctilionoid bats.

Although evolvability of genes and traits may promote specialization during species diversification, how ecology subsequently restricts such variation remains unclear. Chemosensation requires animals to decipher a complex chemical background to locate fitness‐related resources, and thus the underlying genomic architecture and morphology must cope with constant exposure to a changing odorant landscape; detecting adaptation amidst extensive chemosensory diversity is an open challenge. In phyllostomid bats, an ecologically diverse clade that evolved plant visiting from a presumed insectivorous ancestor, the evolution of novel food detection mechanisms is suggested to be a key innovation, as plant‐visiting species rely strongly on olfaction, supplementarily using echolocation. If this is true, exceptional variation in underlying olfactory genes and phenotypes may have preceded dietary diversification. We compared olfactory receptor (OR) genes sequenced from olfactory epithelium transcriptomes and olfactory epithelium surface area of bats with differing diets. Surprisingly, although OR evolution rates were quite variable and generally high, they are largely independent of diet. Olfactory epithelial surface area, however, is relatively larger in plant‐visiting bats and there is an inverse relationship between OR evolution rates and surface area. Relatively larger surface areas suggest greater reliance on olfactory detection and stronger constraint on maintaining an already diverse OR repertoire. Instead of the typical case in which specialization and elaboration are coupled with rapid diversification of associated genes, here the relevant genes are already evolving so quickly that increased reliance on smell has led to stabilizing selection, presumably to maintain the ability to consistently discriminate among specific odorants—a potential ecological constraint on sensory evolution.

While evolvability of genes and traits may promote specialization during species diversification, how ecology subsequently restricts such variation remains unclear. Chemosensation requires animals to decipher a complex chemical background to locate fitness-related resources, and thus the underlying genomic architecture and morphology must cope with constant exposure to a changing odorant landscape; detecting adaptation amidst extensive chemosensory diversity is an open challenge. Phyllostomid bats, an ecologically diverse clade that evolved plant-visiting from an insectivorous ancestor, suggest the evolution of novel food detection mechanisms is a key innovation: phyllostomids behaviorally rely strongly on olfaction, while echolocation is supplemental. If this is true, exceptional variation in underlying olfactory genes and phenotypes may have preceded dietary diversification. We compared olfactory receptor ( OR ) genes sequenced from olfactory epithelium transcriptomes and olfactory epithelium surface area of bats with differing diets. Surprisingly, although OR evolution rates were quite variable and generally high, they are largely independent of diet. Olfactory epithelial surface area, however, is relatively larger in plant-visiting bats and there is an inverse relationship between OR evolution rates and surface area. Relatively larger surface areas suggest greater reliance on olfactory detection and stronger constraint on maintaining an already diverse OR repertoire. Instead of the typical case in which specialization and elaboration is coupled with rapid diversification of associated genes, here the relevant genes are already evolving so quickly that increased reliance on smell has led to stabilizing selection, presumably to maintain the ability to consistently discriminate among specific odorants — a potential ecological constraint on sensory evolution. Significance Statement: The evolutionary relationship between genes and morphology is complex to decipher, and macroevolutionary trends are often measured independently; this is especially challenging to quantify in unstable genomic regions or hypervariable traits. Odorant cues are detected by proteins encoded by the largest and fasted-evolving gene family in the mammalian genome and expressed in epithelia distributed on elaborate bony structures in the nose, posing a challenge to quantification. Yet, the direct interaction of the olfactory system with environmental signals strongly suggest that selection shapes its immense diversity. In neotropical bats, where reliance on plant-visiting evolved from an insectivorous ancestor, we discovered clear dietary differences amongst species, but only after considering morphological and molecular data simultaneously, emphasizing the power of a coupled analysis.

Molecular Biology and Evolution, msab028, https://doi.org/10.1093/molbev/msab028

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.

Changes in behaviour may initiate shifts to new adaptive zones, with physical adaptations for novel environments evolving later. While new mutations are commonly considered engines of adaptive change, sensory evolution enabling access to new resources might also arise from standing genetic diversity, and even gene loss. We examine the relative contribution of molecular adaptations, measured by positive and relaxed selection, acting on eye‐expressed genes associated with shifts to new adaptive zones in ecologically diverse bats from the superfamily Noctilionoidea. Collectively, noctilionoids display remarkable ecological breadth, from highly divergent echolocation to flight strategies linked to specialized insectivory, the parallel evolution of diverse plant‐based diets (e.g., nectar, pollen and fruit) from ancestral insectivory, and—unusually for echolocating bats—often have large, well‐developed eyes. We report contrasting levels of positive selection in genes associated with the development, maintenance and scope of visual function, tracing back to the origins of noctilionoids and Phyllostomidae (the bat family with most dietary diversity), instead of during shifts to novel diets. Generalized plant visiting was not associated with exceptional molecular adaptation, and exploration of these novel niches took place in an ancestral phyllostomid genetic background. In contrast, evidence for positive selection in vision genes was found at subsequent shifts to either nectarivory or frugivory. Thus, neotropical noctilionoids that use visual cues for identifying food and roosts, as well as for orientation, were effectively preadapted, with subsequent molecular adaptations in nectar‐feeding lineages and the subfamily Stenodermatinae of fig‐eating bats fine‐tuning pre‐existing visual adaptations for specialized purposes.

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of these methods to non-model organisms. A series of protocols to optimize tissue collection from bats has been developed for a series of high-throughput sequencing approaches. Outlined here are protocols for the capture of bats, desired demographics to be collected for each bat, and optimized methods to minimize stress on a bat during tissue collection. Specifically outlined are methods for collecting and treating tissue to obtain (i) DNA for high molecular weight genomic analyses, (ii) RNA for tissue-specific transcriptomes, and (iii) proteins for proteomic-level analyses. Lastly, also outlined is a method to avoid lethal sampling by creating viable primary cell cultures from wing clips. A central motivation of these methods is to maximize the amount of potential molecular and morphological data for each bat and suggest optimal ways to preserve tissues so they retain their value as new methods develop in the future. This standardization has become particularly important as initiatives to sequence chromosome-level, error-free genomes of species across the world have emerged, in which multiple scientific parties are spearheading the sequencing of different taxonomic groups. The protocols outlined herein define the ideal tissue collection and tissue preservation methods for Bat1K, the consortium that is sequencing the genomes of every species of bat.

Multigene families evolve from single‐copy ancestral genes via duplication, and typically encode proteins critical to key biological processes. Molecular analyses of these gene families require high‐confidence sequences, but the high sequence similarity of the members can create challenges for sequencing and downstream analyses. Focusing on the common vampire bat, Desmodus rotundus, we evaluated how different sequencing approaches performed in recovering the largest mammalian protein‐coding multigene family: olfactory receptors (OR). Using the genome as a reference, we determined the proportion of intact protein‐coding receptors recovered by: (a) amplicons from degenerate primers sequenced via Sanger technology, (b) RNA‐Seq of the main olfactory epithelium, and (c) those genes captured with probes designed from transcriptomes of closely‐related species. Our initial re‐annotation of the high‐quality vampire bat genome resulted in >400 intact OR genes, more than doubling the original estimate. Sanger‐sequenced amplicons performed the poorest among the three approaches, detecting 50% of the annotated genomic ORs, and targeted sequence capture recovered nearly 75% of annotated genes. Each sequencing approach assembled high‐quality sequences, even if it did not recover all receptors in the genome. While some variation may be due to limitations of the study design (e.g., different individuals), variation among approaches was mostly caused by low coverage of some receptors rather than high rates of assembly error. Given this variability, we caution against using the counts of intact receptors per species to model the birth‐death process of multigene families. Instead, our results support the use of orthologous sequences to explore and model the evolutionary processes shaping these genes.

In mammals, social and reproductive behaviors are mediated by chemical cues encoded by hyperdiverse families of receptors expressed in the vomeronasal organ. Between species, the number of intact receptors can vary by orders of magnitude. However, the evolutionary processes behind variation in receptor number, and its link to fitness-related behaviors are not well understood. From vomeronasal transcriptomes, we discovered the first evidence of intact vomeronasal type-1 receptor (V1r) genes in bats, and we tested whether putatively functional bat receptors were orthologous to those of related taxa, or whether bats have evolved novel receptors. Instead of lineage-specific duplications, we found that bat V1rs show high levels of orthology to those of their relatives, and receptors are under comparative levels of purifying selection as non-bats. Despite widespread vomeronasal organ loss in bats, V1r copies have been retained for >65 million years. The highly conserved nature of bat V1rs challenges our current understanding of mammalian V1r function and suggests roles other than conspecific recognition or mating initiation in social behavior.