Stephen J. Rossiter

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.

Convergent sequence evolution between echolocating bats and dolphins

Cases of convergent evolution - where different lineages have evolved similar traits independently - are common and have proven central to our understanding of selection. Yet convincing examples of adaptive convergence at the sequence level are exceptionally rare [1]. The motor protein Prestin is expressed in mammalian outer hair cells (OHCs) and is thought to confer high frequency sensitivity and selectivity in the mammalian auditory system [2]. We previously reported that the Prestin gene has undergone sequence convergence among unrelated lineages of echolocating bat [3]. Here we report that this gene has also undergone convergent amino acid substitutions in echolocating dolphins, which group with echolocating bats in a phylogenetic tree of Prestin. Furthermore, we find evidence that these changes were driven by natural selection.

The evolution of color vision in nocturnal mammals

Nonfunctional visual genes are usually associated with species that inhabit poor light environments (aquatic/subterranean/nocturnal), and these genes are believed to have lost function through relaxed selection acting on the visual system. Indeed, the visual system is so adaptive that the reconstruction of intact ancestral opsin genes has been used to reject nocturnality in ancestral primates. To test these assertions, we examined the functionality of the short and medium- to long-wavelength opsin genes in a group of mammals that are supremely adapted to a nocturnal niche: the bats. We sequenced the visual cone opsin genes in 33 species of bat with diverse sensory ecologies and reconstructed their evolutionary history spanning 65 million years. We found that, whereas the long-wave opsin gene was conserved in all species, the short-wave opsin gene has undergone dramatic divergence among lineages. The occurrence of gene defects in the short-wave opsin gene leading to loss of function was found to directly coincide with the origin of high-duty-cycle echolocation and changes in roosting ecology in some lineages. Our findings indicate that both opsin genes have been under purifying selection in the majority bats despite a long history of nocturnality. However, when spectacular losses do occur, these result from an evolutionary sensory modality tradeoff, most likely driven by subtle shifts in ecological specialization rather than a nocturnal lifestyle. Our results suggest that UV color vision plays a considerably more important role in nocturnal mammalian sensory ecology than previously appreciated and highlight the caveat of inferring light environments from visual opsins and vice versa.

Accelerated FoxP2 Evolution in Echolocating Bats

FOXP2 is a transcription factor implicated in the development and neural control of orofacial coordination, particularly with respect to vocalisation. Observations that orthologues show almost no variation across vertebrates yet differ by two amino acids between humans and chimpanzees have led to speculation that recent evolutionary changes might relate to the emergence of language. Echolocating bats face especially challenging sensorimotor demands, using vocal signals for orientation and often for prey capture. To determine whether mutations in the FoxP2 gene could be associated with echolocation, we sequenced FoxP2 from echolocating and non-echolocating bats as well as a range of other mammal species. We found that contrary to previous reports, FoxP2 is not highly conserved across all nonhuman mammals but is extremely diverse in echolocating bats. We detected divergent selection (a change in selective pressure) at FoxP2 between bats with contrasting sonar systems, suggesting the intriguing possibility of a role for FoxP2 in the evolution and development of echolocation. We speculate that observed accelerated evolution of FoxP2 in bats supports a previously proposed function in sensorimotor coordination.

Phylogeography of the greater horseshoe bat, Rhinolophus ferrumequinum : contrasting results from mitochondrial and microsatellite data

Phylogeographical studies are typically based on haplotype data, occasionally on nuclear markers such as microsatellites, but rarely combine both. This is unfortunate because the use of markers with contrasting modes of inheritance and rates of evolution might provide a more accurate and comprehensive understanding of a species’ history. Here we present a detailed study of the phylogeography of the greater horseshoe bat, Rhinolophus ferrumequinum, using 1098 bp of the mitochondrial ND2 gene from 45 localities from across its Palaearctic range to infer population history. In addition, we re‐analysed a large microsatellite data set available for this species and compared the results of both markers to infer population relationships and the historical processes influencing them. We show that mtDNA, the most popular marker in phylogeography studies, yielded a misleading result, and would have led us to conclude erroneously that a single expansion had taken place in Europe. Only by combining the mitochondrial and microsatellite data sets are we able to reconstruct the species’ history and show two colonization events in Europe, one before the Last Glacial Maximum (LGM) and one after it. Combining markers also revealed the importance of Asia Minor as an ancient refugium for this species and a source population for the expansion of the greater horseshoe bat into Europe before the LGM.

The hearing gene Prestin reunites echolocating bats

The remarkable high-frequency sensitivity and selectivity of the mammalian auditory system has been attributed to the evolution of mechanical amplification, in which sound waves are amplified by outer hair cells in the cochlea. This process is driven by the recently discovered protein prestin, encoded by the gene Prestin. Echolocating bats use ultrasound for orientation and hunting and possess the highest frequency hearing of all mammals. To test for the involvement of Prestin in the evolution of bat echolocation, we sequenced the coding region in echolocating and nonecholocating species. The resulting putative gene tree showed strong support for a monophyletic assemblage of echolocating species, conflicting with the species phylogeny in which echolocators are paraphyletic. We reject the possibilities that this conflict arises from either gene duplication and loss or relaxed selection in nonecholocating fruit bats. Instead, we hypothesize that the putative gene tree reflects convergence at stretches of functional importance. Convergence is supported by the recovery of the species tree from alignments of hydrophobic transmembrane domains, and the putative gene tree from the intra- and extracellular domains. We also found evidence that Prestin has undergone Darwinian selection associated with the evolution of specialized constant-frequency echolocation, which is characterized by sharp auditory tuning. Our study of a hearing gene in bats strongly implicates Prestin in the evolution of echolocation, and suggests independent evolution of high-frequency hearing in bats. These results highlight the potential problems of extracting phylogenetic signals from functional genes that may be prone to convergence.

Genetic variation and population structure in the endangered greater horseshoe bat Rhinolophus ferrumequinum

Following a dramatic decline last century, the British population of the endangered greater horseshoe bat Rhinolophus ferrumequinum is highly fragmented. To examine the consequences of fragmentation and limited dispersal on patterns of genetic structure and variation, we used microsatellite markers to screen bats from around 50% of the known maternity colonies in Britain, and two areas from continental Europe. Analyses revealed that Welsh and English colonies were genetically isolated. This, and lower variability in Britain than north France, may result from either genetic drift, or the species’ colonization history. Gene flow among most neighbouring colonies was not generally restricted, with one exception. These findings have important implications for the ongoing conservation management of this species.

Outbreeding increases offspring survival in wild greater horseshoe bats (Rhinolophus ferrumequinum)

The factors influencing the survival of greater horseshoe bat (Rhinolophus ferrumequinum) offspring born over seven years at a maternity colony in south-west Britain were studied. The effects of a range of phenotypic and maternal variables were analysed using a historical data set. In addition, the influence of two genetic measures on mortality, individual heterozygosity and a new measure of outbreeding, termed mean d2, was assessed. Logistic regressions were undertaken with survival modelled as a binary response variable. Survival to two life stages was studied for each variable and all models were developed for both sexes separately and together. Only one variable, mean d2, was significantly associated with survival. Male offspring with high mean d2 scores were more likely to survive to their first and second summers. The influence of mean d2 was not due to a single locus under selection but a wider multilocus effect and probably represents heterosis as opposed to solely inbreeding depression. Therefore, the extent to which an individual is outbred may determine survival more than widely used phenotypic characteristics such as size and mass. Mean d2 may reflect immunocompetence, which influences mortality. Protection of mating sites in order to facilitate gene flow and, therefore, outbreeding may help to promote population stability and growth.

Parallel signatures of sequence evolution among hearing genes in echolocating mammals: an emerging model of genetic convergence

Recent findings of sequence convergence in the Prestin gene among some bats and cetaceans suggest that parallel adaptations for high-frequency hearing have taken place during the evolution of echolocation. To determine if this gene is an exception, or instead similar processes have occurred in other hearing genes, we have examined Tmc1 and Pjvk, both of which are associated with non-syndromic hearing loss in mammals. These genes were amplified and sequenced from a number of mammalian species, including echolocating and non-echolocating bats and whales, and were analysed together with published sequences. Sections of both genes showed phylogenetic signals that conflicted with accepted species relationships, with coding regions uniting laryngeal echolocating bats in a monophyletic clade. Bayesian estimates of posterior probabilities of convergent and divergent substitutions provided more direct evidence of sequence convergence between the two groups of laryngeal echolocating bats as well as between echolocating bats and dolphins. We found strong evidence of positive selection acting on some echolocating bat species and echolocating cetaceans, contrasting with purifying selection on non-echolocating bats. Signatures of sequence convergence and molecular adaptation in two additional hearing genes suggest that the acquisition of high-frequency hearing has involved multiple loci.

Continent-wide panmixia of an African fruit bat facilitates transmission of potentially zoonotic viruses

The straw-coloured fruit bat, Eidolon helvum, is Africa’s most widely distributed and commonly hunted fruit bat, often living in close proximity to human populations. This species has been identified as a reservoir of potentially zoonotic viruses, but uncertainties remain regarding viral transmission dynamics and mechanisms of persistence. Here we combine genetic and serological analyses of populations across Africa, to determine the extent of epidemiological connectivity among E. helvum populations. Multiple markers reveal panmixia across the continental range, at a greater geographical scale than previously recorded for any other mammal, whereas populations on remote islands were genetically distinct. Multiple serological assays reveal antibodies to henipaviruses and Lagos bat virus in all locations, including small isolated island populations, indicating that factors other than population size and connectivity may be responsible for viral persistence. Our findings have potentially important public health implications, and highlight a need to avoid disturbances which may precipitate viral spillover.