Normal embryonic development of the greater horseshoe bat Rhinolophus ferrumequinum,with special reference to nose leaf formation

The order Chiroptera (bats) is the second largest group of mammals, composed of more than 1,300 species. Although powered flight and echolocation in bats have attracted many biologists, diversity in bat facial morphology has been almost neglected. Some bat species have a “nose leaf,” a leaf-like epithelial appendage around their nostrils. The nose leaf appears to have been acquired at least three times independently in bat evolution, and its morphology is highly diverse among bats species. Internal tissue morphology of nose-leaves has been investigated through histological analyses of late-stage fetuses of some bat species possessing the nose leaf. However, the proximate factors that bring about chiropteran nose-leaves have not been identified. As an initial step to address the question above, we describe the normal embryonic development of the greater horseshoe bat Rhinolophus ferrumequinum, and examine development of the tissues associated with their nose leaf during embryogenesis through histological analyses. We found that the nose leaf of R. ferrumequinum is formed through two phases. First, the primordium of the nose leaf appears as two tissue bulges aligned top and bottom on the face at embryonic stages 15–16. Second, the sub-regions of the nose leaf are differentiated through ingrowth as well as outgrowth of the epithelium at stage 17. In embryogenesis of Carollia perspicillata, a phyllostomid species with a nose leaf, the nose leaf primordium is formed as a small tissue bulge on the nostril at stage 17. This tissue bulge grows into a dorsally projected thin epithelial structure. Such differences in the nose leaf developmental process between chiropteran lineages may suggest that distinct developmental mechanisms have been employed in each lineage's nose leaf evolution.     

The voltage-gated potassium channel subfamily KQT member 4 (KCNQ4) displays parallel evolution in echolocating bats

Bats are the only mammals that use highly developed laryngeal echolocation, a sensory mechanism based on the ability to emit laryngeal sounds and interpret the returning echoes to identify objects. Although this capability allows bats to orientate and hunt in complete darkness, endowing them with great survival advantages, the genetic bases underlying the evolution of bat echolocation are still largely unknown. Echolocation requires high-frequency hearing that in mammals is largely dependent on somatic electromotility of outer hair cells. Then, understanding the molecular evolution of outer hair cell genes might help to unravel the evolutionary history of echolocation. In this work, we analyzed the molecular evolution of two key outer hair cell genes: the voltage-gated potassium channel gene KCNQ4 and CHRNA10, the gene encoding the α10 nicotinic acetylcholine receptor subunit. We reconstructed the phylogeny of bats based on KCNQ4 and CHRNA10 protein and nucleotide sequences. A phylogenetic tree built using KCNQ4 amino acid sequences showed that two paraphyletic clades of laryngeal echolocating bats grouped together, with eight shared substitutions among particular lineages. In addition, our analyses indicated that two of these parallel substitutions, M388I and P406S, were probably fixed under positive selection and could have had a strong functional impact on KCNQ4. Moreover, our results indicated that KCNQ4 evolved under positive selection in the ancestral lineage leading to mammals, suggesting that this gene might have been important for the evolution of mammalian hearing. On the other hand, we found that CHRNA10, a gene that evolved adaptively in the mammalian lineage, was under strong purifying selection in bats. Thus, the CHRNA10 amino acid tree did not show echolocating bat monophyly and reproduced the bat species tree. These results suggest that only a subset of hearing genes could underlie the evolution of echolocation. The present work continues to delineate the genetic bases of echolocation and ultrasonic hearing in bats.     

The Evolution of Bat Vestibular Systems in the Face of Potential Antagonistic Selection Pressures for Flight and Echolocation

The vestibular system maintains the body’s sense of balance and, therefore, was probably subject to strong selection during evolutionary transitions in locomotion. Among mammals, bats possess unique traits that place unusual demands on their vestibular systems. First, bats are capable of powered flight, which in birds is associated with enlarged semicircular canals. Second, many bats have enlarged cochleae associated with echolocation, and both cochleae and semicircular canals share a space within the petrosal bone. To determine how bat vestibular systems have evolved in the face of these pressures, we used micro-CT scans to compare canal morphology across species with contrasting flight and echolocation capabilities. We found no increase in canal radius in bats associated with the acquisition of powered flight, but canal radius did correlate with body mass in bat species from the suborder Yangochiroptera, and also in non-echolocating Old World fruit bats from the suborder Yinpterochiroptera. No such trend was seen in members of the Yinpterochiroptera that use laryngeal echolocation, although canal radius was associated with wing-tip roundedness in this group. We also found that the vestibular system scaled with cochlea size, although the relationship differed in species that use constant frequency echolocation. Across all bats, the shape of the anterior and lateral canals was associated with large cochlea size and small body size respectively, suggesting differential spatial constraints on each canal depending on its orientation within the skull. Thus in many echolocating bats, it seems that the combination of small body size and enlarged cochlea together act as a principal force on the vestibular system. The two main groups of echolocating bats displayed different canal morphologies, in terms of size and shape in relation to body mass and cochlear size, thus suggesting independent evolutionary pathways and offering tentative support for multiple acquisitions of echolocation.     

Evolutionary insights into Rhinolophus episcopus (Chiroptera,Rhinolophidae) in China: Isolation by distance,environment, or sensory system?

Evolutionary processes can be influenced by several factors, such as geographic isolation, environmental selection, and sensory variation. For most nocturnal bats, echolocation is the primary sensory system used to prey and communicate, and plays important roles in chiropteran diversification and evolution. Understanding the relative contribution of geography, the environment, and this sensory system to population genetic divergence can elucidate the processes involved in bat incipient speciation and evolution. In this study, we collected spatial and environmental information, echolocation calls, as well as the previously published genetic data (six microsatellite loci and the mitochondrial cytochrome b gene) of widely distributed Rhinolophus episcopus populations to test three hypotheses for nuclear and mitochondrial divergence (isolation by distance, isolation by environment, and isolation by sensory variation) and unveil the factors that drive intraspecific genetic differentiation. The moderate level of nuclear differentiation was correlated with geographic/spatial distance and acoustic variation, whereas the relatively high level of mitochondrial differentiation was mainly associated with acoustic divergence. No significant correlation was observed between genetic divergence and environmental variables. Among the three factors, acoustic divergence explained the highest percentage of both nuclear and mitochondrial divergence. Thus, our results indicate that sensory variation may have played important roles in driving population isolation early in bat speciation, which is consistent with the hypothesis of isolation by sensory variation. Our study emphasizes the need to consider more factors, especially sensory traits, and combine multiple statistical methods in landscape genetic studies to test their potential contributions to driving population divergence.     

Things that go bump in the night: evolutionary interactions between cephalopods and cetaceans in the tertiary

Echolocation has evolved independently in several vertebrate groups, and hypotheses about the origin of echolocation in these groups often invoke abiotic mechanisms driving morphological evolution. In bats, for example, the ecological setting associated with the origin of echolocation has been linked to global warming during the Palaeocene–Eocene; similarly, the origin of toothed whales (odontocetes) has been broadly correlated with the establishment of the circum-Antarctic current. These scenarios, and the adaptational hypotheses for the evolution of echolocation with which they are associated, neglect a consideration of possible biotic mechanisms. Here we propose that the origin of echolocation in odontocetes was initially an adaptation for nocturnal epipelagic feeding – primarily on diel migrating cephalopods. We test this hypothesis using data on the temporal, geographical, and water column distributions of odontocetes and cephalopods, and other global events from their respective tertiary histories. From this analysis, we suggest that echolocation in early odontocetes aided nocturnal feeding on cephalopods and other prey items, and that this early system was exapted for deep diving and hunting at depths below the photic zone where abundant cephalopod resources were available 24 h a day. This scenario extends to the evolution of other cephalopod feeding (teuthophagous) marine vertebrates such as pinnipeds and Mesozoic marine reptiles.     

Foraging shifts and visual preadaptation in ecologically diverse bats

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.     

‘No cost of echolocation for flying bats’ revisited

Echolocation is energetically costly for resting bats, but previous experiments suggested echolocation to come at no costs for flying bats. Yet, previous studies did not investigate the relationship between echolocation, flight speed, aerial manoeuvres and metabolism. We re-evaluated the 'no-cost' hypothesis, by quantifying the echolocation pulse rate, the number of aerial manoeuvres (landings and U-turns), and the costs of transport in the 5-g insectivorous bat Rhogeessa io (Vespertilionidae). On average, bats (n = 15) travelled at 1.76 ± 0.36 m s?1 and performed 11.2 ± 6.1 U-turns and 2.8 ± 2.9 ground landings when flying in an octagonal flight cage. Bats made more U-turns with decreasing wing loading (body weight divided by wing area). At flight, bats emitted 19.7 ± 2.7 echolocation pulses s?1 (range 15.3-25.8 pulses s?1), and metabolic rate averaged 2.84 ± 0.95 ml CO? min?1, which was more than 16 times higher than at rest. Bats did not echolocate while not engaged in flight. Costs of transport were not related to the rate of echolocation pulse emission or the number of U-turns, but increased with increasing number of landings; probably as a consequence of slower travel speed when staying briefly on ground. Metabolic power of flight was lower than predicted for R. io under the assumption that energetic costs of echolocation call production is additive to the aerodynamic costs of flight. Results of our experiment are consistent with the notion that echolocation does not add large energetic costs to the aerodynamic power requirements of flight in bats.     

Trapped in the darkness of the night: thermal and energetic constraints of daylight flight in bats

Bats are one of the most successful mammalian groups, even though their foraging activities are restricted to the hours of twilight and night-time. Some studies suggested that bats became nocturnal because of overheating when flying in daylight. This is because--in contrast to feathered wings of birds--dark and naked wing membranes of bats efficiently absorb short-wave solar radiation. We hypothesized that bats face elevated flight costs during daylight flights, since we expected them to alter wing-beat kinematics to reduce heat load by solar radiation. To test this assumption, we measured metabolic rate and body temperature during short flights in the tropical short-tailed fruit bat Carollia perspicillata at night and during the day. Core body temperature of flying bats differed by no more than 2°C between night and daytime flights, whereas mass-specific CO(2) production rates were higher by 15 per cent during daytime. We conclude that increased flight costs only render diurnal bat flights profitable when the relative energy gain during daytime is high and risk of predation is low. Ancestral bats possibly have evolved dark-skinned wing membranes to reduce nocturnal predation, but a low degree of reflectance of wing membranes made them also prone to overheating and elevated energy costs during daylight flights. In consequence, bats may have become trapped in the darkness of the night once dark-skinned wing membranes had evolved.     

Evidence for Repeated Independent Evolution of Migration in the Largest Family of Bats

Background

How migration evolved represents one of the most poignant questions in evolutionary biology. While studies on the evolution of migration in birds are well represented in the literature, migration in bats has received relatively little attention. Yet, more than 30 species of bats are known to migrate annually from breeding to non-breeding locations. Our study is the first to test hypotheses on the evolutionary history of migration in bats using a phylogenetic framework.

Methods and Principal Findings

In addition to providing a review of bat migration in relation to existing hypotheses on the evolution of migration in birds, we use a previously published supertree to formulate and test hypotheses on the evolutionary history of migration in bats. Our results suggest that migration in bats has evolved independently in several lineages potentially as the need arises to track resources (food, roosting site) but not through a series of steps from short- to long-distance migrants, as has been suggested for birds. Moreover, our analyses do not indicate that migration is an ancestral state but has relatively recently evolved in bats. Our results also show that migration is significantly less likely to evolve in cave roosting bats than in tree roosting species.

Conclusions and Significance

This is the first study to provide evidence that migration has evolved independently in bat lineages that are not closely related. If migration evolved as a need to track seasonal resources or seek adequate roosting sites, climate change may have a pivotal impact on bat migratory habits. Our study provides a strong framework for future research on the evolution of migration in chiropterans.     

Rain increases the energy cost of bat flight

Similar to insects, birds and pterosaurs, bats have evolved powered flight. But in contrast to other flying taxa, only bats are furry. Here, we asked whether flight is impaired when bat pelage and wing membranes get wet. We studied the metabolism of short flights in Carollia sowelli, a bat that is exposed to heavy and frequent rainfall in neotropical rainforests. We expected bats to encounter higher thermoregulatory costs, or to suffer from lowered aerodynamic properties when pelage and wing membranes catch moisture. Therefore, we predicted that wet bats face higher flight costs than dry ones. We quantified the flight metabolism in three treatments: dry bats, wet bats and no rain, wet bats and rain. Dry bats showed metabolic rates predicted by allometry. However, flight metabolism increased twofold when bats were wet, or when they were additionally exposed to rain. We conclude that bats may not avoid rain only because of sensory constraints imposed by raindrops on echolocation, but also because of energetic constraints.     

The earliest Asian bats (Mammalia: Chiroptera) address major gaps in bat evolution

Bats dispersed widely after evolving the capacity for powered flight, and fossil bats are known from the early Eocene of most continents. Until now, however, bats have been conspicuously absent from the early Eocene of mainland Asia. Here, we report two teeth from the Junggar Basin of northern Xinjiang, China belonging to the first known early Eocene bats from Asia, representing arguably the most plesiomorphic bat molars currently recognized. These teeth combine certain bat synapomorphies with primitive traits found in other placental mammals, thereby potentially illuminating dental evolution among stem bats. The Junggar Basin teeth suggest that the dentition of the stem chiropteran family Onychonycteridae is surprisingly derived, although their postcranial anatomy is more primitive than that of any other Eocene bats. Additional comparisons with stem bat families Icaronycteridae and Archaeonycteridae fail to identify unambiguous synapomorphies for the latter taxa, raising the possibility that neither is monophyletic as currently recognized. The presence of highly plesiomorphic bats in the early Eocene of central Asia suggests that this region was an important locus for the earliest, transitional phases of bat evolution, as has been demonstrated for other placental mammal orders including Lagomorpha and Rodentia.     

Calls in the Forest: A Comparative Approach to How Bats Find Tree Cavities

Although tree cavities are a particularly critical resource for forest bats, how bats search for and find new roosts is still poorly known. Building on a recent study on the sensory basis of roost finding in the noctule (Ruczynski et al. 2007), here we take a comparative approach to how bats find roosts. We tested the hypothesis that species' flight abilities and echolocation call characteristics play important roles in how well and by which cues bats find new tree roosts. We used the very manoeuvrable, faintly echolocating brown long-eared bat ( Plecotus auritus ) and the less manoeuvrable, louder Daubenton's bat ( Myotis daubentonii ) as study species. The species are sympatric in European temperate forests and both roost in tree cavities. We trained bats in short-term captivity to find entrances to tree cavities and experimentally manipulated the sensory cues available to them. In both species, cue type influenced the search time for successful cavity detection. Visual, olfactory and temperature cues did not improve the bats' performance over the performance by echolocation alone. Eavesdropping on conspecific echolocation calls played back from inside the cavity decreased search time in Daubenton's bat ( M. daubentonii ), underlining the double function of echolocation signals – orientation and communication. This was not so in the brown long-eared bat ( P. auritus ) that has low call amplitudes. The highly manoeuvrable P. auritus found cavities typically from flight and the less manoeuvrable M. daubentonii found more entrances during crawling. Comparison with the noctule data from Ruczyński et al. (2007) indicates that manoeuvrability predicts the mode of cavity search. It further highlights the importance of call amplitude for eavesdropping and cavity detection in bats.     

The Divergence of Echolocation Frequency in Horseshoe Bats: Moth Hearing,Body Size or Habitat?

A phylogenetic approach was used to test three hypotheses regarding the evolution of diversity in the echolocation frequencies used by horseshoe bats (family Rhinolophidae, genus Rhinolophus): 1) Allotonic Frequency Hypothesis (high frequency echolocation in the Rhinolophidae resulted from coevolution with moth hearing); 2) Allometry Hypothesis (echolocation frequency is negatively scaled with body size and evolutionary changes in echolocation frequencies are correlated with changes in body size in the Rhinolophidae); and 3) Foraging Habitat Hypothesis (evolution of echolocation frequency is associated with changes in habitat type). Both discrete and continuous character sets were used for ancestral state reconstructions and for investigating patterns of evolution between frequency and body size, and frequency and habitat type. Contrary to the prediction of the Allotonic Frequency Hypothesis, echolocation frequency in the Rhinolophidae did not increase over time, which would be expected if moth hearing and bat echolocation frequency coevolved. The number of extant species that exhibit calls within moth hearing ranges was not significantly different from the number of species that echolocate outside of moth hearing range. There was also no correlation between changes in frequency and changes in habitat type as predicted by the Foraging Habitat Hypothesis. Instead, the evolution of echolocation frequency within the Rhinolophidae was correlated with changes in body size as predicted by the Allometry Hypothesis.     

食虫蝙蝠与昆虫之间的相互作用和协同进化关系

食虫蝙蝠与昆虫之间是捕食和被捕食的关系,夜行性昆虫是食虫蝙蝠主要的食物来源。在漫长的协同进化中,蝙蝠施加的捕食压力导致夜行性昆虫一系列特征的进化,其中一部分昆虫进化出能听到蝙蝠的超声波信号并采取逃跑行为或者能通过其它方式躲避蝙蝠,同时昆虫的适应性特征同样影响着蝙蝠的回声定位和捕食策略。本文从蝙蝠捕食昆虫的种类、昆虫对蝙蝠捕食的反应和食虫性蝙蝠对昆虫防卫的适应对策等三个方面对食虫蝙蝠与昆虫之间的相互关系进行了概述。     

The communicative potential of bat echolocation pulses

Ecological constraints often shape the echolocation pulses emitted by bat species. Consequently some (but not all) bats emit species-specific echolocation pulses. Because echolocation pulses are often intense and emitted at high rates, they are potential targets for eavesdropping by other bats. Echolocation pulses can also vary within species according to sex, body size, age, social group and geographic location. Whether these features can be recognised by other bats can only be determined reliably by playback experiments, which have shown that echolocation pulses do provide sufficient information for the identification of sex and individual in one species. Playbacks also show that bats can locate conspecifics and heterospecifics at foraging and roost sites by eavesdropping on echolocation pulses. Guilds of echolocating bat species often partition their use of pulse frequencies. Ecology, allometric scaling and phylogeny play roles here, but are not sufficient to explain this partitioning. Evidence is accumulating to support the hypothesis that frequency partitioning evolved to facilitate intraspecific communication. Acoustic character displacement occurs in at least one instance. Future research can relate genetic population structure to regional variation in echolocation pulse features and elucidate those acoustic features that most contribute to discrimination of individuals.     

Comparing aerodynamic efficiency in birds and bats suggests better flight performance in birds

Flight is one of the energetically most costly activities in the animal kingdom, suggesting that natural selection should work to optimize flight performance. The similar size and flight speed of birds and bats may therefore suggest convergent aerodynamic performance; alternatively, flight performance could be restricted by phylogenetic constraints. We test which of these scenarios fit to two measures of aerodynamic flight efficiency in two passerine bird species and two New World leaf-nosed bat species. Using time-resolved particle image velocimetry measurements of the wake of the animals flying in a wind tunnel, we derived the span efficiency, a metric for the efficiency of generating lift, and the lift-to-drag ratio, a metric for mechanical energetic flight efficiency. We show that the birds significantly outperform the bats in both metrics, which we ascribe to variation in aerodynamic function of body and wing upstroke: Bird bodies generated relatively more lift than bat bodies, resulting in a more uniform spanwise lift distribution and higher span efficiency. A likely explanation would be that the bat ears and nose leaf, associated with echolocation, disturb the flow over the body. During the upstroke, the birds retract their wings to make them aerodynamically inactive, while the membranous bat wings generate thrust and negative lift. Despite the differences in performance, the wake morphology of both birds and bats resemble the optimal wake for their respective lift-to-drag ratio regimes. This suggests that evolution has optimized performance relative to the respective conditions of birds and bats, but that maximum performance is possibly limited by phylogenetic constraints. Although ecological differences between birds and bats are subjected to many conspiring variables, the different aerodynamic flight efficiency for the bird and bat species studied here may help explain why birds typically fly faster, migrate more frequently and migrate longer distances than bats.     

Bat echolocation calls: adaptation and convergent evolution

Bat echolocation calls provide remarkable examples of 'good design' through evolution by natural selection. Theory developed from acoustics and sonar engineering permits a strong predictive basis for understanding echolocation performance. Call features, such as frequency, bandwidth, duration and pulse interval are all related to ecological niche. Recent technological breakthroughs have aided our understanding of adaptive aspects of call design in free-living bats. Stereo videogrammetry, laser scanning of habitat features and acoustic flight path tracking permit reconstruction of the flight paths of echolocating bats relative to obstacles and prey in nature. These methods show that echolocation calls are among the most intense airborne vocalizations produced by animals. Acoustic tracking has clarified how and why bats vary call structure in relation to flight speed. Bats using broadband echolocation calls adjust call design in a range-dependent manner so that nearby obstacles are localized accurately. Recent phylogenetic analyses based on gene sequences show that particular types of echolocation signals have evolved independently in several lineages of bats. Call design is often influenced more by perceptual challenges imposed by the environment than by phylogeny, and provides excellent examples of convergent evolution. Now that whole genome sequences of bats are imminent, understanding the functional genomics of echolocation will become a major challenge.     

Extinction of the acoustic startle response in moths endemic to a bat-free habitat

Most moths use ears solely to detect the echolocation calls of hunting, insectivorous bats and evoke evasive flight manoeuvres. This singularity of purpose predicts that this sensoribehavioural network will regress if the selective force that originally maintained it is removed. We tested this with noctuid moths from the islands of Tahiti and Moorea, sites where bats have never existed and where an earlier study demonstrated that the ears of endemic species resemble those of adventives although partially reduced in sensitivity. To determine if these moths still express the anti-bat defensive behaviour of acoustic startle response (ASR) we compared the nocturnal flight times of six endemic to six adventive species in the presence and absence of artificial bat echolocation sounds. Whereas all of the adventive species reduced their flight times when exposed to ultrasound, only one of the six endemic species did so. These differences were significant when tested using a phylogenetically based pairwise comparison and when comparing effect sizes. We conclude that the absence of bats in this habitat has caused the neural circuitry that normally controls the ASR behaviour in bat-exposed moths to become decoupled from the functionally vestigial ears of endemic Tahitian moths.     

Prestin Shows Divergent Evolution Between Constant Frequency Echolocating Bats

The gene Prestin encodes a motor protein that is thought to confer the high-frequency sensitivity and selectivity that characterizes the mammalian auditory system. Recent research shows that the Prestin gene has undergone a burst of positive selection on the ancestral branch of the Old World horseshoe and leaf-nosed bats (Rhinolophidae and Hipposideridae, respectively), and also on the branch leading to echolocating cetaceans. Moreover, these two groups share a large number of convergent amino acid sequence replacements. Horseshoe and leaf-nosed bats exhibit narrowband echolocation, in which the emitted calls are based on the second harmonic of a predominantly constant frequency (CF) component, the frequency of which is also over-represented in the cochlea. This highly specialized form of echolocation has also evolved independently in the neotropical Parnell's mustached bat (Pteronotus parnellii). To test whether the convergent evolution of CF echolocation between lineages has arisen from common changes in the Prestin gene, we sequenced the Prestin coding region (~2,212?bp, >99% coverage) in P. parnellii and several related species that use broadband echolocation calls. Our reconstructed Prestin gene tree and amino acid tree showed that P. parnellii did not group together with Old World horseshoe and leaf-nosed bats, but rather clustered within its true sister species. Comparisons of sequences confirmed that P. parnellii shared most amino acid changes with its congeners, and we found no evidence of positive selection in the branch leading to the genus of Pteronotus. Our result suggests that the adaptive changes seen in Prestin in horseshoe and leaf-nosed bats are not necessary for CF echolocation in P. parnellii.