Evolution In Bats Research Articles

Digest: Repeated body size evolution in island bats.

Island ecosystems represent outstanding natural laboratories for studying the interplay between ecology and evolution. Lavery et al., (2024) use genomic approaches to identify a remarkable example of repeated evolution in Hipposideros bats across the Solomon Islands archipelago. They show that larger-bodied bats have independently evolved on different islands, highlighting an exciting new system for exploring the ecological and evolutionary drivers of repeated evolution in mammals.

Detecting SARS-CoV-2 in bats of New Mexico using immunohistochemistry on formalin- fixed, paraffn-embedded tissues

Objectives: The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV-2) and its zoonotic origin have raised critical questions regarding the potential role of local bat populations as reservoirs or intermediates for the transmission of this or related coronaviruses to humans. The goal of this project is to investigate the presence and diversity of coronaviruses in bat species native to the Southwest USA. Broadly, this study has two primary aims: (1) to detect antigens of coronaviruses in tissues of local bat populations via histological methods and (2) to perform a molecular screen of coronaviruses in these bats. Methods: Individuals from five bat species were evaluated: Artibeus jamaicensis, Myotis velifer, Antrozous pallidus, Tadarida brasiliensis, and Eptesicus fuscus. All species were local, wild-caught bats, with the exception of Artibeus jamaicensis, which is from a colony established at New Mexico State University (NMSU). Tissue was sourced from bats collected by TJO at NMSU. Bat tissue was fixed with formalin and samples subsequently taken of lung, kidney, nasal epithelium, and uterus. Tissue was further processed and embedded in paraffn wax. The embedded tissue was sectioned via a rotary microtome at 4 μm thickness and mounted onto slides. Slides were stained with general coronavirus anti-nucleocapsid primary antibody and a hematoxylin counterstain to visualize cellular structures. Slides were cover-slipped with DPX mounting medium and observed via light microscopy. Results: The immunohistochemistry protocol was performed on tissue from each of the five bat species investigated and on human placenta samples known to be positive for SARS-CoV-2. The latter was used as positive controls. Putative positive staining was seen in both human placenta and bat tissues. Interestingly, coronavirus was detected in Artibeus jamaicensis samples, an unexpected finding given its status as a laboratory colony. Verification of this putative finding is ongoing. Further work on our samples will determine the genetic identity of the coronaviruses detected and contribute to developing a molecular screen for more effcient detection of coronavirus antigens in local bat tissue. This work will serve as a springboard for further understanding coronavirus ecology and evolution in bats of the US southwest, and it can be used to further inform both local surveillance efforts and public policy surrounding the interaction between humans and animals in the natural environment and in controlled settings. Conclusions: Putative detection of coronavirus antigens was found in tissues from bat samples from the Southwestern USA. Implications for this work include broadening our understanding of the diversity and abundance of coronaviruses in the US. Burrell College; US National Science Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Convergent deployment of ancestral functions during the evolution of mammalian flight membranes.

Lateral flight membranes, or patagia, have evolved repeatedly in diverse mammalian lineages. While little is known about patagium development, its recurrent evolution may suggest a shared molecular basis. By combining transcriptomics, developmental experiments, and mouse transgenics, we demonstrate that lateral Wnt5a expression in the marsupial sugar glider (Petaurus breviceps) promotes the differentiation of its patagium primordium. We further show that this function of Wnt5a reprises ancestral roles in skin morphogenesis predating mammalian flight and has been convergently used during patagium evolution in eutherian bats. Moreover, we find that many genes involved in limb development have been redeployed during patagium outgrowth in both the sugar glider and bat. Together, our findings reveal that deeply conserved genetic toolkits contribute to the evolutionary transition to flight in mammals.

Physical constraints on thermoregulation and flight drive morphological evolution in bats

Body size and shape fundamentally determine organismal energy requirements by modulating heat and mass exchange with the environment and the costs of locomotion, thermoregulation, and maintenance. Ecologists have long used the physical linkage between morphology and energy balance to explain why the body size and shape of many organisms vary across climatic gradients, e.g., why larger endotherms are more common in colder regions. However, few modeling exercises have aimed at investigating this link from first principles. Body size evolution in bats contrasts with the patterns observed in other endotherms, probably because physical constraints on flight limit morphological adaptations. Here, we develop a biophysical model based on heat transfer and aerodynamic principles to investigate energy constraints on morphological evolution in bats. Our biophysical model predicts that the energy costs of thermoregulation and flight, respectively, impose upper and lower limits on the relationship of wing surface area to body mass (S-MR), giving rise to an optimal S-MR at which both energy costs are minimized. A comparative analysis of 278 species of bats supports the model’s prediction that S-MR evolves toward an optimal shape and that the strength of selection is higher among species experiencing greater energy demands for thermoregulation in cold climates. Our study suggests that energy costs modulate the mode of morphological evolution in bats—hence shedding light on a long-standing debate over bats’ conformity to ecogeographical patterns observed in other mammals—and offers a procedure for investigating complex macroecological patterns from first principles.

Natural selection in the evolution of SARS-CoV-2 in bats created a generalist virus and highly capable human pathogen.

Virus host shifts are generally associated with novel adaptations to exploit the cells of the new host species optimally. Surprisingly, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has apparently required little to no significant adaptation to humans since the start of the Coronavirus Disease 2019 (COVID-19) pandemic and to October 2020. Here we assess the types of natural selection taking place in Sarbecoviruses in horseshoe bats versus the early SARS-CoV-2 evolution in humans. While there is moderate evidence of diversifying positive selection in SARS-CoV-2 in humans, it is limited to the early phase of the pandemic, and purifying selection is much weaker in SARS-CoV-2 than in related bat Sarbecoviruses. In contrast, our analysis detects evidence for significant positive episodic diversifying selection acting at the base of the bat virus lineage SARS-CoV-2 emerged from, accompanied by an adaptive depletion in CpG composition presumed to be linked to the action of antiviral mechanisms in these ancestral bat hosts. The closest bat virus to SARS-CoV-2, RmYN02 (sharing an ancestor about 1976), is a recombinant with a structure that includes differential CpG content in Spike; clear evidence of coinfection and evolution in bats without involvement of other species. While an undiscovered "facilitating" intermediate species cannot be discounted, collectively, our results support the progenitor of SARS-CoV-2 being capable of efficient human-human transmission as a consequence of its adaptive evolutionary history in bats, not humans, which created a relatively generalist virus.

Molecular evolution of prolactin in Chiroptera: Accelerated evolution and a large insertion in vespertilionid bats

Pituitary prolactin (PRL) shows an episodic pattern of evolution in mammals, with a slow underlying rate (near stasis) and periods of rapid change in some groups. PRL evolution in bats, the second most speciose mammalian order, has not previously been studied, and is examined here. Slow basal evolution of PRL is seen in some bats, particularly megabats, but in most microbat groups evolution of PRL is more rapid. Accelerated evolution of PRL is particularly notable in the family Vespertilionidae, where analysis of nonsynonymous and synonymous substitutions indicates that it reflects adaptive evolution/positive selection. Remarkably, vespertilionid bats also show a large sequence insertion, of variable length, into exon 4 of PRL, giving a protein sequence 18–60 amino acids longer than normal, with the longest insertions in bats of the genus Myotis. An equivalent insertion has not been reported in PRL of any other vertebrate group. In the 3-dimensional structure of the complex between PRL and the extracellular domain (ecd) of its receptor (PRL:PRLR2) the inserted sequence is seen to be introduced in the short loop between helices 2 and 3 of PRL; it is far removed from the receptor-binding sites, and may not interfere with binding. The ecd of the receptor also shows variable rates of evolution, with a higher rate in the Vespertilionidae, but this is much less marked than for the hormone. The distribution of substitutions introduced into PRL during vespertilionid evolution appears to be non-random, and this and the evidence for positive selection suggests that the rapid evolution and insert sequence introduction were associated with a significant change in the biological properties of the hormone.

Molecular Evolution of the Nuclear Factor (Erythroid-Derived 2)-Like 2 Gene Nrf2 in Old World Fruit Bats (Chiroptera: Pteropodidae).

Mammals developed antioxidant systems to defend against oxidative damage in their daily life. Enzymatic antioxidants and low molecular weight antioxidants (LMWAs) constitute major parts of the antioxidant systems. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2, encoded by the Nrf2 gene) is a central transcriptional regulator, regulating transcription, of many antioxidant enzymes. Frugivorous bats eat large amounts of fruits that contain high levels of LMWAs such as vitamin C, thus, a reliance on LMWAs might greatly reduce the need for antioxidant enzymes in comparison to insectivorous bats. Therefore, it is possible that frugivorous bats have a reduced need for Nrf2 function due to their substantial intake of diet-antioxidants. To test whether the Nrf2 gene has undergone relaxed evolution in fruit-eating bats, we obtained Nrf2 sequences from 16 species of bats, including four Old World fruit bats (Pteropodidae) and one New World fruit bat (Phyllostomidae). Our molecular evolutionary analyses revealed changes in the selection pressure acting on Nrf2 gene and identified seven specific amino acid substitutions that occurred on the ancestral lineage leading to Old World fruit bats. Biochemical experiments were conducted to examine Nrf2 in Old World fruit bats and showed that the amount of catalase, which is regulated by Nrf2, was significantly lower in the brain, heart and liver of Old World fruit bats despite higher levels of Nrf2 protein in Old World fruit bats. Computational predictions suggest that three of these seven amino acid replacements might be deleterious to Nrf2 function. Therefore, the results suggest that Nrf2 gene might have experienced relaxed constraint in Old World fruit bats, however, we cannot rule out the possibility of positive selection. Our study provides the first data on the molecular adaptation of Nrf2 gene in frugivorous bats in compensation to the increased levels of LWMAs from their fruit-diet.

Phosphoenolpyruvate carboxykinase 1 gene (Pck1) displays parallel evolution between Old World and New World fruit bats.

Bats are an ideal mammalian group for exploring adaptations to fasting due to their large variety of diets and because fasting is a regular part of their life cycle. Mammals fed on a carbohydrate-rich diet experience a rapid decrease in blood glucose levels during a fast, thus, the development of mechanisms to resist the consequences of regular fasts, experienced on a daily basis, must have been crucial in the evolution of frugivorous bats. Phosphoenolpyruvate carboxykinase 1 (PEPCK1, encoded by the Pck1 gene) is the rate-limiting enzyme in gluconeogenesis and is largely responsible for the maintenance of glucose homeostasis during fasting in fruit-eating bats. To test whether Pck1 has experienced adaptive evolution in frugivorous bats, we obtained Pck1 coding sequence from 20 species of bats, including five Old World fruit bats (OWFBs) (Pteropodidae) and two New World fruit bats (NWFBs) (Phyllostomidae). Our molecular evolutionary analyses of these sequences revealed that Pck1 was under purifying selection in both Old World and New World fruit bats with no evidence of positive selection detected in either ancestral branch leading to fruit bats. Interestingly, however, six specific amino acid substitutions were detected on the ancestral lineage of OWFBs. In addition, we found considerable evidence for parallel evolution, at the amino acid level, between the PEPCK1 sequences of Old World fruit bats and New World fruit bats. Test for parallel evolution showed that four parallel substitutions (Q276R, R503H, I558V and Q593R) were driven by natural selection. Our study provides evidence that Pck1 underwent parallel evolution between Old World and New World fruit bats, two lineages of mammals that feed on a carbohydrate-rich diet and experience regular periods of fasting as part of their life cycle.

Unusual influenza A viruses in bats.

Influenza A viruses infect a remarkably diverse number of hosts. Two completely new influenza A virus subtypes were recently discovered in bats, dramatically expanding the host range of the virus. These bat viruses are extremely divergent from all other known strains and likely have unique replication cycles. Phylogenetic analysis indicates long-term, isolated evolution in bats. This is supported by a high seroprevalence in sampled bat populations. As bats represent ~20% of all classified mammals, these findings suggests the presence of a massive cryptic reservoir of poorly characterized influenza A viruses. Here, we review the exciting progress made on understanding these newly discovered viruses, and discuss their zoonotic potential.

Differential expression of Meis2, Mab21l2 and Tbx3 during limb development associated with diversification of limb morphology in mammals.

Bats are the only mammals capable of self-powered flight using wings. Differing from mouse or human limbs, four elongated digits within a broad wing membrane support the bat wing, and the foot of the bat has evolved a long calcar that spread the interfemoral membrane. Our recent mRNA sequencing (mRNA-Seq) study found unique expression patterns for genes at the 5′ end of the Hoxd gene cluster and for Tbx3 that are associated with digit elongation and wing membrane growth in bats. In this study, we focused on two additional genes, Meis2 and Mab21l2, identified from the mRNA-Seq data. Using whole-mount in situ hybridization (WISH) we validated the mRNA-Seq results for differences in the expression patterns of Meis2 and Mab21l2 between bat and mouse limbs, and further characterize the timing and location of the expression of these two genes. These analyses suggest that Meis2 may function in wing membrane growth and Mab21l2 may have a role in AP and DV axial patterning. In addition, we found that Tbx3 is uniquely expressed in the unique calcar structure found in the bat hindlimb, suggesting a role for this gene in calcar growth and elongation. Moreover, analysis of the coding sequences for Meis2, Mab21l2 and Tbx3 showed that Meis2 and Mab21l2 have high sequence identity, consistent with the functions of genes being conserved, but that Tbx3 showed accelerated evolution in bats. However, evidence for positive selection in Tbx3 was not found, which would suggest that the function of this gene has not been changed. Together, our findings support the hypothesis that the modulation of the spatiotemporal expression patterns of multiple functional conserved genes control limb morphology and drive morphological change in the diversification of mammalian limbs.

Prestin Expression in Cochlea of Bats Using Different Echolocation Systems

Outer hair cells (OHCs) have a unique ability to contract and elongate in response to changes in intracellular potential, and Prestin is the motor protein of the cochlea of the OHCs. It is the first time to invest the Prestin expression in different bat species. To invest Prestin expression in different bat species, which have different frequency, we did the coronal sections’ staining of the cochlea using immunhistochemistry. Experiment was designed to determine if the high-frequency bats’ OHCs have more expression than the low-frequency bats’OHCs. We found that the expression in three species was similar and had no obvious difference. Though the study of bats Prestin evolution suggested that Prestin has accelerating evolution in echolocation bats with high frequency, our we showed that the Prestin expression has nothing to do with the frequency, and the Prestin expression in high-frequency bats and low-frequency bats is similar.

Parallel evolution of the glycogen synthase 1 (muscle) gene Gys1 between Old World and New World fruit bats (Order: Chiroptera).

Glycogen synthase, which catalyzes the synthesis of glycogen, is especially important for Old World (Pteropodidae) and New World (Phyllostomidae) fruit bats that ingest high-carbohydrate diets. Glycogen synthase 1, encoded by the Gys1 gene, is the glycogen synthase isozyme that functions in muscles. To determine whether Gys1 has undergone adaptive evolution in bats with carbohydrate-rich diets, in comparison to insect-eating sister bat taxa, we sequenced the coding region of the Gys1 gene from 10 species of bats, including two Old World fruit bats (Pteropodidae) and a New World fruit bat (Phyllostomidae). Our results show no evidence for positive selection in the Gys1 coding sequence on the ancestral Old World and the New World Artibeus lituratus branches. Tests for convergent evolution indicated convergence of the sequences and one parallel amino acid substitution (T395A) was detected on these branches, which was likely driven by natural selection.

Relaxed evolution in the tyrosine aminotransferase gene tat in old world fruit bats (Chiroptera: Pteropodidae).

Frugivorous and nectarivorous bats fuel their metabolism mostly by using carbohydrates and allocate the restricted amounts of ingested proteins mainly for anabolic protein syntheses rather than for catabolic energy production. Thus, it is possible that genes involved in protein (amino acid) catabolism may have undergone relaxed evolution in these fruit- and nectar-eating bats. The tyrosine aminotransferase (TAT, encoded by the Tat gene) is the rate-limiting enzyme in the tyrosine catabolic pathway. To test whether the Tat gene has undergone relaxed evolution in the fruit- and nectar-eating bats, we obtained the Tat coding region from 20 bat species including four Old World fruit bats (Pteropodidae) and two New World fruit bats (Phyllostomidae). Phylogenetic reconstructions revealed a gene tree in which all echolocating bats (including the New World fruit bats) formed a monophyletic group. The phylogenetic conflict appears to stem from accelerated TAT protein sequence evolution in the Old World fruit bats. Our molecular evolutionary analyses confirmed a change in the selection pressure acting on Tat, which was likely caused by a relaxation of the evolutionary constraints on the Tat gene in the Old World fruit bats. Hepatic TAT activity assays showed that TAT activities in species of the Old World fruit bats are significantly lower than those of insectivorous bats and omnivorous mice, which was not caused by a change in TAT protein levels in the liver. Our study provides unambiguous evidence that the Tat gene has undergone relaxed evolution in the Old World fruit bats in response to changes in their metabolism due to the evolution of their special diet.

Ontogeny of vocalization in Jamaican fruit bats with implications for the evolution of echolocation

AbstractA long‐standing question in bat biology is if the evolution of echolocation and flight are associated or if they evolved independently, and if so, which evolved first. We seek to use ontogeny as a surrogate for understanding linkages between flight evolution and echolocation in bats. To do this we quantify the onset of recognizable sonar calls in newborn Artibeus jamaicensis and the tempo of growth and development across several different postnatal flight stages. By dropping individuals from a perch beginning on day 1 postpartum, we recorded vocalizations and quantified their flight ability into five developmental stages (flop, flutter, flap, flight and adult). One‐day‐old individuals were capable of emitting sonar‐like frequency‐modulated (FM) calls during free‐fall that were not significantly different from adult sonar calls in high and low frequency (kHz). However, bandwidth (kHz) did increase significantly with age as did sweep rate (kHz ms−1), whereas call duration significantly decreased. Few bats older than 18 days emitted communication calls as they fell and measured parameters of communication calls did not change significantly with age. Our data support the hypothesis that communication and sonar calls are discrete and independently derived at birth and thus have different evolutionary pathways as well.

The Glycogen Synthase 2 Gene (Gys2) Displays Parallel Evolution Between Old World and New World Fruit Bats

Frugivorous and nectarivorous bats rely largely on hepatic glycogenesis and glycogenolysis for postprandial blood glucose disposal and maintenance of glucose homeostasis during short time starvation, respectively. The glycogen synthase 2 encoded by the Gys2 gene plays a critical role in liver glycogen synthesis. To test whether the Gys2 gene has undergone adaptive evolution in bats with carbohydrate-rich diets in relation to their insect-eating sister taxa, we sequenced the coding region of the Gys2 gene in a number of bat species, including three Old World fruit bats (OWFBs) (Pteropodidae) and two New World fruit bats (NWFBs) (Phyllostomidae). Our results showed that the Gys2 coding sequences are highly conserved across all bat species we examined, and no evidence of positive selection was detected in the ancestral branches leading to OWFBs and NWFBs. Our explicit convergence test showed that posterior probabilities of convergence between several branches of OWFBs, and the NWFBs were markedly higher than that of divergence. Three parallel amino acid substitutions (Q72H, K371Q, and E666D) were detected among branches of OWFBs and NWFBs. Tests for parallel evolution showed that two parallel substitutions (Q72H and E666D) were driven by natural selection, while the K371Q was more likely to be fixed randomly. Thus, our results suggested that the Gys2 gene has undergone parallel evolution on amino acid level between OWFBs and NWFBs in relation to their carbohydrate metabolism.

Cloning and Molecular Evolution of the Aldehyde Dehydrogenase 2 Gene (Aldh2) in Bats (Chiroptera)

Old World fruit bats (Pteropodidae) and New World fruit bats (Phyllostomidae) ingest significant quantities of ethanol while foraging. Mitochondrial aldehyde dehydrogenase (ALDH2, encoded by the Aldh2 gene) plays an important role in ethanol metabolism. To test whether the Aldh2 gene has undergone adaptive evolution in frugivorous and nectarivorous bats in relation to ethanol elimination, we sequenced part of the coding region of the gene (1,143 bp, ~73 % coverage) in 14 bat species, including three Old World fruit bats and two New World fruit bats. Our results showed that the Aldh2 coding sequences are highly conserved across all bat species we examined, and no evidence of positive selection was detected in the ancestral branches leading to Old World fruit bats and New World fruit bats. Further research is needed to determine whether other genes involved in ethanol metabolism have been the targets of positive selection in frugivorous and nectarivorous bats.

Chromosome Evolution in Bats as Revealed by FISH: The Ongoing Search for the Ancestral Chiropteran Karyotype

Chiroptera, the second largest order of mammals, comprises more than 1,000 species in 18 highly morphologically diverse families. Chromosome painting with human probes has been applied to 10 bat species from 8 families. Except for the combination 10/12pq/22q, all syntenic segmental associations proposed for the mammalian ancestor have been found in Chiroptera. Bat-specific painting probes, established from 4 species of 3 families, have been used in whole chromosome painting experiments in 29 species from 8 families. The results show that the prevailing mode of chromosomal evolution in bats is Robertsonian translocation with a large number of convergent events. Given our present knowledge of chiropteran karyotypes, only a few elements of the ancestral chiropteran karyotype can be reconstructed with confidence.

Adaptive Evolution in the Glucose Transporter 4 Gene Slc2a4 in Old World Fruit Bats (Family: Pteropodidae)

Frugivorous and nectarivorous bats are able to ingest large quantities of sugar in a short time span while avoiding the potentially adverse side-effects of elevated blood glucose. The glucose transporter 4 protein (GLUT4) encoded by the Slc2a4 gene plays a critical role in transmembrane skeletal muscle glucose uptake and thus glucose homeostasis. To test whether the Slc2a4 gene has undergone adaptive evolution in bats with carbohydrate-rich diets in relation to their insect-eating sister taxa, we sequenced the coding region of the Slc2a4 gene in a number of bat species, including four Old World fruit bats (Pteropodidae) and three New World fruit bats (Phyllostomidae). Our molecular evolutionary analyses revealed evidence that Slc2a4 has undergone a change in selection pressure in Old World fruit bats with 11 amino acid substitutions detected on the ancestral branch, whereas, no positive selection was detected in the New World fruit bats. We noted that in the former group, amino acid replacements were biased towards either Serine or Isoleucine, and, of the 11 changes, six were specific to Old World fruit bats (A133S, A164S, V377F, V386I, V441I and G459S). Our study presents preliminary evidence that the Slc2a4 gene has undergone adaptive changes in Old World fruit bats in relation to their ability to meet the demands of a high sugar diet.

Independent evolution of functional MHC class II DRB genes in New World bat species

Genes of the major histocompatibility complex (MHC) play a pivotal role in the vertebrate immune system and are attractive markers for functional, fitness-related, genetic variation. Although bats (Chiroptera) represent the second largest mammalian order and are prone to various emerging infectious diseases, little is known about MHC evolution in bats. In the present study, we examined expressed MHC class II DRB sequences (exons 1 to 4) of New World bat species, Saccopteryx bilineata, Carollia perspicillata, Noctilio albiventris and Noctilio leporinus (only exon 2). We found a wide range of copy number variation of DRB loci with one locus detected in the genus Noctilio and up to ten functional loci observed in S. bilineata. Sequence variation between alleles of the same taxa was high with evidence for positive selection. We found statistical support for recombination or gene conversion events among sequences within the same but not between bat species. Phylogenetic relationships among DRB alleles provided strong evidence for independent evolution of the functional MHC class II DRB genes in the three investigated species, either by recent gene duplication, or homogenization of duplicated loci by frequent gene conversion events. Phylogenetic analysis of all available chiropteran DRB exon 2 sequences confirmed their monophyletic origin within families, but revealed a possible trans-species mode of evolution pattern in congeneric bat species, e.g. within the genera Noctilio and Myotis. This is the first study investigating phylogenetic relationships of MHC genes within bats and therefore contributes to a better understanding of MHC evolution in one of the most dominant mammalian order.

Modelling the prey detection performance of Rhinonicteris aurantia (Chiroptera: Hipposideridae) in different atmospheric conditions discounts the notional role of relative humidity in adaptive evolution

We examined a recent notion that differences in echolocation call frequency amongst geographic groups of constant frequency (CF)-emitting bats is the result of a trade-off between maximising prey detection range at lower frequencies and enhancing small-prey resolution at higher frequencies in different atmospheric (relative humidity; RH) environments. Isolated populations of the endemic Australian orange leaf-nosed bat Rhinonicteris aurantia were used as an example since geographic isolation in different environments has been a precursor to differences in their characteristic echolocation call frequencies (mean difference c. 6kHz; means of 114.64 and 120.99kHz). The influence of both atmospheric temperature and RH on maximum prey detection range was explored through mathematical modelling. This revealed that temperature was of similar importance to relative humidity and that under certain circumstances, each could reduce the effect of the other on ultrasound attenuation rates. The newly developed models contain significant conceptual improvements in method compared to other recent approaches, and can be applied to the situation of any other species of bat. For a given set of atmospheric conditions, the prey detection range of R. aurantia was reduced slightly when call frequency increased by 6kHz, but an increase in RH, temperature or both reduced detection range significantly. A similar trend was also evident in prey detection volume ratios calculated for the same conditions. Spatial volume ratios were applied to assess the impact of changed atmospheric conditions and prey size on foraging ecology. Reductions in detection range associated with increases in RH and/or temperature also varied in relation to the size (cross sectional area) of insect prey. Modelling demonstrated that small (6kHz) movements in call frequency could not compensate for the changes in prey detection range and spatial detection volumes that result from significant changes in atmospheric temperature or RH. The notion that differences in RH are the primary cause leading to adaptive evolution and speciation in CF-emitting bats by precipitating intraspecific differences in the mean call frequency of geographically isolated bat populations was not supported by the results of this case study.