Mustached Bat Research Articles

The genome sequence of the endemic Mexican common mustached Bat, Pteronotus mexicanus. Miller, 1902 [Mormoopidae; Pteronotus]

We describe here the first characterization of the genome of the bat Pteronotus mexicanus, an endemic species of Mexico, as part of the Mexican Bat Genome Project which focuses on the characterization and assembly of the genomes of endemic bats in Mexico. The genome was assembled from a liver tissue sample of an adult male from Jalisco, Mexico provided by the Texas Tech University Museum tissue collection. The assembled genome size was 1.9 Gb. The assembly of the genome was fitted in a framework of 110,533 scaffolds and 1,659,535 contigs. The ecological importance of bats such as P. mexicanus, and their diverse ecological roles, underscores the value of having complete genomes in addressing information gaps and facing challenges regarding their function in ecosystems and their conservation.

Bat cellular immunity varies by year and dietary habit amidst land conversion.

Monitoring the health of wildlife populations is essential in the face of increased agricultural expansion and forest fragmentation. Loss of habitat and habitat degradation can negatively affect an animal's physiological state, possibly resulting in immunosuppression and increased morbidity or mortality. We sought to determine how land conversion may differentially impact cellular immunity and infection risk in Neotropical bats species regularly infected with bloodborne pathogens, and to evaluate how effects may vary over time and by dietary habit. We studied common vampire bats (Desmodus rotundus), northern yellow-shouldered bats (Sturnira parvidens) and Mesoamerican mustached bats (Pteronotus mesoamericanus), representing the dietary habits of sanguivory, frugivory and insectivory respectively, in northern Belize. We compared estimated total white blood cell count, leukocyte differentials, neutrophil to lymphocyte ratio and infection status with two bloodborne bacterial pathogens (Bartonella spp. and hemoplasmas) of 118 bats captured in a broadleaf, secondary forest over three years (2017-2019). During this period, tree cover decreased by 14.5% while rangeland expanded by 14.3%, indicating increasing habitat loss and fragmentation. We found evidence for bat species-specific responses of cellular immunity between years, with neutrophil counts significantly decreasing in S. parvidens from 2017 to 2018, but marginally increasing in D. rotundus. However, the odds of infection with Bartonella spp. and hemoplasmas between 2017 and 2019 did not differ between bat species, contrary to our prediction that pathogen prevalence may increase with land conversion. We conclude that each bat species invested differently in cellular immunity in ways that changed over years of increasing habitat loss and fragmentation. We recommend further research on the interactions between land conversion, immunity and infection across dietary habits of Neotropical bats for informed management and conservation.

Long-Read Sequencing Reveals Rapid Evolution of Immunity- and Cancer-Related Genes in Bats.

Bats are exceptional among mammals for their powered flight, extended lifespans, and robust immune systems and therefore have been of particular interest in comparative genomics. Using the Oxford Nanopore Technologies long-read platform, we sequenced the genomes of two bat species with key phylogenetic positions, the Jamaican fruit bat (Artibeus jamaicensis) and the Mesoamerican mustached bat (Pteronotus mesoamericanus), and carried out a comprehensive comparative genomic analysis with a diverse collection of bats and other mammals. The high-quality, long-read genome assemblies revealed a contraction of interferon (IFN)-α at the immunity-related type I IFN locus in bats, resulting in a shift in relative IFN-ω and IFN-α copy numbers. Contradicting previous hypotheses of constitutive expression of IFN-α being a feature of the bat immune system, three bat species lost all IFN-α genes. This shift to IFN-ω could contribute to the increased viral tolerance that has made bats a common reservoir for viruses that can be transmitted to humans. Antiviral genes stimulated by type I IFNs also showed evidence of rapid evolution, including a lineage-specific duplication of IFN-induced transmembrane genes and positive selection in IFIT2. In addition, 33 tumor suppressors and 6 DNA-repair genes showed signs of positive selection, perhaps contributing to increased longevity and reduced cancer rates in bats. The robust immune systems of bats rely on both bat-wide and lineage-specific evolution in the immune gene repertoire, suggesting diverse immune strategies. Our study provides new genomic resources for bats and sheds new light on the extraordinary molecular evolution in this critically important group of mammals.

Predation of a mustached bat, Pteronotus sp. (Mormoopidae), by an Amazon tree boa, Corallus hortulanus (Boidae), in the Brazilian Amazon

ABSTRACT Several bat species use caves as roosts and some of these caves can harbor high concentrations of individuals. Such caves may represent opportunities for certain predators, benefiting from the concentration of potential prey. Here, we report the predation of a Pteronotus bat by a Corallus hortulanus in the Brazilian Amazon. On three occasions over a year, individuals of C. hortulanus were observed around a cave that harbors populations containing tens of thousands of bats. On one occasion, an individual of this snake species was observed preying on a Pteronotus sp. as it left the cave. Our record extends the known diet for C. hortulanus, documenting the predation of a mormoopid bat by this snake species for the first time. This record reinforces the need for longitudinal studies at sites with exceptional bat populations to gain a deeper understanding of the ecological predator-prey relationships involving this diverse group of mammals.

Acoustic clue: bringing echolocation call data into the distribution dilemma of Pteronotus (Chiroptera: Mormoopidae) complexes in Central America

Abstract In Central America, the distributional limits and the contact zones of some Pteronotus species, such as the naked-backed bats and the lesser mustached bats, are unclear. To elucidate the distributional dilemma of the species groups Pteronotus fulvus + Pteronotus davyi and Pteronotus psilotis + Pteronotus personatus in Central America, we studied the acoustic variation of their echolocation calls along the range of possible contact zones and the existence of distinct phonic groups. We performed hierarchical k-means clustering on principal components (HCPC) using acoustic samples from Mexico, Honduras, El Salvador, Nicaragua and Costa Rica to describe the global acoustic diversity, possibly overlooking differences between species groups. We assessed whether those acoustic differences were related to the geographical distance and geographical location. We found evidence of sympatry for three phonic groups within each species complex, without a clear correspondence to the known distribution of species. The changes in frequency of their echolocation calls seem to follow a similar pattern to the geographical variation in body size of the species. Future studies in Central America should include an integrative sampling of individually captured, tagged and recorded bats to help in resolution of the distribution dilemma raised here.

Hemispheric and Sex Differences in Mustached Bat Primary Auditory Cortex Revealed by Neural Responses to Slow Frequency Modulations.

The mustached bat (Pteronotus parnellii) is a mammalian model of cortical hemispheric asymmetry. In this species, complex social vocalizations are processed preferentially in the left Doppler-shifted constant frequency (DSCF) subregion of primary auditory cortex. Like hemispheric specializations for speech and music, this bat brain asymmetry differs between sexes (i.e., males>females) and is linked to spectrotemporal processing based on selectivities to frequency modulations (FMs) with rapid rates (>0.5 kHz/ms). Analyzing responses to the long-duration (>10 ms), slow-rate (<0.5 kHz/ms) FMs to which most DSCF neurons respond may reveal additional neural substrates underlying this asymmetry. Here, we bilaterally recorded responses from 176 DSCF neurons in male and female bats that were elicited by upward and downward FMs fixed at 0.04 kHz/ms and presented at 0-90 dB SPL. In females, we found inter-hemispheric latency differences consistent with applying different temporal windows to precisely integrate spectrotemporal information. In males, we found a substrate for asymmetry less related to spectrotemporal processing than to acoustic energy (i.e., amplitude). These results suggest that in the DSCF area, (1) hemispheric differences in spectrotemporal processing manifest differently between sexes, and (2) cortical asymmetry for social communication is driven by spectrotemporal processing differences and neural selectivities for amplitude.

First record of albinism in a mustached bat (Chiroptera, Mormoopidae) from South America

Notas sobre Mamíferos Sudamericanos es una revista de la Sociedad Argentina para el Estudio de los Mamíferos (SAREM). Publica información acotada donde se describen nuevas localidades de registro, datos sobre comportamiento y aspectos ecológicos de mamíferos neotropicales.

Correlating Cochlear Morphometrics from Parnell's Mustached Bat (Pteronotus parnellii) with Hearing.

Morphometric analysis of the inner ear of mammals can provide information for cochlear frequency mapping, a species-specific designation of locations in the cochlea at which different sound frequencies are encoded. Morphometric variation occurs in the hair cells of the organ of Corti along the cochlea, with the base encoding the highest frequency sounds and the apex encoding the lowest frequencies. Changes in cell shape and spacing can yield additional information about the biophysical basis of cochlear tuning mechanisms. Here, we investigate how morphometric analysis of hair cells in mammals can be used to predict the relationship between frequency and cochlear location. We used linear and geometric morphometrics to analyze scanning electron micrographs of the hair cells of the cochleae in Parnell's mustached bat (Pteronotus parnellii) and Wistar rat (Rattus norvegicus) and determined a relationship between cochlear morphometrics and their frequency map. Sixteen of twenty-two of the morphometric parameters analyzed showed a significant change along the cochlea, including the distance between the rows of hair cells, outer hair cell width, and gap width between hair cells. A multiple linear regression model revealed that nine of these parameters are responsible for 86.9% of the variation in these morphometric data. Determining the most biologically relevant measurements related to frequency detection can give us a greater understanding of the essential biomechanical characteristics for frequency selectivity during sound transduction in a diversity of animals.

Binaural Response Properties and Sensitivity to Interaural Difference of Neurons in the Auditory Cortex of the Big Brown Bat, Eptesicus fuscus

This study examines binaural response properties and sensitivity to interaural level difference of single neurons in the primary auditory cortex (AC) of the big brown bat, Eptesicus fuscus under earphone stimulation conditions. Contralateral sound stimulation always evoked response from all 306 AC neurons recorded but ipsilateral sound stimulation either excited, inhibited or did not affect their responses. High best frequency (BF) neurons typically had high minimum threshold (MT) and low BF neurons had low MT. However, both BF and MT did not correlate with their recording depth. The BF of these AC neurons progressively changed from high to low along the anteromedial-posterolateral axis of the AC. Their number of impulses and response latency varied with sound level and inter-aural level differences (ILD). Their number of impulses typically increased either monotonically or non-monotonically to a maximum and the latency shortened to a minimum at a specific sound level. Among 205 AC neurons studied at varied ILD, 178 (87%) and 127 (62%) neurons discharged maximally and responded with the shortest response latency at a specific ILD, respectively. Neurons sequentially isolated within an orthogonal electrode puncture shared similar BF, MT, binaurality and ILD curves. However, the response latency of these AC neurons progressively shortened with recording depth. Species-specific difference among this bat, the mustached bat and the pallid bat is discussed in terms of frequency and binaurality representation in the AC.

Activity Records of the Endangered Paraguaná Moustached Bat, Pteronotus paraguanensis, in the Main Vegetation Types of the Paraguaná Peninsula, Venezuela

The Paraguana moustached bat, Pteronotus paraguanensis (Mormoopidae), is one of the four species of bats endemic to Venezuela. Besides having a geographic distribution restricted to the Paraguana Peninsula, it is currently classified as Endangered in the IUCN Red List of Threatened Species. Changes in land use, frequent human disturbance at diurnal roosts and poisoning with agro-pesticides from farming operations are some of the main threats that affect this species. Despite its conservation status and the pressing need to protect its feeding areas, habitat use of this species has not been well documented. The aim of this study was to record this bat's activity using acoustic monitoring to identify the vegetation types most frequently used throughout the year. We recorded echolocation calls over the course of eight months between 2015 and 2016, using walking transects distributed along the vegetation types most representative of the peninsula. Activity of P. paraguanensis differed significantly among vegetation types, reaching highest relative activity in thorn woodlands (45%), followed by premontane woodlands (20%), disturbed habitats (19%), thorn scrubs (11%), and columnar cactus forests (4%). Activity (mean number of bat passes/h) was comparatively higher in premontane woodlands (78.17 ± 49.82 SD) than in the other vegetation units (range: 4.39 ± 5.71 SD – 15.51 ± 16.11 SD) where this species was detected. This result is indicative that this species is mainly associated with forest habitats; however, it can also be present at lower frequency in disturbed lands. Our bat call recordings also help confirm that the Paraguana Peninsula's isthmus represents an ecological barrier that precludes dispersion of P. paraguanensis to the mainland. Based on our findings, we highlight the need to focus conservation actions for this species on protection of the remnant patches of forest vegetation still present in the peninsula.

Mole senses.

Many people are familiar with the meandering tunnels of moles, far fewer are familiar with the creatures that made them. Such secrecy is, of course, one of the great benefits of burrowing through the earth where few predators can follow. But it's not the only benefit. Soil contains a smorgasbord of nutritious invertebrate prey - hence all the tunneling. In fact the richness of the soil niche has given rise to a diversity of mole species that could be said to resemble the evolutionary diversification of bats, though on a smaller scale. In both cases, mammals have evolved a suite of unique anatomical traits (coincidentally involving modification of the forelimb) that allows them to exploit a huge resource of invertebrate prey that is largely inaccessible to their competitors. For bats the thin, delicate wings were the key innovation, for moles the forelimbs have undergone a similarly dramatic structural shift, but in the opposite direction - the bones have become short, stout, and powerful to act as shovels.

Bat species richness in the region of the Central Valleys of Oaxaca, Mexico

We present a revised checklist of bat species occurring in the semi–urbanized region of the Central Valleys of Oaxaca, Mexico. The checklist is based on surveys using mist nets, recordings of echolocation calls, data from literature, and museum databases. Results show that the Central Valleys of Oaxaca have a species richness of 33 bat species belonging to 22 genera and five families. Species like the Mustached bat Pteronotus parnellii, the Western Red bat Lasiurus blossevillii and the Free–tailed bat Promops centralis were recorded after 32, 30 and 19 years respectively according to the records of the literature. We also recorded four species classified in some risk category according to either the Mexican government’s red list (NOM–059) or the IUCN red list. The recordings of the echolocation calls are the first from the region of Central Valleys of Oaxaca. Bat diversity in the Central Valleys of Oaxaca was underestimated and emphasizes the need for further research

Inhibitory mechanisms shaping delay-tuned combination-sensitivity in the auditory cortex and thalamus of the mustached bat

Delay-tuned auditory neurons of the mustached bat show facilitative responses to a combination of signal elements of a biosonar pulse-echo pair with a specific echo delay. The subcollicular nuclei produce latency-constant phasic on-responding neurons, and the inferior colliculus produces delay-tuned combination-sensitive neurons, designated “FM-FM” neurons. The combination-sensitivity is a facilitated response to the coincidence of the excitatory rebound following glycinergic inhibition to the pulse (1st harmonic) and the short-latency response to the echo (2nd–4th harmonics). The facilitative response of thalamic FM-FM neurons is mediated by glutamate receptors (NMDA and non-NMDA receptors). Different from collicular FM-FM neurons, thalamic ones respond more selectively to pulse-echo pairs than individual signal elements. A number of differences in response properties between collicular and thalamic or cortical FM-FM neurons have been reported. However, differences between thalamic and cortical FM-FM neurons have remained to be studied. Here, we report that GABAergic inhibition controls the duration of burst of spikes of facilitative responses of thalamic FM-FM neurons and sharpens the delay tuning of cortical ones. That is, intra-cortical inhibition sharpens the delay tuning of cortical FM-FM neurons that is potentially broad because of divergent/convergent thalamo-cortical projections. Compared with thalamic neurons, cortical ones tend to show sharper delay tuning, longer response duration, and larger facilitation index. However, those differences are statistically insignificant.

Isolation and characterization of microsatellite marker loci in the Wagner’s mustached bat Pteronotus psilotis (Chiroptera: Mormoopidae) and cross-amplification in other related species

Pteronotus psilotis, a mormoopid bat, is an insectivorous, gregarious and strict cave-dwelling species that is found areas between the sea level and an elevation of about 1000 masl. This species is present in diverse habitats ranging from rain forest to dry deciduous forest. Nine microsatellite loci were developed for Wagner's mustached bat, Pteronotus psilotis using the next-generation sequencing approach, and their utility for population genetics studies was assessed. All loci were polymorphic (7-15 alleles) and characterized in 30 individuals from three P. psilotis populations, with the levels of observed and expected heterozygosity ranging from 0.280 to 0.867 and 0.584 to 0.842, respectively. One locus showed significant departures from Hardy-Weinberg expectations after Bonferroni correction. Cross-amplification in 11 other bat species was tested, for which eight microsatellites were successfully amplified, and of these seven were polymorphic. The development of these new microsatellite loci will contribute to investigations of genetic population structure, genetic diversity and gene flow in P. psilotis populations, as well as in other closely related bat species.

A three-dimensional digital neurological atlas of the mustached bat (Pteronotus parnellii)

Substantial knowledge of auditory processing within mammalian nervous systems emerged from neurophysiological studies of the mustached bat (Pteronotus parnellii). This highly social and vocal species retrieves precise information about the velocity and range of its targets through echolocation. Such high acoustic processing demands were likely the evolutionary pressures driving the over-development at peripheral (cochlea), metencephalic (cochlear nucleus), mesencephalic (inferior colliculus), diencephalic (medial geniculate body of the thalamus), and telencephalic (auditory cortex) auditory processing levels in this species. Auditory researchers stand to benefit from a three dimensional brain atlas of this species, due to its considerable contribution to auditory neuroscience. Our MRI-based atlas was generated from 2 sets of image data of an ex-vivo male mustached bat's brain: a detailed 3D-T2-weighted-RARE scan [(59 × 63 x 85) μm3] and track density images based on super resolution diffusion tensor images [(78) μm3] reconstructed from a set of low resolution diffusion weighted images using Super-Resolution-Reconstruction (SRR). By surface-rendering these delineations and extrapolating from cortical landmarks and data from previous studies, we generated overlays that estimate the locations of classic functional subregions within mustached bat auditory cortex. This atlas is freely available from our website and can simplify future electrophysiological, microinjection, and neuroimaging studies in this and related species.

Natural echolocation sequences evoke echo-delay selectivity in the auditory midbrain of the FM bat, Eptesicus fuscus.

Echolocating bats must process temporal streams of sonar sounds to represent objects along the range axis. Neuronal echo-delay tuning, the putative mechanism of sonar ranging, has been characterized in the inferior colliculus (IC) of the mustached bat, an insectivorous species that produces echolocation calls consisting of constant frequency and frequency modulated (FM) components, but not in species that use FM signals alone. This raises questions about the mechanisms that give rise to echo-delay tuning in insectivorous bats that use different signal designs. To investigate whether stimulus context may account for species differences in echo-delay selectivity, we characterized single-unit responses in the IC of awake passively listening FM bats, Eptesicus fuscus, to broadcasts of natural sonar call-echo sequences, which contained dynamic changes in signal duration, interval, spectrotemporal structure, and echo-delay. In E. fuscus, neural selectivity to call-echo delay emerges in a population of IC neurons when stimulated with call-echo pairs presented at intervals mimicking those in a natural sonar sequence. To determine whether echo-delay selectivity also depends on the spectrotemporal features of individual sounds within natural sonar sequences, we studied responses to computer-generated echolocation signals that controlled for call interval, duration, bandwidth, sweep rate, and echo-delay. A subpopulation of IC neurons responded selectively to the combination of the spectrotemporal structure of natural call-echo pairs and their temporal patterning within a dynamic sonar sequence. These new findings suggest that the FM bat's fine control over biosonar signal parameters may modulate IC neuronal selectivity to the dimension of echo-delay. NEW & NOTEWORTHY Echolocating bats perform precise auditory temporal computations to estimate their distance to objects. Here, we report that response selectivity of neurons in the inferior colliculus of a frequency modulated bat to call-echo delay, or target range tuning, depends on the temporal patterning and spectrotemporal features of sound elements in a natural echolocation sequence. We suggest that echo responses to objects at different distances are gated by the bat's active control over the spectrotemporal patterning of its sonar emissions.

Intraspecific Evolutionary Relationships and Diversification Patterns of the Wagner's Mustached Bat, Pteronotus personatus (Chiroptera: Mormoopidae)

Wagner's mustached bat (Pteronotus personatus) is an insectivorous bat distributed throughout America from Mexico to Brazil, which inhabits a range of habitats from rain forests to dry deciduous forests. There are two currently recognized species within the P. personatus complex, for which we examined 235 cytochrome oxidase I (COI) sequences and 138 cytochrome b (Cytb) sequences in order to explore its genetic variation in Mexico as well as in Central and South America. Our results reveal considerable differences in the genetic structure inside this species complex, indicating five genetic lineages: 1) Gulf of Mexico and the Mexican Pacific coastal plain to the Isthmus of Tehuantepec, 2) Southeastern Mexico, 3) Guatemala, 4) Guyana and Suriname-COI/Guyana and Venezuela-Cytb, and 5) Guyana, Suriname, French Guiana and Brazil. In addition, we used the isolation-with-migration coalescent method to estimate divergence times. The results indicate that vicariant events occurred roughly 1,624,000–2,450,000 years ago during the Early Pleistocene, wherein Central America was the center of two separate diversification processes, one toward Mexico and the other South America. The intraspecific lineages obtained for P. personatus demonstrate the need to reevaluate the species complex limits of this taxon.

Doppler shifts in bat biosonar: The good, the bad, and the unexpected

Most models for explaining bat biosonar are based on linear systems theory. A notable exception are frequency shifts due to the Doppler effect that occur whenever there is relative motion between the wave emitter and receiver. Bat species such as horseshoe bats, Old World leaf-nosed bats, and mustached bats have evolved particularly sophisticated biosonar systems that are able to exploit Doppler shifts for detecting prey in clutter. In this case, “good Doppler shifts” induced by the wing beat velocity of an insect prey give the prey echoes unique signatures that stand out among echoes from stationary targets such as reflecting facets in dense vegetation. Since these “good Doppler shifts” are small, bats exploiting them had to evolve specialized biosonar mechanisms to detect them. However, these mechanism are also sensitive to “bad Doppler shifts” that result, e.g,. from a bat's own flight velocity. It has been generally assumed that bats with Doppler-based biosonar will eliminate such nuisance Doppler shifts through compensation behaviors to allow them to detect the good Doppler shifts. However, a complete picture of Doppler shifts in bat biosonar systems may also have to account for other sources of Doppler shifts that do not fall into the above categories.

Genetic diversity of the ghost-faced bat Mormoops megalophylla Peters, 1864 (Chiroptera: Mormoopidae) in Ecuador; implications for its conservation

Mormoops megalophylla is a cave-dwelling bat distributed from southern United States across Central America to northern Peru. Its conservation status at a global level is of Least Concern, according to the IUCN Red List of Threatened Species; in Ecuador, however, it is included under the Vulnerable category due to the threats faced by the only two viable populations known. Individuals from each locality (Carchi and Pichincha) were captured and marked. The D-loop of the mitochondrial control region was obtained from wing membrane tissue samples, in order to analyze the geographic distribution of nucleotide and haplotype diversity of the populations, as well as gene flow between them. The molecular variation within and between populations was evaluated through a molecular variance analysis. A high haplotype diversity and a low nucleotide diversity were observed. The gene-flow estimator revealed that Carchi and Pichincha make up a single population coming from a single lineage. The network of haplotypes indicated that those with the highest frequency are shared in both localities; the largest number of unique haplotypes, however, was observed in Pichincha. The high haplotype diversity and low nucleotide diversity values in Ecuador are due to the fact that the ghost-faced bat populations may have experienced a fast-growing period from a low effective population size, with sufficient time to accumulate haplotype diversity, but insufficient to increase nucleotide diversity. The low genetic variability between both localities indicates the existence of a panmictic population that may have been split by factors such as habitat transformation, leading to isolated colonies. The preservation of this vulnerable species will depend on conservation efforts and studies that seek to supplement the analysis of genetic variability with other molecular markers, a continued monitoring of migratory processes, and inventorying of intermediate sites and localities with historical records.

Geographical variation in the high-duty cycle echolocation of the cryptic common mustached bat Pteronotus cf. rubiginosus (Mormoopidae)

ABSTRACTThe use of bioacoustics as a tool for bat research is rapidly increasing worldwide. There is substantial evidence that environmental factors such as weather conditions or habitat structure can affect echolocation call structure in bats and thus compromise proper species identification. However, intraspecific differences in echolocation due to geographical variation are poorly understood, which poses a number of issues in terms of method standardization. We examined acoustic data for Pteronotus cf. rubiginosus from the Central Amazon and the Guiana Shield. We provide the first evidence of intraspecific geographic variation in bat echolocation in the Neotropics, with calls significantly differing in almost all standard acoustic parameters for the two lineages of this clade. We complement our bioacoustic data with molecular and morphological data for both species. Considerable overlap in trait values prevents reliable discrimination between the two sympatric Pteronotus based on morphological characters. On the other hand, significant divergence in the frequency of maximum energy suggests that bioacoustics can be used to readily separate both taxa despite extensive intraspecific variability in their echolocation across the Amazon. Given the relative lack of barriers preventing contact between bat populations from the Central Amazon and French Guiana, the documented acoustic variation needs to be further studied in geographically intermediate locations to understand the potential isolation processes that could be causing the described divergence in echolocation and to determine whether this variation is either discrete or continuous.