Variation in the frequency of the echolocation calls of Hipposideros ruber in the Gulf of Guinea: an exploration of the adaptive meaning of the constant frequency value in rhinolophoid CF bats

A. GUILL E’ N, * t J. JUSTE B . t & C . I B A’

N EZ t*Department of Biology, University of Missouri-St. Louis, 8001 Natural Bridge Road, St. Louis, MO 63121—4499, USAtEstacio n Biolo gica de Don ana, CSIC, Apartado 1056, 41080 Sevilla, Spain Departamento de Biouu mica y Biolog a Molecular I , Universidad Complutense de Madrid, aacultad de eterinaria, 28040 Madrid, Spain

Keywords:

adaptation; Chiroptera;constant frequency; echolocation; geographical variation; Hipposideros ruber; humidity;speciation.

Abstract

This study describes variation patterns in the constant frequency (CF) segment of echolocation calls of the bat Hipposideros ruber within and among populations across the region of the Gulf of Guinea. Correlations of variation in CF with variation in body size, body condition, environmental humidity and presence of ecologically similar species are studied in an attempt to identify the forces driving the evolution of CF. We found that bats may adapt the frequency to humidity, and that CF may evolve under interspecific interactions, either of ecological or of social nature. The results support an adaptive value for the high values of CF, and challenge the Allotonic Frequency Hypothesis’. We found correlation between frequency and a body condition index, which may trigger social selection processes in this species sexually dimorphic in CF. Combined social and environmental selection on CF could trigger diversification of bats along ecotones separating habitats with contrasting air humidity.

Introduction

Horseshoe (Rhinolophidae) and roundleaf (Hipposider- idae) bats use an echolocation system that relies on calls containing constant frequency segments (hereafter CF; Griffin, 1958). Constant frequency segments of these calls have much higher pitch than calls used by other bats, a characteristic associated with a shift from the first to the second harmonic as the information carrier. The bats construct complex sensorial images of their prey after micromodulations produced in the echo of the originally CF segment by the differential movements of the body parts of the fluttering insects they hunt (Kober& Schnitzler, 1990; von der Emde & Schnitzler, 1990). Neurological, behavioural and ecological studies have recently increased our understanding of this narrow

Correspondence: Dr A. Guille& n, Department of Biology, University of Missouri-St. Louis, Saint Louis, MO 63121, USA.

Tel.: +1 314 516 6207; fax: +1 314 516 6233;e-mail: aguill e n@admiral. u msl.edu

J . E V O L . B I O L . 1 3 ( 2 0 0 0 ) 7 0 — 8 0 © 2 0 0 0 B L A C K W E L L S C I E N C E L T D

analysis sonar system, although the reason for using such high frequencies remains unclear (Fenton & Fullard,1979; Fullard, 1987; Schnitzler, 1987; Vater, 1987; Heller& Helversen, 1989; Neuweiler, 1989, 1990a; Ru” bsamen & Scha” fer, 1990a,b; Jones, 1995, 1996, 1997).

Echolocation requires high-frequency sounds that are highly directional and limit perception to unobstructed objects located at relatively short distance. Signal char- acteristics will evolve primarily in response to factors causing distortion of the signal in the direct pathway from the emitter to the target and back (attenuation, absorption), and factors affecting echo formation (char- acteristics and position of prey with respect to the solid background). Other factors important to the evolution of social signals, such as pattern loss by scattering, reflection and refraction of sound by objects in the transmission medium (Bradbury & Vehrencamp, 1998), should have little significance in evolution of echolocation.

Because echolocation is a short-range detection sys- tem, it is expected that bats that hunt for insects flying in open air will use, in calls or parts of the calls whose main

function is prey detection, the frequencies that give them the largest detection distances for the preferred prey. While the absolute value of the detection distance changes with air temperature and humidity, maximum echo strength in the aerial detection of a spherical target is always achieved when using a sound with wavelength n times the diameter of the target ([wavelength n X diameterj; Pye, 1983; Hartley, 1989). Then, an inverse relationship of frequency vs. ideal prey diameter is expected; and because predator and prey body sizes are directly related (Peters, 1983), an inverse relationship between frequency and bat body size is also expected. The main function of the ending quasi-CF segment of calls produced by aerial hawking insectivorous bats is prey detection (Simmons & Grinnell, 1988; Neuweiler,1989, 1990). Frequency of this segment shows a negative trend towards body size with an elevation that suggests an adaptive match between frequency and prey size in those bats (Pye, 1983; Barclay & Brigham, 1991; Jones,1996, 1997). Horseshoe and roundleaf bats also show an inverse relationship between CF and body size, although the intercepts are much higher (Heller & Helversen,1989; Barclay & Brigham, 1991; Jones, 1996). It is clear from the studies cited above that these bats neither use the CF segment for mere detection nor hunt in open spaces (i.e. Neuweiler, 1989). Higher frequencies may be necessary when the goal instead is to encode vibrational characteristics of prey. The inverse trend could then arise when the resolving power of smaller moving structures increases with the frequency of the call carrying the information.

An alternative explanation, framed as the Allotonic Frequency Hypothesis’, states that high frequencies used by horseshoe and roundleaf bats are a way to circumvent the auditory defences of moths, which may make up the bulk of the bats’ diet (Fenton & Fullard, 1979; Fullard,1987; Jones, 1992). Since frequency response of theauditory organs of moths does not seem to correlate with moth size (Fenton & Fullard, 1979; Surlykke, 1988), the allotonic frequency hypothesis denies a functional mean- ing of the relationship between CF and body size. A developmental constraint (i.e. the coupling of devel- opment of structures for sound production or reception with the development of the skull) might then explain the trend (longer vocal chords and larger cavities produce and resonate sounds of lower frequencies).

A simultaneous demonstration of an adaptive value of lower CF and the absence of a tight constraint between frequency and body size would favour the functional meaning of the trend vs. the allotonic frequency expla- nation. Selective pressures favouring lower frequencies would lead, through adaptive evolution, to a clustering of the CF of all rhinolophid and hipposiderid bats just above the upper frequency threshold of the hearing system of tympanate moths. The existence of the developmental constraint may be investigated by comparing patterns of covariation between CF and body size among sex,

populations and species living under different social or ecological conditions.

Insights into the potential adaptive meaning of CF may be obtained from the evolutionary response of the character to changes in the assemblage of immediate ecological competitors. If CF value were related to prey size, its mean and/or variance would likely respond to changing interspecific competitive interactions for resource partitioning (Van Valen, 1965; Arthur, 1982; Endler, 1986; Jones, 1995, 1997), as would any other ecomorphological attribute (Grant, 1994).

High-frequency sounds are attenuated in the atmo- sphere at higher rate than deeper ones, and the rate at which absorption of sound energy increases is directly related to both air humidity and frequency (Lawrence & Simmons, 1982; Hartley, 1989). Perceptive ranges of rhinolophids and hipposiderids might be rather short, potentially limiting and bats experience great losses in range with increasing humidity (Hartley, 1989). When frequencies higher than needed for mere detection are used for other perceptive purpose, humidity may deter- mine a levelling point in a trade-off between selection towards higher frequencies to obtain more resolution in prey classification, and towards lower frequencies to achieve longer range. Correlation of a trait (frequency) with an environmental variable (air humidity at the hunting grounds) would also suggest the existence of natural selection on the trait (Endler, 1986).

Bats with CF calls, including rhinolophids and hippo- siderids, use echolocation calls, or signals structurally similar to the echolocation calls, in communication (Fenton, 1985). Given the formidable capacity of these bats for discriminating frequencies around the emitted CF (Vater, 1987), social information might be encoded in minute differences or modulations in frequency. This situation may exert strong selection on individuals to maintain their CF frequencies close to the population average in order to interact socially. Heller & von Helversen (1989) have suggested the existence of acoustic communication channels’ in these bats. Fre- quency might also represent a species-specific recogni- tion signal (Butlin, 1995). Information on the health of a bat might also be encoded in the frequency of its sounds (Huffman & Henson, 1993), which may allow social selection processes. Social selection may influence with- in-and between-population variation in CF, potentially confounding the effects of ecological factors, and should be considered in the study of the selective universe operating on the evolution of characteristics of echo- location calls in bats.

In this paper we study intraspecific patterns of varia- tion in the CF of Hipposideros ruber (Noack 1893) in the islands of the Gulf of Guinea (Central Africa) and the immediate mainland in an attempt to understand factors underlying variation in CF. These populations live syntopically with different numbers of congeneric species and offer the opportunity to test for the existence of

J . E V O L . B I O L . 1 3 ( 2 0 0 0 ) 7 0 — 8 0 © 2 0 0 0 B L A C K W E L L S C I E N C E L T D

(1) a constraint of body size on CF, (2) shifts in means or changes in the variance or sexual dimorphism of CF indicating an adaptive variation of the trait associated with changes in the composition of the ecological assemblage, (3) adaptation of CF to local humidity and (4) covariation between CF and a body condition index, since a social role of the CF value could be rooted in the information about phenotypic or genotypic quality pro- vided by CF as a signal. Possible social and ecological causes underlying the patterns found and their evolu- tionary implications are discussed.

Materials and methods

The species and the geographical settings

The colonial bat Hipposideros ruber is widely distributed and abundant in the Central African rainforest belt and surrounding forest—savanna mosaics. On the mainland, this species lives syntopically with at least 10 other species of rhinolophid and hipposiderid bats. Hipposideros ruber is also present on the larger islands of the Gulf of Guinea (Hayman & Hill, 1971). The large land-bridge island of Bioko holds an important subset (six species) of the taxa present in the immediate mainland; whereas, only one congeneric species coexists with H. ruber on the oceanic island of Sa o Tome& , and none on the smaller oceanic island of Pri&ncipe (Juste B. & Iba& n ez, 1994). Although the whole region is included in the rainforest biome, local populations of H. ruber experience rather different environmental conditions. Conspicuous con- trasts occur between the southern slopes and the driest northern slopes of the central volcanic massifs of the islands with dramatic differences in sunshine, humidity and rainfall due to foehn’s effect (Fig. 1). These climate differences are impressively reflected in the vegetation, which changes from hyperhumid rainforest in the southern areas to deciduous forest in the northern areas, with baobab savannahs on some of the islands (Juste B.& Fa, 1994). It may be inferred that the impressive differences in rainfall and vegetation structure are reflected in differences in average air humidity in the hunting grounds of bats living in different areas.

Data collection and sound analysis

Bats were netted in different expeditions from October1992 to February 1994. A total of 437 bats from 16 colonies were sampled in R&io Muni (Equatorial Guinea, Western central Africa) and the islands of Bioko, Pri&ncipe and Sa o Tome& (Fig. 1). We intended to sample colonies across the environmental range that the different popu- lations experience, but due to logistic difficulties this plan was realized fully only on Sa o Tome& (Table 1, Fig. 1). Only fully grown specimens, with complete epiphysial fusion of finger joints (Anthony, 1988), were recorded for the study. Forearm length (FA), measured to the

nearest 0.1 mm with dial calipers, was used as a simple measure of body size. Body mass was measured to the nearest 0.5 g with Pesola’ spring scales for individuals from colonies 3, 5, 7 and 12.

Recordings of calls with resting CF frequency were obtained from hand-held bats restrained motionless15 cm in front of the microphone of a Lars PetterssonElektronik D960 ultrasound detector. Bats with narrow analysis echolocation systems broadcast in this situation true echolocation calls, structurally indistinguishable from the calls produced in hunting flight. The resting frequency’ is the individually characteristic and stable CF emitted by the motionless bat, which coincides with the response frequency of the cochlear acoustic fovea’ (a greatly expanded segment of the basilar membrane specialized to resolve frequencies in a narrow interval around the CF of the call; Ru” bsamen & Scha” fer, 1990a). In contrast, flying bats broadcast calls with variable frequency compensated for the Doppler effect caused by movement, so the echo returns with a carrier frequency around the resting frequency’, allowing the narrow frequency analysis (Schnitzler, 1970). Signals were slowed-down 10 times with the detector built-in A/D— D/A converter and recorded onto metal-XR Sony tapes with a Sony WM-D6C cassette recorder. Recordings were analysed on a Kay DSP 5500 Sonagraph with the sampling frequency set to 40 kHz. The bats broadcast typical hipposiderid echolocation calls, composed of an initial CF segment 6—9 ms long followed by a steep downward frequency modulated sweep (Fig. 2). The frequency of the CF component in the second harmonic (the functional one) was measured from an average power spectrum built with 512-point fast Fourier trans- forms and taken over the complete CF segment (Fig. 2). The resolution attainable with this process was 400 Hz. For each individual, 10 calls were measured and the mean value was used in the analysis. Within-individual variation was very small, the SD averaging 0.3 kHz. For controlling shifts in frequency due to equipment failure or power instability, an 880-Hz reference sound from a musical tuner was recorded at intervals interspersed with data recordings. No significant departures from the reference frequency were detected in the analysis.

Hypotheses and statistical analyses

Variable names are typed in uppercase to avoid confusion with their general biological meaning. As a factor, POPULATION has four levels and it refers to the four different populations of the species sampled (three islands and the mainland). COLONY refers to each of the 16 colonies sampled (Fig. 1).

Because the distribution of both CF and FA did not depart significantly from normality, but data were unbalanced among cells, general linear models (GLM) were used for the analyses with SAS statistical package (Littell et al., 1991). Since some cells missed data, type IV

Fig. 1 Study area with the location of sampled localities. Altitude and precipitation iso-lines are according to Tera& n (1962) and Jones et al. (1991).

sums of squares were used to build the tests (Shaw & Mitchell-Olds, 1993). Full models were built initially, but they were simplified subsequently by removing nonsig- nificant terms. Parameter estimates produced by the option SOLUTION of GLM procedure of SAS were inspected to determine how factor levels contributed to the patterns detected.

Changes in means of CF related to the sexual and geographical structure of the populations (potentially related to changes in ecological assemblage) were studied with a linear model that included SEX and POPULATION as main fixed effects and COLONY as a random effect nested within POPULATION (nested effect typed as COLONY[POPj).

Since longer vocal chords and larger cavities produce and resonate sounds of lower frequencies, changes in body size could cause changes in CF if this trait lacked adaptive value or if it were severely constrained during ontogeny by body size. Variation in FA, a trait considered under strong selection and tight developmental control because of its relation with body size (Williams, 1992) and its fundamental role for wing performance (Gummer& Brigham, 1995), was used as reference for assessing the importance of variation in CF. A GLM with the same structure as the one presented above was used for studying variation in FA. The relation between general body size and CF was assessed within colonies by studying the correlation between the residuals from the

1 1 5 144.64 ± 1.32 51.08 ± 1.02 11 146.93 ± 0.86 51.28 ± 0.73 2.35 (MAR, in m), at the location of the colonie1 2 10 146.66 ± 2.90 50.09 ± 1.05 5 148.24 ± 1.58 48.94 ± 1.36 1.75 Column Pop indicates population (1 =

Ri&o2 3 27 133.84 ± 1.36 51.53 ± 1.52 19 135.92 ± 1.44 50.59 ± 0.80 1.90 Muni, 2 = Bioko, 3 = Sao Tome& , 4 = Pr&in-2 4 25 135.11 ± 1.41 50.26 ± 0.90 26 136.25 ± 1.85 49.38 ± 1.10 2.40 cipe); column C shows colony number.

2 5 1 134.62 50.00 8 138.17 ± 2.13 49.31 ± 0.89 2.103 6 20 136.48 ± 1.02 51.65 ± 0.75 0 — — 3.503 7 0 — — 21 140.65 ± 1.22 50.70 ± 0.68 0.803 8 24 138.03 ± 1.40 50.51 ± 0.91 20 140.78 ± 1.16 50.28 ± 0.76 0.803 9 7 136.55 ± 1.67 49.96 ± 1.04 26 139.30 ± 2.29 49.96 ± 0.77 1.403 10 6 135.71 ± 1.72 50.90 ± 1.05 19 138.93 ± 1.26 49.95 ± 0.69 6.503 11 9 136.29 ± 2.18 50.40 ± 0.80 19 138.70 ± 2.11 50.01 ± 0.56 7.004 12 15 136.79 ± 1.50 50.16 ± 0.47 19 138.46 ± 1.10 50.03 ± 0.96 2.404 13 1 136.34 50.10 2 136.37 ± 0.64 49.15 ± 1.48 1.704 14 21 136.74 ± 1.36 50.18 ± 0.83 18 137.67 ± 2.60 49.79 ± 0.94 1.704 15 14 134.18 ± 2.07 50.69 ± 0.94 13 135.78 ± 1.80 49.85 ± 0.46 1.604 16 25 136.33 ± 1.89 50.40 ± 0.80 8 135.18 ± 2.56 50.06 ± 0.76 2.50

Females Males

Pop C N mCF mFA N mCF mFA MAR

Table 1 Sample size (N) and mean ± SD of resting frequency (mCF, in kHz), and fore- arm length (mFA, in mm), per colony and

sex. Last column shows mean annual rainfalls.

Fig. 2 Sonogram (left) and power spectrum (right) of a typical echolocation call of Hipposideros ruber. Maximum energy is on the second harmonic, which is used as the information carrier.

CF and FA GLM models described above, both for all data and within sex.

The effects of POPULATION, COLONY and SEX on the variances of CF and FA were studied with a multilevel— multifactorial adaptation of Levene’s test (Van Valen,1978). The absolute difference between each measure-ment and the median of the corresponding cell (POPU- LATION X COLONY X SEX) was divided by the cell mean. The new variables (unsigned relative deviations) were used as response variables for two linear models with the same structure as those used for studying the population structure of original variables CF and FA. The

amount of variation in CF was compared with that in FA by a Wilcoxon paired-sample test on the corresponding unsigned relative deviations.

Since parallelism in development could yield correla- tion patterns between traits that are actually evolution- arily independent, it is relevant to check whether correlations detected within populations also hold across populations under different selective regimes. We simul- taneously studied the effects of FA and environmental humidity across populations with an A N C O V A model in which the means of CF and FA per colony (Table 1) were the response variable and covariate, respectively (mCF and mFA), and POPULATION was the main fixed effect. In the analyses described above we found significant correlation between CF and FA, but different slopes for each sex; therefore, SEX was also included as a main fixed effect in the model and a separate-slopes by SEX effect was specified for mFA (mFA[SEXj). To test for the effect of environmental humidity we included local mean annual rainfall (hereafter MAR) as a second covariate in the model. MAR was obtained (Table 1) from isoyet lines published in Tera& n (1962) and Jones et al. (1991). We consider this a rough index of the target variable: humidity at the hunting grounds of bats.

A social role of the CF value could be rooted in the potential phenotypic or genotypic quality information provided by the signal. Following Krebs & Singleton (1993), a body condition index (BCON) was calculated as the ratio between the actual body mass and the mass predicted by a ln—ln regression of body mass vs. FA for all individuals with available data (colonies 3, 5, 7 and 12). Because we were interested in the individual effect of body condition, the variation due to SEX and COLONY was removed by including these explanatory variables in the initial A N C O V A model. We then tested for a correla- tion between the residuals of the first GLM model for CF with BCON, for all data and within sex.

Table 2 Effect of POPULATION, COLONY and SEX on the resting frequency (CF) of Hipposideros ruber in the Gulf of Guinea.

Effect d.f. MS F P

POPULATION 3 896.301 49.74 <0.001COLONY[POP] 12 25.501 8.53 <0.001SEX 1 199.187 66.63 <0.001POPULATION* SEX 3 17.081 5.71 0.001ERROR 411 2.989

Tests were built with type IV sums of squares. COLONY[POPj was considered as a random effect, and the adjusted denominator’s d.f. and MS for POPULATION were, respectively, 13.44 and 18.021. The interaction term SEX X COLONY[POPj was removed from the final model, since it was nonsignificant (a10,401 = 1.51, P = 0.134). The model is significant and explains a large proportion of data variance (a19,411 = 71.48, P < 0.001, r2 = 0.77).

Table 3 Effect of POPULATION, COLONY and SEX on the forearm length (FA) of Hipposideros ruber in the Gulf of Guinea.

Effect d.f. MS F P

POPULATION 3 5.039 0.91 0.468COLONY[POP] 12 8.022 9.96 <0.001SEX 1 25.122 31.21 <0.001ERROR 411 0.805

Tests were built with type IV sums of squares. COLONY[POPj was considered as a random effect, and the adjusted denominator’s d.f. and MS for POPULATION were, respectively, 13.30 and 5.532. The interaction terms POPULATION X SEX and SEX X COLONY[POPj were nonsignificant (a3,32.17 = 0.47, P = 0.710, and a10,408 = 0.97,P = 0.470), so they were removed from the final model. The model issignificant, but it explains a relatively low proportion of data variance (a16,411 = 11.37, P < 0.001, r2 = 0.30).

Results

POPULATION, COLONY and SEX had a significant effect on CF (Table 2). Differences among insular populations, although significant, were small, but the colonies on the mainland (Ri&o Muni) show noticeably higher values of CF (Table 1). A conspicuous sexual dimorphism exists: males use higher pitched calls in all colonies except for Sundi& (colony 16) in Pri&ncipe, where the dimorphism was reversed (Table 1). However, the importance of this dimorphism changed among populations (POPULA- TION X SEX term significant), but not among colonies within populations (Table 2). The smallest sexual dimor- phism was found in Pri&ncipe (0.8 kHz), while Sao Tome& population had the largest (2.7 kHz), and Ri&o Muni and Bioko showed intermediate and similar values (1.9 and1.7 kHz).

The variation in FA was not as structured across social and geographical units as it was in CF. Only COLONY and SEX had a significant effect on FA, and the model explained a much lower proportion of the data variance than the model for CF. Females had slightly but consis- tently longer forearms across colonies and populations (Tables 1 and 3).

Correlation between residuals of the two upper models was close to zero (r 0.02, N 426, P 0.705). Separate analyses by sex revealed a marginally nonsignificant negative trend in females (r —0.13, N 202, P 0.068) and a significant positive relationship in males (r 0.17, N 224, P 0.012).

There were no differences in the amount of variation in CF among sexes or social and geographical units. No effect except COLONY[POPj (a15,415 2.36, P 0.003,r2 0.008) was significant for the variance of CF, and thiseffect explained less than 1% of the variance of the relative deviations from CF cell medians. The importance and direction of the difference in FA variation between sexes varied among colonies (SEX X COLONY[POPj effect significant: a13,408 1.83, P 0.036). However,

separate models for each sex revealed no significant effects on the amount of variation. The overall variation was noticeably and significantly larger in FA than in CF (W 13 475, [N 0j 419, P < 0.001).

None of the interaction terms or forearm length (mFA[SEXj; a2,22 2.00, P 0.159) explained variation in mCF across colonies and populations. After removing all these terms from the model, MAR had an almost significant negative effect on mCF (r —0.26, a1,24 4.06, P 0.055). Inspection of the estimates of the parameters of the model and regression models for each population revealed a significant negative trend in the island of Sao Tome& (r —0.25, a1,7 13.71, P 0.008), but nonsignificant trends in the other three populations where variation in MAR among sampled sites was much lower.

No interaction term involving the covariate ln(FA) had a significant effect on ln(BODY MASS). Main effects and their interaction were significant, so they were main- tained in the model used for calculating BCON. The model adjusted to obtain predicted body mass (ln[BODY MASSj [1.36 ± 0.38j X ln[LAj — C; where C is a con- stant corresponding to each COLONY X SEX cell) was significant, and the proportion of variance explained washigh (a8,108 22.15, P <0.001, r2 0.64). Correlation ofBCON with the residuals of the initial model for studyingvariation of CF (that described in Table 2) was positive and significant, for the whole dataset (r 0.29, N 116, P 0.002) and within sex (females: r 0.34, N 57, P 0.010; males r 0.27, N 59, P 0.041).

Discussion

Populations of H. ruber in the Gulf of Guinea have a noticeable partitioning of the variation in CF between sexes, and among colonies and populations, while vari- ation within populations and sexes is extremely narrow. All the colonies on islands showed similar CF values, which were markedly lower than those of colonies on

the mainland. Populations in the islands and the main- land might belong to different sibling species, but molecular data show that the populations from Ri&o Muni and Bioko are genetically very similar (1.6% Kimura two-parameter distance in mitochondrial cytochrome b sequences; A. Guille& n, unpublished data). Some patterns in our dataset show that changes in CF do not merely result from general changes in body size. Frequency and body size are correlated within colonies, but negatively among females and positively among males. Moreover, despite the large differences in CF existing between populations on the islands and the mainland, there were no significant differences in body or skull size among them (Juste B., 1990), and the slight significant differ- ences in size among colonies were not correlated with differences in CF.

Large shifts in CF between populations of CF rhino- lophoid bats separated by geographical barriers may be common, since the few studies that have dealt with this issue have detected them. Francis & Habersetzer (1998) report large shifts, over 10 kHz, in two out of three species recorded both in Borneo and Peninsular Malaysia. Hipposideros cervinus living in the Malayan peninsula used higher pitched calls, although they had slightly larger body size than bats from Borneo. Hippo- sideros galeritus from Malaya were similar in size to those living in Borneo, but used much deeper calls. Some populations isolated by distance also exhibit large differ- ences (e.g. R. ferrumeuuinum between Europe & Japan, Heller & von Helversen, 1989; Hipposideros fulvus between India & Malaysia, Heller & von Helversen, 1989; Jones et al., 1994). Although, in this study body size and CF covary between sexes according to what is expected from physical laws (longer chords and larger cavities produce and resonate deeper sounds), this is not the rule in other rhinolophoid CF bats for which sexual dimorphism in CF has been reported. In Hipposideros speoris, females produce sounds 3 kHz deeper than males, but differences in forearm length between sexes are not significant (Jones et al., 1994). Females of Rhinolophus hipposideros are larger than males, but produce calls with higher CF (Jones et al., 1992; Guille& n, 1996). Females of Rhinolophus rouxi, R. creaghi and R. thomasi use higher pitched sounds than males, but there are no differences between sexes in forearm length (Neuweiler et al., 1987; Francis & Habersetzer, 1998; Francis & Guille& n, unpublished data). Hipposideros commersoni, an African hipposiderid bat, shows a notorious dimorphism in body size, reflected in many skull structures not involved in echolocation. However, the CF of the echolocation call does not differ significantly between sexes (Guille& n, 1996). These fre- quent observations of intraspecific shifts in CF value independent of changes in body size imply that body size does not strongly constrain CF in horseshoe and round- leaf bats.

Differences between populations from the islands and the mainland may be due to genetic or cultural drift, or

selection under different environmental conditions. The similarity of the island populations is difficult to explain under pure drift, because some of them are historically closer to the mainland than to other island populations (the population on Bioko is genetically much closer to the populations in the mainland than to the populations in other islands; A. Guille& n, unpublished data). Clime differences are probably not the causal factors of the differences, since rainfall and temperatures on the mainland are close to the average values from the islands (despite the higher spatial environmental heterogeneity on islands). Shifts reported by Francis & Habersetzer (1998) hardly correlate with environmental differences between Borneo and Malaya, since the two species reported experience changes in opposite directions.

The shift in CF between populations could be a result of ecological displacement or release (Arthur, 1982). However, the invariable intrapopulational variance of CF and the absence of increased sexual dimorphism in the simpler ecological assemblages studied, contrary to the expectations of the adaptive variation hypothesis’ (Van Valen, 1965; Selander, 1966; Grant & Price, 1981), would not support an ecological explanation relating the diver- gence to interspecific interactions. Alternatively, the extreme narrowness of the variance of CF in every population suggests that strong stabilizing selection may be in action, and it may overcome effects of ecological release on the variance of CF. Other populations of H. ruber recorded elsewhere in Africa have a CF value close to the island populations studied here (Pye, 1972; Fenton & Fullard, 1979; Heller, 1992; Jones et al., 1993), except for bats recorded by Pye (1972) in Uganda which show similar CF to the bats recorded by us in the mainland. Among the recording sites, these two main- land localities (Pye’s and ours) are probably the only ones where H. ruber lives in sympatry with Hipposideros fuliginosus, a close relative with a somewhat larger body size (Hayman & Hill, 1971). The shift between mainland and island populations might be related to the release from an interspecific interaction, either ecological or social, with the latter species.

We found a significant inverse correlation between CF colony means and environmental humidity measured as local MAR in the island of Sao Tome& . When all popu- lations were analysed together, the trend was at the limit of significance. Geographical variation of a phenotypic character, genetically or culturally inherited, is the combined result of processes of drift, natural and sexual selection, and gene flow (Slatkin, 1987). Average values and geographical variation shown in one population are highly dependent on the interaction of the geographical structure of the selective environment with gene flow patterns and may not be directly comparable to other populations. The island of Sao Tome& presents more environmental variation (wider range in MAR) than other islands. Our sample of localities in that island also captures more geographical and environmental variation

than samples from other islands (in fact, the combined range of MAR in all other localities together is less than half the range available in Sao Tome& ). The potential effect on the geographical variation of CF may be counteracted by gene flow through migration in other islands with more moderate selective landscapes. Some interspecific patterns support the idea that adaptation of CF to the average humidity in the hunting grounds may occur. Heller & von Helversen (1989) noticed that horseshoe bats living in dry habitats have higher CF relative to body size than those living in wet environ- ments. A coincident pattern exists for the African groups of Hipposideros with representatives in both the rainforest belt and the surrounding dry environments. Rain forest dwellers (H. ruber and H. commersoni thomensis and H. commersoni gigas) use lower relative CF than their relatives in the dry areas (H. caffer, the sibling species of H. ruber, and H. commersoni marungensis, a relative of the other two forms; Jones et al., 1993; Guille& n, 1996). These patterns are expected if higher frequencies have an actual cost in ecological performance by reducing the detection space for the bats in humid environments.

High frequencies used by CF rhinolophoid bats may be necessary for extracting information about prey charac- teristics through the analysis of the micromodulations caused by moving prey in the CF segment of the call. Higher frequencies experience larger differences in Doppler shifts when reflected in objects moving at different speed (Pye, 1983), and may be required for discriminating smaller prey having a shorter range of linear speed of their moving parts. But very high frequencies are heavily attenuated in humid air (Hartley,1989), and that may establish a trade-off between higher frequencies for enhancing resolution in prey classifica- tion and lower frequencies for longer range detection. Fixation of the trade-off point in the population may explain the absence of response in the variance of CF to changes in the composition of the competitive environ- ment represented by the ecological assemblage.

These results challenge the validity of the Allotonic Frequency Hypothesis’ as an explanation of the evolu- tionary achievement of high frequencies by rhinolophids and hipposiderids (Fenton & Fullard, 1979; Fullard, 1987; Jones, 1992; Rydell et al., 1995). Empirical evidence of the CF value being positively correlated with the incidence of moths in the diet of rhinolophid and hipposiderid bats has been used to support this hypoth- esis (Jones, 1992; Rydell et al., 1995). However, higher consumption of tympanate moths by bats with frequen- cies above the hearing frequency range of the insects does not prove that moth hearing was the selective factor underlying a shift to higher frequencies. These bats may have shifted to the second higher harmonic during the origin of this echolocation system because of the func- tional requirements of narrow frequency analysis. Linked use of high frequencies and the second harmonic is prevalent among horseshoe and roundleaf bats, and it

seems to be the ancestral condition for the group. All hipposiderids for which sounds have been described use the second harmonic. Most horseshoe bats also use the second harmonic, except a few recently derived species with particular ecology that have reverted to using the first harmonic, lower in frequency (A. Guille& n, unpub- lished data). After adopting high frequencies, the bats could find easy access to the resource represented by tympanate insects, partially inaccessible to other bats. This being the case, the allotonic frequency would have the meaning of a spandrel’ attribute useful for hunting tympanate insects, in the sense of Gould & Lewontin (1979) and Williams (1992), while not being an adapta- tion for that function.

Sexual differences in echolocation calls commented above might be related to a social function of the echolocation call. The absence of sexual dimorphism in CF in some species with large dimorphism in body size, such as the case of H. commersoni commented above, suggest that a social evolutionary pressure exists for keeping the frequency within a narrow range. The conspicuously narrow within-colony variation in CF found here points to a strong selective mechanism acting on this character. Extremely narrow intrapopulational variations in CF, with CVs close to 1%, are typical in rhinolophoid CF bats (Jones et al., 1992, 1993, 1994; Guille& n, 1996). According to some views, intrapopula- tional phenotypic variation in mating signals is typically low when compared with other quantitative characters (Butlin, 1995; but see an opposite view in Møller & Swaddle, 1997).

Given the significant correlation between CF and body condition index, females of H. ruber could obtain infor- mation about ecophysiological performance of potential mates after the sounds they broadcast. Relationships between CF and body condition indexes have been reported for H. fulvus (Jones et al., 1994) and R. fer- rumeuuinum (Guille& n, 1996). Numerous studies have shown that tuning properties of the mammalian cochlea are altered by changes in physiological variables (tem- perature, blood pressure, hormonal levels, etc.) that may ultimately affect the micromechanical properties of the inner ear (Bell, 1992; Huffman & Henson, 1993). Given the nature of the echolocation system used by CF rhinolophoidea, any shift in cochlear tuning must be followed by a concomitant shift in the CF of the broadcasted pulses (Huffman & Henson, 1993). This may well facilitate the development of a sexual selection mechanism with female preferences based on phenotypic quality of males detected by minute differences in frequency. Simultaneous selection on CF and body size may result in the positive correlation between these two variables found in males in our study. Once all females in a population have evolved a preference, the selected characteristic of the call (i.e. frequency) may become part of the specific mate recognition system of the population (Butlin, 1995). However, the reversed dimorphism of the

colony at Sundi& may indicate that sexual dimorphism is under dynamic evolution in H. ruber. Heller & von Helversen (1989) found that the CF values of the horseshoe and roundleaf bat species reported from a relatively small region in Malaysia (and thus regarded as the total set of species in a syntopic ecological assemblage or guild) were distributed significantly more evenly on a log scale than expected at random. This even spacing makes little sense as a result of ecological displacement of a character related to prey size because the species involved belong to two different families (Rhinolophidae and Hipposideridae) with somewhat different echoloca- tion systems with probably different functional proper- ties. However, an evolutionary accommodation of frequency channels for social communication may result in the spacing of CF among species with different phylogenetic origin and ecomorphological characteristics.

A possible interaction between call frequency and speciation in bats has recently been considered. Barrat et al. (1997) suggest that acoustic divergence followed genetic isolation in the recently discovered sibling species corresponding to two phonic types of the European bat Pipistrellus pipistrellus. For the same sibling species, Jones (1995, 1997) has proposed a speciation model based on sexual selection on social calls. Patterns of variation in frequency found in this study would point to a potential social selection mechanism directly acting on the echo- location calls of H. ruber. This study and the comparative database also show that the frequency of the echolo- cation calls is affected by the environmental humidity (at the hunting grounds). The inferred adaptive meaning of the CF value implies that frequency is also affected by the size of the available or preferred prey (bats preying on larger insects should have deeper CF). A combined social and environmental selection on CF could produce divergence and assortative mating, and lead to speciation in which dispersal barriers appear among populations living under different environments or when new adap- tive zones become available. H. caffer, a sister species of H. ruber living in the African dry forests surrounding the rainforest block (Hayman & Hill, 1971), produces sexu- ally dimorphic calls with CF higher than H. ruber (Heller,1992; Jones et al., 1993). We may envisage a speciation scenario proceeding from habitat specialization, to dis- ruptive ecological selection on frequency, and assortative mating at both sides of the dry—wet/savanna—forest contact line surrounding the African rainforest belt, which has been considered a key element for the diversification of African vertebrates (Grubb, 1978).

Acknowledgments

We thank the Agriculture Ministries of the Republics of Equatorial Guinea and Sao Tome& and Pri&ncipe for their support; Pedro Jordano, Robert E. Ricklefs, Erpur Hansen and Zuleyma Tang-Marti&nez for comments on drafts in different stages of elaboration; Angus Gascoigne and

Carlos Ru&iz for their help during field work. Damond Kyllo helped with the language. This research was partially supported by the Spanish DGICYT (PB90-0143), the Junta de Andaluci&a (research group RNM-158), and by predoctoral (UNICAJA-Junta de Andaluci&a) and postdoctoral (Ministerio de Educacio& n y Ciencia) grants to A.G. The European Union (ECOFAC project) and the World Bank covered partially travel expenses to J.J.B.

ReferencesAnthony, E.L.P. 1988. Age determination in bats. In: Ecological

and Behavioral Methods for the Study of Bats (T. H. Kunz, ed.), pp. 47—58. Smithsonian Institution Press, Washington, DC.

Arthur, W. 1982. The evolutionary consequences of interspecific competition. In: Advances in Ecological Research, Vol. 12. (A. F. Macfadyen & E. D. Ford, eds), pp. 127—187. Academic Press, London.

Barclay, R.M.R. & Brigham, R.M. 1991. Prey detection, dietary niche breadth, and body size in bats: why are aerial insectiv- orous bats so small? Amer. Nat. 137: 693—703.

Barrat, E.M., Bruford, M.W., Burland, T.M., Jones, G., Racey, P.A. & Wayne, R.K. 1997. DNA answers the call of pipistrelle bat species. Nature 387: 138—139.

Bell, A. 1992. Circadian and menstrual rhythms in frequency variations of spontaneous otoacoustic emissions from human ears. Hearing Res. 58: 91—100.

Bradbury, J.W. & Vehrencamp, S.L. 1998. Principles of AnimalCommunication. Sinauer Associates Inc., Sunderland.

Butlin, R. 1995. Genetic variation in mating signals and responses. In: Speciation and the Recognition Concept Theory and Application (D. M. Lambert & H. G. Spencer, eds), pp. 327—366. The Johns Hopkins University Press, Baltimore.

Endler, J.A. 1986. Natural Selection in the Wild. PrincetonUniversity Press, Princeton.

Fenton, M.B. 1985. Communication in the Chiroptera. IndianaUniversity Press, Bloomington.

Fenton, M.B. & Fullard, J.H. 1979. The influence of moth hearing on bat echolocation strategies. J. Comp. Physiol.132: 77—86.

Francis, C.M. & Habersetzer, J. 1998. Inter- and intra-specific variation in echolocation call frequency and morphology of horseshoe bats, Rhinolophus and Hipposideros. In: Bat Biology and Conservation (T. H. Kunz & P. A. Racey, eds), pp. 169—179. Smithsonian Institution Press, Washington D.C.

Fullard, J.H. 1987. Sensory ecology and neuroethology of mothsand bats: interactions in a global perspective. In: Recent Advances in the Study of Bats (M. B. Fenton, P. Racey & J. M. V. Rayner, eds), pp. 244—272. Cambridge University Press, Cambridge.

Gould, S.J. & Lewontin, R.C. 1979. The spandrels of San Marco and the Panglossian paradigm: a critique of the adaptationist programme. Proc. R. Soc. Lond. B 205: 581—598.

Grant, P.R. 1994. Ecological character displacement. Science266: 746—747.

Grant, P.R. & Price, T.D. 1981. Population variation in contin- uously varying traits as an ecological genetics problem. Amer. Zool. 21: 795—811.

Griffin, D.R. 1958. Listening in the Dark. Yale University Press, New Haven.

Grubb, P. 1978. Patterns of speciation in African mammals. Bull. Carnegie Mus. Nat. Hist. 6: 152—167.

Guille& n, A. 1996. Ecolocacion en murcielagos: estudios desde un punto de vista ecologico y evolutivo. PhD Dissertation, Universidad Complutense de Madrid, Madrid.

Gummer, D.L. & Brigham, R.M. 1995. Does fluctuating asym- metry reflect the importance of traits in little brown bats (Myotis lucifigus)? Can. J. Zool. 73: 990—992.

Hartley, D.J. 1989. The effect of atmospheric sound absorption on signal bandwidth and energy and some consequences for bat echolocation. J. Acoust. Soc. Am. 85: 1338—1347.

Hayman, R.W. & Hill, J.E. 1971. Order Chiroptera. In: The Mammals of Africa. An Identification Manual. Parts 1—15 (J. Meester & H. W. Setzer, eds), pp. 1—73. Smithsonian Institution Press, Washington, DC.

Heller, K.-G. 1992. The echolocation calls of Hipposideros ruber and Hipposideros caffer. In: Prague Studies in Mammalogy (I. Hora& cek & V. Vohral&ik, eds), pp. 75—77. Charles University Press, Praha.

Heller, K.-G. & von Helversen, O. 1989. Resource partitioning of sonar frequency bands in rhinolophoid bats. Oecologia 80:178—186.

Huffman, R.F. & Henson, O.W. Jr. 1993. Labile cochlear tuning in the mustached bat. I. Concomitant shifts in biosonar emission frequency. J. Comp. Physiol. A 171: 725—734.

Jones, G. 1992. Bats vs moths: studies on the diets of rhinolophidand hipposiderid bats support the allotonic frequency hypothesis. In: Prague Studies in Mammalogy (I. Hora& cek & V. Vohral&ik, eds), pp. 87—92. Charles University Press, Praha.

Jones, G. 1995. Variation in bat echolocation: implications forresource partitioning and communication. Le Rhinolophe 11:53—59.

Jones, G. 1996. Does echolocation constrain the evolution of body size in bats? In: Miniature ertebrates: the Implications of Small Size (P. J. Miller, ed.), pp. 111—128. Symposia of the Zoological Society of London 69. Oxford Science Publications, Oxford.

Jones, G. 1997. Acoustic signals and speciation: the roles of natural and sexual selection in the evolution of cryptic species. Adv. Stud. Behav. 26: 317—354.

Jones, G., Gordon, T. & Nightindale, J. 1992. Sex and agedifferences in the echolocation calls of the lesser horseshoe bat, Rhinolophus hipposideros. Mammalia 56: 189—193.

Jones, G., Morton, M., Hughesand, P.M. & Budden, R.M. 1993.Echolocation, flight morphology and foraging strategies of some West African hipposiderid bats. J. Zool. Lond. 230:385—400.

Jones, G., Sripathi, K., Waters, D.A. & Marimuthu, G. 1994. Individual variation in the echolocation calls of three sym- patric indian hipposiderid bats, and an experimental attempt to jam bat echolocation. aolia Zool. 43: 347—361.

Jones, P.J., Burlisonand, J.P. & Tye, A. 1991. Conservac ao DosEcosistemas alorestais Na Republica Democratica de Sao Tome E Pr ncipe. UICN, Gland, Switzerland.

Juste, B. & J. 1990. Sistematica de los murcielagos de las islas oceanicas del Golfo de Guinea. PhD Dissertation, Universidad de Santiago, Santiago de Compostela, Spain.

Juste, B., J. & Fa, J. 1994. Biodiversity conservation in the Gulf of Guinea islands: taking stock and preparing action. Biodiver. Conserv. 3: 759—771.Juste, B., J. & Iba& nez, C. 1994. Bats of the Gulf of Guinea

islands:faunal composition and origins. Biodiver. Conserv. 3: 837—850.

Kober, R. & Schnitzler, H.-U. 1990. Information in sonar echoes of fluttering insects available for echolocating bats. J. Acoust. Soc. Am. 87: 882—896.

Krebs, C.J. & Singleton, G.R. 1993. Indices of condition for smallmammals. Austral. J. Zool. 41: 317—323.

Lawrence, B.D. & Simmons, J.A. 1982. Measurements of atmo- spheric attenuation at ultrasonic frequencies and the signifi- cance for echolocation by bats. J. Acoust. Soc. Am. 71: 585—590.

Littell, R.C., Freundand, R.J. & Spector, C. 1991. SAS System forLinear Models, 3rd edn. SAS Institute Inc., Cary, NC.

Møller, A.P. & Swaddle, J.P. 1997. Asymmetry, DevelopmentalStability, and Evolution. Oxford University Press, Oxford.

Neuweiler, G. 1989. Foraging ecology and audition in echo-locating bats. TREE 4: 160—166.

Neuweiler, G. 1990. Auditory adaptations for prey capture in echolocating bats. Physiol. Rev. 70: 615—641.

Neuweiler, G., Metzner, W., Heilmann, U., Ru” bsamen, R., Eckrichand, M. & Costa, H.H. 1987. Foraging behaviour and echolocation in the rufous horsoeshoe bat (Rhinolophus rouxi) of Sri Lanka. Behav. Ecol. Sociobiol. 20: 53—67.

Peters, R.H. 1983. The Ecological Implications of Body Size. Cam- bridge University Press, Cambridge.

Pye, J.D. 1972. Bimodal distribution of constant frequency in some hipposiderid bats (Mammalia: Hipposideridae). J. Zool. Lond. 166: 323—335.

Pye, J.D. 1983. Echolocation and countermeasures. In: Bioacous-tics. A Comparative Approach (B. Lewis, ed.), pp. 407—429. Academic Press, New York.

Ru” bsamen, R. & Scha” fer, M. 1990a. Ontogenesis of auditory fovea representation in the inferior colliculus of the Sri Lankan horseshoe bat, Rhinolophus rouxi. J. Comp. Physiol. A167: 757—769.

Ru” bsamen, R. & Scha” fer, M. 1990b. Audiovocal interactions during development? Vocalisation in deafened young horse- shoe bats vs. audition in vocalisation-impaired bats. J. Comp. Physiol. A 167: 771—784.

Rydell, J., Jones, G. & Waters, D. 1995. Echolocating bats and hearing moths: who are the winners? Oikos 73: 419—424.

Schnitzler, H.-U. 1970. Comparison of the echolocation behavior in Rhinolophus ferrumeuuinum and Chilonycteris rubiginosa. Bijdragen Tot Dierkunde 40: 77—80.

Schnitzler, H.-U. 1987. Echoes of fluttering insects: information for echolocating bats. In: Recent Advances in the Study of Bats (M. B. Fenton, P. Racey & J. M. V. Rayner, eds), pp. 226—243. Cambridge University Press, Cambridge.

Selander, R. 1966. Sexual dimorphism and niche utilization in birds. Condor 68: 113—151.

Shaw, R.G. & Mitchell-Olds, T. 1993. Anova for unbalanceddata: an overview. Ecology 74: 1638—1645.

Simmons, J.A. & Grinnell, A.D. 1988. The performance of echolocation: acoustic images perceived by echolocating bats. In: Animal Sonar: Processes and Performance (P. E. Nachtigall & P. W. B. Moore, eds), pp. 353—385. Plenum Press, New York.

Slatkin, M. 1987. Gene flow and the geographic structure of natural populations. Science 236: 787—792.

Surlykke, A. 1988. Interaction between echolocating bats andtheir prey. In: Animal Sonar: Processes and Performance (P. E. Nachtigall & P. W. B. Moore, eds), pp. 551—566. Plenum Press, New York.

Tera& n, M. 1962. S ntesis Geografica de aernando Poo. Instituto de Estudios Africanos e Instituto Juan Sebastia& n Elcano (CSIC), Madrid.

Van Valen, L. 1965. Morphological variation and width of ecological niche. Amer. Nat. 99: 377—390.

Van Valen, L. 1978. The statistics of variation. Evol. Theory4: 33—43.

Vater, M. 1987. Narrow-band frequency analysis in bats. In: Recent Advances in the Study of Bats (M. B. Fenton, P. Racey & J. M. V. Rayner, eds), pp. 200—225. Cambridge University Press, Cambridge.

von der Emde, G. & Schnitzler, H.-U. 1990. Classification of insects by echolocating greater horseshoe bats. J. Comp. Physiol. A 167: 423—430.

Williams, C.G. 1992. Natural Selection. Domains, Levels andChallenges. Oxford University Press, New York.

Received 9 March 1999; accepted 14 June 1999