Variation in the Frequency of the Echolocation Calls of Hipposideros Ruber in the Gulf

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, USA tEstacio 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: Abstract adaptation; This study describes variation patterns in the constant frequency (CF) segment Chiroptera; of echolocation calls of the bat Hipposideros ruber within and among constant frequency; populations across the region of the Gulf of Guinea. Correlations of variation echolocation; in CF with variation in body size, body condition, environmental humidity geographical variation; and presence of ecologically similar species are studied in an attempt to Hipposideros ruber; identify the forces driving the evolution of CF. We found that bats may adapt humidity; the frequency to humidity, and that CF may evolve under interspecific speciation. 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.

e-mail: aguill e n@admiral. u msl.edu 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; 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 characteristics 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 (characteristics 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 system, it is expected that bats that hunt for insects flying in open air will use, in calls or parts of the calls whose main

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 function is prey detection, the frequencies that give them populations and species living under different social or the largest detection distances for the preferred prey. ecological conditions. While the absolute value of the detection distance Insights into the potential adaptive meaning of CF may changes with air temperature and humidity, maximum be obtained from the evolutionary response of the echo strength in the aerial detection of a spherical target character to changes in the assemblage of immediate is always achieved when using a sound with wavelength ecological competitors. If CF value were related to prey n times the diameter of the target ([wavelength size, its mean and/or variance would likely respond n X diameterj; Pye, 1983; Hartley, 1989). Then, an to changing interspecific competitive interactions for inverse relationship of frequency vs. ideal prey diameter resource partitioning (Van Valen, 1965; Arthur, 1982; is expected; and because predator and prey body sizes are Endler, 1986; Jones, 1995, 1997), as would any other directly related (Peters, 1983), an inverse relationship ecomorphological attribute (Grant, 1994). between frequency and bat body size is also expected. High-frequency sounds are attenuated in the atmo- The main function of the ending quasi-CF segment of sphere at higher rate than deeper ones, and the rate at calls produced by aerial hawking insectivorous bats is which absorption of sound energy increases is directly prey detection (Simmons & Grinnell, 1988; Neuweiler, related to both air humidity and frequency (Lawrence & 1989, 1990). Frequency of this segment shows a negative Simmons, 1982; Hartley, 1989). Perceptive ranges of trend towards body size with an elevation that suggests rhinolophids and hipposiderids might be rather short, an adaptive match between frequency and prey size in potentially limiting and bats experience great losses in those bats (Pye, 1983; Barclay & Brigham, 1991; Jones, range with increasing humidity (Hartley, 1989). When 1996, 1997). Horseshoe and roundleaf bats also show an frequencies higher than needed for mere detection are inverse relationship between CF and body size, although used for other perceptive purpose, humidity may deter- the intercepts are much higher (Heller & Helversen, mine a levelling point in a trade-off between selection 1989; Barclay & Brigham, 1991; Jones, 1996). It is clear towards higher frequencies to obtain more resolution in from the studies cited above that these bats neither use prey classification, and towards lower frequencies to the CF segment for mere detection nor hunt in open achieve longer range. Correlation of a trait (frequency) spaces (i.e. Neuweiler, 1989). Higher frequencies may be with an environmental variable (air humidity at the necessary when the goal instead is to encode vibrational hunting grounds) would also suggest the existence of characteristics of prey. The inverse trend could then arise natural selection on the trait (Endler, 1986). when the resolving power of smaller moving structures Bats with CF calls, including rhinolophids and hippo- increases with the frequency of the call carrying the siderids, use echolocation calls, or signals structurally information. similar to the echolocation calls, in communication An alternative explanation, framed as the Allotonic (Fenton, 1985). Given the formidable capacity of these Frequency Hypothesis’, states that high frequencies used bats for discriminating frequencies around the emitted by horseshoe and roundleaf bats are a way to circumvent CF (Vater, 1987), social information might be encoded in the auditory defences of moths, which may make up the minute differences or modulations in frequency. This bulk of the bats’ diet (Fenton & Fullard, 1979; Fullard, situation may exert strong selection on individuals to 1987; Jones, 1992). Since frequency response of the maintain their CF frequencies close to the population auditory organs of moths does not seem to correlate with average in order to interact socially. Heller & von moth size (Fenton & Fullard, 1979; Surlykke, 1988), the Helversen (1989) have suggested the existence of allotonic frequency hypothesis denies a functional mean- acoustic communication channels’ in these bats. Fre- ing of the relationship between CF and body size. quency might also represent a species-specific recogni- A developmental constraint (i.e. the coupling of devel- tion signal (Butlin, 1995). Information on the health of a opment of structures for sound production or reception bat might also be encoded in the frequency of its sounds with the development of the skull) might then explain (Huffman & Henson, 1993), which may allow social the trend (longer vocal chords and larger cavities produce selection processes. Social selection may influence with- and resonate sounds of lower frequencies). in-and between-population variation in CF, potentially A simultaneous demonstration of an adaptive value of confounding the effects of ecological factors, and should lower CF and the absence of a tight constraint between be considered in the study of the selective universe frequency and body size would favour the functional operating on the evolution of characteristics of echo- meaning of the trend vs. the allotonic frequency expla- location calls in bats. nation. Selective pressures favouring lower frequencies In this paper we study intraspecific patterns of varia- would lead, through adaptive evolution, to a clustering of tion in the CF of Hipposideros ruber (Noack 1893) in the the CF of all rhinolophid and hipposiderid bats just above islands of the Gulf of Guinea (Central Africa) and the the upper frequency threshold of the hearing system of immediate mainland in an attempt to understand factors tympanate moths. The existence of the developmental underlying variation in CF. These populations live constraint may be investigated by comparing patterns of syntopically with different numbers of congeneric species covariation between CF and body size among sex, and offer the opportunity to test for the existence of (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 provided by CF as a signal. Possible social and ecological causes underlying the patterns found and their evolutionary 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 October 1992 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 populations 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

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 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 motionless 15 cm in front of the microphone of a Lars Pettersson Elektronik 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

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 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 & Since longer vocal chords and larger cavities produce Mitchell-Olds, 1993). Full models were built initially, but and resonate sounds of lower frequencies, changes in they were simplified subsequently by removing nonsig- body size could cause changes in CF if this trait lacked nificant terms. Parameter estimates produced by the adaptive value or if it were severely constrained during option SOLUTION of GLM procedure of SAS were ontogeny by body size. Variation in FA, a trait considered inspected to determine how factor levels contributed to under strong selection and tight developmental control the patterns detected. because of its relation with body size (Williams, 1992) Changes in means of CF related to the sexual and and its fundamental role for wing performance (Gummer geographical structure of the populations (potentially & Brigham, 1995), was used as reference for assessing the related to changes in ecological assemblage) were studied importance of variation in CF. A GLM with the same with a linear model that included SEX and structure as the one presented above was used for POPULATION as main fixed effects and COLONY as a studying variation in FA. The relation between general random effect nested within POPULATION (nested body size and CF was assessed within colonies by effect typed as COLONY[POPj). studying the correlation between the residuals from the

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

sex. Last column shows mean annual rainfall 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 colonie s. 1 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&o 2 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 = 2 4 25 135.11 ± 1.41 50.26 ± 0.90 26 136.25 ± 1.85 49.38 ± 1.10 2.40 cipe);Pr&in- column C shows colony number. 2 5 1 134.62 50.00 8 138.17 ± 2.13 49.31 ± 0.89 2.10 3 6 20 136.48 ± 1.02 51.65 ± 0.75 0 — — 3.50 3 7 0 — — 21 140.65 ± 1.22 50.70 ± 0.68 0.80 3 8 24 138.03 ± 1.40 50.51 ± 0.91 20 140.78 ± 1.16 50.28 ± 0.76 0.80 3 9 7 136.55 ± 1.67 49.96 ± 1.04 26 139.30 ± 2.29 49.96 ± 0.77 1.40 3 10 6 135.71 ± 1.72 50.90 ± 1.05 19 138.93 ± 1.26 49.95 ± 0.69 6.50 3 11 9 136.29 ± 2.18 50.40 ± 0.80 19 138.70 ± 2.11 50.01 ± 0.56 7.00 4 12 15 136.79 ± 1.50 50.16 ± 0.47 19 138.46 ± 1.10 50.03 ± 0.96 2.40 4 13 1 136.34 50.10 2 136.37 ± 0.64 49.15 ± 1.48 1.70 4 14 21 136.74 ± 1.36 50.18 ± 0.83 18 137.67 ± 2.60 49.79 ± 0.94 1.70 4 15 14 134.18 ± 2.07 50.69 ± 0.94 13 135.78 ± 1.80 49.85 ± 0.46 1.60 4 16 25 136.33 ± 1.89 50.40 ± 0.80 8 135.18 ± 2.56 50.06 ± 0.76 2.50

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 correlation 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 Fig. 2 Sonogram (left) and power spectrum (right) of a typical published in Tera& n (1962) and Jones et al. (1991). echolocation call of Hipposideros ruber. Maximum energy is on the We consider this a rough index of the target second harmonic, which is used as the information carrier. variable: humidity at the hunting grounds of bats. A social role of the CF value could be rooted in the CF and FA GLM models described above, both for all data potential phenotypic or genotypic quality information and within sex. provided by the signal. Following Krebs & Singleton The effects of POPULATION, COLONY and SEX on the (1993), a body condition index (BCON) was calculated as variances of CF and FA were studied with a multilevel— the ratio between the actual body mass and the mass multifactorial adaptation of Levene’s test (Van Valen, predicted by a ln—ln regression of body mass vs. FA for all 1978). The absolute difference between each measure- individuals with available data (colonies 3, 5, 7 and 12). ment and the median of the corresponding cell (POPU- Because we were interested in the individual effect of LATION X COLONY X SEX) was divided by the cell body condition, the variation due to SEX and COLONY mean. The new variables (unsigned relative deviations) was removed by including these explanatory variables in were used as response variables for two linear models the initial A N C O V A model. We then tested for a correla- with the same structure as those used for studying the tion between the residuals of the first GLM model for CF population structure of original variables CF and FA. The with BCON, for all data and within sex.

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 Table 2 Effect of POPULATION, COLONY and SEX on the resting Table 3 Effect of POPULATION, COLONY and SEX on the forearm frequency (CF) of Hipposideros ruber in the Gulf of Guinea. length (FA) of Hipposideros ruber in the Gulf of Guinea.

Effect d.f. MS F P Effect d.f. MS F P

POPULATION 3 896.301 49.74 <0.001 POPULATION 3 5.039 0.91 0.468 COLONY[POP] 12 25.501 8.53 <0.001 COLONY[POP] 12 8.022 9.96 <0.001 SEX 1 199.187 66.63 <0.001 SEX 1 25.122 31.21 <0.001 POPULATION* SEX 3 17.081 5.71 0.001 ERROR 411 0.805 ERROR 411 2.989 Tests were built with type IV sums of squares. COLONY[POPj was Tests were built with type IV sums of squares. COLONY[POPj was considered as a random effect, and the adjusted denominator’s d.f. considered as a random effect, and the adjusted denominator’s d.f. and MS for POPULATION were, respectively, 13.30 and 5.532. The and MS for POPULATION were, respectively, 13.44 and 18.021. The interaction terms POPULATION X SEX and SEX X COLONY[POPj interaction term SEX X COLONY[POPj was removed from the final were nonsignificant (a3,32.17 = 0.47, P = 0.710, and a10,408 = 0.97, model, since it was nonsignificant (a10,401 = 1.51, P = 0.134). The P = 0.470), so they were removed from the final model. The model is model is significant and explains a large proportion of data variance significant, but it explains a relatively low proportion of data (a = 71.48, P < 0.001, r2 = 0.77). 2 19,411 variance (a16,411 = 11.37, P < 0.001, r = 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 dimorphism 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 and 1.7 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 this effect 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,

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 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 constant corresponding to each COLONY X SEX cell) was significant, and the proportion of variance explained was 2 high (a8,108 22.15, P <0.001, r 0.64). Correlation of BCON with the residuals of the initial model for studying variation 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 variation 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

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 the mainland. Populations in the islands and the main- selection under different environmental conditions. The land might belong to different sibling species, but similarity of the island populations is difficult to explain molecular data show that the populations from Ri&o under pure drift, because some of them are historically Muni and Bioko are genetically very similar (1.6% closer to the mainland than to other island populations Kimura two-parameter distance in mitochondrial (the population on Bioko is genetically much closer to cytochrome b sequences; A. Guille& n, unpublished the populations in the mainland than to the populations data). Some patterns in our dataset show that changes in other islands; A. Guille& n, unpublished data). in CF do not merely result from general changes in Clime differences are probably not the causal factors body size. Frequency and body size are correlated of the differences, since rainfall and temperatures on within colonies, but negatively among females and the mainland are close to the average values from the positively among males. Moreover, despite the large islands (despite the higher spatial environmental differences in CF existing between populations on the heterogeneity on islands). Shifts reported by Francis & islands and the mainland, there were no significant Habersetzer (1998) hardly correlate with environmental differences in body or skull size among them (Juste differences between Borneo and Malaya, since the two B., 1990), and the slight significant differences in species reported experience changes in opposite size among colonies were not correlated with directions. differences in CF. The shift in CF between populations could be a result Large shifts in CF between populations of CF rhino- of ecological displacement or release (Arthur, 1982). lophoid bats separated by geographical barriers may be However, the invariable intrapopulational variance of CF common, since the few studies that have dealt with this and the absence of increased sexual dimorphism in the issue have detected them. Francis & Habersetzer (1998) simpler ecological assemblages studied, contrary to the report large shifts, over 10 kHz, in two out of three expectations of the adaptive variation hypothesis’ (Van species recorded both in Borneo and Peninsular Valen, 1965; Selander, 1966; Grant & Price, 1981), would Malaysia. Hipposideros cervinus living in the Malayan not support an ecological explanation relating the diver- peninsula used higher pitched calls, although they had gence to interspecific interactions. Alternatively, the slightly larger body size than bats from Borneo. Hippo- extreme narrowness of the variance of CF in every sideros galeritus from Malaya were similar in size to those population suggests that strong stabilizing selection may living in Borneo, but used much deeper calls. Some be in action, and it may overcome effects of ecological populations isolated by distance also exhibit large differ- release on the variance of CF. Other populations of ences (e.g. R. ferrumeuuinum between Europe & Japan, H. ruber recorded elsewhere in Africa have a CF value Heller & von Helversen, 1989; Hipposideros fulvus between close to the island populations studied here (Pye, 1972; India & Malaysia, Heller & von Helversen, 1989; Jones Fenton & Fullard, 1979; Heller, 1992; Jones et al., 1993), et al., 1994). Although, in this study body size and CF except for bats recorded by Pye (1972) in Uganda which covary between sexes according to what is expected from show similar CF to the bats recorded by us in the physical laws (longer chords and larger cavities produce mainland. Among the recording sites, these two main- and resonate deeper sounds), this is not the rule in other land localities (Pye’s and ours) are probably the only ones rhinolophoid CF bats for which sexual dimorphism in CF where H. ruber lives in sympatry with Hipposideros has been reported. In Hipposideros speoris, females produce fuliginosus, a close relative with a somewhat larger body sounds 3 kHz deeper than males, but differences in size (Hayman & Hill, 1971). The shift between mainland forearm length between sexes are not significant (Jones and island populations might be related to the release et al., 1994). Females of Rhinolophus hipposideros are larger from an interspecific interaction, either ecological or than males, but produce calls with higher CF (Jones social, with the latter species. et al., 1992; Guille& n, 1996). Females of Rhinolophus We found a significant inverse correlation between CF rouxi, R. creaghi and R. thomasi use higher pitched colony means and environmental humidity measured as sounds than males, but there are no differences local MAR in the island of Sao Tome& . When all between sexes in forearm length (Neuweiler et al., populations were analysed together, the trend was at 1987; Francis & Habersetzer, 1998; Francis & Guille& n, the limit of significance. Geographical variation of a unpublished data). Hipposideros commersoni, an African phenotypic character, genetically or culturally inherited, hipposiderid bat, shows a notorious dimorphism in is the combined result of processes of drift, natural and body size, reflected in many skull structures not involved sexual selection, and gene flow (Slatkin, 1987). Average in echolocation. However, the CF of the echolocation values and geographical variation shown in one call does not differ significantly between sexes (Guille& population are highly dependent on the interaction of n, 1996). These fre- quent observations of the geographical structure of the selective environment intraspecific shifts in CF value independent of changes with gene flow patterns and may not be directly in body size imply that body size does not strongly comparable to other populations. The island of Sao constrain CF in horseshoe and roundleaf bats. Tome& presents more environmental variation (wider Differences between populations from the islands and range in MAR) than other islands. Our sample of the mainland may be due to genetic or cultural drift, or localities in that island also captures more geographical and environmental variation

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 than samples from other islands (in fact, the combined seems to be the ancestral condition for the group. All range of MAR in all other localities together is less than hipposiderids for which sounds have been described use half the range available in Sao Tome& ). The the second harmonic. Most horseshoe bats also use the potential effect on the geographical variation of CF second harmonic, except a few recently derived species may be counteracted by gene flow through migration with particular ecology that have reverted to using the in other islands with more moderate selective first harmonic, lower in frequency (A. Guille& n, landscapes. Some interspecific patterns support the idea unpublished data). After adopting high frequencies, that adaptation of CF to the average humidity in the the bats could find easy access to the resource hunting grounds may occur. Heller & von Helversen represented by tympanate insects, partially (1989) noticed that horseshoe bats living in dry inaccessible to other bats. This being the case, the habitats have higher CF relative to body size than allotonic frequency would have the meaning of a those living in wet environments. A coincident spandrel’ attribute useful for hunting tympanate pattern exists for the African groups of Hipposideros with insects, in the sense of Gould & Lewontin (1979) and representatives in both the rainforest belt and the Williams (1992), while not being an adaptation for surrounding dry environments. Rain forest dwellers (H. that function. ruber and H. commersoni thomensis and H. commersoni Sexual differences in echolocation calls commented gigas) use lower relative CF than their relatives in the above might be related to a social function of the dry areas (H. caffer, the sibling species of H. ruber, and echolocation call. The absence of sexual dimorphism in H. commersoni marungensis, a relative of the other two CF in some species with large dimorphism in body size, forms; Jones et al., 1993; Guille& n, 1996). These patterns such as the case of H. commersoni commented above, are expected if higher frequencies have an actual cost in suggest that a social evolutionary pressure exists for ecological performance by reducing the detection space keeping the frequency within a narrow range. The for the bats in humid environments. conspicuously narrow within-colony variation in CF High frequencies used by CF rhinolophoid bats may be found here points to a strong selective mechanism acting necessary for extracting information about prey charac- on this character. Extremely narrow intrapopulational teristics through the analysis of the micromodulations variations in CF, with CVs close to 1%, are typical in caused by moving prey in the CF segment of the call. rhinolophoid CF bats (Jones et al., 1992, 1993, 1994; Higher frequencies experience larger differences in Guille& n, 1996). According to some views, Doppler shifts when reflected in objects moving at intrapopulational phenotypic variation in mating different speed (Pye, 1983), and may be required for signals is typically low when compared with other discriminating smaller prey having a shorter range of quantitative characters (Butlin, 1995; but see an linear speed of their moving parts. But very high opposite view in Møller & Swaddle, 1997). frequencies are heavily attenuated in humid air (Hartley, Given the significant correlation between CF and body 1989), and that may establish a trade-off between higher condition index, females of H. ruber could obtain infor- frequencies for enhancing resolution in prey classifica- mation about ecophysiological performance of potential tion and lower frequencies for longer range detection. mates after the sounds they broadcast. Relationships Fixation of the trade-off point in the population may between CF and body condition indexes have been explain the absence of response in the variance of CF to reported for H. fulvus (Jones et al., 1994) and R. fer- changes in the composition of the competitive environ- rumeuuinum (Guille& n, 1996). Numerous studies ment represented by the ecological assemblage. have shown that tuning properties of the mammalian These results challenge the validity of the Allotonic cochlea are altered by changes in physiological Frequency Hypothesis’ as an explanation of the evolu- variables (temperature, blood pressure, hormonal tionary achievement of high frequencies by rhinolophids levels, etc.) that may ultimately affect the and hipposiderids (Fenton & Fullard, 1979; Fullard, 1987; micromechanical properties of the inner ear (Bell, 1992; Jones, 1992; Rydell et al., 1995). Empirical evidence of Huffman & Henson, 1993). Given the nature of the the CF value being positively correlated with the echolocation system used by CF rhinolophoidea, any incidence of moths in the diet of rhinolophid and shift in cochlear tuning must be followed by a hipposiderid bats has been used to support this hypoth- concomitant shift in the CF of the broadcasted pulses esis (Jones, 1992; Rydell et al., 1995). However, higher (Huffman & Henson, 1993). This may well facilitate consumption of tympanate moths by bats with frequen- the development of a sexual selection mechanism with cies above the hearing frequency range of the insects female preferences based on phenotypic quality of males does not prove that moth hearing was the selective factor detected by minute differences in frequency. underlying a shift to higher frequencies. These bats may Simultaneous selection on CF and body size may result have shifted to the second higher harmonic during the in the positive correlation between these two variables origin of this echolocation system because of the func- found in males in our study. Once all females in a tional requirements of narrow frequency analysis. Linked population have evolved a preference, the selected use of high frequencies and the second harmonic is characteristic of the call (i.e. frequency) may become part prevalent among horseshoe and roundleaf bats, and it of the specific mate recognition system of the population (Butlin, 1995). However, the reversed dimorphism of the

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 colony at Sundi& may indicate that sexual dimorphism is Carlos Ru&iz for their help during field work. Damond under dynamic evolution in H. ruber. Heller & von Kyllo helped with the language. This research was Helversen (1989) found that the CF values of the partially supported by the Spanish DGICYT (PB90- horseshoe and roundleaf bat species reported from a 0143), the Junta de Andaluci&a (research group RNM- relatively small region in Malaysia (and thus regarded as 158), and by predoctoral (UNICAJA-Junta de Andaluci&a) the total set of species in a syntopic ecological assemblage and postdoctoral (Ministerio de Educacio& n y or guild) were distributed significantly more evenly on a Ciencia) grants to A.G. The European Union (ECOFAC log scale than expected at random. This even spacing project) and the World Bank covered partially travel makes little sense as a result of ecological displacement of expenses to J.J.B. a character related to prey size because the species involved belong to two different families (Rhinolophidae and Hipposideridae) with somewhat different echoloca- References tion systems with probably different functional proper- Anthony, E.L.P. 1988. Age determination in bats. In: Ecological ties. However, an evolutionary accommodation of and Behavioral Methods for the Study of Bats (T. H. Kunz, ed.), frequency channels for social communication may result pp. 47—58. Smithsonian Institution Press, Washington, DC. in the spacing of CF among species with different Arthur, W. 1982. The evolutionary consequences of interspecific phylogenetic origin and ecomorphological characteristics. competition. In: Advances in Ecological Research, Vol. 12. (A. F. A possible interaction between call frequency and Macfadyen & E. D. 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