Microcebus Murinus) and Rufous Mouse Lemurs (Microcebus Rufus)*

International Journal of Primatology, Vol. 21, No. 5, 2000

Use of Vocal Fingerprinting for Speciﬁc Discrimination of Gray (Microcebus murinus) and Rufous Mouse Lemurs (Microcebus rufus)*

Elke Zimmermann,1,2 Ekaterina Vorobieva,1 Dorothea Wrogemann,1 Thomas Hafen1 Received April 28, 1999; revision September 14, 1999; 2nd revision October 19, 1999; accepted November 2, 1999

Advertisement calls are often important noninvasive tools for discriminating cryptic species and for assessing specific diversity and speciation patterns in nature. We investigated the contribution of these calls to uncover specific diversity in nocturnal Malagasy lemurs. We compared sexual advertisement and predator advertisement calls of two mouse lemur species, western gray and eastern rufous mouse lemurs (Microcebus murinus and M. rufus, respectively) living in two contrasting habitats (dry deciduous vs. rain forest), and analyzed them statistically. Both species emitted several highly variable whistle calls in the context of predator-avoidance. Intrapopulation variation was high and overlapped interspecific variation. Sexual advertisement calls, given in the mating context, displayed a totally distinct, species-specific acoustic structure. Whereas gray mouse lemurs produced rapidly multifrequency modulated, long trill calls, rufous mouse lemurs gave slowly frequency- modulated short chirp calls. Our results suggest specific status for gray and rufous mouse lemurs and indicate the importance of predation and social needs in shaping vocal communication. KEY WORDS: vocalization; sexual advertisement; predator advertisement; taxonomy; evolution; mouse lemur; primate; Madagascar.

1Institut fu¨ r Zoologie, Tiera¨rztliche Hochschule Hannover, Germany. 2Address for correspondence: Elke Zimmermann, Institut fu¨ r Zoologie, Tiera¨rztliche Hochschule Hannover, Bu¨ nteweg 17, 30559 Hannover, Germany; Tel: xx49-511-9538740; Fax: xx49-511-9538586; e-mail: [email protected] *Dedicated to Prof. Kuhn on his 65th anniversary.

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0164-0291/00/1000-0837$19.50/0  2000 Plenum Publishing Corporation 838 Zimmermann, Vorobieva, Wrogemann, and Hafen

INTRODUCTION

Nocturnal primates are often difficult to distinguish in size, pelage coloration and other gross visual bodily characteristics. Consequently, species diagnosis by noninvasive methods is extremely difficult. Bioacoustic studies on African bush babies (Zimmermann et al., 1989; Zimmermann, 1990; Masters, 1991) and on Asian tarsiers (Niemitz, 1994; Nietsch and Kopp, 1998) have shown that calls used for social communication can be employed reliably as a vocal fingerprint to discriminate sibling species. The most prominent differences in the acoustic structure of homologous calls—given in the same context—between species occur in advertisement calls. Sexual advertisement calls are given most frequently during the breeding season. They seem to be involved in mate recognition and mate choice (Zimmermann, 1995) and therefore may act as a premating isolating mechanism. Other contact, attention and alarm calls with harmonic structure show less distinct differences. Based on bioacoustics, Bearder et al. (1995) proposed that the diversity of the African bush babies is highly underesti- mated and comprises many more than the 11 species already described. Analyzing the duets of Asian tarsiers, Nietsch (1999) found geographically distinct structural differences between populations which indicate the same might be true for this nocturnal primate taxon. To date, there is no information whether this method will also work in the highly endangered nocturnal Malagasy lemurs, wherein it could be an important noninvasive tool for conservation, e.g. for specific identification or for monitoring specific diversity. Furthermore it might give new insights into speciation patterns of this threatened primate group. Mouse lemurs (Microcebus spp.) are the world’s smallest primates. They live in a wide range of forest habitats in Madagascar from sea level up to a height Յ1300 m above sea level (Mittermeier et al., 1994; Rakotoari- son et al., 1997). Due to their overall similarity in external appearance, their taxonomy is controversial. Detailed information regarding their geographic range and their biology is lacking. It is unclear, whether the described species belong to a metapopulation or to different biological species (sensu Mayr, 1973). Schwarz (1931) lumped together all the lesser mouse lemurs in a single species, Microcebus murinus. Later, Martin (1972) recognized two separate species based mainly on differences in ear length and cranial morphology: A gray long-eared form with a more rounded head (Micro- cebus murinus) typically inhabiting dry forests from west to southeast Mada- gascar, and a rufous short-eared form with a smaller head (Microcebus rufus), typically inhabiting rain forest areas in eastern Madagascar. The species are known to occur sympatrically in a transient forest in southeastern Madagascar. However, the limits of geographic distribution of these two Vocal Fingerprinting of Mouse Lemurs 839 forms are not yet known (Ganzhorn et al., 1997). Schmid and Kappeler (1994) rediscovered the smallest species, the dwarf mouse lemur (Micro- cebus myoxinus), in a dry deciduous forest in western Madagascar, and Zimmermann et al. (1998) described a new species with the greatest known body length, the golden brown mouse lemur (Microcebus ravelobensis), from northwestern Madagascar. Karyological investigations have not revealed any difference among Microcebus murinus, M. rufus and M. myoxinus (Rumpler et al., 1998). RAPD-fingerprinting studies show differences among them (Tomiuk et al., 1998), but they also show differences even between different populations of Microcebus murinus (Hafen, 1998). Neither morphology nor genetics have clarified whether individuals of populations have developed species- specific signals by which they recognize each other as mating partners and therefore belong to the same species. According to Paterson (1993), individuals of populations belonging to the same species should reproduce successfully with each other. Reproductive success depends on the recognition of a species-specific mate. Selection should therefore favor signals, nervous systems and behaviors guaranteeing successful mate recognition. Thus, it can be hypothesized that signals evolved in the mating context (and probably acting as a premating isolating mechanism) should be of higher diagnostic value for specific identification than signals evolved in other social situations. The aims of this study are to test this hypothesis, to give primary quantitative bioacoustic data on rufous mouse lemurs, to identify and quan- tify differences in advertisement calls between rufous and gray mouse lemurs in order to assess their specific status, and to check which part of their vocal repertoire provides the most reliable tool for noninvasive species diagnosis. Furthermore, we discuss the effect of habitat structure, predation and social needs on signal structure.

MATERIAL AND METHODS

Subjects

We maintained and bred mouse lemurs in a colony at the animal house of the Institute of Zoology, Hannover (formerly based at Stuttgart, Konstanz and Go¨ ttingen). Wrogemann et al. (in press) provide details of their husbandry and maintenance. The gray mouse lemurs (Microcebus murinus) are descendants of a population living in the dry deciduous forests of southeast Madagascar (Mandena) imported to Europe and the United States more than 30 years ago (Hafen, 1998). We included 12 male and 4 840 Zimmermann, Vorobieva, Wrogemann, and Hafen female captive-born gray mouse lemurs in our study. Rufous mouse lemurs (Microcebus rufus) were captured in the highland humid rain forests of eastern Madagascar in the area of Andasibe (Zimmermann et al., 1998). Three males and three females were imported to Germany in 1995 to establish a breeding colony (under CITES No. E 0599/95, E 0388/95 and E 0055/95). We included 2 male and 3 female wild-born and one captive- born male (F1-generation) rufous mouse lemurs in our study.

Data Collection

We collected data shortly before and during the mating seasons in 1990–1998. We observed pairs during the first 2 hours of their activity period. Sexes could be reliably identified by pelage markings. We conti- nously recorded behaviors before and after call emission, as well as time and date on one channel of a tape recorder per Martin and Bateson (1993). Procedures corresponded to those already described (Zimmermann et al., 1993; Zimmermann, 1996). To conduct interspecific comparison of vocalizations, we considered the following behavioral situations: 1. Predator situation: Mouse lemur male–female pairs. The pair was confronted visually with unknown or potentially dangerous objects such as a bottle or a terrestrial (living python or python dummy) or an aerial predator (barn owl dummy or barn owl silhouette) in front of the enclosure. 2. Mating situation: Mouse lemur pairs with estrous female. Estrus was detected by vaginal morphology and cytology according to Bu¨ sching et al. (1998) and Radespiel (1998).

Sound Analysis and Statistics

We recorded vocalizations with a NAGRA-IV-SJ tape recorder, using Bruel and Kjaer microphones (Type 4133) with preampliﬁers and BASF tapes. Tape speed was 38 cm/s. We digitized calls and analyzed them at half-speed (sampling rate 44 kHz) via a Digital Sound Analyzing System (Avisoft-SAS LabPro 3.4d, Specht; FFT-length 256; 50% overlap, Hamming window). Frequency resolution of the appropriate frequency range was 172 Hz. In Table I, we describe the acoustic variables measured for whistle, chirp and trill calls. Figure 1a illustrates the measured variables for whistle calls, Fig. 1b for chirp calls and Fig. 1c for trill calls. We calculated means of individuals or groups for all variables of whis- Vocal Fingerprinting of Mouse Lemurs 841

Table I. Description of acoustic characteristics used to characterize whistle, chirp and trill calls Whistle calls: cd Call duration, sec ici Intercall interval, sec F1 Onset frequency of fundamental, kHz F2 End frequency of fundamental, kHz Fmax Maximum frequency of fundamental, kHz Fmin Minimum frequency of fundamental, kHz Fmax/Fmin Division Fmax to Fmin N of calls per series Number of calls per series I/I in series Intercall interval in series, sec Chirp calls: cd Call duration, sec d1 Duration of the 1st call unit, sec d2 Duration of the 2nd call unit, sec iui Interunit interval F1 Onset frequency of fundamental of the 1st call unit, kHz F2 End frequency of fundamental of the 1st call unit, kHz F3 Onset freuency of fundamental of the 2nd call unit, kHz F4 End frequency of fundamental of the 2nd call unit, kHz Fmax Maximum frequency of fundamental, kHz Fmin Minimum frequency of fundamental, kHz Fmax/Fmin Division Fmax to Fmin F2/F1 Division F2 to F2 F3/F4 Division F3 to F4 Trill calls: LGES Call duration, sec Rate Modulation rate ϭ (sum of duration of the first 7 modulations (from F1EN to F7EN)/7)Ϫ1 ϫ 1000, Hz FAAN Onset frequency of fundamental, kHz FSEN Maximum frequency of fundamental, kHz AFAN Average start frequency of the first 7 modulations ϭ 7 ͚ FnAN/7, kHz nϭ1 AFEN Average end frequency of the first 7 modulations ϭ 7 ͚ FnEN/7, kHz nϭ1 ABB Average bandwidth of the first 7 modulations ϭ 7 ͚ (FnEN-FnAN)/7, kHz nϭ1

tle, chirp and trill calls, respectively, using STATISTICA software (StatSoft, 1996 Inc.), and tested the acoustic variables characterizing whistle calls for significant intergroup and interspecific differences with nested ANOVA with groups nested in species (general linear model procedure, mixed model analysis of variance) using SAS software (SAS 6.12). We consider a level of p Ͻ 0.05 to be significant. We did not compare chirp and trill call variables statistically because of their clearly distinct acoustic structure. 842 Zimmermann, Vorobieva, Wrogemann, and Hafen

Fig. 1. (a) Measured characteristics of whistle calls: F1—onset frequency of fundamental; F2—end frequency of fundamental; Fmax—maximum frequency of fundamental; Fmin— minimum frequency of fundamental; cd—call duration; ici—intercall interval. (b) Measured Vocal Fingerprinting of Mouse Lemurs 843

RESULTS

Vocalizations Given in the Predator Context

Predator-advertisement or alarm calls are produced by rufous and gray mouse lemurs in response to unknown or potentially dangerous objects. Both species emitted series of narrow-band, almost constant-frequency harmonic whistle calls in the range between 11 and 22 kHz (Fig. 2; Table II). Whistle calls can be tentatively classified into very short, short and long whistle calls in Microcebus murinus and short and long whistle calls in M. rufus, though there was a continuous transition among all of them. Differences in call structure between the two species occur in fundamental frequency and temporal pattern. Mean fundamental frequency is higher and mean whistle duration shorter in Microcebus rufus than in M. murinus. Intercall interval in series is shorter in Microcebus rufus than in M. murinus. Series of whistle calls contain fewer calls in Microcebus rufus than in M. murinus. According to the results of a nested ANOVA, six variables showed significant group-specific variability. Because of a high intra- and interspecific variability, however, these differences are not significant at the species level (Table III). Consequently, there is no clear species separation.

Vocalizations Given in the Mating Context

Sexual advertisement calls are used very frequently during the breeding season (Zimmermann and Lerch, 1993; Hafen et al., 1998; Bu¨ sching et al., 1998). Vocalizations of both species were broadband frequency-modulated, extending on average from 14 to 34 kHz. Strong and obvious divergences in the acoustic structure of advertisement calls between the species were revealed (Fig. 3, Table IV a,b). Microcebus rufus produced a two-compo- nent chirp call with a relatively stereotypic structure. The ﬁrst unit of the call was upward frequency modulated. An interunit interval separated it

Fig. 1. (Continued). characteristics of chirp calls: F1—onset frequency of fundamental of the first call unit; F2—end frequency of fundamental of the first call unit; F3—onset frequency of fundamental of the second call unit; F4—end frequency of fundamental of the second call unit; Fmax—maximum frequency of fundamental; Fmin—minimum frequency of fundamental; cd—call duration; d1—duration of the first call unit; d2—duration of the second call unit; iui—interunit interval. (c) Measured characteristics of trill calls: FAAN—onset frequency of fundamental; FSEN—maximum frequency of fundamental; F1AN—start frequency of the first modulation; F1EN—end frequency of the first modulation; F2AN—start frequency of the second modulation, etc.; LGES—call duration. 844 Zimmermann, Vorobieva, Wrogemann, and Hafen

Fig. 2. Representative sonograms of predator advertisement calls: a–b—short and long whistles of Microcebus, rufus, c–e—very short, short and long whistles of M. murinus. Vocal Fingerprinting of Mouse Lemurs 845

Table II. Comparison of acoustic characteristics of whistle calls of 3 groups of Microcebus rufus (29–687 calls per group) and 4 groups of M. murinus (34–718 calls per group)

Sound Standard parameter Species Mean deviation Minimum Maximum cd, sec rufus .084 .055 .038 .145 murinus .128 .049 .078 .193 F1, kHz rufus 18.3 3.4 15.6 22.1 murinus 14.3 1.6 12.5 16.4 F2, kHz rufus 17.4 3.9 14.8 21.9 murinus 14.3 2.0 12.0 16.8 Fmax, kHz rufus 18.7 3.4 16.1 22.6 murinus 15.6 1.5 14.2 17.5 Fmin, kHz rufus 17.2 3.8 14.7 21.6 murinus 13.8 1.7 11.7 15.9 Fmax/Fmin rufus 1.1 .0 1.0 1.1 murinus 1.1 .1 1.1 1.2 N of calls rufus 3.3 1.2 2.1 4.5 per series murinus 6.4 3.3 2.6 9.2 I/I in series rufus .043 .009 .038 .054 sec murinus .095 .020 .084 .125

from the second downward frequency-modulated unit. The whole series was given as a single call. It lasted Յ140 ms on average. Microcebus murinus emitted an acoustically complex designed multifrequency modulated trill call with a mean call duration of 700 ms that is about 5-fold the duration of that of its sibling species.

DISCUSSION

Sexual advertisement, but not predator advertisement, calls showed clear species-speciﬁc differences in vocal structure between small nocturnal Malagasy lemurs such as western gray and the eastern rufous mouse lemurs

Table III. Interspeciﬁc and intragroup differences in variability of whistle call structure between gray and rufous mouse lemurs as indicated by a nested ANOVA

Species Groups

F1 F:3.46 Pr Ͼ F:0.1216 F:3.82 Pr Ͼ F:0.0001 F2 F:0.2650 Pr Ͼ F:0.2650 F:422.2920 Pr Ͼ F:0.0001 Fmax F:2.1848 Pr Ͼ F:0.1993 F:333.3525 Pr Ͼ F:0.0001 Fmin F:2.1802 Pr Ͼ F:0.1997 F:422.4138 Pr Ͼ F:0.0001 cd F:1.4734 Pr Ͼ F:0.2785 F:70.1841 Pr Ͼ F:0.0001 Fmax/Fmin F:1.1475 Pr Ͼ F:0.3328 F:165.8423 Pr Ͼ F:0.0001 846 Zimmermann, Vorobieva, Wrogemann, and Hafen

Fig. 3. Representative sonograms of sexual advertisement calls: (a) chirp call of Microcebus rufus and (b) trill call of M. murinus. confirming our hypothesis that signals evolved in the mating context are of higher diagnostic value for species identification than signals evolved in other social situations. Thus, vocal fingerprinting of sexual advertisement calls can be an important noninvasive tool for species diagnosis in small nocturnal Malagasy lemurs and may also help to clarify specific status. The revealed species-specificity in vocal structure between gray and rufous mouse lemurs agrees with less obvious differences in morphology, genetics and reproduction (Martin, 1972; Atsalis et al., 1996; Tomiuk et al., 1998; Zimmermann et al., 1998; Pastorini et al., submitted; Wrogemann et al., 2000). They suggest species status for the investigated populations. Rufous and gray mouse lemurs differ in the habitats they occupy. Whereas rufous mouse lemurs live in evergreen and humid rain forests of eastern Madagascar, gray mouse lemurs inhabit dry and deciduous forests of western Madagascar. It can be assumed that sound transmission properties between these two habitats are different, though there is no datum on habitat acoustics from Madagascar. According to the acoustic adaptation hypothesis (Daniel and Blumstein, 1998), selection should modify the acoustic structure of advertisement calls according to habitat properties in order to optimize their transmission (Morton, 1975; Hansen, 1979; Rothstein and Fleischer, 1987; Bradbury and Vehrencamp, 1998). However, contrary to this hypothesis the two species have evolved predator advertisement calls with a similar overall acoustic pattern. The whistle calls have a repetitive, Vocal Fingerprinting of Mouse Lemurs 847

Table IVa. Acoustic characteristics of chirp calls of 3 males Microcebus rufus (7–75 calls per individual)

Sound Standard parameter Mean deviation Minimum Maximum

cd, sec .136 .011 .124 .147 d1, sec .048 .011 .037 .059 iui, sec .063 .018 .044 .080 d2, sec .026 .009 .020 .036 F1, kHz 15.3 1.0 14.5 16.4 F2, kHz 26.6 1.9 25.2 28.7 F3, kHz 27.5 5.1 22.0 32.1 F4, kHz 16.5 .5 16.1 17.0 Fmax, kHz 28.5 3.4 25.2 32.1 Fmin, kHz 15.1 .7 14.5 15.9 Fmax/Fmin 1.9 .2 1.8 2.2 F2/F1 1.8 .2 1.6 2.0 F3/F4 1.7 .3 1.4 2.0

narrow-band, high-frequency harmonic structure extending from approxi- mately 10 to 20 kHz. Due to high intraspecific variation, species-specificity was lacking. The lack of specific distinctiveness in this part of their vocal repertoire may be caused by similar predator pressures and therefore similar environmental constraints. Snakes, viverrids, dogs, cats and nocturnal raptors, such as owls, are the major predators on small mammals in both habitats (Goodman et al., 1993). Snakes cannot hear at all; birds, including owls, cannot hear beyond 10 kHz; and, the other predators can perceive these frequencies but have difficulties locating their prey by narrow-band sounds in this high-frequency range (Fay, 1990; Hauser, 1996). It is therefore unlikely that predator advertisement calls in closely related species diverge. Most interestingly, not only do whistle calls of the two species show a great deal of overlap; the same is true for whistle calls of all small members of

Table IVb. Acoustic Characteristics of Trill Calls of 9 Male Microcebus murinus (19–100 Calls per Individual).

Standard Sound parameter Mean deviation Minimum Maximum

LGES, sec 0.699 0.058 0.607 0.784 Rate, Hz 42.3 3.6 37.2 49.3 FAAN, kHz 19.4 2.1 15.2 23.1 FSEN, kHz 29.6 2.4 26.2 33.9 AFAN, kHz 16.8 1.5 13.7 19.6 AFEN, kHz 25.7 2.2 22.1 29.3 ABB, kHz 9.0 1.6 6.8 10.9 848 Zimmermann, Vorobieva, Wrogemann, and Hafen the Cheirogaleidae, regardless of their respective habitats (Rakotoarison et al. (1997): Allocebus trichotis and Zimmermann (1995): Cheirogaleus major of the rain forest and Stanger (1995): C. medius and Mirza coquereli of the dry forest). Even unrelated species evolve similarly structured calls under these circumstances, as evidenced by bioacoustic studies on birds, squirrels and anthropoid primates (Marler, 1955, 1973; Koeppl et al., 1978; Alcock, 1998). In contrast to the acoustic similarity of predator advertisement calls, sexual advertisement calls of rufous and gray mouse lemurs exhibit strong species-specific differences, implying that not only habitat acoustics and predation pressure but also social needs may trigger specific distinctiveness. The high-frequency broadband frequency-modulated sound structure char- acterizes both species and may be an adaptation to be localizable for conspe- cifics without the risk of being disturbed by other sound-producing animals and of being captured by nocturnal raptors. Both species, however, clearly diverge in overall acoustic pattern, e.g. in frequency modulation and call duration. Whereas the chirp call of the rain-forest dwelling species, Micro- cebus rufus, is short and shows only one single slow-frequency modulation, the trill call of the dry forest-dwelling specific, M. murinus, displays a complex, multifrequency-modulated structure with long call duration. These differences might be explained by differences in degradation dis- cussed to affect temporal patterns of amplitude or intensity modulation between forested and more open habitats (Wiley and Richards, 1978). Reverberations are more severe in forested habitats primarily because they mask rapidly repeated notes such as those in the trill call of dry forest Microcebus murinus. In contrast, irregular amplitude fluctuations in open habitats favor calls with such a call structure. Calls in open habitats are masked by wind at irregular intervals. Since calls that contain rapidly repeated, short notes can be detected in short spaces of time, they should be favored in more open areas. These habitat constraints seem to fit well with the trill call structure of Microcebus murinus in the more open dry forest and that of the chirps of M. rufus in the more closed rain forest. Similar, phenomena occur in song birds wherein frequency modulation is slower in forest-dwelling species than in species living in more open habitats. Nottebohm (1975), Wiley and Richards (1982), Hunter and Krebs (1979) and Bradbury and Vehrencamp (1998) explained them as adaptations for optimal transmission. There is also evidence that sound transmission properties in dry deciduous forests favor the evolution of relatively long, multifrequency-modulated trill advertisement calls of small Malagasy lemurs. Thus, populations of gray mouse lemurs in dry deciduous forests of northwestern (Ampijoroa), western (Kirindy) and southeastern Madagascar (Mandena) all produce Vocal Fingerprinting of Mouse Lemurs 849 trill calls that have a similar overall acoustic pattern. They show only slight, but significant, differences in modulation rate and call duration (Hafen, 1998; Hafen and Zimmermann, submitted; Zietemann, 1998). Even closely related Coquerel’s mouse lemurs (Mirza coquereli) produces a similar call under similar circumstances (Stanger, 1995). However, the structure of advertisement calls may be influenced not only by habitat structure and predation pressure but also by social needs. Gray mouse lemurs live in a dispersed multimale-multifemale network (Radespiel et al., 1998; Rades- piel, 1998) with a male-biased sex ratio and home ranges that overlap intra- and intersexually. They are promiscous; usually males compete for access to an estrous female (Fietz, 1998; Radespiel, 1998). Sexually active males gave trill calls as a sexual display, most frequently when sexual competition is highest in courting an estrous female (Zimmermann, 1995; Peters, 1999). Trill calling encodes cues on individuality and social status (Zimmermann and Lerch, 1993; Zimmermann, 1996) and attracts the attention of neigh- boring males. They seem to be a criterion for mate choice by females (Zimmermann, 1995). How much these calls are modified by sexual selection and act as a premating isolation mechanism is currently tested by field and laboratory playback experiments. Besides sexual selection, spatial proximity as assessed by population density may affect sound structure. Nothing is known to date about the social organization of rufous mouse lemurs. However, their population densities in tropical rain forests are usually much lower than in dry deciduous forests (Ganzhorn, 1988). If rufous mouse lemurs have a much lower population density than gray mouse lemurs, the lower competition among males and between sexes may favor a sexual advertisement call with a shorter duration that is less costly to produce than long trill calls are.

CONCLUSIONS

Predator-advertisement calls of two nocturnal sibling specific of Mala- gasy mouse lemurs display a high similarity in overall vocal pattern. Intra- specific variation in acoustic structure is high and overlaps interspecific variation, making species identification unreliable. Differences in transmission qualities of habitats—evergreen rain forest versus dry deciduous forest—seem to have a lesser impact on call structure than that of similar predation pressures. Sexual advertisement calls have clear species-specific differences in frequency and temporal pattern. Whereas rain forest-dwelling rufous mouse lemurs emit high-frequency slowly frequency-modulated short chirp calls, dry forest dwelling gray mouse lemurs produce high-frequency rapid and 850 Zimmermann, Vorobieva, Wrogemann, and Hafen multifrequency-modulated long trill calls. The species-specific characteristics may be explained by divergences in habitat structure and social needs. They may act as a premating isolating mechanism. Results suggest that rufous and gray mouse lemurs form distinct biological species, which can be noninvasively identified by their vocal fingerprint. They do not belong to the same metapopulation.

ACKNOWLEDGMENTS

We thank the Ministe´re des Eaux et Foreˆts de Madagascar and Mme. Prof. Berthe Rakotosamimanana, Department of Anthropology, University of Antananarivo, Madagascar and the Bundesamt fu¨ r Naturschutz, Bonn, for logistical support; Sandra Bunte, Mu¨ nchen, and Perris Flu¨ gge, Kirsti Falkenhagen and Brigitte Lohmeyer, Hannover, for their help in data re- cording; Prof. Kreienbrock and Dr. Rohn (Institute of Biometry, TiHo) for their help in applying and interpreting of the nested ANOVA; and Drs. Judith Masters (Natal Museum, South Africa), Ute Radespiel (Cardiff University, Great Britain) and Sabine Schmidt (Laboratory of Psychoacous- tics, TiHo) for critical discussions and two anonymous reviewers for further remarks and suggestions. This study was supported ﬁnancially by grants of the German Academic Exchange Council (DAAD-Forschungsstipendium to EW), the German Research Council (DFG AZ Wr 34/1-1) and Stiftung Volkswagen to EZ (Zusammenarbeit mit Entwicklungsla¨ndern). Prof. Kuhn, Institute of Anatomy, University of Go¨ ttingen, the Deutsches Pri- matenzentrum (DPZ) and the Tiera¨rztliche Hochschule Hannover (TiHo) provided the facilities for animal maintenance and breeding.

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