Anogenital gland secretions of Lemur catta and Propithecus verreauxi coquereli A preliminary chemical examination.код для вставкиСкачать
American Journal of Primatology 63:49–62 (2004) RESEARCH ARTICLE Anogenital Gland Secretions of Lemur catta and Propithecus verreauxi coquereli : A Preliminary Chemical Examination R.A. HAYES1n, T.L. MORELLI2, and P.C. WRIGHT3 1 School of Natural Resource Sciences, Queensland University of Technology, Brisbane, Australia 2 Department of Ecology and Evolution, State University of New York at Stony Brook, Stony Brook, New York 3 Department of Anthropology, State University of New York at Stony Brook, Stony Brook, New York Although prosimians are greatly olfaction-oriented, little is known about the specifics of how they use scent to communicate. In this preliminary study we attempted to delineate intra- and interspecific differences among the anogenital gland secretions of two lemur species (Lemur catta and Propithecus verreauxi coquereli) using gas chromatography-mass spectrometry (GC-MS). The results indicate that the two species are discernible through scent. Furthermore, we were able to identify reproductive status using this technique. The anogenital secretions of the different sexes in L. catta, though perhaps not P. v. coquereli, are chemically distinguishable. Given this information, it appears that at least some lemur species can use scent marks to determine species, sex, and reproductive status. Am J Primatol 63:49–62, 2004. r 2004 Wiley-Liss, Inc. Key words: lemur; scent marking; chemical fingerprint; olfaction; gas chromatography-mass spectrometry; prosimian primate INTRODUCTION Although olfactory communication is known to be important in many mammalian species, researchers rarely study its use, not to mention its evolution, in primates. Sensory faculties in general, with the exception of vision, have been largely overlooked in primate species [Epple, 1986]. Although most primates are believed to have a visual and auditory focus, prosimians, along with many New Contract grant sponsor: DUPC; Contract grant sponsor: Wenner Gren Foundation; Contract grant sponsor: Sokal/Slobodkin. n Correspondence to: R.A. Hayes, School of Natural Resource Sciences, Queensland University of Technology, Brisbane, QLD 4001 Australia. E-mail: email@example.com Received 3 March 2003; revision accepted 1 April 2004 DOI 10.1002/ajp.20038 Published online in Wiley InterScience (www.interscience.wiley.com). r 2004 Wiley-Liss, Inc. 50 / Hayes et al. World monkeys, have retained olfactory complexity in addition to developing other sensory modalities [Jolly, 1966]. Olfactory messages (particularly scent marks) offer a relatively prolonged signal for communicating dominance status, sexual condition, and territorial ownership [Epple, 1986]. An animal may use odor to advertise key information about itself, including its species, group, or family membership; long- or short-term breeding or social status; and sex [Albone, 1984]. In addition, the scent mark can potentially communicate genetic information for use in decisions regarding mate choice or nepotism [Jordan & Bruford, 1998; Penn & Potts, 1999; Zavazava & Eggert, 1997]. Previous investigations of olfaction in prosimians concentrated on the animals’ response to scent, or the role of tradition in scent placement in the wild, but did not attempt to explore the chemical nature of the scent itself, or to distinguish chemical differences between individuals [e.g., Epple et al., 1987; Evans, 1980; Harrington, 1974, 1977; Mertl-Millhollen, 2000; Price & Feistner, 1994; Ramsay & Giller, 1996; Vick & Conley, 1976]. With this preliminary study we begin to fill that gap by investigating olfactory communication in prosimians, using the ring-tailed lemur (Lemur catta) and Coquerel’s sifaka (Propithecus verreauxi coquereli) as models. By analyzing the chemistry of scent through coupled gas chromatography-mass spectrometry (GC-MS), we hope to learn more about the information contained in the scent mark, as well as to begin to discover how disparate life histories may affect the kind of information communicated. Since a comparable investigation has not been conducted in prosimians, this study also aims to test the predictive power of this methodology. Both of the species studied here are classified in the IUCN Red List of Threatened Species as ‘‘Vulnerable’’ (L. catta: VU A1c; P. v. coquereli: VU A2cd). Coquerel’s sifaka (Propithecus verreauxi coquereli) is a diurnal, primarily folivorous primate. The ring-tailed lemur (Lemur catta) is also diurnal, but it feeds mainly on fruit, is not quite as large as P. v. coquereli, and is much more terrestrial than other lemurs. Neither species displays significant sexual dimorphism in their appearance [Kappeler, 1990b], and both are considered to live in female-dominated groups [Jolly, 1966; Kubzdela et al., 1992; Pereira et al., 1990]. Both species live in multi-male, multi-female groups; however, P. v. coquereli has a smaller mean group size than L. catta [Richard et al., 2002]. The two taxa studied here are allopatric: Coquerel’s sifakas inhabit the forests of northwestern Madagascar, while ring-tailed lemurs are found in the forests and arid, open areas of southern and southwestern Madagascar. Social groups of both species have overlapping home ranges [Gould & Overdorff, 2002; Richard et al., 2001]. However, while ring-tailed lemur females in the wild at Berenty have been observed to aggressively defend their territory, with some groups having frequent intergroup encounters, intergroup encounters between sifakas appeared to be rare and nonconfrontational [Jolly, 1966; Kubzdela et al., 1992; Richard, 1974; Richard et al., 2002]. In another sifaka field site, where intertroop interactions were more common, these interactions consisted of threat displays, such as chasing and rushing at opponents, but no biting [Mertl-Millhollen, 1979]. Both species are strongly seasonal breeders [Rasmussen, 1985; Wright, 1999]. Both L. catta and P. verreauxi, like most prosimians, have specialized glands for scent-marking their surroundings. Ring-tailed lemur males have antebrachial organs, located on the flexor surface of each forearm near a keratinized spur, and brachial organs just above each clavicle. In addition, there are glands on the surface of the scrotum and perianal region [Montagna & Soonyun, 1962]. The spur is used to gouge the surface of a tree, to deposit scent. Often the male mixes the secretion from the antebrachial organ with that of the brachial organ, and Chemistry of Gland Secretions of Two Lemurs / 51 then scent-marks [Evans & Guy, 1968]. During ‘‘stinkfights,’’ males rub their tails with these two secretions and wave them at other males [Jolly, 1966; Mertl, 1976]. Both males and females scent-mark by rubbing the surface of their genital and/or anal region against a tree [Mertl, 1977]. Similarly, male sifakas possess specialized throat glands that they use to mark surrounding branches and tree trunks. Both males and females also mark anogenitally [Mertl-Millhollen, 1979]. Although little is known about the olfactory behavior of P. v. coquereli [Brockman & Whitten, 1996; Mertl-Millhollen, 1979], more data have been published concerning the other subject of this study, L. catta–especially with respect to the glands of the anogenital region. The relationship between the anogenital marking rate and social status in males is ambiguous: two studies reported finding no difference between high- and low-ranking males [Gould & Overdorff, 2002; Oda, 1999], whereas another study showed that higher-ranking males scent-mark more often than lower-ranking males [Ramsay & Giller, 1996]. However, the social status of females does not appear to have any effect on their rate of scent-marking [Kappeler, 1990a; Oda, 1999]. Moreover, Oda  found no significant effect of age (subadult vs. adult) on the marking rate for either sex. It is likely that the female L. catta anogenital mark conveys information about reproductive status, since the marking rate increases as females approach estrus, with a high peak during and after estrus [Jolly, 1972; Kappeler, 1990a, 1998]. In addition, male ring-tailed lemurs mark more frequently [Gould & Overdorff, 2002] and are more interested in female secretion in the breeding season than in the nonbreeding season [Dugmore et al., 1984]. On the other hand, the marks placed by males may be deposited for the information of other males, rather than females [Kappeler, 1990a, 1998; Oda, 1999]. However, the anogenital gland is used by both male (in addition to other glands) and female L. catta to mark the borders of group territories [Mertl-Millhollen, 1988, 2000]. Male ringtailed lemurs respond differently to scent marks from male and female donors [Dugmore et al., 1984; Ramsay & Giller, 1996], although there is no difference in the frequency with which males and females scent-mark with the anogenital region [Oda, 1999]. In an interesting study, Mertl  performed a series of habituation experiments on L. catta, in which a male lemur was exposed to the scent of another lemur’s glandular secretion until it became habituated to the new animal. The male was then exposed to the secretion of a new animal, and any difference in response between the two scents was measured. These experiments showed that male L. catta can distinguish between the secretions of different individuals. If olfaction, and thus scent-marking behavior, is an important part of a species’ behavior and ecology, what is it about this secretion that functions to communicate so much information? What are the components of the secretion, and how do they vary among individuals? Does each individual possess a unique ‘‘chemical fingerprint’’ that allows the animals to distinguish between the glandular secretions? Furthermore, what, if any, are the interspecific differences of the secretion? MATERIALS AND METHODS Samples were collected in February 2002 from two species of lemur that were held in captivity at the Duke University Primate Center (DUPC), Durham, NC (Table I). All of the animals were captive-born, with the exception of the oldest 52 / Hayes et al. TABLE I. Details of the Species, Sex and Age of Each of the Animals Sampled Name Species Sex Cleis Cleomenis Dorieus Dory Katina Hector Licinius Selacius Teres Thrasun Alexianus Antonia Drusilla Livia II Paulina Brutus Nero Philip Trajan Zeno Lemur catta Lemur catta Lemur catta Lemur catta Lemur catta Lemur catta Lemur catta Lemur catta Lemur catta Lemur catta Propithecus verreauxi Propithecus verreauxi Propithecus verreauxi Propithecus verreauxi Propithecus verreauxi Propithecus verreauxi Propithecus verreauxi Propithecus verreauxi Propithecus verreauxi Propithecus verreauxi F F F F F M M M M M F F F F F M M M M M coquereli coquereli coquereli coquereli coquereli coquereli coquereli coquereli coquereli coquereli Age 17 15 2 13 15 13 9 17 7 6 5 4 9 5 11 1 7 3 22 2 P. v. coquereli, Trajan. The L. catta at DUPC, including the test subjects, were allowed to range in semi-free-range enclosures (9.1 ha) during the warmer months (approximately April–October). During the winter they were housed in indoor/outdoor enclosures with other L. catta, separated from the P. v. coquereli. The P. v. coquereli were housed in open-air enclosures that still admitted natural light when the animals were closed in for the winter months. The L. catta were fed vegetables and commercial primate pellets (Lab Diet Product 5038 Monkey Diet; PMI Feeds Inc., Purina, Richmond, IN), whereas the P. v. coquereli were fed vegetables, nuts, leaves, and a different type of commercial primate pellet (Leaf Eater Primate Diet Mini-Biscuit #5672, Mazuri, Richmond, IN). The analysis included sexually mature and immature animals of both species. Although the age of sexual maturity is variable, it is considered to be 2 years for L. catta and 3–5 years for P. v. coquereli (Hess, personal communication) [Richard et al., 2001]. In analyses of skin gland secretion in mammals, purity is important, volumes are generally small, and high volatility is common. Given these concerns, we chose to use gas-chromatography-mass spectrometry, since it is considered by many researchers in the field to be the most practical analytical method [e.g., Buesching et al., 2002a, b; Gassett et al., 1997; Hayes et al., 2001, 2002a, b]. Duplicate swabs were taken of the anogenital region of 10 individuals of each species (five males and five females). Swabs were also collected from the other scent glands of the male, the results of which are discussed elsewhere [Hayes et al., in press]. We collected secretions by rubbing the area of the scent gland of a hand-captured lemur with 1 cm 4 cm glass filter paper [Salamon, 1994]. The swabs were then sealed in an airtight vial and stored in the laboratory at a reduced temperature (approximately –751C). The samples were air-freighted at ambient temperature to Australia, and then immediately returned to –751C before they were analyzed at the University of Western Sydney. Chemistry of Gland Secretions of Two Lemurs / 53 In the laboratory, the vial containing the swab was opened and the filter paper was inserted into the liner of the cooled injector port (451C) of a gas chromatograph (GC; 5890 Series II; Hewlett Packard) coupled to a mass spectrometer (MS; model 5971A; Hewlett Packard) and fitted with a silica capillary column (model DB5-HT, no. 122-5731; J&W; 30 m long 0.25 mm ID 0.1 mm film thickness). We cryofocused the sample onto the front of the GC column by maintaining the column at low temperature (21C) and ramping up the injector to 1501C at 271C/min. Data were acquired under the following GC conditions: inlet temperature: 1501C; carrier gas: helium at 1.1 mL/min; split ratio = 1:2; detector temperature = 2801C; temperature program: initial temperature = 21C; initial time = 4 min; rate = 201C/min to 501C and then 51C/min; final temperature = 2501C; final time = 2 min. The MS was held at 1901C in the ion source with an ionization energy of 70 eV and a scan rate of 0.9 scans/sec. The two swabs collected from each animal were analyzed and compared. Tentative identities were assigned to peaks with respect to the Wiley mass spectral library. Mass spectra of peaks from different samples with the same retention time were compared to ensure that the compounds were indeed the same. An arbitrary threshold of peak size of an abundance of 100,000 relative to background was used to define the presence or absence of a peak. We used this figure in an attempt to reduce the number of analyzed peaks to a manageable level without excluding important components. We statistically assessed the presence of peaks in the chromatograms, and their relative areas by nonparametric methods (Bray-Curtis cluster analysis and multidimensional scaling (MDS) ordination) to ascertain whether any differences could be detected between the animals. It can be difficult to analyze chromatographic data statistically, because they typically show high levels of variation and very right-skewed distributions. In this way they are similar to ecological data regarding species composition [Clarke & Warwick, 1994a, b]. The use of informal display methods, such as clustering and MDS, may be a more valid approach for such data compared to other commonly used techniques, such as principal components analysis (PCA) [Hayes et al., 2002b]. MDS works on a sample dissimilarity matrix rather than the original data array, makes fewer assumptions about the nature and quality of the data, and does not assume normality of the data [Clarke & Warwick, 1994a, b]. MDS uses the dissimilarity matrix of the data to produce a graphical representation. Each point in the MDS represents an individual lemur, and points that are close together (clumped) correspond to individuals with similar peak composition (presence and abundance). Since they represent relative differences between samples, the axes of an MDS plot are dimensionless. MDS has been used successfully in previous studies to analyze chromatographic data [Hayes et al., 2002a, b, 2003]. To determine whether clusters of individual lemurs were significantly different from each other, we used an analysis of similarity (ANOSIM). The ANOSIM tests are a range of Mantel-type permutations of randomization procedures, which make no distributional assumptions. These tests depend only upon rank similarities, and thus are appropriate for this type of data. We used a similarity percentages (SIMPER) analysis to determine which peaks were the most important in contributing to any differences between groups, and to assess similarity between individuals within each group. The software used for the multivariate analysis was Primer 5 for Windows (V 5.2.4, 2001). 54 / Hayes et al. RESULTS Several compounds found in the secretion of the lemurs were identified by MS (Table II). These compounds were primarily identified as straight and branched long-chain hydrocarbons with alcohol and aldehyde components. In addition, several of the components were oxygenated compounds, particularly esters. The percentage of individuals in each group from which the component was identified is shown in Table II. It should be noted that there is a contaminant peak at approximately 51 min that appears in all chromatograms, including blank samples of paper without secretion. This peak acted as an internal standard, and was thus invaluable in allowing comparisons of retention times to be made between chromatograms. The chromatograms produced by the two species of lemurs are distinctly different from each other. Swabs from the anogenital region of L. catta demonstrate many more peaks compared to those from P. v. coquereli (Figs. 1 and 2). In addition to looking different, the samples from the two species are statistically distinguishable (ANOSIM: global R = 0.492, P = 0.001); in other words, it is possible to determine from which species an unknown sample was collected. In the case of L. catta, it is also possible to determine the sex of the animal from which a swab was obtained (ANOSIM: R = 0.248, P = 0.032). In contrast, however, swabs from male and female P. v. coquereli are statistically indistinguishable (ANOSIM: R = 0.14, P = 0.135). There is more variability in the P. v. coquereli samples compared to the L. catta samples, with larger distances between individuals. This larger variability may account for the lack of statistically significant difference between the sexes in P. v. coquereli. The SIMPER analysis shown in Table III is a measure of the similarities and differences of samples within a defined grouping, in this case based on species and sex. One can see from a comparison of the within-group similarities (Table IIIa) that samples from P. v. coquereli females are much less similar to each other than are samples from the other three groups. In addition, one can see that the average dissimilarity between all of the groupings is very high (Table IIIb), which suggests that each group is distinct from the others, as described above. The MDS output is shown in Fig. 3, which gives a visual representation of the data described by the ANOSIM (above). Each point on the figure represents an individual lemur. Points that are close together are more similar, and those farther away are more different. One can see that individuals from each of the two species clump together, that male and female L. catta are different from each other, and that the sexes of P. v. coquereli are not well differentiated. DISCUSSION The objective of this pilot study was to investigate the use of GC-MS to reveal specific information encoded in prosimian scent marks, and to compare this information intra- and interspecifically. We collected swabs from the surface of the skin directly overlying scent glands in the anogenital region of manually restrained lemurs, and equated this with the scent mark. This is most likely an oversimplification of a natural scent mark, which may be chemically more complex than that found on a collected swab. For example, when a scent mark is placed by a free-ranging animal, the glandular secretion may be mixed with materials from other sources, such as urine. Although the results presented here should be interpreted with caution, it is likely that the observed patterns in the collected anogenital gland secretions are important and worthy of note. Chemistry of Gland Secretions of Two Lemurs / 55 TABLE II. Tentative Identity of Compounds Identified From Swabs of the Glandular Regions of the Two Species of Lemurn Name Cyclooctane 1-dodecanol Methyl-cyclodecane (Z)-2-decene 4-decene 1-butyl-1-methyl2-propyl-cyclopropane 6-tridecene Hydrocarbon Cyclododecane 3-methyl-6-octen-1-ol Hydrocarbon Long chain alcohol 1-tridecene 1-pentadecanol 1-hexadecanol Long chain alcohol Long chain alcohol 1-heptadecanol (Z)-7-hexadecene Long chain ester 1-octadecanol Long chain alcohol Long chain alcohol Long chain alcohol Hydrocarbon Long chain ester Long chain alkene Hydrocarbon (E)-b-farnesene Long chain ester Long chain ester Long chain ester Octanoic acid, hexadecyl ester Hydrocarbon Decanoic acid, decyl ester Long chain ester Long chain ester 1-hexadecanal, acetate Long chain alcohol Long chain alcohol Long chain alcohol Long chain alcohol Long chain alcohol Long chain alcohol Long chain alcohol L. catta, f. anogenital L. catta, m. anogenital P. v. coquereli, f. anogenital P. v. coquereli, m. anogenital 0 60 60 60 80 80 0 60 60 60 60 60 20 0 0 0 40 0 60 0 0 0 100 20 80 100 100 100 100 80 80 80 80 100 100 100 60 100 100 80 100 40 60 60 80 80 80 80 100 60 80 60 60 60 60 60 60 60 60 60 60 60 20 40 40 60 20 40 40 40 40 60 60 40 40 40 80 40 0 0 40 0 0 0 0 0 0 20 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 80 0 0 0 60 0 20 80 80 0 80 60 0 80 80 0 0 0 0 0 0 80 0 0 0 60 20 60 20 40 40 40 40 40 20 40 40 60 80 60 80 80 100 100 100 80 40 80 80 40 0 0 0 20 40 0 20 0 60 0 0 80 0 0 0 60 20 20 20 20 20 20 20 n The number indicates the percentage of individuals sampled in which the given compound was identified in retention time order. 56 / Hayes et al. L. catta female (Cleomenis) - anogenital gland 7500000 7000000 6500000 6000000 Abundance 5500000 5000000 4500000 4000000 3500000 3000000 2500000 2000000 1500000 1000000 500000 0 10.00 20.00 30.00 (a) 40.00 50.00 60.00 Time (min) L. catta male (Thrason) - anogenital gland 6000000 5500000 5000000 Abundance 4500000 4000000 3500000 3000000 2500000 2000000 1500000 1000000 500000 0 10.00 (b) 20.00 30.00 40.00 50.00 60.00 Time (min) Fig. 1. Chromatograms produced by swabs from the anogenital region of L. catta. A typical female swab is shown on top (a), and a typical male swab is shown below (b). These two animals were selected at random. The peak at approximately 51 min is a contaminant that appears in all of the chromatograms, including blanks. The collected anogenital gland secretions appear to reveal specifics regarding species, sex, age, and reproductive status. Other information indicating group and family membership may also be communicated, but this is ambiguous because of the small sample size. The observed differences between the anogenital gland secretions of the two study species may reflect how these scent marks are used in the wild. Behavioral observations suggest that these scent marks are used to mark the boundaries of the territory defended by the social group. Since the major use of these secretions may be for intraspecific communication [Kappeler, 1998], an animal should be able to distinguish a mark placed by a member of its own species from a less relevant mark derived from a different species. Where the Chemistry of Gland Secretions of Two Lemurs / 57 Abundance P. v. coquereli female (Drusilla) - anogenital gland 3.4e+07 3.2e+07 3e+07 2.8e+07 2.6e+07 2.4e+07 2.2e+07 2e+07 1.8e+07 1.6e+07 1.4e+07 1.2e+07 1e+07 8000000 6000000 4000000 2000000 0 10.00 20.00 30.00 (a) 40.00 50.00 60.00 Time (min) P. v. coquereli male (Zeno) - anogenital gland 4500000 4000000 Abundance 3500000 3000000 2500000 2000000 1500000 1000000 500000 0 10.00 20.00 30.00 (b) 40.00 50.00 60.00 50.00 60.00 Time (min) P. v. coquereli male (Nero) - anogenital gland 6e+07 5.5e+07 5e+07 Abundance 4.5e+07 4e+07 3.5e+07 3e+07 2.5e+07 2e+07 1.5e+07 1e+07 5000000 0 10.00 (c) 20.00 30.00 40.00 Time (min) Fig. 2. Chromatograms produced by swabs from the anogenital region of P. v. coquereli. A typical female swab selected at random is shown at the top (a), with two different types of male swab below (b and c). The peak at approximately 51 min is a contaminant that appears in all of the chromatograms, including blanks. 58 / Hayes et al. TABLE III. Results of the SIMPER Analysis, a Measure of the Average Similarity/ Dissimilarities Between the Anogenital Swabs From Lemursn Group/s Similarity/dissimilarity (%) a. Similarity within group L. catta female L. catta male P. v. coquereli female P. v. coquereli male 49.6 34.5 5.7 25.8 b. Dissimilarity between groups L. catta female/L. catta male L. catta female/P. v. coquereli female L. catta female/P. v. coquereli male L. catta male/P. v. coquereli female L. catta male/P. v. coquereli male P. v. coquereli female/P. v. coquereli male 69.0 92.5 77.5 92.6 83.3 87.7 n Comparisons are made with samples grouped according to species and sex. The results shown are: a) the average percentage similarity within groups, and b) the average percentage dissimilarity of samples between groups. One can see that the swabs from the P. v. coquereli females are much less similar than are the swabs from the other groups. It can also be seen that the average percentage dissimilarity between groupings is very high, demonstrating that the groups are discrete. Stress: 0.12 Fig. 3. Two-dimensional MDS ordination of the 20 lemur anogenital samples. The plot is based on square root-transformed abundances and a Bray-Curtis similarity matrix. Note that the two species are separate, and the sexes are separate for L. catta. SymbolsFm: L. catta females (n = 5); n: L. catta males (n = 5); ’: P. v. coquereli females (n = 5); &: P. v. coquereli males (n = 5). distribution of L. catta overlaps with that of another P. verreauxi subspecies (P. verreauxi verreauxi), although there is some vertical zonation of marking location, the vertical distribution of marks overlap extensively [Millhollen, 1986]. Chemistry of Gland Secretions of Two Lemurs / 59 In addition to being used to mark the boundaries of social group territory (an action performed by all individuals in the group [Mertl-Millhollen, 1988, 2000]), the secretions of the anogenital gland are used to determine sexual condition in female L. catta [Jolly, 1972; Kappeler, 1990a, 1998]. The results from the present study indicate that L. catta scent marks may communicate detailed information to the recipient, including the sex of the marker. However, male and female sifaka anogenital gland secretions were not distinguishable by our tests. There are several possible explanations for this. P.v. coquereli may not use scent marks in the same way as the ring-tailed lemurs. On the other hand, it may be that sex is encoded by some information other than that found in the volatile chemicals of the anogenital mark. For example, the secretion from other scent glands (such as the throat gland in the P. v. coquereli males) may be used in contexts in which sex differentiation is important, since they are not present in females [Hayes et al., in press]. We will soon be conducting habituation tests and behavioral bioassays to determine whether P. v. coquereli can in practice differentiate between the scent marks of males and females from the different glands [after Mertl, 1975; Epple, 1986]. There are some complicating factors that arise when one attempts to interpret the results of this preliminary study. The two species were fed similar, but not identical, diets. The animals were also housed differently. Although the housing at the time of the study was very similar, L. catta may exhibit long-term effects from their semi-free-range allowances during the warmer months. This, rather than genetic causes, may explain the differences observed between the two species. However, it would not explain the differences observed between individuals or between the sexes within species, and thus we suspect that these interspecific differences are real. The handling procedures were stressful for most of the animals; however, since there was only a short period of time between the capture of the animal and the collection of the secretion, it is difficult to imagine how handling could have affected the results. In addition, because of the comparative nature of the study, it is likely that stress had little effect on the results. Similarly, although manual expression of the secretion may have affected the results [Belcher et al., 1988], such manipulation does not easily explain the observed differences, since both species were treated similarly. The shipping process may have caused a decrease in variation through a potential loss of some of the more volatile constituents from the airtight vials in which the samples were transported, or as a result of bacterial activity. Consequently, samples that were actually different may have become indistinguishable. However, since differences were still apparent between samples, the conclusions drawn in the current study are likely to be correct, or to err on the conservative side. A similar issue is that concerning loss of information throughout the collection and analysis. It may be that although the animals themselves are able to gain information from the anogenital gland secretions, the analytical technique employed in this study may not be sensitive enough to detect the subtle nuances that are used as cues. As in all such studies, one cannot assume that absence in the results means absence in nature. Although it was originally believed that semiochemical messages could only be composed of highly volatile compounds, this is no longer thought to be the case. Research has shown that proteins are vital components of the scent-mark secretions of a variety of organisms, including the golden hamster (Mesocricetus auratus) [Singer et al., 1986], meadow vole (Microtus pennsylvanicus) [Ferkin et al., 1997], cotton-top tamarin (Saguinus fuscicolis) [Belcher et al., 1990], and 60 / Hayes et al. house mouse (Mus domesticus) [Hurst et al., 1998]. It is possible that there are other important components to the scent mark, such as high-molecular-weight, nonvolatile compounds (e.g., proteins) that are not detected by GC-MS, and thus were not examined in this study. Given that prosimians possess a functioning vomeronasal system, it may be worthwhile in future studies to examine these compounds to determine whether there is other information that is not being captured by the present analytical technique. However, using GC-MS to analyze the information found in the glandular secretions of prosimians appears to be a meaningful and efficient way to better understand this important sensory modality. This research demonstrates that the lemur anogenital gland secretions encode information on sex, reproductive status, and individuality, as well as species. The methodological complications in this work may cause differences to be underestimated; however, the evidence of categorical distinctions indicates that important information can be gleaned from the technique used. Forthcoming habituation experiments and behavioral bioassays, such as those described above, will help to clarify the context in which these scent marks are used by these species, and the importance of the differences between these and other lemur species. It is hoped they will also lead to a better understanding of the evolution of olfactory communication in prosimians. ACKNOWLEDGMENTS The authors thank Associate Professor S. Grant Wylie and Dr. Sonia Claus for assistance with technical aspects of this paper, and Bill Hess and David Haring for their advice and help with the sample collections. This research was carried out under SUNY Stony Brook University IACUC registry no. A026-02-01, and Duke University IACUC registry no. A026-02-01. The samples were imported into Australia under AQIS permit no. 200205460. This research was supported in part by a grant from the Wenner Gren Foundation to P.C.W., and a Sokal/Slobodkin travel award to T.L.M. This is DUPC publication #767. REFERENCES Albone ES. 1984. Mammalian semiochemistry: the investigation of chemical signals between mammals. New York: Wiley. p 372. Belcher AM, Epple G, Kuderling I, Smith III AB. 1988. Volatile components of scent material from cotton-top tamarin (Saguinus o. oedipus): a chemical and behavioral study. J Chem Ecol 14:1367–1384. Belcher AM, Epple G, Greenfield KL, Richards LE, Kuderling I, Smith III AB. 1990. Proteins: biologically relevant components of the scent marks of a primate (Saguinus fuscicollis). Chem Senses 15:432–446. Brockman DK, Whitten PL. 1996. Reproduction in free-ranging Propithecus verreauxi– estrus and the relationship between multiple partner matings and fertilization. Am J Phys Anthropol 100:57–69. Buesching CD, Waterhouse JS, MacDonald DW. 2002a. Gas-chromatographic analyses of the subcaudal gland secretion of the European badger (Meles meles). Part I: chemical differences related to individual parameters. J Chem Ecol 28:41–56. Buesching CD, Waterhouse JS, MacDonald DW. 2002b. Gas-chromatographic analyses of the subcaudal gland secretion of the European badger (Meles meles). Part II: time-related variation in the individualspecific composition. J Chem Ecol 28:57–69. Clarke KR, Warwick RM. 1994a. Similaritybased testing for community patterns: the two-way layout with no replication. Marine Biol 118:167–176. Clarke KR, Warwick RM. 1994b. Changes in marine communities: an approach to statistical analysis and interpretation. Plymouth: Plymouth Marine Laboratory. p 144. Dugmore SJ, Bailey K, Evans CS. 1984. Discrimination by male ring-tailed lemurs Chemistry of Gland Secretions of Two Lemurs / 61 (Lemur catta) between the scent marks of males and those of female conspecifics. Int J Primatol 5:235–245. Epple G. 1986. Communication by chemical signals. In: Mitchell G, Erwin J, editors. Comparative primate biology. New York: A.R. Liss. p 531–580. Epple G, Alveario MC, Belcher AM, Smith III AB. 1987. Species and subspecies specificity in urine and scent marks of saddle-back tamarins (Saguinus fuscicollis). Int J Primatol 8:663–681. Evans CS, Guy RW. 1968. Social behaviour and reproductive cycles in captive ringtailed lemurs (Lemur catta). J Zool Lond 156:181. Evans CS. 1980. Diosmic response to scentsignals in Lemur catta. In: MüllerSchwarze D, Silverstein RM, editors. Chemical signals. New York: Plenum Press. p 417–420. Ferkin MH, Sorokin ES, Johnston RE, Lee CJ. 1997. Attractiveness of scent varies with protein content of the diet of meadow voles. Anim Behav 53:133–141. Gassett JW, Wiesler DP, Baker AG, Osborn DA, Miller KV, Marchinton RL, Novotny M. 1997. Volatile compounds from the forehead region of male white-tailed deer (Odocoileus virginianus). J Chem Ecol 23:569–578. Gould L, Overdorff DJ. 2002. Adult male scent-marking in Lemur catta and Eulemur fulvus rufus. Int J Primatol 23:575–586. Harrington JE. 1974. Olfactory communication in Lemur fulvus. In: Martin RD, Doyle GA, Walker AC, editors. Prosimian biology. London: Duckworth. p 331–346. Harrington JE. 1977. Discrimination between males and females by scent in Lemur fulvus. Anim Behav 25:147–151. Hayes RA, Richardson BJ, Wyllie SG. 2001. Increased social dominance in male rabbits, Oryctolagus cuniculus, is associated with increased secretion of 2-phenoxyethanol from the chin gland. In: Marchlewska-Koj A, Lepri JJ, Müller-Schwarze D, editors. Chemical signals in vertebrates. Vol. IX. New York: Kluwer Academic/Plenum Publishers. p 335–342. Hayes RA, Richardson BJ, Wyllie SG. 2002a. Semiochemicals and social signaling in the wild European rabbit in Australia. I. Scent profiles of chin gland secretion from the field. J Chem Ecol 28:363–384. Hayes RA, Richardson BJ, Claus SC, Wyllie SG. 2002b. Semiochemicals and social signaling in the wild European rabbit in Australia. II. Variations in the chemical composition of the chin gland secretion across sampling sites. J Chem Ecol 28: 2613–2625. Hayes RA, Richardson BJ, Wyllie SG. 2003. To fix or not to fix. The role of 2– phenoxyethanol in rabbit (Oryctolagus cuniculus) chin gland secretion. J Chem Ecol 29:1051–1064. Hayes RA, Morelli T-L, Wright PC. The chemistry of scent marking in two lemurs: Lemur catta and Propithecus verreauxi coquereli. In: Mason RT, LeMaster MP, Müller-Schwarze D, editors. Chemical signals in verebrates. Vol. X. New York: Kluwer/Plenum/Academic Press (in press). Hurst JL, Robertson DHL, Tolladay U, Beynon RJ. 1998. Proteins in urine scent marks of male house mice extend longevity of olfactory signals. Anim Behav 55: 1289–1297. Jolly A. 1966. Lemur behavior: a Madagascar field study. Chicago: University of Chicago Press. p 187. Jolly A. 1972. Troop continuity and troop spacing in Propithecus verreauxi and Lemur catta at Berenty (Madagascar). Folia Primatol 17:335–362. Jordan WC, Bruford MW. 1998. New perspectives on mate choice and the MHC. Heredity 81:239–245. Kappeler PM. 1990a. Social status and scent marking behaviour in Lemur catta. Anim Behav 40:774–776. Kappeler PM. 1990b. The evolution of sexual size dimorphism in prosimian primates. Am J Primatol 21:201–214. Kappeler PM. 1998. To whom it may concern: the transmission and function of chemical signals in Lemur catta. Behav Ecol Sociobiol 42:411–421. Kubzdela KS, Richard AF, Pereira ME. 1992. Social relations in semi-free-ranging sifakas (Propithecus verreauxi coquereli) and the question of female dominance. Am J Primatol 28:139–145. Mertl AS. 1975. Discrimination of individuals by scent in a primate. Behav Biol 14: 505–509. Mertl AS. 1976. Olfactory and visual cues in social interactions of Lemur catta. Folia Primatol 26:151–161. Mertl AS. 1977. Habituation to territorial scent marks in the field by Lemur catta. Behav Biol 21:500–507. Mertl-Millhollen AS. 1979. Olfactory demarcation of territorial boundaries by a primate–Propithecus verreauxi. Folia Primatol 32:35–42. Mertl-Millhollen AS. 1988. Olfactory discrimination of territorial but not range boundaries by Lemur catta. Folia Primatol 50:175–187. Mertl-Millhollen AS. 2000. Tradition in Lemur catta. Behavior at Berenty Reserve, Madagascar. Int J Primatol 21:287–298. 62 / Hayes et al. Millhollen AS. 1986. Territorial scent marking by two sympatric lemur species. In: Duvall D, Müller-Schwarze D, Silverstein RM, editors. Chemical signals in vertebrates 4th. New York. Plenum Press. p 647–652. Montagna W, Soonyun J. 1962. Skin of primates 10. Skin of ring-tailed lemur (Lemur catta). Am J Phys Anthropol 20:95. Oda R. 1999. Scent marking and contact call production in ring-tailed lemurs (Lemur catta). Folia Primatol 70:121–124. Penn DJ, Potts WK. 1999. The evolution of mating preferences and major histocompatibility complex genes. Am Nat 153: 145–164. Pereira ME, Kaufman R, Kappeler PM, Overdorff DJ. 1990. Female dominance does not characterize all of the Lemuridae. Folia Primatol 55:96–103. Price EC, Feistner TC. 1994. Responses of captive aye-ayes (Daubentonia madagascarensis) to the scent of conspecifics: a preliminary investigation. Folia Primatol 62:170–174. Ramsay NF, Giller PS. 1996. Scent-marking in ring-tailed lemurs: responses to the introduction of ‘‘foreign’’ scent in the home range. Primates 37:13–23. Rasmussen DT. 1985. A comparative study of breeding seasonality and litter size in eleven taxa of captive lemurs (Lemur and Varecia). Int J Primatol 6:501–511. Richard AF. 1974. Patterns of mating in Propithecus verreauxi. In: Martin RD, Walker AC, Doyle G, editors. Prosimian biology. London: Duckworth. p 49–74. Richard AF, Dewar RE, Schwatrz M, Ratsirason J. 2001. Mass change, environmental variability and female fertility in wild Propithecus verreauxi. J Hum Evol 39: 381–391. Richard AF, Dewar RE, Schwatrz M, Ratsirason J. 2002. Life in the slow lane? Demography and life histories of male and female sifaka (Propithecus verreauxi verreauxi). J Zool 256:421–436. Salamon M. 1994. Seasonal, sexual and dietary induced variations in the sternal scent secretion in the brushtail possum (Trichosurus vulpeculai). In: Apfelbach R, MüllerSchwarze D, Reutier K, Weiber E, editors. Chemical signals in vertebrates. Vol. VII. Oxford: Pergamon Press. p 211–222. Singer AG, Macrides F, Clancy AN, Agosta WC. 1986. Purification and analysis of a proteinaceaous aphrodisiac pheromone from hamster vaginal discharge. J Biol Chem 261:13323–13326. Vick LG, Conley JM. 1976. An ethogram for Lemur fulvus. Primates 17:125–144. Wright PC. 1999. Lemur traits and Madagascar ecology: coping with an island environment. Yearb Phys Anthropol 42:31–72. Zavazava N, Eggert F. 1997. MHC and behavior. Immunol Today 18:8–10.