This document is a thesis submitted by Maryna Jordaan investigating whether male impala use dung for olfactory communication. The study aimed to determine if there were differences in the volatile organic compounds emitted from the dung of territorial versus non-territorial male impala. Gas chromatography-mass spectrometry analysis identified 52 volatile compounds from dung samples. Statistical analysis found no significant difference in the scent profiles between territorial and non-territorial males, suggesting dung may be used to communicate age rather than territorial status. Observations of female behavior also indicated olfactory communication via dung could be used for female-male communication.

1. 1
Do male impala use dung for olfactory communication?
by
Maryna Jordaan
Submitted in partial fulfilment of requirements for the degree of Bachelor of Science
(Honours)
In the
School of life sciences
College of Agriculture, Engineering and Science
University of KwaZulu-Natal, Pietermaritzburg
November 2014
Supervisor: Dr Shrader & Dr Jürgens

2. 2
Do male impala use dung for olfactory communication?
M. Jordaan, 211520647
School of Life Sciences, University of KwaZulu-Natal, Private Bag x01, Scottsville, 3209
Formatted according to the South African Journal of Botany
Some mammals communicate using olfactory signals. A key advantage of this is that
olfactory cues can persist for long periods of time, even in the absence of the sender.
Mammals may send signals through specialised glands or through excretory products such as
urine and faeces. Some species even defecate communally in middens, which then
concentrates olfactory information spatially. Impala are one such species. Males, especially
territorial males, tend to use middens, however, it is unknown if olfactory information is
transferred through the scent of their dung. One possibility is that they may advertise their
territorial status. To investigate this, I 1) identified the chemical compounds emitted from the
dung of adult male impala, and 2) determined if there was any difference in the volatile
compounds given off by the dung of territorial (i.e. males with female harems) and similar
aged bachelor-herd males. I hypothesised that there would be differences between the scent
profiles of territorial and non-territorial male impala. If information on reproductive status is
transmitted in dung, then I predicted that the dung of territorial males would have different
volatile chemicals or proportions in their dung scent, compared to similarly aged males in
bachelor herds. In contrast, if age of the individual determined the volatiles being given off,
then there should be no difference between the overall scent profiles from these similar-aged
males despite their differing status. I found no significant difference in the scent profiles
between territorial and non-territorial males. This suggests that dung may be used to
communicate age between males, because I only sampled similar aged males. Moreover,
during the field work, I observed female impala defecating and urinating in middens and
territorial males displaying a Flehmen response. These observations suggest that olfactory
communication via dung may also be used for female-male communication where females
may use volatile organic compounds to indicate their reproductive status.
Key words: territoriality, impala, volatile organic compounds, olfactory communication

3. 3
Introduction
Animals can send and receive information visually, acoustically and/or olfactorally (Seyfarth
and Cheney, 2003). For example, birds have elaborate calls and colourful feathers to attract
mates, while some frogs use bright colours to warn predators of their toxicity (Darst et al.,
2006; Orians, 1969). In contrast, olfactory communication is the process whereby a chemical
signal is produced by a sender and transmitted to the receiver (Eisenberg and Kleinman,
1972). A benefit of this form of communication is that chemical signals can persist in the
environment even in the absence of the sender (Eisenberg and Kleinman, 1972). However, as
chemical scent naturally diffuses into the environment, it is greatly affected by wind speed
and direction and thus generally cannot be directed (Bossert and Wilson, 1963; Eisenberg and
Kleinman, 1972).
Pheromones and general odourants are the two main classes of olfactory stimuli
(Touhara and Vosshall, 2009). Pheromones are molecules that are released from individuals
for intraspecies communication. In contrast, general odourants are volatile chemical
compounds with a molecular weight less than 300 (Touhara and Vosshall, 2009). They can be
found anywhere in the environment, including the by-products of metabolism or excreted
waste products (Touhara and Vosshall, 2009). Such volatile organic compounds (VOCs) may
influence a wide array of vertebrate behaviours, such as feeding site selection, stress and
alarm, species recognition and selection, copulatory behaviour, detection of oestrus, gender
identification and individual recognition and preference (Doty, 1986; Lledo et al., 2005;
Miller et al., 2003). Animals can obtain information about individuals just by smelling
particular odours from scent (Rich and Hurst, 1998). For example, information can be
acquired about an individual’s position in the social hierarchy, age and diet (Blaustein, 1981;
Rich and Hurst, 1998).
Olfactory communication can occur through specialised materials from scent glands
or excreted waste products (Grau, 1976). Using waste products is a key form of olfactory
communication in many ungulates and is common in demarcating territories (Grau, 1976).
Ungulates that provide specific olfactory information via dung are often found to have
localised defecation sites (i.e. middens) (Apio et al., 2006). Within these middens, species
may deposit dung alone, add additional scent from the pre-orbital scent gland and/or urinate
(Jarmen, 1974). The frequency in which individuals use these middens is what makes them
effective markers of territorial boundaries (Jarmen, 1974). Moreover, by defecating
communally, olfactory signals are concentrated spatially across the landscape.

4. 4
Many artiodactyl species have a ritualized performance when they urinate or defecate
(Grau, 1976). These behaviours have been documented for Grant’s gazelle (Nanger granti) ,
springbok (Antidorcas marsupialis) and impala (Aepyceros melampus) (Coblentz, 1976).
Middens are used more often by female bushbuck than males (Wronski et al., 2006). This
suggests that females may use these defecation sites to communicate to males when they are
in oestrus (Wronski et al., 2006). In a recent study conducted on the white rhino
(Ceratotherium simum), specific chemicals within the scent of dung identified age, sex, the
territorial status of males and the reproductive status of females (Marneweck, 2013). This
suggests that for white rhinos, middens may act as a sign post from which to pass on
information.
Impala are territorial throughout the year (Jarmen, 1974). Territorial males maintain
breeding herds that are separate to the bachelor herds (Zaloumis, 2005). The territories of a
breeding herds may overlap with territories of other breeding herds, but at the end of the
breeding season (March to June), these herds are no longer kept separate by the males (Stuart
and Stuart, 1997). Within bachelor herds, males organise themselves into a hierarchy, such
that the most dominant males will ultimately challenge a territorial male for his breeding herd
(Jarmen, 1974). Essentially, the social status of an individual determines whether or not they
will be able to breed (Bramley and Neaves, 1972). Social status and reproductive activity are
positively correlated, therefore a territorial male will be more reproductively successful
(Bramley and Neaves, 1972).
Territorial male impalas may urinate and defecate in response to the scent of another
male (Jarman, 1979). Territorial males may also defecate after they have been challenged by
a subordinate male to breed with the female herd that the territorial male guards (Jarman,
1979). For impala, middens are unlikely to act as visual cues, as they cannot be seen from a
distance (personal observation). In contrast, the scent of a midden appears to be enough to
stimulate a male into defecating and urinating (Jarman, 1979). In order to increase chemical
information, males may also rub their scent glands in the area surrounding the midden
(Jarman, 1979). Ultimately, the combination of these olfactory cues may pass on a large
amount of information (Jarman, 1979).
Despite observations of male impala using middens, there are no data on what types
of information they may be transmitting. Moreover, it is uncertain whether information is
even transmitted via dung. As only males have been observed to defecate in middens, the
broad aim of this study was to determine if impala males use dung to communicate
information to other males. I hypothesised that territorial males would have a different

5. 5
chemical composition in their dung scent compared to non-territorial males. To do this, I
identified the chemical compounds found in the dung of similar-aged male impalas that
differed in territorial status (i.e. territorial and non-territorial bachelor males). If territorial
status (i.e. territorial vs. bachelor males) is transmitted in dung, then I predicted that territorial
males would have different volatile chemicals or chemical proportions within the scent of
their dung compared to similarly aged males in bachelor herds. In contrast, as I focussed on
similar-aged males, no difference between the scent profiles could suggest that dung is used
to transmit information on age between males.
Methods
The study was conducted during June 2014 in the Hluhluwe-iMfolozi Park, South Africa
(28.2198° S, 31.9519° E). Ten scent samples were collected from the dung of both adult
territorial males (i.e. dominant male with a harem) and 10 from similarly aged non-territorial
males living in bachelor herds (N= 20 dung samples). Adult males were determined by the
length of their horns, with adults being considered to have an average horn length of ~50 cm
(Stuart and Stuart, 1997). Territoriality is related to dominance and essentially mating success
(Owen-Smith, 1977). Non-territorial bachelor herds may consist of only sub-adults or young
males, or may include fully grown prime adults (Owen-Smith, 1977). It was difficult to
determine whether a male was territorial or non-territorial when there was more than one
male integrated in a female herd. When this occurred, the location of where the male in
question defecated was noted, for example, whether it was in a midden or not. It was assumed
that males that defecated in or near an obvious midden were territorial. To determine whether
a male was territorial or non-territorial in an integrated herd, how the male behaved and
interacted with the surrounding males was also noted. Sampling was random and even though
the location of each sample was taken, it was still possible that some males were sampled
more than once. This likelihood increased when samples were collected from males in
relatively the same area.
To collect the scent samples, I observed an individual and waited for it to defecate.
Within 5 minutes of defecation I collected a scent sample, which was extracted using
dynamic headspace (Augusto et al., 2003). For each sample, I collected a handful of dung (~6
g) using a polyacetate bag. The bag was then closed, and air was then sucked from the bag for
25 minutes using a micro pump with a realized flow rate of 150 ml/min. Scent samples were
collected in a small filter filled with 1 mg of Tenax® and 1 mg of Carbotrap® (thermo-
desorption cartridges).

6. 6
Identification of scent samples via GC-MS
I used gas chromatography coupled with mass spectrometry (GC-MS) to analyse the scent
samples that were collected in the field. The GC-MS that was used was a Bruker 450 GC-MS
with a Varian VF-5ms column (30 m x 0.25 mm internal diameter; film thickness 0.25 μm),
connected via Quick-Switch, to a Varian VF-1ms column (10m; film thickness 0.25 μm, 0.25
mm internal diameter) which is attached to a Bruker 300 quadrupole mass spectrometer in
electron-impact ionization mode at 70 eV (detector set to Extended Dynamic Range, EDR). I
placed the thermo-desorption cartridges in the Bruker 1079 injector equipped with a
ChromatoProbe thermal desorbtion device (Amirav and Dagan, 1997, Dötterl et al., 2005).
The flow rate of helium carrier gas was 1.8 ml min-1. The injector was held at 40 °C for 2
minutes with a 20:1 split. The temperature was then increased to 200 °C at 200 °C min-1,
held for 10 minutes, increased to 250 °C at 200 °C min-1 and held for a further 10 minutes.
After the initial 2 minutes, the split was removed for thermal desorption and then a 100:1
split was introduced after 4.2 minutes to flush the injector. The GC oven was held at 40 °C
for 3 minutes and then increased to 240 °C at 10 °C min-1
and held there for 12 minutes.
Compounds were identified using the Varian Workstation software with the NIST
2011 mass spectral library mass spectral library (NIST/EPA/NIH Mass Spectral Library, data
version: NIST 2011; MS search software version 2.0 d). In addition, identifications of
tentatively identified compounds were verified by using the absolute retention time plotted
against the relative retention time of authentic standards and published Kovats indices
(KOVATS index; e.g. as provided in the NIST 2011 library).
Statistical Analysis
I analysed the dung scent data using the software package PRIMER v6 (Clarke and Gorley,
2006; Clarke and Warwick, 2001). All scent compounds considered potential contaminants
were excluded prior to the statistical analysis. These contaminants were from residue
compounds from the GC-MS and compounds found in similar abundance from the control
sample. As a result, the analysis was limited to those compounds that could potentially
convey information. Fifty one compounds were identified, three of which were unidentified.
These compounds were grouped according to their chemical structure (Knudsen et al.,
2006). Benzenoids and phenylpropanoids, terpenes and nitrogen compounds are undigested
plant material and waste (Knudsen et al., 2006). Thus, they are unlikely to directly convey
information about status or age of an individual. Rather, they better reflect diet. Thus, these
classes, including the three unidentified compounds, were excluded from statistical analyses.

7. 7
In contrast, the remaining compound groups have been shown to convey information. The
differences in scent composition between different sample groups were assessed using the
proportion of individual compounds (compositional data) in the scent. Proportion amounts
were preferred over the absolute amounts since the volatile emission totals varied greatly
among individual samples (Dötterl et al., 2005).
I used non-linear multidimensional scaling (NMDS) based on Bray–Curtis similarities
to detect similarities among the samples. The significance level of differences in scent
profiles of territorial and non-territorial males was assessed with an analysis of similarities
(procedure ANOSIM, (Clarke and Gorley, 2006) with 10,000 random permutations. The
ANOSIM test calculates the test statistic R as well as a level of significance. The R value
ranges between 0 (no separation of groups) and 1 (complete separation of the sample groups)
(Clarke and Warwick, 2001). I used SIMPER (factor: species) in PRIMER v6 to identify the
compounds responsible for dissimilarities among territorial and non-territorial males (Clarke
and Warwick, 2001).
Results
From the gas chromatography-mass spectrometry (GC-MS) analyses, 52 volatile organic
compounds were identified from the dung scents of territorial and non-territorial male impala
(Table 1). Within these, I identified nine main compound classes: terpenoids (T), benzenoids
and phenylpropanoids (BC), aliphatic esters, aliphatic alcohols, sulphur containing
compounds (SCC), aliphatic aldehydes, aliphatic alkanes, aliphatic ketones and nitrogen
containing compounds (NCC) (Table 1).
Territorial and non-territorial males did not share all volatiles in common. From the
aliphatic compound group, proportions of 2,6-Dimethyl-7-octene, isobutyl acetate, ethyl
butyrate, isobutyl butyrate, 2-butoxyethanol and 2-butanone with a percentage proportion
below one were found in territorial males, but were not found in non-territorial males (Table
2). However, hexanal and octan-1-ol were only found in non-territorial males, both with
percentage proportion below one (Table 2). Isoamyl alcohol was found in both territorial and
non-territorial males, but non-territorial males had a higher proportion of 4.48%, (Table 2).
Sulphur containing compounds such as 2,4-Dithiapentane were only found in territorial males
(Table 2). Isopropyl isothiocyanate had the highest proportion of 18.15% in territorial males
(Table 2). However, these differences were non-significant (Fig 1).
An analysis of similarity (ANOSIM) showed that the overall scent profile of territorial
and non-territorial males were not well separated (R = 0.006; p = 0.374) (Figure 2).

8. 8
Moreover, using NMDS based on Bray-Curtis similarities no clear distinction between the
volatile compounds emitted from the dung of territorial and non-territorial males was found.
Thus, the sample clusters are not related to the territorial status of the individuals in the
cluster. In addition, when calculating similarity percentages (SIMPER) based on Bray-Curtis
similarities, I found that for non-territorial males the compound decanal had the highest
contribution to the similarity of this group (Table 3). Specifically, decanal, isobutyl
isothiocyanate, octanal, dimethyl sulfone, isoamyl alcohol and octan-3-ol contributed 91.70%
to the cumulative percentage (Table 3). In territorial males, isobutyl isothiocyanate had the
highest contribution percentage (Table 4). In addition to this compound, decanal, isopropyl,
isothiocyanante, dimethyl sulfide, isoamyl alcohol and oct-1-en-3-ol contributed 91.92% to
the cumulative percentage similarity of this group (Table 4). The isopropyl isothiocyanate
was the compound that explained most of the difference between territorial and non-territorial
males (Table 5). This was likely as it was only found in territorial males (Tables 3 & 4).
Discussion
There was no significant difference between the overall scent profile of territorial and non-
territorial male impalas. However, there was also a high degree of variation in the percentage
proportions of compounds between territorial and non-territorial males. As seen in the
multidimensional scaling based on bray-curtis similarities, there was a high degree of
variance amongst the samples. For example, territorial males had a higher percentage
proportion of isopropyl isothiocyanate than non-territorial males did.
As I focussed on similar-aged males, it is possible that dung is used to transmit
information about the age of an individual between males. As predicted, if there was no
significant difference between the scent profile of territorial and non-territorial male impala,
then scent may be driven by age. Interactions between male impala are strongly affected by
their age (Murray, 1981). Same-aged males that are both young (1-3 years old) and old (7
years and older) are generally more closely associated with each other (Murray, 1981).
However, these younger and older individuals avoid males that are socially mature (4-6 years
old) (Murray, 1981). Impala males aged four and half to six and half years usually occupy
territories during the height of the breeding season (Owen-Smith, 1977). Young males often
associate with old males so that they may learn and develop skills for adult dominance
interactions (Murray, 1981). Socially mature males are solitary throughout the year or
accompanied by female harems during the breeding season (Murray, 1981). This segregation
and avoidance of bachelor males can be explained by their territoriality that is maintained

9. 9
with their rank of dominance (Murray, 1981). With regards to olfactory communication,
immature and subordinate red deer (Cervus elaphus), have significantly different volatile
proportions in their urine, as compared to adults (Bakke and Figenschou, 1990). Therefore, it
is possible then, that scent may be an olfactory cue that dominant territorial males can use to
identify potential rivals.
Unfortunately, I collected my samples towards the end of the breeding season, when
testosterone hormone levels may have been similar between territorial and non-territorial
males (Agosta, 1992). During rut, territorial males have significantly higher levels of
testosterone than non-territorial males in (Bramley and Neaves, 1972). This is possibly due to
the fact that they have increased sexual activity, as compared to bachelor males (Bramley and
Neaves, 1972). Urine and faeces are primary mediums where metabolized hormones, such as
testosterone, can be excreted, and therefore aid in determining an individual’s social rank
(Miller et al., 1998). However, it is unlikely that a males hormone level controls the
proportions of volatile organic compounds in an individual’s dung scent (Miller et al., 1998).
In wolves, it was found that higher testosterone levels resulted in increased concentrations of
sulphur compounds (Raymer et al., 1986).
Territorial male impala may defecate in response to females that defecate in the
male’s territory or after confronting non-territorial bachelor males that had challenge them for
breeding rights (Jarman, 1979). These volatile organic compounds that are emitted from their
waste may aid in recognising individuals and establishing territories and dominance (Miller et
al., 1998). For white rhinos, territorial males have a higher percentage proportion of aliphatic
aldehydes in their dung compared to non-territorial males (Marneweck, 2013). These
aliphatic aldehyde compounds were also found to indicate territoriality in male moose (Alces
alces) and feral horses (Equus) (Rikako, 2001; Whittle et al., 2000). Aliphatic compounds are
found in numerous mammals around the world, and may therefore be a universal sign of an
individual’s status. The presence of these compounds suggests that dung scent of a dominant
male is used to indicate his territorial status. Because I did not collect samples during the
height of the rutting season, it is possible that there may be significant differences in aliphatic
classes and sulphur containing compounds between territorial and non-territorial male impala
(Figure 1). However, they may only be noticed during the rutting season.
Another possibility is that in contrast to what is reported in the literature, the impala
middens may be used for female-male communication. It has been suggested that octan-3-ol
may be a possible indicator of an individual’s sex (Burger et al., 1999). For example, this
volatile organic compound was only found in female Bontebok (Damaliscus dorcas dorcas)

10. 10
and blesbok (Damaliscus dorcas phillipsi) (Burger et al., 1999). During my study, I observed
female impala defecating in middens. Although it was beyond the scope of the study, this
behaviour may be to communicate their reproductive status. For example, one study found
that female bushbuck used middens more often than males (Wronski et al., 2006). This
suggests that females use these defecation sites to communicate to males when they are in
oestrus (Wronski et al., 2006). In addition, a recent study conducted on the white rhino, found
that the scent profiles of dung transmitted information on the age, sex, the territorial status of
a male, and the reproductive state of a female (Marneweck, 2013). Also, it has been
documented in the male Mohor gazelle (Gazella dama mhorr) use the females urinary and
faecal olfactory signals to determine their reproductive status (Pickard et al., 2003). As a
result, unlike my initial predictions, the use of middens by impala may not be limited to male-
male communication, but rather female-male communication. This however, needs to be
explored further.
In conclusion, there was no difference found in the scent profile between territorial
and non-territorial males. However, due to the limitations of this project, future sampling
should be carried out throughout the year, especially during the height of the rut. In addition
to determining differences in scent profiles of different ages and sexes.
Acknowledgements
I would like to thank Ezemvelo KZNWildlife for allowing me to collect data in the
Hluhluwe-iMmfolozi Park. Many thanks go to my supervisors Dr A. Shrader and Dr A.
Jürgens for helping me with my project and for their guidance throughout the year. Special
thanks go to Dr A. Shrader, for without whom; this project would not have been possible. I
would also like to thank Mrs C. Marneweck for supervising my fieldwork and reviewing
endless drafts of my report. Many thanks go to Mr. J. R. Schofield for helping me collect my
data and keeping me company while waiting for males to leave me freshly defecated samples.
Lastly, I thank my family and friends for their support and encouragement throughout the
year.

11. 11
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13. 13
Tables and Figures
Table 1. Volatile organic compounds identified and grouped according to biosynthetic pathway. T= terpenoids, BC= benzenoids, A. = aliphatic
compounds, SCC= sulphur containing compounds, NCC= nitrogen containing compounds
T BC A. ester A. alcohol SCC
A.
aldehyde
A. alkane A. ketone NCC
Carvone Phenol Ethyl butyrate Octan-3-ol Dimethyl sulfide Octanal
2,6-Dimethyl-7-
octene
2-
Butanone
Indole
β-Pinene p-Cresol Isoamyl acetate Octan-1-ol Dimethyl sulfone Decanal
Linalool m-Cresol Butyl hexanoate Oct-1-en-3-ol 2,4-Dithiapentane Hexanal
Limonene 3-Ethylphenol Isobutyl acetate 1-Hexadecanol
Isobutyl
isothiocyanate
Undecanal
γ-Terpinen Benzaldehyde
2-
Butoxyethanol
Isoamyl alcohol
Isopropyl
isothiocyanate
α-Farnesene Benzyl alcohol
Isoamyl
butyrate
Levomenthol 2-Pentanol acetate
Isobutyl
butyrate
Camphen-6-ol 2-Phenylethyl alcohol
2-Methyl-6-heptanone Hexyl 2-methylbutyrate
6-Methyl-5-hepten-2-ol
2-Methylbutyl
isobutyrate
6-Methyl-5-heptene-2-one
3-Methylbutyl 3-
methylbutyrate
2,7-Dimethyl-2,7-octanediol
1,6-Octadiene, 3,7-dimethyl-
3,7,11-Trimethyl-2,4-
dodecadiene
6,10-Dimethyl-5,9-
undecadien-2-one
4-Oxoisophorone (2,6,6-
Trimethyl-2-cyclohexene-
1,4-dione)

14. 14
Table 2: Average percentage proportion and compound classes of volatile organic
compounds identified for territorial and non-territorial male impala. SCC = sulphur
containing compounds.
Compound Territorial Non-territorial Compound class
Isopropyl isothiocyanate 18.15 2.48 Sulphur
Isobutyl isothiocyanate 10.13 5.28 Sulphur
Decanal 2.62 4.41 Aliphatic aldehyde
Octanal 1.86 3.39 Aliphatic aldehyde
Dimethyl sulfide 1.74 5.61 Sulphur
Isoamyl alcohol 1.55 4.48 Aliphatic alcohol
Isoamyl acetate 1.47 0.85 Aliphatic ester
Isoamyl butyrate 0.80 0.18 Aliphatic ester
2-Butanone 0.52 0 Aliphatic ketone
2,6-Dimethyl-7-octene 0.44 0 Aliphatic alkane
Isobutyl acetate 0.43 0 Aliphatic ester
Isobutyl butyrate 0.26 0 Aliphatic ester
Oct-1-en-3-ol 0.18 1.19 Aliphatic alcohol
Dimethyl sulfone 0.10 0.43 Sulphur
Octan-3-ol 0.10 0.64 Aliphatic alcohol
Ethyl butyrate 0.08 0 Aliphatic ester
Butyl hexanoate 0.02 0 Aliphatic ester
2,4-Dithiapentane 0.02 0 Sulphur
2-Butoxyethanol 0.01 0 Aliphatic ester
Undecanal 0.01 0.14 Aliphatic aldehyde
Hexanal 0 0.45 Aliphatic aldehyde
Octan-1-ol 0 0.19 Aliphatic alcohol
1-Hexadecanol 0 0.01 Aliphatic alcohol

15. 15
Table 3: The average abundance and contribution to similarity for volatile organic
compounds in non-territorial male impala. Similarity percentages (SIMPER) based on Bray-
Curtis similarities. Volatile organic compounds were selected with the highest cumulative
percentage. A. = aliphatic compounds; SCC = sulphur containing compounds.
Volatile organic
compound
Compound
Class
Average
Abundance
Contribution
%
Cumulative
%
Decanal A. aldehyde 1.63 26.28 26.28
Isobutyl isothiocyanate SCC 1.68 24.36 50.64
Octanal A. aldehyde 1.35 20.96 71.6
Dimethyl sulfone SCC 0.6 9.41 81.01
Isoamyl alcohol A. aldehyde 0.89 6.22 87.23
Octan-3-ol A. alcohol 0.42 4.48 91.7
Table 4: The average abundance and contribution to similarity for volatile organic
compounds in territorial male impala. Similarity percentages (SIMPER) based on Bray-Curtis
similarities. Volatile organic compounds were selected with the highest cumulative
percentage. A. = aliphatic compounds; SCC = sulphur containing compounds.
Volatile organic
compound
Compound class
Average
Abundance
Contribution
%
Cumulative
%
Isobutyl isothiocyanate SCC 2.5 33.9 33.9
Decanal A. aldehyde 1.42 17.92 51.82
Isopropyl
isothiocyanate
SCC 2.54 16.6 68.42
Dimethyl sulfide SCC 1.54 8.96 77.37
Isoamyl alcohol A. aldehyde 1.01 7.77 85.14
Oct-1-en-3-ol A. alcohol 0.67 6.78 91.92

16. 16
Table 5: Similarity percentages (SIMPER) based on Bray-Curtis similarities Average
contribution to dissimilarity between samples of territorial and non-territorial male impala. A.
= aliphatic compounds; SCC = sulphur containing compounds.
Volatile organic
compound
Compound
Classes
Contribution
%
Cumulative
%
Isopropyl isothiocyanate SCC 16.97 16.97
Isobutyl isothiocyanate SCC 12.53 29.5
Dimethyl sulfide SCC 10.56 40.06
Octanal A. aldehyde 8.06 48.12
Decanal A. aldehyde 7.82 55.94
Isoamyl alcohol A. aldehyde 7.62 63.57
Oct-1-en-3-ol A. alcohol 4.57 68.14
Isoamyl acetate A. ester 4.48 72.62
Dimethyl sulfone SCC 2.93 78.54
Octan-3-ol A. alcohol 2.86 81.4
Isoamyl butyrate A. ester 2.18 83.58
Pentacosane A. alkane 2.13 85.7
Isobutyl acetate SCC 1.36 87.07
Hexanal A. aldehyde 1.23 88.3
Octan-1-ol A. alcohol 1.2 89.5
2-Butanone A. ketone 1.19 90.69

17. 17
Figure 1. Total percentage mean proportions of volatile organic compounds for territorial
(white) non-territorial (grey) male impala. A. = aliphatic groups
Figure 2. Non-metric multi-dimensional based on Bray Curtis similarities between territorial
(circle) and non-territorial (triangle) male impala. 2-D stress value 0.15.
-10
0
10
20
30
40
50
60
Meanpercentageproportion(%)
Compound groups