Vocalization As an Indicator of Individual Quality in the Rock Hyrax
Vocalization as an indicator of individual quality in the rock hyrax
THESIS SUBMITTED FOR THE DEGREE "DOCTOR OF PHILOSOPHY"
BY
Lee Koren
SUBMITTED TO THE SENATE OF TEL-AVIV UNIVERSITY
October 2006
This work was carried out under the supervision of
Dr. Eli Geffen and Dr. Ofer Mokady
Abstract
Vocal communication is used in various systems within and between species. Animals may vocally communicate such messages as an imminent danger, or advertise their social or mating status. Song is almost synonymous with birds and is rarely linked to mammals. In a variety of birds, anurans, insects and a few mammals, different elements of singing have been linked to hormonal levels, social status, body condition, and with reproductive success. Various models have addressed risk taking, decision making and hierarchy formation in social groups. Singing involves high costs in terms of time, energy and predation risk, warranting consequent rewards, such as social status and ultimately reproductive success. The rock hyrax ( Procavia capensis ) is an ideal model to test theories regarding honest signaling and advertising, since hyraxes live in multi-male and female social groups, with polyandrous mating and a low female reproductive skew. Hyraxes are also diurnal, facilitating behavioural observations, and in the Ein Gedi Nature Reserve can also be studied in their natural habitat, which includes predators, competitors, diseases and harsh weather conditions. Acoustic communication constitutes a widely used means of information transfer amongst hyraxes. Both males and females produce alarm calls, yet only some sexually mature males sing. Singers sing almost year round in individually distinct voices, and often counter-sing with neighbours. The purpose of this research was to investigate the relationships between male hyrax songs and individual attributes, such as survival, body condition, hormonal state and social status. This research includes data I have collected over five years of observations and experiments in the field and in the laboratory. I used an interdisciplinary approach, which involved physiological, behavioural and ecological tools to assess the above relationships. Over two hundred and thirty animals were captured and individually marked. Each animal was measured, weighed, and photographed. Hair samples were cut for the hormonal analysis. Field observations of agonistic interactions allowed me to rank animals in terms of social status in five groups. Surprisingly, results showed that in most groups, the most dominant members of the group were females. Females also ranked on average higher than males, and they lived longer than them. Despite that, adult male hyraxes were bigger and in a better physical condition than adult females. Hormones were extracted from hair samples. I essayed the levels of two androgens (testosterone and androstenedione; 'male' hormones), estradiol (a 'female' hormone) and cortisol (a stress hormone). I discovered that females had similar levels as males of both androgens tested. This result is the first of its kind, since female mammals usually have significantly lower testosterone levels than males. The elevated levels in female hyrax may be associated with the female-dominated social organization. Hyraxes seem to defy most mammalian hormonal and social rules, challenging perceptions and necessitating new explanations. Adult cortisol levels were found to be higher in females than in males, perhaps due to the extensive reproductive efforts, which include approximately 230 days of gestation each year. Female social ranks were positively related to cortisol levels, and negatively related to androgen levels and to body condition. Intersexual differences in hormonal strategies arise in pups. Pup mortality in Ein Gedi hyraxes is high for both sexes, yet male pups that survived their first year of life had lower cortisol levels, while females had higher cortisol and testosterone levels, than those who died. Both low male cortisol levels and high female testosterone levels are related to subordination in adults. It is possible that pups, in order to survive, need to signal subordination. Only a third of adult males sing. Singers are on average older and more dominant than non-singers. Singers also have higher testosterone levels and lower estradiol levels, than non-singers. Their cortisol levels, which were related to their social status, were also higher than in non-singers. High ranking singers had higher cortisol levels, similar to the case in females and to other social mammals, possibly due to the risk and stress involved with increased agonistic interactions. I recorded songs from 17 males. Songs were digitalized and analyzed. I focused on three main song elements: wails, chucks and snorts. All songs contained wails and all but one contained chucks, but only songs belonging to 11 (out of 17) males contained snorts. I measured time and frequency related parameters for each song and element. Using discriminate function analysis, each singer was individually recognized, based on these song parameters. I used multiple regressions in order to determine the relationship between specific song elements and male attributes. I found that one song component corresponded to every aspect of male quality I measured, possibly advertising it. For example, age, androgen levels and social ranks, were all related to the snort component. Body size and cortisol levels were associated with the chuck component. Formant frequencies, which are produced by the vocal tract, are associated in hyraxes with fur coverage and social ranks. Taken together, my results suggest that male hyraxes in Ein Gedi advertise their age, body size and condition, hormonal levels and social ranks using song. Singing is expensive in terms of time expenditure and predation risks, and factors that enhance it, such as high hormonal levels, are expensive as well. The benefits expected, in terms of reproductive success, have yet to be assessed, but they must be considered, as well as the potential audiences and their response to the singing.
Acknowledgements
Thank you To my supervisors: Dr. Eli Geffen and Dr. Ofer Mokady, who held my hand, lent a hand and let go when necessary. Who were brave and secure enough to allow me to find my own way. To my committee: Prof. Uzi Motro, Prof. Yoram Yom-Tov and Prof. Arnon Lotem for their support. To the project students (Liat, Gill, Nati, Modi and Leah), technicians (Beth, Itamar, Oren, Yotam and Enbal), field guides (Ynon, Dafna, Ta'ir, Shimi) and park rangers, for their help in the field. To friends who bravely restrained northern hyraxes (Anat, Tamar and Ofir). To the Israeli Nature and Parks authority for their permission to work in The Ein Gedi Nature Reserve. To the Ein Gedi Field School for their warm hospitality and logistic help. To the Ein Gedi Emergency Rescue Crew (Alon, Eran, and Yakov), for lending me carrying equipment and for securing my ropes. To the beautiful Ein Gedi and Mizpe Shalem women, who danced, shared their lives, friendship and support (especially to Rohik, Saraleh, Chaya and Orna). To Hanita and Yael, who babysat Nour. To Michal, Doron and Yael for their friendship and support while working in the field. To the molecular crew (Zlil, Michal, Ronit, Adar, Tamar and Haggar), for guarding the flame. To my friends in the lab (i.e. the salad club: Anat, Sigal, Sharon, Vered, Tamar, Inbar and Hagar). To Rachel Paz for the support, advice, help and chocolates (and reprimands for eating them). To members of the ex- Institute for Nature Conservation Research, especially to Prof. Dan Eisikowitch, who accepted me for a MSc, and to Dr. Sarig Gafni for the hospitality. To my dad, Prof. Gideon Koren for the help, advice, encouragement and the idea for the hormone analyses. To Tatyana Karaskov and Julia Klein, for their help with the hormone analysis and for teaching me the hair-testing protocol. To Dr. Yuval Zohar, who skillfully dissected the run-over hyraxes with me. To Dr. Noam Leader, Dr. Ofer Amir and Dr. Noam Amir, for their help diving into the acoustic world. To Dr. T. W. Fitch, for helpful formant analyses advice. To Dr. Hans deVries, for his generosity, patience and help with the rank determination, steepness and linearity testing. To Dr. Charlotte K. Hemerlijk, for sharing with me her Matrix Tester program. To my mother, for encouraging me to trust my intuition. To my wonderful sister Tal, who is always there for me, and to her partner Yuval, for all the help. To Amir, who came through whenever no one else could. To all of my extended family, especially to my grandmother who came out and helped me set up Nour in Ein Gedi, and to friends who are part of my family, especially to my 'sisters' Anat and Ronit and my teacher, friend and guide Fifi. To my partner, Avi, for the great love, for the help in the field, for the support at home, and for Nour. To my daughter Nour, who allowed me to work in the field throughout the pregnancy and during her first 2 years of life. Who shares my love of Ein Gedi, helped me capture pups, and asks good questions. To the hyraxes, who emit such funny, fun to work on vocalizations. Also to the leopards, wolves, and hyaenas, and to the floods, that helped make every day in the field a celebration… Thank you all!
The extensive field work in this research was very logistically complex. Loads of help was extended to me in every junction. I humbly and truly apologize if I forgot to mention anyone who helped along the way…Thank you very much… Table of Contents
Prelude ...... 2 General Introduction...... 2 Rock Hyrax...... 3 General background ...... 3 Vocalization ...... 4 Objectives ...... 5 Chapter 1: Who sings? ...... 7 Introduction...... 7 Age ...... 7 Body condition ...... 8 Social hierarchy ...... 9 Methods...... 11 Morphometrics ...... 11 Survival ...... 12 Body Condition ...... 12 Social hierarchy ...... 13 Statistical Analysis ...... 14 Permits ...... 15 Results...... 16 Age Determination ...... 16 Survival ...... 17 Morphometrics ...... 18 Weight controlled for length ...... 21 Fur coverage ...... 22 Social hierarchy ...... 23 Males: who sings? ...... 28 Discussion ...... 29 Age and survival ...... 29 Body condition ...... 30 Social hierarchy ...... 31 Singers ...... 32 Chapter 2: Hormonal profiles ...... 34 Introduction...... 34 Background ...... 34 Androgens, estrogens and behaviour ...... 38 Cortisol and behaviour ...... 40 The cost of hormones ...... 41 Hormones in hyraxes ...... 43 Methods...... 44 Hormone levels ...... 44 Statistical Analysis ...... 44 Results...... 45 Sex and Class ...... 45 Hormone correlates ...... 50 Fur coverage ...... 51 Hormones and social hierarchy ...... 52 Males: who sings? ...... 55
Discussion ...... 58 Intersexual comparisons: levels, fitness and correlates ...... 58 Fur coverage ...... 62 Social hierarchy ...... 63 Singing ...... 65 Chapter 3: Why Sing? ...... 67 Introduction...... 67 Vocal recognition ...... 67 Signaling information honestly ...... 68 The source-filter theory ...... 70 Hormones and song ...... 71 Methods...... 73 Equipment ...... 73 Song analysis ...... 73 Filter estimation ...... 74 Statistical Analysis ...... 76 Results...... 77 Vocal recognition ...... 77 Factor structure ...... 86 Anatomical measurements ...... 88 Anatomical measurements ...... 88 Signaling information ...... 89 Discussion ...... 96 Vocal recognition ...... 96 Signaling information honestly ...... 97 Nonlinearities ...... 101 More to choose from… ...... 103 General Discussion...... 105 Female choice ...... 106 More hyrax thoughts and further research ...... 108 Postlude...... 113 Appendix...... 114 Further validations for hair-testing protocol determining hormonal levels ...... 114 References ...... 118
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Prelude
In my master's thesis (Koren, 2000), I explored the hypothesis that rock hyraxes ( Procavia capensis ) in Ein Gedi live in a female dominated association. During two consecutive years (1999-2000), I identified and marked eighty-four hyraxes, and studied three hyrax groups. I found that female hyraxes guard more than males, take turn babysitting the pups, and vocalize when predators approach. While collecting behavioural observations, I noticed an extensive singing behaviour that differed from the alarm calls that I studied. Hyrax songs resemble bird songs in many aspects: they are rich, complex, and mostly harmonic. Each singer has a personally unique voice (which I have learned to recognize), and sings distinctive, non repeated songs (which progress with time in their complexity). This form of sophisticated acoustic communication, along with its dynamic features, intrigues me. An interactive approach combining behavioural work in the field with molecular and hormonal analyses in the laboratory can be utilized in order to examine this unique signal, in the exceptional hyrax system.
General Introduction
In social mammals, males expand vast amounts of energy in order to appear attractive and to advertise themselves as successful mates. Advertising strength and stamina help deter other males from a physical challenge. Displays using visual, behavioural, scent and auditory cues are used in communication. Animals that use elaborate displays and thus endanger themselves, advertise their quality to both male and female conspecifics (Zahavi, 2003). Singing serves as such an advertisement. It can be expensive in terms of time and energy investment and in terms of increased predation risk (Tuttle and Ryan, 1982).
A song is defined by Collin's dictionary (1999) as 'the characteristic tuneful call or sound made by certain birds or insects'. An exhaustive review of the term song, pertaining to birds, was not successful in uniting definitions and concepts (Spector, 1994). The significance of vocal communication, especially male vocalization, has been addressed in many animal systems. Andersson (1994) lists more than 60 species where aspects of male singing or calling have been demonstrated to be associated with higher reproductive success. Most of them are birds and anurans, followed by insects and by one mammal. In mammals, usually the olfactory sense is the most developed. Some mammals use acoustic communication
2 termed 'calling', which consists of repeated, atonal notes (Waser, 1977b; Clutton- Brock and Albon, 1979; Zimmermann and Lerch, 1993; Potter et al., 1994; McElligott and Hayden, 1999; Insley, 2000; Ghazanfar et al., 2001; Uster and Zuberbuhler, 2001; Charrier et al., 2003; Frommolt et al., 2003; Kitchen et al., 2003; Wich et al., 2003; Fischer et al., 2004; Soltis et al., 2005a; Harris et al., 2006). Unlike the case in birds, mammals rarely sing per definition (i.e. long, complex vocalizations used for long distance communication; Spector, 1994). Song has only been attributed to a few primates (Howler's, mouse lemurs, orangutans, and gibbons; Tenaza, 1976; Zimmermann and Lerch,1993; Delgado, 2006; Geissmann and Nijman, 2006), to marine mammals (Payne and McVay, 1971; Hanggi and Schusterman, 1994; VanParijs et al., 1997) and to bats (reviewed in Davidson and Wilkinson, 2004). In mice too, males sing if they detect a female, or her pheromones (Holy and Guo, 2005). Following my research (Koren 2000 and the current thesis) I wish to add another mammal to the exclusive list of singing mammals, an ancestral pro ungulate (Kleinschmidt et al ., 1986), the rock hyrax ( Procavia capensis ).
Rock Hyrax General background Rock hyraxes are abundant across Africa and the Middle East, wherever appropriate shelter (i.e. rocks) and food exist. The Ein Gedi Nature Reserve (see below in the methods section) supports a large hyrax population. Hyraxes belong to the hyracoidea (Afrotheria; Murata et al ., 2003; Springer et al ., 1997), a sister taxon of the African elephant. Rock hyraxes are cooperative breeders that live in large, mixed-sex group units, which include, in Ein Gedi, several males (one mature and several juvenile late dispersers), and between ten and twenty females with their pups. They are diurnal, basking in the morning to warm up (Meltzer, 1976) before feeding and social activities (Koren, 2000). Observations of agonistic interactions in hyrax groups in Ein Gedi suggest female dominance (Koren, 2000; Koren et al ., 2006). Dominance is shown by staring, approaching, grinding molars, biting, pushing and chasing. Their tusks can inflict fatal wounds (LK personal observation).
Rock hyrax are seasonal breeders (Mendelssohn, 1965; Millar, 1971; Millar, 1972; Neaves, 1973), with synchronized births (Mendelssohn, 1965; Sale, 1965). Mate guarding and manipulation by the territorial male must therefore be limited, offering subordinates an opportunity to sire offspring as well (Emlen and Oring, 1977). Gestation period is considerably long for such small mammals, about 230 days,
3 with the litter size ranging from one to six (Mendelssohn, 1965; Sale, 1965; Millar, 1971). The pups are very large at birth, fully haired, and are born with their eyes open (Mendelssohn, 1965; Sale, 1965; Millar, 1971). Immediately after birth, the young pups interact with each other and with the sub-adults in the group. Pups nurse in their first year of life until a new litter receives priority, enforced by the mothers (LK personal observation). Sexual maturity is at 16 months of age, and longevity is up to 12 years (Mendelssohn, 1965; Glover and Sale, 1968). Adolescent males between 17 and 24 months old are forced to disperse (Hoeck et al ., 1982). They live in the periphery colonies, in Ein Gedi, in bachelor groups (Koren, 2000). In rare cases, when females emigrate, they are eventually accepted into new groups. Adult male hyraxes reside in a group for only a few years before they are evicted. They must therefore respond to any challenge by strange males, yet be careful not to get injured or lose their fitness in unnecessary battles. In the Judean desert, hyraxes constitute a substantial component in leopards’ (Panthera pardus ) diet (Ilani, 1980). Large eagles occasionally eat hyrax (O. Bahat, Per. Comm.), while foxes and other terrestrial carnivores hunt their young. Thus, predation is a significant factor. Antipredatory behaviour mostly consists of remaining close to cover and responding to alarm calls.
Vocalization Acoustic communication constitutes the most widely used means of information transfer amongst rock hyrax (Fourie, 1977). Both male and female hyraxes produce loud repetitive warning trills, yet mostly adult males, but not all, engage in diverse singing vocalization. All singers are sexually mature and out of their natal groups. They sing most of the year in individually distinct voices (Fourie, 1977; Hoeck et al ., 1982; Koren, 2000) and, like in birds, bats and forest monkeys (Tenaza, 1976; Waser, 1977a; Hyman, 2003; Behr et al., 2006), countersing with neighbouring males (Koren, 2000). Males can be ranked by the complexity of their vocal repertoire; males that live with a group of females sing elaborately, using different song elements, while their bachelor counterparts use mostly atonal calls, composed of a few notes at a time (Koren, 2000).
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Objectives The objective of this research was to decipher the messages sent out by males in their singing behavior. I examined who sings, the motivation for singing and the information that is vocally broadcasted.
The following three hypotheses were tested (Fig. 1):
1. Singing is a reflection of social status.
2. Male age, body size, condition, dominance rank and hormonal state are all reflected in the song.
3. Social status, body condition and hormone levels are interrelated.
In order to examine the above trends I divided the results of this long-term study into 3 chapters:
1. Who Sings?
The objective of this chapter is to examine the unique profile of males that sing and to compare them to males that do not sing and to females, on the basis of:
• the demographic data collected and analyzed for the Ein Gedi hyrax population
• morphometric measurements
• body condition parameters
• social rank
2. Hormonal profile of singers
The objective of this chapter is to define the hormonal profiles of singers and of non-singers in relation to their age class, body condition parameters and social rank.
3. Why sing? Does singing advertise key information?
The objective of this chapter is to look at hyrax vocal recognition and vocal components, and their relationship with the age, morphometric, body condition, social rank and hormonal parameters.
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Hormonal levels Vocalization
Reproductive Success
Survival (age)
Body condition Social
status
Figure 1: Schematic diagram representing the relationships that were tested between male hyrax parameters. The arrows symbolize possible associations. Some parameters influence others unidirectionally, and other relationships (i.e. arrows) are expected to be bidirectional. The objective of this study was to examine the relationships symbolized by the arrows. Male reproductive success in the study population is not completed and is therefore not included in this thesis.
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Chapter 1: Who sings? Introduction The object of this chapter is to draw a profile of the singing hyrax. I want to characterize and define singers in terms of their age, body size and condition and social ranks, in order to asses whether, by singing, hyrax advertise fitness and quality. The ultimate measure of individual quality in nature is reproductive success. Since not all individuals are equal, assessment of potential mates is important. Mates may be chosen for benefits they provide either directly (i.e. food, shelter or protection) or indirectly (i.e. attractive or beneficial genes that will be passed on to offspring). The benefits I am attempting to asses are indirect, mediated through male phenotypes, advertising individual quality, such as singing. In this chapter I will attempt to link the singing phenotype to longevity and to body condition parameters, as well as to social status; and to asses each parameter as a potential quality indicator for 'good genes'.
Age The relationship between survival (longevity) and fitness may explain females' preference based on age (Brooks and Kemp, 2001). The idea behind the age-based indicator mechanism (AIM) is that older males are more adapted than younger males and can therefore pass their genetic superiority to their offspring. This concept unites sexual selection and life-history theories, yet it may overlap with the viability- indicator model, which is based on females' preference for males that advertise breeding value of survival (Zahavi, 1975; Brooks and Kemp, 2001). Several studies linked male age and attractiveness to females (reviewed in Kokko, 1998; Kokko and Lindstrom, 1996). A negative relationship between age and mate quality include the possible accumulation of mutations, lower fertility, possible conflicts between early life history and late life parameters and the fact that offspring of younger parents come from a more updated gene-pool, hence better adapted to the current conditions (reviewed by Hansen and Price, 1995). Kokko et al . (2002a) proposed that it is possible that high quality males may advertise so intensely that their survival is worse than lower quality males. Under this assumption, their offspring will possess high reproductive value, disassociating viability and age from good genes (Kokko, 2001).
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When a females' preference for older males exists, it may not necessarily be under AIM but rather because of older males' tendency to invest more in their offspring (Preault et al ., 2005) or because of direct benefits their accumulated life experience can help provide.
Body condition Under the handicap principle, an honest signal is one that is expensive to maintain and thus indicates genetic quality (Zahavi, 1975). Females of many species prefer to mate with males that have the fewest parasites (Moller, 1993). The upholding of a strong, healthy body can be an indicator of individual quality, as explained by the immunocompetence handicap hypothesis (ICHH), which discusses the price of maintaining an immune system (Hamilton and Zuk, 1982; Norris and Evans, 2000). Under this model, a male’s appearance is an honest signal, indicating genetic quality. Only high quality males, possessing ‘good genes’, will not suffer reduced fitness from pathogens or parasite attacks while displaying a healthy fur, for example (Folstad and Karter, 1992; see chapter 2 for more on the ICHH). Ectoparasites load serves as an indication of the overall condition of individuals. Since ectoparasites are transmitted through interaction between animals, they are influenced and may influence the social structure and behaviour of groups. Mating can also be affected by ectoparasites load, since monogamous animals can be expected to contact less ectoparasites than polygamous animals (as the case is with sexually transmitted diseases; Kokko et al ., 2002b). High ectoparasites loads have been experimentally related to slower growth, lower winter survival, and reduced reproduction in marmots ( Marmota flaviventris ), suggesting that ectoparasites are a fitness cost (VanVuren, 1996). In birds as well, ectoparasites have been demonstrated to impose an energy cost on their hosts by elevating metabolism levels (Moller et al ., 1994), and lowering reproductive success in infested birds (Delope and Moller, 1993). Most Ein Gedi hyraxes have ‘mange’, which is a skin condition that leads to extensive scratching, secondary bacterial infections and other responses. Mange is the common name of a suit of skin conditions known from a wide variety of mammals (e.g. cats, Leone et al ., 2003; dogs, Rodriguez-Vivas et al ., 2003; and camels, Kinne and Wernery, 2003). In most animal models it is caused by various parasitic mites (Order Acari). The precise causes of the pathology is unknown, although it is assumed that it may depend on products derived from the digestive system of the mite (Hamilton et al ., 2003). The infection readily spreads by contact within groups of
8 animals (Kinne and Wernery, 2003), and in most models involves severe scratching and hair loss within a few weeks (e.g. Skerratt, 2003). Later clinical responses include lameness, loss of weight and some biochemical responses (e.g. neutrophilia; Skerratt, 2003). The specific agents that are responsible for the mange in the Ein Gedi hyrax are currently unknown. However, collections of ectoparasites from trapped animals indicate the prevalence of mites and the lack of fleas (Koren, 2000), although they are known to be found on hyrax (Theodor and Costa, 1967). Other possible agents are flies, which can bite and fly off without being directly observed. Hippoboscid flies (Lipoptena chalcomelaena ) are found on the Nubian ibex ( Capra ibex nubiana ; Yeruham et al ., 1999), which share the hyraxes' habitat in Ein Gedi. Lipoptena 's bites cause irritations and itching, whereas scratching can cause hair lost. Hippoboscids are also found on house cats and domestic goats ( L. capreoli ) and on dogs and badgers ( L. longipennis ). Hippoboscidae flies also play a role in the transmission of Bartonella among ruminants (Halos et al ., 2004). Bartonella spp . are small Gram-negative bacteria transmitted by blood-sucking arthropods and are considered to be emerging pathogens in humans and animals (reviewed by Halos et al ., 2004).
Social hierarchy An established dominance hierarchy saves group members the energy of repeated agonistic interactions and its fitness reducing implications (Clutton-Brock et al ., 1979). Dominance relations involve an encounter between individuals of the same species, acting in a manner adopted as a result of fights, or early stages of fights (Theraulaz et al ., 1992). In hyrax, dominance is determined mainly by displacement interactions. Aggressive interactions are rare though once they escalate they can lead to severe wounds and death (LK personal observation). An individual’s position in the group is determined by its relationship with others in the group, and their relationships with one another (Richards, 1974). Dominance is associated with benefits, especially in priority to mates, food and shelter (Hemelrijk, 1999). Vehrencamp (1983) introduced the term 'egalitarian' species, in which benefits are equally distributed, as opposed to 'despotic' species, in which benefits are biased towards dominant animals. In an egalitarian society, the dominance hierarchy is 'shallow', meaning that 'steepness' or the differences between social ranks for group members is low (De Vries et al ., 2006). Species that exploit dispersed, abundant resources, like the Ein Gedi hyrax, will tend to live in egalitarian
9 societies. Alternatively, species that exploit clumped resources, that can be usurped by a single individual, will tend to live in despotic societies with well-differentiated relationships and strong linear dominance hierarchies (Vehrencamp, 1983), but there are exceptions, such as among female lionesses (Packer et al ., 2001). In most mammals, males are the dominant sex. Male dominance ranks have been found to correlate with both age and body size (reviewed by Malyon and Healy, 1994), as well as with male androgen levels (see chapter 2), all of which affect fighting ability. Subordination can decrease male reproductive success (e.g. slower ejaculation; Hemelrijk, 1999). Female dominance is rare and is typical of only lemurs (Kappeler, 1990; VanSchaik and Kappeler, 1996; Radespiel and Zimmermann, 2001; Pochron et al., 2003) and spotted hyenas (Kruuk, 1972; Frank et al., 1985). Female dominance can inhibit male copulatory behaviour. In light of preliminary results from hyrax groups in Ein Gedi suggesting female dominance (Koren, 2000; Koren et al ., 2006), it is interesting to investigate typecast male singing behaviour in this system. The objective of this chapter is to answer the question: who sings? I wish to draw a profile of singing hyrax, in terms of age, body size and condition and social ranks, contrasting them to non singers.
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Methods Rock hyraxes (Procavia capensis) were studied in the Ein Gedi Nature Reserve (31°28’N, 35°24’E). Two deep gorges, David and Arugot, constitute the Reserve, which is located west of the Dead Sea in the Judean Desert (Fig. 2). Field seasons averaged six months a year, from February to August, over seven consecutive years (1999-2005). The Arugot gorge population, between the 'Hidden Fall' and the 'Upper Pools', was studied through all seven seasons. The David gorge population, in the 'closed reserve', was studied from 2002 to 2005. The two study sites will be referred to as 'Arugot' and 'David'.
Figure 2: Location of the study sites in the Ein Gedi Nature Reserve in Israel. The top (right) circle locates the David gorge study site, which includes two social groups. The bottom (left) circle is the Arugot gorge study site, which supports four social groups. Morphometrics 233 individual rock hyraxes were studied. Animals were caught using live box traps and anesthetized with Ketamine hydrochloride (0.1ml/kg intramuscular injection). Each animal was individually marked using ear tags or a collar (adults only; collar weight 5g). Body length, from the base of the skull to tip of the tail, and head diameter (at eye level) were measured for each animal as size indicators. Weight and body girth were also measured, but were not used on their own as size indicators for intersexual comparisons since females are pregnant almost two-thirds of the year
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(Sale, 1965). For a more detailed explanation on the methods and measurements see Koren, 2000.
Survival All births in Ein Gedi are in the spring, with pups first observed mid-March. In order to estimate age of individuals, I standardized all individuals born on a specific year by setting their age to March 1st. Age (in months) was normalized using Box-Cox transformation ( λ=0.2). Separate regressions for males and females were plotted in SPSS (nonlinear estimation), using morphometric measurements from individuals whose birth year was known (born after 1998 in Arugot and 2001 in David). In this study, morphometric measurements (weight, body length, body girth and head diameter) highly correlate with each other (p<0.0001). The weight measurements were the most significant when compared to known ages for young hyraxes of both sexes, and were therefore used for age determination (see results). Linear lines were fitted to the data and were used to estimate the age for all hyrax whose birth year was unknown. Given ages are in months, with an expected error of approximately 1 month. Since growth curves are more accurate for young ages, hyraxes' ages were estimated from the earliest available data. Ages for animals that were sampled on multiple, consecutive years were calculated from the earliest record I had for each individual. Age classes were defined as 'pup' (born during the field season; i.e. the first year of life, or age category 0), 'juvenile' (born in the previous field season; i.e. the second year of life, or age category 1), and 'adult' (over 23 months old, age categories 2-10 years).
Body Condition Many hyraxes in Ein Gedi have only partial fur coverage, which is caused by a skin condition termed 'mange'. The cause of the mange is unknown, and no evidence of scabies or flea bites was found. Mange is known to influence body condition, and is most likely contracted through physical contact. Fur coverage, in percentage, was assessed at the time of capture for treated animals. In order to use this value in parametric tests and in linear models, the fur coverage, where needed, was expressed as a proportion, and was transformed by calculating the arcsines of its squared root (arcsine √p). In order to evaluate the hyraxes' physical condition, I calculated the magnitude and the direction of the residuals from the regression of body weight on body length.
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This analysis has been used as an index for body condition and composition in different mammals (Cavallini, 1996; Jakob et al ., 1996), despite its limitations (Green, 2001). Using the JMP6 software, I divided adult hyraxes into three categories. Hyraxes that were within one standard deviation of Student's residuals, to either direction were assigned to category 0 (i.e. 'normal'), animals beyond the ±1SD range were assigned to category 1(i.e. 'over weight') and animals below the ±1SD range were assigned to category -1 (i.e. 'under weight'). Differences between hyrax belonging to the different categories were tested using one way ANOVA (SPSS; v. 13.0).
Social hierarchy Behavioural observations on six focal groups were recorded over a five year period (2000-2004; a total of 600 observation days and approximately 3000 hours). Most observations were in the morning from first light to noon; thereafter the hyraxes retreated from the heat to a period of inactivity. Afternoon sessions were shorter. Ein Gedi hyraxes are active again once the canyon is shaded from the sun, and stay active 2-3 hours after sunset. On average, morning sessions were 6 hours long and afternoon sessions were 2.5 hours long. Agonistic and avoidance interactions as well as guarding behaviour, babysitting, mating, nursing, cuddling and all loud vocalizations were noted. A matrix of encounters was prepared for each group using all pair wise dominance and avoidance interactions observed during a given field season. Agonistic interactions that involved display (i.e. charging, biting, pushing, staring or growling) by one hyrax and evasive action (i.e. retreat) by a second were recorded. Overall, 10 unique matrices were produced from 166 interactions. For each interaction, the winner (remaining hyrax) was placed in the row, and the loser (retreating hyrax) in the column. In the past, I ranked hyrax groups using the BBS ranking method (Jameson et al ., 1999). Several recent articles critiqued the method (e.g. De Vries and Appleby, 2000), which places individuals that did not participate in agonistic interactions high in the hierarchy. This is true for the hyrax data as well. I decided to re-rank the hyrax using two alternative methods: average dominance index (ADI) and the recently recommended David's score (Gammell et al. , 2003). The ADI is a calculation of the average percentage that an individual wins interactions with its group members. ADI= 1 / N Σj wij (where Σj wij is the summary of frequencies with which an individual i attacks or wins an opponent j). I calculated ADI using 'Matrix
13 tester' (C. K. Hemelrijk). This simple method has been proven to be a good indicator of the hierarchy, based on internal dominance values and is compatible with more complex ranking methods (Hemelrijk et al ., 2005). David's score (Ds) is more sophisticated than ADI in that it takes into account opponents' social ranks. It also has the advantage of considering interaction outcomes proportionately, so that in groups where members repeatedly interact with each other (i.e. more than once), ranking is logical (Gammell et al ., 2003). Ds=w+w 2-l-l2 (where w is the sum of winning per row and l is the sum of winning per column. Each ratio is weighed by the summed winning ratio of the loser, summed per row-individual w 2, and per column-individual l2). David's scores were calculated by H. deVries using macros in Excel. In addition, I ranked each animal using David's scores and ADI. The animal with the highest score was ranked first (i.e. 1), the next was ranked second (2), etc. I also categorized ranked animals as either 'high ranking' (first and second most dominant animals) or 'low ranking' (animals ranked third or lower). All scores and ranks show normal distribution. For each group matrix, I also calculated steepness and linearity. Steepness is the degree to which individuals differ from each other in winning dominance encounters. It is calculated by measuring the slope of the straight line fitted to normalized David's scores (corrected for chance) plotted against the animals' ranks (De Vries et al ., 2006). Normalized David's scores and the randomization procedure for determining the statistical significance of the steepness were calculated using macros in Excel (H. deVries). Linearity is the degree of established binary dominance relationships and the degree of transitivity in these relationships. Since my data contains many unknown or tied relationships, I used deVries' improved linearity test (h'), implemented on MatMan1.1 (Devries, 1995).
Statistical Analysis The numerous field seasons, which allowed me to study hyrax social behaviour in depth and repeatedly sample the same individuals over the years, posed a challenge when I attempted to use conventional statistical tools. The 233 hyrax were sampled 443 times, an average of 1.9±1.3 samples per animal (±SD). Range is 1-9 samples per animal. In order to avoid pseudo-replication, I used only a single entry per hyrax (Sokal and Rohlf, 1995). The operational data set includes the first complete data set derived from each animal. Principal component analysis (PCA; SPSS; v. 13.0) was used to combine body length, head diameter and body girth into a
14 single morphometric variable (morphometric component). That first component in the PCA explained 90.5% of the variance in the morphometric data (Eigenvalue = 2.7). Normalized variables were used for parametric tests (Sokal and Rohlf, 1995). In order to compare equality of means, 2 sample, 2-tailed t-tests were used, without assuming equal variances (SPSS; v. 13.0). In order to test association between two variables I computed correlation by permutations (Stat xact v. 6 and Rundom). To test the relationship between social status and morphometric variables I used stepwise multiple regressions by permutation on Permute! v. 3.4 (Casgrain, 2002).
Correction for age Size (morphometric component) and body condition index (weight corrected for length) are significantly related to normalized hyrax age (see Table 1). The residuals of their regression on age were therefore used for all statistics and analyses. Testosterone and transformed estradiol were also significantly related to normalized age, but since age contributed less than 5% to their variance, they were not corrected.
Table 1: Significant statistics from the linear regression of hyrax morphometrics, body condition and hormonal measurements on normalized age. *Only cases where r2 >0.05 were corrected. Dependant Factor r2 F df p Morphometric component 0.338 65.765 1,129 <0.0001 Weight controlled for length 0.162 24.661 1,128 <0.0001 Testosterone* 0.045 5.515 1,118 0.021 Estradiol (normalized)* 0.046 4.394 1,92 0.039 Linear Regression (Independent: Age)
Permits Permits for capturing hyrax, a protected species, at the Ein Gedi Nature Reserve were issued annually by the Nature and Park Authority. The permit numbers are: 1999/5983, 2000/8871, 2001/8871, 2001/11116 (for the Korazin population), 2002/14674, 2004/17687, 2005/20737.
15
Results Age Determination Most hyraxes' birth years are known, since they were captured as pups or as juveniles. Linear equations were fitted to Box-Cox transformed known age and 2 2 weight data (males r =0.9255, F (1,88) =1092.6, p<0.0001; Fig. 3 and females r =0.943,
F(1,130) =2147.35, p<0.0001; Fig. 4), in order to calculate the ages for immigrants, and to extrapolate and estimate the ages of hyraxes that were born before 1998.
7
6
5
4
3
normalized age normalized 2
1
0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 weight
Figure 3: Normalized known age (years) as a function of weight (Kg) in Ein Gedi male hyraxes 2 (r =0.9255; F (1,88)=1092.6; p<0.0001). Fitted curve equation: normalized age =0.9711+1.681 (weight ) was used to estimate the ages of male hyraxes whose ages were unknown.
16
8
7
6
5
4
3 normalized age normalized 2
1
0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 weight
Figure 4: Normalized known age (years) as a function of weight (Kg) in Ein Gedi female hyraxes 2 (r =0.943; F (1,130) =2147.35; p<0.0001). Fitted curve equation: normalized age =0.7205+1.9893 (weight ) was used to estimate the ages of female hyraxes whose ages were unknown.
Survival Hyrax sex proportion at birth is equal (i.e. not significantly different than 1:1 ratio; χ2=5.68; df=1; p=0.46), for all study years (1998-2005). Chances of survival, to reach one year of age, are not different for males than they are for females. In fact, well into adulthood, up to 5 years of age, female biased sex ratio is not significant (Fig. 5). Once hyraxes pass the age of 6 years (age category 6+), the difference between males and females deviates from that expected by random (z=2.16; df=6; p=0.031). In my study area, 5 females reached the age of 10, while no males older than eight (8 years old) were present. Since females out live males, and because age (and experience) can significantly interact with other variables, age was removed from related size indicators (see methods).
17
80 male female 70
60
50
40
30 Number of hyrax Number
20
10
0 0 1 2 3 4 5 6+
Age
Figure 5: Ein Gedi hyrax population. The number of hyraxes counted in each age group in years. Total number of hyraxes in each age category shows survival decline for both sexes. At the oldest age category, females are significantly more numerous than males (z=2.16; df=6; p=0.031). In my study, older males (6+) are scarce, with the oldest being 7 years old. A handful of females lived to 10 years of age.
Morphometrics Pups (31 males and 45 female) and juveniles (33 males and 43 females) are sexually monomorphic. Male and female pups are the same weight (t=0.616; df=67; p=0.54) and size (morphometric component; t=0.796; df=63; df=0.4). Juveniles also do not differ between the sexes in their weight (t=0.019; df=77; p=0.985) or their morphometric component (t=0.629; df=71; p=0.532). At adulthood, however, males are on average heavier (t=3.6; df=137; p<0.0001; Fig. 6), and bigger than females (i.e. morphometric component corrected for age; t=8.098; df=120; p<0.0001; Fig. 7). Growths curves show that hyraxes cease to grow, reaching full adult size, at approximately the age of 50 months in males (4 years old; where the curves level off; Fig. 8), and 40 months in females (approximately 3 years old; Fig. 9). Multiple regression analyses showed that pup size is a good predictor of adult size (based on 2 morphometric components: R =0.782; F (1,3) =10.7; p=0.046).
18
n = 68 3 n = 71
2.5
2
Weight 1.5
1
0.5
0 male female
Figure 6: Average adult male and female weights (Kg±SD; ♂=2.6±0.4; ♀=2.3±0.4). Adult males are significantly heavier than adult females (t=3.6; df=137; p<0.0001). Sample size is above SD bars.
0.6
n=65 0.4
0.2 n=66
0
-0.2 Morphometric component -0.4
-0.6 male female
Figure 7: Average log morphometric component corrected for transformed age (±SD) in adult hyrax. Adult males are significantly larger than adult females (t=8.098; df=120; p<0.0001). Sample size is above SD bars.
19
2
1.5
1
0.5
0
-0.5
-1
-1.5 Morphometric component -2
-2.5 0 10 20 30 40 50 60 70 Age
Figure 8: Male morphometric component as a function of hyrax age. Male hyraxes cease to grow, reaching full adult size, at approximately the age of 50 months (4 years old; where the curve level 2 off). Morphometric component=1.097 ln (age)-3.102 (r =0.954, F (1,126) =2614.4; p<0.001).
2 1.5 1
0.5 0 -0.5 -1 -1.5 -2 Morphometric component Morphometric
-2.5 -3 0 10 20 30 40 50 60 70 80 90 Age
Figure 9: Female morphometric component as a function of hyrax age. Female hyraxes cease to grow, reaching full adult size, at approximately the age of 40 months (approximately 3 years old; where the curve level off). Morphometric component=0.915 ln (age)-2.764 (r 2=0.941, F(1,146) =2308.9; p<0.001).
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Weight controlled for length Weight and length, in the Ein Gedi hyrax, are related parameters (r2=0.455; n=122; p<0.0001; Fig. 10).
Figure 10: Adult hyrax weight (Kg) as a function of body length (cm). Linear fit 2 (weight =0.077 length -0.517; r =0.463; F (1,123) =106.123; p<0.0001) helps divide animals into body condition categories. Hyraxes that are within one standard deviation (SD) unit (of studendized residuals) to either direction are considered 'normal' (marked by x). Hyraxes that are beyond 1SD are 'over weight' (marked by full circles) and hyraxes that are below are 'under weight' (marked by open circles).
On average, adult males are in a 'better' (i.e. more positive) body condition than females (t=2.12; df=127; p=0.036; Fig. 11). No differences are found between male and female 'body condition' in pups (t=0.61; df=72; p=0.544) nor in juveniles (t=1.453; df=68; p=0.151).
21
0.4 n=64 0.3 n=66 0.2
0.1
0
-0.1
-0.2
Weight controlled forWeight controlled length -0.3
-0.4 male female
Figure 11: Average weight controlled for length corrected for age (±SD) for adult male and female hyraxes. Males are, on average, in a better body condition than females are (t=2.12; df=127; p=0.036).
Fur coverage Hair growth in the hyrax is rapid. Within two months, bold spots that I created by cutting hair for the hormonal analyses have completely re-covered. Fur condition in a given year is not an indicator of its past or future states. No sexual differences in fur coverage are seen in pups (t=1.712; df=37; p=0.095), nor in juveniles (t=0.071; df=58; p=0.944). In adults, however, male fur coverage is, on average, significantly fuller than females' (t=2.4; df=95; p=0.02; Fig. 12). Fur coverage is not correlated with weight on length residuals.
22
1.8 n = 24 n = 28 n = 29 n = 35 1.6 n = 45 n = 51
1.4
1.2
1
0.8
0.6 Fur coverage Fur
0.4
0.2
0 Male Pups Female Juveniles Adults
Figure 12: Average transformed fur coverage (±SD). Pups and juveniles have similar proportion of fur coverage in both sexes. Adult males have, on average, fuller fur than adult females (t=2.4; df=95; p=0.02). Sample size is above SD bars.
Social hierarchy Hyrax group size, in my study, fluctuated between 6-13 juvenile and adult females and 2-6 juvenile and adult males. Agonistic interactions included displacements from favoured resting and feeding locations, growling, huffing, biting and chasing. Based on these interactions, I used two methods to rank the hyrax: average dominance index (ADI) and David's score. Since the two methods yielded lowly related scores (r=0.08, p=0.04; Fig. 13) and highly correlated ranks (r=0.8, p<0.0001; Fig. 14), I used both. I will present the results using David's score (see methods section). Animals that are higher in the group hierarchy have higher David's scores and lower ranks (the most dominant individual is ranked 1 st , etc.).
23
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1 Average DominanceAverage Index
0 0 1 2 3 4 5 6 7 David's score
Figure 13: Relatedness of two social dominance indices: Average Dominance Index (ADI) and David's score (r=0.08; p=0.04; n=55).
14
12
10
8
6 ADI ranks ADI
4
2
0 0 2 4 6 8
David's score ranks
Figure 14: Relatedness of rank derived from two social dominance indices: Average Dominance Index (ADI) and David's score (r=0. 86; p<0.0001; n=55).
24
Overall, adult females hold more dominant positions, represented by lower rank orders, than adult males (using David's score t=2; df=49; p=0.05; Fig. 15).
female n = 28
male n = 27
0 1 2 3 4 5 6 7 8 9 10
Rank position
Figure 15: Average rank order (±SD) for adult hyrax. The animals on top of the hierarchy are ranked 1 st (lowest in rank order). Females are more dominant, ranking on average lower than males (t=1.998; df =49 ; p=0.05). Order is determined using David's score. Sample size is beside SD bars.
I collected sufficient behavioral data to create hierarchy scales for 4 social groups spanning 5 years. In most social groups ranked, females occupy the highest hierarchy positions in the group (Table 2). Six out of nine of each of the top positions in the social rank (1 st , 2 nd , and 3 rd place) is occupied by females. Out of all of the agonistic dyad interactions observed, 29% were between females, 32% between males and 39% between a female and a male hyrax.
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Table 2: Dominant sex for Ein Gedi social groups. Social rank order is determined using David's score. Rank 1 designates the most dominant group member, 2 the next and 3 is subordinate to 1 and 2. Subordinate ranks 4-9 are not displayed. The sex of ranked hyrax is indicated by the symbols ♀ (female) and ♂ (male). Scores (Average Dominance Index, followed by David's) are shown in parentheses. In group 1, the second position was shared by 3 females in 2002, and by a female and a male in 2003. Social Rank Group Year 1 2 3 1 2000 ♂ (0.81; 4.64) ♀ (0.63; 4.3) ♀ (0.56; 4.2) 1 2002 ♀(0.67; 3.17) 3♀ (0.58; 3.1) ♂ (0.42; 2.9) 1 2003 ♀ (0.64; 5.7) ♀/♂ (0.55; 5.6) ♀ (0.5; 5.4) 3 2000 ♀ (0.6; 5.2) ♂ (0.57; 5.1) ♀ (0.5; 5.05) 3 2001 ♀ (0.85; n/a) ♀ (0.5; n/a) ♂ (0.33; n/a) 3 2002 ♀ (0.61; 4.67) ♀ (0.56; 4.6) ♀ (0.5; 4.5) 3 2004 ♂ (0.6; 5.1) ♀ (0.55; 5.05) ♂ (0.5; 5) 4 2002 ♀ (0.56; 4.16) ♂ (0.5; 4.1) ♀ (0.44; 4) 5 2003 ♂ (0.71; 3.9) ♂ (0.57; 3.7) ♀ (0.5; 3.5)
I used multiple regressions to test whether size or body condition indices are related to social status. In both sexes, rank derived using David's score is related to 2 2 weight (males: R =0.218; F (1,21)=5.861; p=0.025; and females: R =0.245;
F(1,25)=8.133; p=0.009; Fig. 16). Also in females, David's score is negatively related to weight controlled for length (corrected for age), so that dominant females are 2 trimmer (R =0.158; F (1,25)=4.695; p=0.04; Fig. 17).
male female
13 9 12 8 11 10 7 9 6 8 7 5 6 4 5 4 3 3 Rank (David's score) 2 Rank Rank (David's score)
2 1 1 0 0 1.5 2 2.5 3 3.5 1.5 2 2.5 3 3.5 Weight Weight
Figure 16: Rank derived using David's score is correlated with weight in both male (left graph; 2 2 R =0.218; F (1,21) =5.861; p=0.025) and female hyraxes (right graph; R =0.245; F (1,25) =8.133; p=0.009). Dominant animals, ranking in the first places on the social scales, are heavier.
26
5.5
5
4.5
4
3.5 David's score David's
3
2.5 -1 -0.5 0 0.5 Weight controlled for length
Figure 17: David's score as a function of weight controlled for length, corrected for age in adult 2 female hyraxes (R =0.158; F (1,25) =4.695; p=0.04). Dominant females, with higher David's scores, are trimmer than subordinate females.
Linearity and Steepness Linearity (h') is not significant in any of the matrices tested. Steepness is also mostly insignificant (Table 3), due to the relatively high number of dyads in which there is bidirectional winning. In group 3, in 2000, linearity was not significant (h' = 0.383, p=0.36) due to the large number of unknown relationships. However, due to the lack of bidirectional dyads (all non-zero dyads are unidirectional), steepness was significant (0.135, p=0.02) for that group and year. Significant steepness (p<0.0001) was also calculated for group 3 in 2003.
Table 3: Summary of steepness of dominance hierarchies in hyrax social groups in Ein Gedi. Groups 1, 3, 4 and 5 are mixed-sex groups and group 2 is a bachelor male group. Steepness was evaluated for each group on its own and in two cases with neighboring groups. Group 3 shows significant steepness in two separate years. Group Number Observed Expected SD P (year) of hyrax Slope Slope 1 (2000) 11 0.045 0.046 0.009 0.568 2 (2000) 12 0.083 0.068 0.013 0.123 1 & 2 (2000) 20 0.025 0.022 0.003 0.188 3 (2000) 9 0.135 0.095 0.020 0.020 2 & 3 (2000) 17 0.039 0.031 0.005 0.067 1 (2002) 10 0.040 0.039 0.005 0.429 3 (2002) 7 0.058 0.057 0.008 0.510 4 (2002) 9 0.049 0.047 0.006 0.418 2 (2003) 8 0.055 0.050 0.005 0.229 3 (2003) 12 0.027 0.023 0.002 <0.0001 5 (2003) 8 0.080 0.066 0.015 0.213 3 (2004) 11 0.022 0.024 0.002 0.772
27
Males: who sings? Among adult males, individuals that sing are significantly older than ones that do not sing ('silent males'; t=2.792; df=32; p=0.009; Fig. 18). Singers are also, on average, physically bigger than non singers (i.e. morphometrics component; t=4; df=18; p<0.0001), but the size difference is due to their age (i.e. morphometrics component corrected for age is not significant).
70 n = 19 60
50 n = 19
40 Age Age
30
20
10
0 silent male singer
Figure 18: Average age (±SD) of adult males. Males that sing are significantly older than males that are 'silent' (t=2.792; df=32; p=0.009). Age is normalized. Sample size is above SD bars.
Males that sing are more dominant (i.e. rank 1 st or 2 nd in the hierarchy using David's score) than males that do not sing (t= 2.8; df=9; p=0.02; Fig. 19). This effect is robust even when rank was corrected for age.
28 singer n = 8
silent n = 14
0 0.2 0.4 0.6 0.8 1 1.2 1.4 Rank position Figure 19: Average rank position (±SD) in adult males. Males that sing are more dominant (i.e. in the 1 st or 2 nd place), and rank on average higher than males that are 'silent' (t= 2.8; df=9; p=0.02). Ranks are determined using David's score. Low rank score, or position, indicate dominance. Sample size is above SD bars.
Discussion Female hyraxes live longer and are more dominant than males, which are bigger and are also in a better body condition, as adults. Singers are males who are older, bigger, and more dominant than non singing males. Below I discuss the significance of age, fur cover and social rank as indicators of male quality in the hyrax social system. I also discuss the possible motivation that promotes male singing, despite the possible dangers and energy lost involved.
Age and survival Male hyraxes reach full growth at approximately age 4, and live up to the age of 7, in Ein Gedi. When an animal attains old age, and has thus survived fights, illnesses and predator attacks, it has attained valuable experience. Since old age is a rarity among male hyraxes, and might also convey inherent, survival-important qualities, a 5 or a 6 year old male hyrax would not 'want' to be confused with a 3 or a 4 year old. Survival success is an important quality, which may indicate a superior genetic quality, and is often a predictor of reproductive success as well (Clutton- Brock, 1988; Brooks and Kemp, 2001). Older males should therefore be sought out by females as preferable mates (Kokko and Lindstrom, 1996; Kokko, 1998). They
29 also often mate earlier, attaining greater reproductive success (Eberle and Kappeler, 2004). Age, under some circumstances, may also be negatively related to breeding value if male advertisement is so intense that it sacrifices its life, though not detracting from its high quality (Kokko et al ., 2002a). A female's preference for older males may also be a by product of a different preference, for an attractive male trait that happens to be correlated with age. Thus females mating with attractive males may also mate with older males (i.e. selection is not for age directly). Male traits may also be correlated with age because they are correlated with body size (e.g. acoustic and morphological traits). Thus, these traits may take time to fully develop, creating a correlation between age and the size of the trait (Hansen and Price, 1995).
Body condition Most Ein Gedi hyraxes suffer from fur loss and bald spots. The cause of their skin 'mange' is yet to be determined. In the future, skin biopsies as well as bacteriological cultures should be taken, in an attempt to isolate the cause. If hyrax mange is caused by Lipoptena flies, it will be complicated to assess and standardize responses since the time animals are bitten and the number of bites, the amount of scratching and its effects are difficult to measure. The individual variance must be great due to individual strategies and personalities (Korte et al ., 2005). Since optimal immune responses are context specific, rather than generic, depending on the infection status of the animal, a maximum immune response is not always optimal (Viney et al ., 2005). While fur coverage may have an important role in protecting hyraxes from strong radiation and from UV light, I wish to consider the possible role it may play in advertisement, in the context of sexual selection. Both body condition indices used in this study show that female hyraxes are in a poorer body condition than male hyraxes. This is contradictory to Moore and Wilson's (2002) study, which examined the role of parasites in mediating sexual selection pressures in mammals, showing an association between sexual size dimorphism and male-biased parasitism across taxa (Moore and Wilson, 2002). Muehlenbein and Bribiescas (2005), in a review of mammalian immunocompetence levels, conclude that despite conflicting data, females seem to be overall more successful in combating parasites than males. The explanation offered by the reviewers is, that in light of the reproduction-survival trade-off, longevity is more important for female fitness whereas mating rate is more important for male
30 fitness (Muehlenbein and Bribiescas, 2005). This explanation might be applicable in the hyrax system as well, where females outlive males. In many systems, however, parasitism has a high reproductive cost attached (Buchanan et al ., 1999). Females that mate with multiple males are also likely to contract sexually transmitted diseases (Kokko et al ., 2002b) and parasites, which negatively affect body condition. The findings that, in hyrax, adult males have significantly fuller furs than adult females can indicate that: 1. maintaining full fur coverage is indeed expensive. Females that expand a vast amount of energy in reproduction cannot afford this added expense. Males, not burdened by pregnancy and nursing year round, may be able to invest more in fur maintenance, despite the costs imposed by promiscuous mating and shared sleeping quarters. 2. maintaining fur coverage is not a necessity for survival, but rather fur coverage is used as a signal of male quality (i.e. 'good genes'), in a form of a handicap. Under this hypothesis, females prefer males with fuller fur coverage and thus males will 'pay' a price to maintain it. The cost should be high enough so that cheaters (i.e. low quality males) will not be able to maintain full fur coverage (Zahavi, 1977). Cost (i.e. the immunocompetence handicap hypothesis; ICHH) is further discussed in chapter 2. In line with the hypotheses that fur coverage may be a sexually selected trait, and in line with the ICHH, I expected that male singers would have fuller furs than males that are silent. On average, male singers do not have fuller furs than non singers; but I found that singers transmit individual vocal characters that are strongly correlated with fur coverage, allowing them to advertise it (see chapter 3).
Social hierarchy Hyrax females are, on average, more dominant than males. This result is robust even when corrected for age, which is associated with social status. Agonistic interactions in the rock hyrax are not restricted to cohorts, like in fallow deer ( Dama dama ; Jennings et al ., 2006). In most Ein Gedi groups, females are the most dominant member of the group (67% are females in the top three rank positions), yet domination is not unconditional, as seen in the spotted hyena or in the lemurs (Kruuk, 1972; Frank et al., 1985; Kappeler, 1990; VanSchaik and Kappeler, 1996; Radespiel and Zimmermann, 2001; Pochron et al., 2003). Dominant hyraxes are older, bigger animals, as seen in other systems as well (e.g. African elephants, Loxodonta Africana ; Archie et al ., 2006). Dominant female bisons ( Bison bison ) are heavier and fatter, siring heavier offspring as well
31
(Vervaecke et al ., 2005). Conversely, dominant female hyraxes are trimmer than subordinate females. If dominant females have more viable offspring (requiring extended nursing) then their costs are substantially higher; especially considering the long hyrax pregnancy, which amounts to over 60% of their adult lives. Dominant females, who invest in agonistic behaviour in order to base their social status in the group, suffer reduced body conditions. Dominant males are exempt from such a cost. Aggression in hyrax groups is low, probably because most females and some males live in the same area for many years, having well established social relations. Eavesdropping on social interactions is also likely to be prevalent in the highly social hyrax, serving to minimize conflict escalation. When conflicts do arise though, aggression is very fierce, likely enhanced by the 'audience effect' (Johnstone, 2001). For most hyraxes, the best strategy is often to approach, assess the situation, and peacefully leave the area without a confrontation. Hyraxes have a fission-fusion social system (e.g. African elephants, lions and chimpanzees; Watts, 1998; Packer et al ., 2005; Archie et al ., 2006). Sub groups separate and regroup at times (Gerlach and Hoeck, 2001). In my study population, groups 1, 4 and 5 went through a few cycles of separation and regrouping. Group 3 was the most stable, and the only group whose steepness was significant. Steepness can be related to the level of social activity in the group (Stevens et al ., 2005). Overall, hyrax groups can be considered, due to their low slopes and loosely defined linearity, egalitarian (De Vries et al ., 2006). This is consistent with the expectation for species that rely on abundant, widely distributed resources (Vehrencamp, 1983). Despite the fact that lionesses live in a competitive environment with clumped resources, they are also egalitarian, with low reproductive skew, secretive parturition and shared mothering (Packer et al ., 2001), similar to female hyraxes. The importance of rank for males in terms of fitness will be interesting to assess in this egalitarian female dominated system. As rank is not traded off with the body condition indices in my study males, costs are likely present on different planes.
Singers Size is not a good category discriminator for adult males since younger and older, subordinates and dominants may all be the same size. Males must therefore find alternative ways to advertise important features. Survival success, life experience and dominance, in male hyraxes, can be communicated acoustically (i.e. by the actual act of singing). Singers can be generally defined as adult males who are older, larger
32 and more dominant than males that do not sing. These attributes have been shown to be associated with female mate choice and with reproductive success in various systems (Clutton-Brock and Albon, 1979; Creel et al ., 1997; Howard and Young, 1998; Reby and McComb, 2003a; Reby and McComb, 2003b).
The main question, once I have established that song is positively related to age, is whether age indicates fitness in the rock hyrax. If sexual attractiveness (i.e. singing) and fitness are positively related, then the 'good-genes' assumptions, where a trait signals a positive quality, are satisfied (Kokko, 1997a). Longevity and fitness are not necessarily linked, for singers, who exert energy singing and endanger themselves by being conspicuous to predators, may be outlived by males who are silent and sit on the periphery of the groups, conserving energy and waiting for mating opportunities. Also, male sexual advertisements often increase with old age (Preault et al ., 2005), not always indicating an increase in quality, but rather because some traits take time to fully develop, creating a correlation between age and the size of the trait (i.e. Fisherian process; Hansen and Price, 1995). On the other hand, honest sexual advertisements are sometimes not correlated, or are negatively correlated, with age. Theoretically, high quality males may invest so much energy in advertising that they actually die younger than low quality males, despite being preferable mates (Kokko, 1998; Kokko, 2001; Kokko et al ., 2002a).
Assessment of the association between attractiveness and lifetime fitness, through the analysis of male reproductive success, will allow me to consider in the future, whether in hyraxes, male singing satisfies 'good genes' assumptions, linking age, body size and dominance to fitness.
33
Chapter 2: Hormonal profiles Hormones are closely tied to, affect and reflect behaviour. Especially when studying behaviour that is likely to be sexually selected, such as male singing in the hyrax, hormones are implicated as probable mediators.
Introduction Background The gonads, adrenal and fat tissues convert cholesterol to a variety of sex steroids and glucocorticoids (Fig. 20). Hormones are also synthesized de novo in the brain, in different pathways, and act locally in it. Hormones are coordinators of physiological processes and behaviours in and between individuals, over time (Adkins-Regan, 2005). The chemical pathway between androgens ('masculinizing' steroids, i.e. DHEA, androstenedione (A4), testosterone (T) and 5 α-DHT), as well as their aromatization to estrogens ('feminizing' steroids, i.e. estrone, estradiol (E 2) and estriol) are significant to social and to reproductive behaviours. Glucocorticoids such as corticosterone and cortisol (C) are related to basic survival behaviours. Hormones help adjust behaviour to circumstances and to contexts: physical, social, and developmental (Adkins-Regan, 2005).
Steroid hormones are identical across species, and need to be present in only tiny amounts to start behavioural cascades. No sex-specific hormones exist. Androgens can have feminizing effects, and estrogens can have masculinizing effects (Balthazart and Ball, 1998). Hormones can also be made in one place, transported to another cell or tissue, and there modified, amplified, or converted into a different steroid. Circulating steroids might therefore not necessarily be the active steroids for behaviour, but they act as indicators, for example, of an increase in reproductive activity (Balthazart and Ball, 1998). Eventually, steroids are metabolized in the liver to forms that, when excreted from the body, can be informative to conspecifics (Adkins-Regan, 2005). A4, for example, is a potent male pheromone in the goldfish (Sorensen et al ., 2005).
Hormones are responsible for differences in social behaviours between and within the sexes, yet hormones do not usually 'cause' behavioural phenotypes, but rather regulate them by changing thresholds for other factors (Adkins-Regan, 2005). Also, more than one hormone is linked to certain behaviours, and each hormone participates in regulating more than one behaviour. To complicate things further, both
34 behaviour and hormonal responses are results of experiences and of Pavlovian conditioning (Adkins-Regan, 2005).
Information from outside the body is translated into hormonal responses via two portal systems: the HPG, which connects the hypothalamus, hypophysial and gonads, and the HPA, which connects the hypothalamus, anterior pituitary, and adrenal cortex. The HPG is responsible, among other things, for reproduction; while the classic stress response is controlled by the HPA. Both HPG and HPA also affect each other. Continuous neural and hormonal feedback controls hormonal levels (Adkins-Regan, 2005). Androgen levels can be subject to biotic factors influenced by seasonality, such as food availability (Millar and Fairall, 1976), and to social factors, such as competitive interactions (Mazur and Booth, 1998; Oyegbile and Marler, 2005), among others.
Androgens are involved in almost every body function, including neuronal growth, muscle and bone development, immune reactions, epidermal gland development and function, communication, somatostatin release, aggressive and sexual behaviour (Staub and DeBeer, 1997). T is also involved in spermatogenesis and in the development of secondary sex characters (Adkins-Regan, 2005).
Testes are the site for the production of both sperm and androgens. Castrated men have lower metabolic rates and live longer. T levels are found to be positively correlated with testes size across 116 bird species (Garamszegi et al ., 2005) and in some within species comparisons (e.g. Denk and Kempenaers, 2006). T level is also likely to be correlated with spermatogenesis, so that males with high T levels are more likely to win sperm competition over males with low T levels. Garamszegi et al . (2005) hypothesized that testes size increases in order to accommodate sperm competition, and that the rise in T levels is its by-product. A4 is considered a weak androgen since it has a low affinity for androgen receptors and weak peripheral androgenic effects. Despite that, A4 can be a potent organizer of anatomical and behavioral differentiation, able to mimic the effects of T on T-responsive neural systems (Villalba et al ., 1999). It is also a precursor of T, DHT (which usually has a higher affinity to androgen receptors than T and is thus considered a more potent androgen), and of estrogens.
The enzyme aromatase converts androgens into estrogens in target organs and in the brain, which allows regulation of physiological and behavioural processes (such as sexual behaviour and brain differentiation) and responsiveness to environmental
35 cues (Balthazart and Ball, 1998). Because significant levels of aromatase are present in the telencephalon of birds and mammals, it is possible that estrogens, in high local concentrations, affect cognitive processes via non-genomic mechanisms (Balthazart and Ball, 1998).
E2 is important for brain development and sexual differentiation and is thought to be in charge of plasticity in adult brains (Soma et al ., 2004). Estrogen has multiple functions in the control of singing, song development, song organization, and song seasonal plasticity (Fusani and Gahr, 2006). It is also thought responsible for aggression in non-breeding birds (Wingfield et al ., 2001), and is found in high levels in male pigs and horses. Glucocorticoids, such as C, are considered one of the major components of the physiological stress response. They can raise as a reaction to various stressors, such as population density (Rogovin et al ., 2003), food shortage (Perez-Rodriguez et al ., 2006), bad weather (Raouf et al ., 2006), social conflicts (Beehner et al ., 2005; Bergman et al ., 2005) or a predator attach (Sapolsky, 2000). They increase the availability of glucose in the bloodstream and mobilize energy storages in response to crisis (i.e. increase in muscle movement, enhanced cardiovascular tone and sharpened cognition while inhibiting anabolic processes such as digestion, energy storage, growth and reproduction). Excess (chronic) stress can have detrimental effects on metabolism, vascular function, growth, tissue repair, immune defenses, reproduction and neuron health (Sapolsky, 2000).
36
Glucocorticoids
Androgens
Estrogens
Figure 20: Pathways for the synthesis of sex steroids and glucocorticoids in gonadal and adrenal (interrenal) tissue. Most arrows are unidirectional while 2 arrows, between androstenedione and testosterone and between estrone and estradiol (17 β) are bidirectional, indicating that reverse transformation occurs. Copied from Adkins-Regan (2005). The four hormones circled are essayed in this study.
37
Androgens, estrogens and behaviour The androgen testosterone (T) is considered 'the male hormone'. This may be the reason why the role of androgens in female vertebrates has been, until recently, mostly ignored (Staub and DeBeer, 1997), despite the fact that both genders produce androgens in the gonads and adrenal tissue and metabolize them in peripheral tissues. T levels were tested in female elephants (Rasmussen et al ., 1984), spotted hyaenas Crocuta crocuta (Frank et al ., 1985; Glickman et al ., 1987; Glickman et al ., 1992b; Goymann et al ., 2001), humans (Longcope, 1986), California ground squirrels Spermophilus beecheyi (Holekamp and Talamantes, 1991), Greylag geese Anser anser (Hirschenhauser et al ., 1999), placental sharks: the carcharhinids (Rasmussen et al ., 1999), European moles Talpa europaea (Whitworth et al ., 1999), ring-tailed lemurs Lemur catta (von Engelhardt et al ., 2000), and fossas Cryptoprocta ferox (Hawkins et al ., 2002). In all of these studies, T levels in adult females were significantly lower than in adult males, year round. I could not find a single report of a placental mammal whose adult female T levels were equal or higher than those of adult males.
Since T is generally assumed to be associated with dominance and aggression, and since hierarchy is often determined by agonistic interactions (Mazur and Booth, 1998), higher female T levels may perhaps be expected in social systems where females are dominant. However, although in ring-tailed lemurs ( Lemur catta ) females dominate males in all social contexts, their androgen levels are significantly lower than those in males (von Engelhardt et al ., 2000). Spotted hyaena ( Crocuta crocuta ) females are also dominant and more aggressive than males (Kruuk, 1972; Frank et al., 1985; Glickman et al., 1992b), and juvenile female T levels in this species are as high as those of the juvenile males. However, after the age of 26.5 months, once the animals are sexually mature, male T levels are significantly higher (Glickman et al ., 1992b). In adult spotted hyaena females, the dominant circulating androgen is A4 and not T. A4 levels in female spotted hyaenas are reported to be the same, or even higher than in males (Glickman et al ., 1987; Glickman et al ., 1992a; Glickman et al ., 1992b; Goymann et al ., 2001). Adult female fossas ( Cryptoprocta ferox ) have elevated DHT levels (Hawkins et al ., 2002), but not T.
In human males, high levels of T promote dominance behavior, sometimes in the form of aggression (Mazur and Booth, 1998). In mammalian males, T both affects and responds to behavior. T levels rise in the face of a challenge and after winning,
38 and decline in losers after a competition (Mazur and Booth, 1998; Oyegbile and Marler, 2005). Animals use information from previous interactive experiences to adjust their behaviour in subsequent social situations. Androgens respond to the social environment and prepare the individual for competitive situations. For example, T levels in men as well as in cichlid fish (Oreochromis mossambicus ), rise while watching a fight, without actively participating in it (Mazur et al ., 1997; Oliveira et al ., 2001). In human females such responses are not detected (Mazur et al ., 1997). T levels in women are significantly lower (one-tenth; Longcope, 1986) than the levels measured in men, and its association with female aggression or status is a matter of dispute (Cashdan, 1995; Mazur and Booth, 1998; Christiansen, 2001; Cashdan, 2003).
E2 is often considered the behaviourally antagonistic hormone to androgens.
Women with high E2 levels report fewer competitive interactions than other women
(Cashdan, 2003). In meerkats ( Suricata suricatta ), E 2 levels are higher in dominant females than in subordinates, and are higher in females with unrelated breeding partners in the group than in females with related males in the group (Carlson et al ., 2004).
The 'challenge hypothesis' predicts varying androgen responses to mating, breeding or territorial behaviour (Wingfield et al ., 1990). Large meta-analysis of vertebrate males showed that independent of the mating system, high T levels correspond to high frequencies of sexual behaviour, with the largest effects being observed in non paternal species, particularly in mammals (Hirschenhauser and Oliveira, 2006). However, the results provided no support for the predicted modulation of either baseline or situational androgen levels in response to high levels of agonistic interactions by mating or by parental care system (Hirschenhauser and Oliveira, 2006). During the mating season, dominant and more active vertebrate males have higher T levels, which regulates territorial and sexual behaviours, such as song, aggressive display and courtship display (Ketterson and Nolan, 1992; Ros et al., 2004; Chastel et al., 2005; Kabelik et al., 2006). In wild baboons, T levels are positively correlated with aggressiveness but not with copulatory success (Sapolsky, 1982). In the African wild dogs ( Lycaon pictus ), dominant males have higher T levels and mate at higher rates than subordinate males (Creel et al ., 1997). In wild chimpanzees, high- ranking males are more aggressive and have higher T levels than low-ranking males
39
(Muller and Wrangham, 2004a). In captive possums ( Trichosurus vulpecula ), dominant males have higher T levels only during periods of hierarchy formations and during the breeding season (Wehi et al ., 2006). Dominant male sifakas ( Propithecus verreauxi ) have higher T levels than subordinate males, year round (Kraus et al ., 1999). Although there is a vast volume of correlational data between aggressive behaviour in males and T levels, it does not prove a cause-effect relationship, neither is it conclusive. In bonobos ( Pan paniscus ), for example, T levels are not related to ranks in either sex (Sannen et al ., 2004). T levels can also reflect body condition, food availability and social conditions, decreasing with a food shortage (Perez- Rodriguez et al ., 2006), and rising with an increase in group size (Smith et al ., 2005). Aggression can be influenced by these or by other factors indirectly.
Cortisol and behaviour A short-term increase in glucocorticoids serves to redirect energy from reproduction and to channel it towards resolution of the stressful situation. Long-term elevations of glucocorticoids can cause reproductive suppression altogether (Sands and Creel, 2004). Wingfield and Sapolsky (2003) suggested mechanisms whereby despite survival pressures, reproduction can proceed. Experimental administration of glucocorticoids cause increased locomotor activity in Gambel’s White-Crowned Sparrows ( Zonotrichia leucophrys gambelii ) (Breuner et al ., 1998). Captive black-legged kittiwake chicks implanted with corticosterone begged more and were more aggressive compared to controls. Even after the implants were removed, growth rate and cognitive abilities were lower than controls (Kitaysky et al ., 2003). In colonial cliff swallows ( Petrochelidon pyrrhonota ), glucocorticoid levels increased with group size and with ectoparasitism levels, which are related to each other (Raouf et al ., 2006). Experimentally stressed tree lizards had higher glucocorticoid levels (but not T levels) and healed slower from an inflicted wound than control lizards (French et al ., 2006).
Variations in stress and in glucocorticoid levels often relate to social status (Sapolsky, 1982; Wirth et al ., 2006). Glucocorticoids in caged animals are higher in subordinates, which usually have low T levels and low expression of sexual traits and sexual displays; or in animals that lose experimental contests, than in dominant or in winners (i.e. the subordination stress paradigm; Jasnow et al ., 2001; Plavicki et al ., 2004). Contrasting this situation, in free ranging social mammals, such as African wild dogs ( Lycaon pictus ), dwarf mongooses ( Helogale parvula ), ring-tailed lemurs
40
(Lemur catta ), chimpanzees ( Pan troglodytes schweinfurthii ), gray wolves ( Canis lupus ), bisons ( Bison bison ), meerkats ( Suricata suricatta ), Japanese macaques (Macaca fuscata ), and in humans, basal glucocorticoid levels are actually higher in dominants than in subordinates (Creel and Creel, 1991; Creel and Waser, 1994; Creel et al., 1996; Creel et al., 1997; Cavigelli, 1999; Creel, 2001; Barrett et al., 2002; Cavigelli et al., 2003; Carlson et al., 2004; Muller and Wrangham, 2004b; Sands and Creel, 2004; Creel, 2005; Gruenewald et al., 2006; Mooring et al., 2006). But there are blatant exceptions, as in the popular case of the Savannah baboon ( Papio anubis ; (Sapolsky, 1993). Studies from other baboon species ( Papio hamadryas ursinus ) show that high C levels are typical of periods of social instability and hierarchy formation (Beehner et al ., 2005; Bergman et al ., 2005). In primates, subordinates have higher C levels when subjected to more stressors and have less kin (social) support (Abbott et al ., 2003). Another exception are systems where queuing conventions are strictly observed, such as in male spotted hyenas (Goymann et al ., 2003).
Creel et al .(1997) assumed that subordinates rarely suffer from chronic social stress in the wild (Creel, 2001; Creel, 2005). Goymann and Wingfield (2004a) introduced the concept of 'allostatic load' to hypothesize for each sex and social system which social rank 'shoulders the burden' of elevated glucocorticoid levels. Allostatic means 'maintaining stability through change'; and it is sustained through glucocorticoids. According to the allostatic model, subordinate free ranging social mammals can have, under certain conditions, higher glucocorticoids levels than dominant conspecifics. The determinant factor is not social rank itself, but rather the physiological costs that are attached to achieving and maintaining social ranks, which vary in different species, populations and sexes (Sapolsky, 1992; Goymann and Wingfield, 2004a). Another factor effecting allostatic load is personality type. Different types of personality (i.e. hawks or doves) chose different behavioural strategies (i.e. fight-flight or freeze-hide) to cope with stressors and adapt to allostasis (Korte et al ., 2005). The benefits of the different stress responses trade-off with health and disease (Korte et al ., 2005).
The cost of hormones The extraordinary activity during the breeding season, which may cause a huge weight loss in males (e.g. courtship display, maintaining a high social status and high levels of T), may also be responsible for an overproduction of corticosteroids that
41 only individuals in good physical condition can afford. Other costs of high T levels include higher energetic demands, reduced fat stores, interference with pair bonds and parental care, and increased injury due to an increase in activity and aggression, that can also expose individuals to predation and increase mortality (Wingfield et al ., 1990; Wingfield et al ., 2001). The cost of behaviour also includes the cost of the hormonal mechanisms that support it. Physiological costs of HPA activation and of chronic glucocorticoids elevation include heart problems, ulcers, immunosuppression resulting in increased disease risk, neuronal damage and HPG (e.g. reproduction) inhibition (Cooper and Faisal, 1990; von Holst, 1998; Salzet et al., 2000; Sapolsky, 2000; Jasnow et al., 2001). Glucocorticoids are also highly oxidized molecules, of the kind that are toxic and mutagenic (Adkins-Regan, 2005). Immunosuppression can occur due to social conditions and may have fitness consequences, not just for survival but also for mate selection (Nelson and Klein, 2000). Folstad and Karter (1992) proposed that T suppresses the immune system, allowing T mediated sexual traits to serve as honest signals of male quality. The immunocompetence handicap hypothesis (ICHH) predicts that each male has his own optimum T levels allowing maximum trait expression, while minimizing immunosuppression (Folstad and Karter, 1992), which allows females to choose quality males on the basis of their secondary sexual characters (Zahavi, 1975). In wild chimpanzees, T and C levels are synergistically positively associated with parasite richness, possibly interfering with the ability to mount an effective immune response against multiple parasites (Muehlenbein, 2006). This physiological trade-off between T, parasite loads and immunocompetence has been tested in a few systems. Olsson et al .(2000) found that experimentally manipulated male sand lizards ( Lacerta agilis ) exposed to elevated T levels, suffered from increased tick load and mass loss but enjoyed higher mating success, compared to control males (Olsson et al ., 2000). Mixed results leave the hypothesis unconfirmed (Roberts et al ., 2004). Another possibility is that T mediates immunocompetence indirectly by elevating glucocorticoids, which are established immunosuppressants (Evans et al., 2000; Casto et al., 2001). Evans et al .(2000) found that in house sparrows ( Passer domesticus ), experimentally elevated T levels increased glucocorticoids and immunosuppression. When the effects of glucocorticoids are controlled, T is actually an immunoenhancer, probably through its effect on dominance and access to resources (Evans et al ., 2000).
The physiological link between T and glucocorticoids may be body condition (Perez-Rodriguez et al ., 2006). If T is immunosuppressive, individuals in a poor body
42 condition should have lower levels of T to minimize the risks of disease or infection (Perez-Rodriguez et al ., 2006). On the other hand, high T levels can also therefore impair body condition. Roberts et al . (2004) suggested that T and C may compete for binding sites, or share binding proteins that keep them from circulating at high levels, deeming them biologically inert. T implanted dark-eyed junco ( Junco hyemalis ) males display more, have higher metabolic rates, loose winter fat deposits quicker, and suffer higher corticosterone levels and higher mortality rates than controls. They also sing more frequently, feed their nestlings less frequently and are generally more 'restless' than controls (Ketterson and Nolan, 1992). A positive correlation between glucocorticoids and T support the stress-mediated version of the ICHH, rather than the subordination stress paradigm (Schoech et al., 1999; Buchanan, 2000; Evans et al., 2000; Mateos, 2005) Estrogens are expensive and can handicap both sexes in a few known ways. In male song birds, a heavy cost of T is the oncogenic effects of its estrogenic metabolites (Wingfield et al ., 2001). Estrogens are oncogenic in mammals as well.
E2 increases activity levels and metabolic rates in female rats (Wade and Schneider, 1992), and along with T increases oxygen consumption in lizards ( Chalcides ocellatus ; Alsadoon et al ., 1990), and in humans (Muehlenbein and Bribiescas, 2005). Estrous deermice ( Peromyscus maniculatus ) are more likely to suffer predation than non-estrous females because of their odour, which is sex steroid dependent, serving to attract males (Cushing, 1985). Despite the fact that some males have high circulating estrogen levels and that some important behaviours, such as courtship, are based on them, little attention has been paid to estrogens in males (the same as for androgens and females).
Hormones in hyraxes Hyraxes have a distinctive hormonal system, similar in some aspects to the African elephant. For example, circulating progesterone is relatively low during pregnancy, an unusual phenomenon in placental mammals (Heap et al ., 1975; Hodges et al ., 1997; Kirkman et al ., 2001). Both are also noted as having exceptionally high levels of androgens compared to other clades (Gustafson and Shemesh, 1976). In the 1970s, a few endocrinological studies, sparked by interest in the hyraxes' intra- abdominal testes, measured seasonal changes in male T levels (Millar and Glover, 1970; Millar, 1972; Neaves, 1973; Millar and Fairall, 1976; Neaves, 1979). Unfortunately, these studies did not include females, whose T levels were not
43 measured. Preliminary results from rock hyraxes from different populations across Israel, show that females have elevated T levels (Koren et al ., 2006). To the best of my knowledge, however, no studies measured A4, E 2 or C levels neither in males nor in female hyraxes. In order to investigate whether hormones are part of the mechanism, or are mediators of singing behaviour in the rock hyrax, I first set out to describe the hormonal profiles of the system, and than specifically examine singers, comparing them hormonally to non singing hyraxes.
Methods Hormone levels Hair samples were cut from trapped hyraxes in the field, without need for anesthesia. The extraction method has been published (Koren et al ., 2002) and refined (see appendix). This non-invasive method for the extraction of hormones has since been used by other laboratories for the study of different mammal species.
Yearly hair samples, from six field seasons were batch processed. Samples were tested in duplicates in a double-blind experimental set-up. The steroid hormones testosterone (T), Androstenedione (A4), Estradiol (E 2) and cortisol (C) were detected using a competitive ELISA method. In order for hormone levels to be compared across areas and years, results obtained from different ELISA-plates were standardized.
Statistical Analysis
Standardized T levels were normal. The hormones C, E 2 and A4 were further transformed by log (hormone) 2 to linearize the data, when necessary, for linear models. Relationships between standardized untransformed hormones are linear. Correlations between them were tested by permutation. In order to test predicted or functional relationships between hormones, social status, body condition and morphometric variables, I used stepwise multiple regressions through randomization (1000 permutations using Permute! software; version 3.4). Since I had more data for T and C (I started essaying them earlier in the research), I ran the multiple regressions in two steps. First, I entered all four steroids and than, if A4 and E 2 were not involved, I reran the test with only T and C, so that more data can be included in the analyses.
44
Results Sex and Class I compared male and female testosterone (T; Fig. 21), androstenedione (A4;
Fig. 22), estradiol (E2; Fig. 23) and cortisol (C; Fig. 24) levels in pups, juveniles and adults. No significant differences are found between the sexes in their mean hormonal levels (Table 4). Adult females have marginally higher E 2 and C levels than adult males.
Table 4: Summary of results for comparisons between male and female standardized hormone levels in pups, juveniles and adults. Statistics were calculated by randomization (Stat xact v.6). Testosterone Androstenedione Estradiol Cortisol
Pups t39 =0.202; p=0.84 t37 =0.6; p=0.56 t37 =1.95; p=0.85 t37 =0.358; p=0.73
Juveniles t74 =0.77; p=0.45 t64 =0.655; p=0.51 t64 =1.53; p=0.14 T66 =0.477; p=0.64
Adults t120 =1.49; p=0.137 t94 =0.068; p=0.95 t94 =1.9; p=0.058 T119 =2.48; p=0.094
1.5
58 23 1.0 36 16 62 0.5 38
0.0
-0.5 Standardized Testosterone Standardized
-1.0
-1.5 Pups Juv eniles Adults Figure 21: Average age class standardized testosterone (T) levels (±SD) in male (open circles) and female (full circles) hyraxes. Sampled 1999-2005. Sample size is above SD bars.
45
1.5 16
47 1.0 47
21
0.5 32 32
0.0
-0.5 Standardized Androstenedione Standardized
-1.0
-1.5 Pups Juven iles Adults Figure 22: Average age class standardized androstenedione (A4) levels (±SD) in male (open circles) and female (full circles) hyraxes. Sampled 2002-2005. Sample size is above SD bars.
1.5 21 16 47 1.0
32 47 0.5 32
0.0
-0.5 Standardized Estradiol Standardized
-1.0
-1.5
Pups Juven iles Adults
Figure 23: Average age class standardized estradiol (E 2) levels (±SD) in male (open circles) and female (full circles) hyraxes. Sampled 2002-2005. Sample size is above SD bars. Adult females have marginally higher E 2 levels than adult males (t=1.9; df=94; p=0.058).
46
1.5 58
1.0 16 21 61 0.5 32 34
0.0
-0.5 Standardized Cortisol Standardized
-1.0
-1.5 Pups Juveniles Adults Figure 24: Average age class standardized cortisol (C) levels (±SD) in male (open circles) and female (full circles) hyraxes. Sampled 2000-2005. Sample size is above SD bars. Adult females have marginally higher C levels than adult males (t=2.48; df=119; p=0.094).
When levels are analyzed in adults per year and per gorge, adult female rock hyraxes have, on average, higher hormonal levels than adult males (Fig. 25-28). Since some animals were sampled more than once in the study (i.e. on different years), separate t-tests were necessary to test each year and location. To avoid type I errors (i.e. obtaining statistically significant results by chance), in order to consider the results from these multiple tests, sequential Bonferroni adjustments were used (Sokal and Rohlf, 1995), setting the experiment-wise error rate to be α<0.005 for T and α <0.006 for C, E2, A4. Under these conditions, no differences are significant between male and female hormone levels.
47
Figure 25: Average standardized testosterone (T) levels ( ±SD) for adult male (open circles) and female (full circles) hyraxes across the study sites and years. Stars indicate significant differences between the sexes. Average female T levels were significantly higher than males in Arugot in 2000 (t=2.5; df=32; p=0.017) and in David in 2004 (t=2.5;df=9; p=0.035). Sample size is above SD bars.
Figure 26: Average standardized androstenedione (A4) levels ( ±SD) for adult male (open circles) and female (full circles) hyraxes across the study sites and years. Sample size is shown above SD bars.
48
Figure 27: Average standardized estradiol (E2) levels ( ±SD) for adult male (open circles) and female (full circles) hyraxes across the study sites and years. Average female E2 levels were significantly higher than males in David in 2004 (t=3.191; df=8; p=0.012; indicated by the star). Sample size is above SD bars.
Figure 28: Average standardized cortisol (C) levels ( ±SD) for adult male (open circles) and female (full circles) hyraxes across the study sites and years. Stars indicate significant differences between the sexes. Average female C levels were significantly higher than males in Arugot in 2002 (t=2.584; df=14; p=0.022), in 2004 (t=2.072; df=23; p=0.05), and in 2005 (t=2.527;df=16; p=0.023). Sample size is above SD bars.
49
Using multiple regression analyses, I tested whether hormones at young ages were associated with hormonal levels and with social ranks in adulthood. No relations were found between juvenile hormone levels and adult status or levels in 2 males. In females, juvenile T levels predict adult T levels (R =0.29; F (1,13) =5.316; 2 p=0.038) and adult ranks (R =0.662; F (1,4)=7.84; p=0.049).
Hormone correlates In males, androgens are correlated with each other, and to a lesser degree with cortisol (Table 5). In females, trends are similar but are much stronger. Androgens are highly correlated with each other (Fig. 29) and with cortisol levels (Fig. 30).
Table 5: Table of correlations for steroid hormones tested in male (left) and in female (right) adult rock hyraxes. Values above the diagonal are the p values, calculated through permutation. Values below the diagonal are the r values. Adult males Adult females p p r T C E A r T C E A T 0.0722 0.0743 0.0149 T 0.0002 0.0924 0.0001 C 0.2423 0.9454 0.0633 C 0.6081 0.8543 0.0020 E 0.2676 -0.0052 0.4261 E 0.2426 0.0295 0.3833 A 0.3464 0.2839 0.1113 A 0.5900 0.4329 0.1326
male female
3 3 3 3 2 2 2 2 1 1 1 1 0 0 -1 -1 -1 -1
Standardized Standardized Androstenedione -2 Standardized Androstenedione Standardized -2 -2 -2 -2 -1 0 1 2 3 4 -2 -1 -1 0 1 1 2 2 3 Standardized Testosterone Standardized Testosterone
Figure 29: Standardized androgen levels are associated in both sexes; more strongly in females (on the right; r=0.59; n=46; p=0.0001) than in males (left graph; r=0.346; n=46; p=0.015).
50
0.8 5 0.6
0.4 4
0.2 3 0.0 2 -0.2
-0.4 1
-0.6 0 Standardized Cortisol Standardized -0.8 Cortisol Standardized -1 -1.0
-1.2 -2 -3 -2 -1 0 1 2 3 -2 -1 0 1 2 3 4 Standardized Testosterone Standardized Testosterone
Figure 30: Cortisol levels are correlated with testosterone levels in adult female hyraxes (on the right; r=0.608; n=58; p=0.0001) and marginally in adult males (left graph; r=0.242; n=59; p=0.0722). Both hormones are standardized. Fur coverage In order to examine whether androgens are associated with the hyraxes' resistance to parasites, I looked at indirect evidence, through fur coverage. Multiple regressions were tested by permutation. In adult males, androgen levels were not significantly related to fur coverage. In adult females, A4 levels were significantly associated with fur coverage (R2=0.114; n=37; p=0.048; Fig. 33). Females with higher androgen levels had less fur coverage.
0.4
0.2
0
-0.2
-0.4 Fur Fur coverage
-0.6
-0.8 -4 -3 -2 -1 0 1 2 Standardized Androstenedione
Figure 31: Fur coverage as a function of androstenedione (A4) levels in adult female hyraxes (R2=0.114; n=37; p=0.048). Females with higher A4 levels have less fur coverage. Fur coverage is transformed and A4 levels are standardized and transformed.
51
Hormones and social hierarchy
Androgens Since the variances within the sexes for the hormone levels in adult hyraxes are profound, I compared the sexes in the context of social status. When comparing only dominant animals (i.e. in the 1 st or 2 nd rank position in the social hierarchy), no differences are seen between male and female androgen levels. Between subordinate hyraxes (ranking 3 rd or lower in the social hierarchy), there are significant differences between male and female androgen levels. Low ranking females have higher T levels than low ranking males (t=2.04; df=31; p=0.05; Fig. 34). Low ranking males, on the other hand, have higher A4 levels than low ranking females (t=2.557; df=18; p=0.02; Fig. 35).
1.5 17
1.0
20 4 0.5
0.0
5 -0.5 Standardized Testosterone Standardized
-1.0
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Subordinate Dominant
Figure 32: Adult average standardized testosterone (T) levels (±SD) in male (open circles) and female (full circles) hyraxes, categorized by rank position. Dominants are all individuals in the 1st and 2 nd hierarchy position and subordinates are in the 3 rd position or lower. Ranks within the groups were scaled using David's score. In high ranking hyraxes (i.e. dominants), there are no differences between the sexes in T levels. In lower ranking hyrax (i.e. subordinates), females have significantly higher T levels than males in the same social class (t=2.04; df=31; p=0.05). Within female hyrax, subordinates have significanly higher T levels than dominant females (t=3.9; df=19 ; p=0.001). Sample size is shown above SD bars.
52
1.0 5 11 0.5
3 11 0.0
-0.5
-1.0
-1.5 Standardized Androstenedione Standardized
-2.0
-2.5 Subordinate Dominant
Figure 33:Adult average standardized androstenedione (A4) levels (±SD) in male (open circles) and female (full circles) hyraxes, categorized by rank position. Dominants are all individuals in the 1 st and 2 nd hierarchy position and subordinates are in the 3 rd position or lower. Ranks within the groups were scaled using David's score. In high ranking hyraxes (i.e. dominants), there are no differences between the sexes in A4 levels. In lower ranking hyrax (i.e. subordinates), males have significantly higher A4 levels than females in the same social class (t=2.557; df=18; p=0.02). Sample size is shown above SD bars.
Androgens play unusual roles in female hyrax. Juvenile T levels, in females, 2 are good predictors of social status in adulthood (David's score; R =0.662; F (1,4) =7.84; p=0.049), so that juvenile females with higher T levels are likely to be more dominant adults. Paradoxically, high ranking adult females (ranked 1 st and 2 nd using David's score) have significantly lower T levels than low ranking females (ranked 3 rd -8th ; t=3.9; df=19; p=0.001; Fig. 34). In females, both androgens T and A4 are inversely related to dominance parameters so that more dominant females have lower androgen levels. Multiple regression by permutation linked rank (derived by David's score) with T levels (R 2=0.213; n=22; p=0.026; Fig. 36), and David's score with A4 levels (R 2=0.277; n=16; p=0.029; Fig. 37).
53
3
2.5
2
1.5
1
0.5
0
-0.5 StandardizedTestosterone -1
-1.5 0 1 2 3 4 5 6 7 8 9 Rank (David's score)
Figure 34: In adult females, ranks (derived by David's score) are associated with standardized T levels (R2=0.213; n=22; p=0.026). The more dominant females rank in the first places (i.e. 1,2) on the social scale, and have lower T levels.
1
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0
-0.5
-1
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-2
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StandardizedAndrostenedione -3
-3.5 2.5 3 3.5 4 4.5 5 5.5 6 David's score
Figure 35: In adult females, David's score is associated with standardized androstenedione (A4) levels (R2=0.277; n=16; p=0.029). As social status, reflected by David's score, increases A4 levels decrease. A4 levels are transformed.
54
Cortisol On average, dominant females have double the concentration of C than subordinates (t=1.76; df=20; p=0.094; Fig. 38).
0.5 n=5
0.0
n=18 -0.5
-1.0
-1.5 Standardized Cortisol Standardized
-2.0
dominant subordinate -2.5
Figure 36: Average standardized cortisol (C) levels (±SD) in adult females, categorized by rank position. Dominants are all individuals in the 1 st and 2 nd hierarchy position and subordinates are in the 3 rd position or lower. Ranks within the groups were scaled using David's score. Dominant females have marginally higher C levels than subordinates (t=1.76; df=20; p=0.094).
Males: who sings? Males that sing have a typical masculine hormonal profile. Singers have higher T levels and lower E2 levels than males that do not sing (i.e. silent males; t=2.4; df=14; p=0.029; Fig. 39 for T and t=2; df=32; p=0.05; Fig. 40 for E2). Singers also have higher C levels (t=2.4; df=38; p=0.02; Fig. 41) than silent males.
55
0.6 12
0.4
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0.0
-0.2
15 -0.4
Standardized Standardized Testosterone -0.6
-0.8
silent singer Figure 37: Average standardized testosterone (T) levels (±SD) in adult male hyraxes. Singers have higher T levels than non singers (i.e. silent males; t=2.4; df=14; p=0.029 ). Sample size is shown above SD bars.
-0.4 20
-0.6
14 -0.8
-1.0
-1.2 Standardized Standardized Estradiol
-1.4
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silent singer
Figure 38: Average standardized estradiol (E2) levels (±SD) in adult male hyrax. Singers have lower E2 levels than non singers (i.e. silent males; t=2; df=32; p=0.05). Sample size is shown above SD bars. E2 levels are transformed.
56
-0.2 19
-0.4
-0.6
-0.8 21
-1.0
-1.2 Standardized Standardized Cortisol
-1.4
-1.6 singer silent Figure 39: Average standardized cortisol (C) levels (±SD) in adult male hyrax. Singers have higer C levels than non singers (i.e. silent males; t=2.4; df=38; p=0.02). Sample size is shown above SD bars. C levels are transformed.
In males that sing, contrary to the general adult male population, rank derived using David's score is negatively correlated with cortisol levels (r=-0.71; n=8; p=0.049; Fig. 42), so that high ranking males (in the 1 st position in the social rank) have the highest C levels. Hormone levels in singers do not correlate with each other, as opposed to the case in all males.
57
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Standardized Cortisol Cortisol Standardized -1
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-1.4 0 1 2 3 4 5 6 Rank (David's score)
Figure 40: Standardized cortisol (C) levels are associated with rank in male singers. Among males that sing, those that rank in the first places in the social scale have higher C levels than singers that rank lower in the group (r=-0.71; n=8; p=0.049).
Discussion Hyraxes are monomorphic in many aspects. Even in their hormonal profiles, no differences are detected between the sexes in any of the age classes. In this study, I focus on adults, in an attempt to link steroid hormones to strategies and behaviours in male and in female hyraxes. I found interesting associations between androgens and social status, and androgens and body condition in females but not in the population of adult males. Despite gross deviations from the expected mammalian pattern in the intersexual hormonal comparisons, synthesis with social factors and comprehension of the system can suggest a few explanations. Singers comply with the typical mammalian norm in their hormonal profile, in comparison to non singing adult males, providing evidence for singing as a sexually selected trait.
Intersexual comparisons: levels, fitness and correlates T and A4 are classified as androgens, which are considered 'male' sex hormones. Despite this definition, in the rock hyrax, adult females do not have lower androgen levels than adult males. In fact, both sexes have, on average, similar hormonal levels. Furthermore, when T levels are compared per year, it is evident that
58 females have, on average, higher levels than males. In two cases, the differences are statistically significant. To my knowledge, no other female mammal has such elevated T levels, compared to males (see introduction to this chapter). A4 is considered a weaker androgen than T. In the spotted hyenas, adult females have elevated A4 levels (Glickman et al ., 1987; Glickman et al ., 1992b; Goymann et al ., 2001). In the rock hyrax, A4 levels in females were no different than in males, across all years tested. In female fossas, another androgen (dihydrotestosterone) is elevated, but not T (Hawkins et al ., 2002). Different receptors or affinities for receptors may be responsible for elevation of different androgens in females of different species.
Female hyraxes breed every year and nurse pups for up to a year (LK personal observation). High androgen levels are expected to interfere with female reproduction. Wingfield et al . (2001) suggested possible mechanisms that may help to alleviate the high costs of T and continue reproduction. These include resistance, social modulation, insensitivity, hypersensitivity, precursor and neurosteroid hypotheses. Female hyraxes may cope with the high cost of high T levels by either having hormone binding proteins that prevent high circulating androgen levels, or by acquiring insensitivity to the behavioural effects of high androgen levels, by down regulating receptors and enzymes (Soma et al ., 1999; Wingfield et al ., 2001). Another possibility is that my hormone collecting method is not suitable to reflect the actual trend in rock hyrax. Since hair-testing samples and averages a few months of hormonal levels, it is unable to discriminate between short steep surges (peaks) and maintenance of longer, medium hormonal levels. It may in fact be that males maintain higher T levels most of the time, and females have low baseline T levels that spike up (i.e. high T level peaks) at parturition and mating (i.e. social modulation of secretion; Wingfield et al ., 2001). Despite this possibility, I do not think it is likely since female rock hyraxes show consistently higher T levels than males even when tested in different locations and in different seasons. Blood samples were consistent with my findings as well (Koren et al ., 2006). Another interesting finding is that the hormonal differences seen between the sexes are not reflected in dominant animals (1 st or 2 nd in the social rank), but rather in subordinates (3 rd and lower). When splitting hyraxes into dominants and subordinates, no differences are seen in androgen levels between high ranking males and females. Between subordinates, though, males and females differ significantly.
59
Low ranking females have higher T levels than low ranking males. Low ranking males, on the other hand, have higher A4 levels than low ranking females.
E2 is considered a 'female hormone', yet differences between male and female adult levels are marginal. In most individual years analyzed, females had no higher
E2 levels than males. Since T and A4 can be aromatized to estrogens, and since estrogens are also responsible for some aspects of male sexual behaviour, it is understandable that both sexes can have a similar range of E 2 levels. In the future, it will be interesting to investigate, in hyraxes, the role of progesterone, which is considered a 'female hormone' as well, but is a precursor to androgens ('upstream' rather than 'downstream' like E 2).
Adult females have marginally higher C levels than adult males. When C levels are compared per year, females have significantly higher average C levels in 3 years. Higher adult female C levels can be due to their higher ranks, as seen in chapter 1, as well as to their extensive reproduction efforts. In all hormones, individual differences are considerable, resulting in big statistical variances. When statistical differences are significant despite the vast intra-sexual variations, then results are robust.
Hyraxes have a distinctive hormonal system (Heap et al ., 1975; Hodges et al ., 1997; Kirkman et al ., 2001; Koren et al ., 2006), noted as also having exceptionally high levels of androgens compared to other clades (Gustafson and Shemesh, 1976). When males have very high androgen production, females of those species often do as well (Staub and DeBeer, 1997). Hyrax may also be similar to African elephants (and spotted hyenas) in that they initially develop male-type external genitalia prior to gonadal differentiation, opposite to the rest of the known mammals (Glickman et al ., 2005). This early development may explain the common matriarchal social systems in these species, as well as hormone eccentricities, such as elevated androgen levels.
Hormones act differently in males than in females. For example, in European stonechats, no sex differences are found in T levels (Schwabl et al ., 2005), yet only in males, and not in females, T, C, and E 2 levels increase following an experimental disturbance (Canoine and Gwinner, 2005). In humans and in dark-eyed juncos ( Junco hyemalis ) as well, contest-related fluctuations in T levels are seen in males but not in females (Mazur et al., 1997; Jawor et al., 2006). Also in juncos, circulating T levels predict dominance status in males and not in females (Jawor et al ., 2006).
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Experimental manipulation of egg yolk corticosterone in Japanese quails ( Coturnix coturnix japonica ), showed delayed growth in male but not in female chicks. In adults, the opposite is seen, whereas HPA response decreased in females but not in males (Hayward et al ., 2006). Generally, female steroid levels are more sensitive to condition than male levels (Adkins-Regan, 2005). Differences between the sexes may be due to a difference in receptor numbers, types, dispersion or affinity, rather than to circulating hormonal levels (Soma et al ., 1999). These differences can be tested with specific or general receptor blockers. In female hyraxes, more so than in males, T and C levels are positively correlated. Christiansen (2001) reports that along with a raise in C levels in a stressful event (or an expectation of such) in humans, there is a sex-typical opposite reaction: a significant decline of T levels in males and an increase in T levels in females. Both reactions though may have the same function, suppressing fertility in order to increase survival (Christiansen, 2001). In male elephants ( Loxodonta africana ), during the musth period, T levels increase while glucocorticoids levels do not (Ganswindt et al ., 2005). Interaction between androgens and glucocorticoids in males vary with social factors. Successful male baboons increase T levels in response to stress while subordinates decrease it (Sapolsky, 1982). Males with high copulation rates had low basal C levels and fast, steep elevations in C levels following stress (Sapolsky, 1982). The relationship between T and glucocorticoids is also condition dependant. Males in good shape that have their T levels experimentally raised have low glucocorticoids and males in low condition react to the same experimental setup with elevated glucocorticoids levels (Evans et al., 2000; Buchanan et al., 2003a).
Hormones at younger ages can shape animal development and behaviour later on in life. Equally, adult behaviour is an indication of its developmental history. In my study, juvenile female T levels predicted both adult T levels and adult social ranks. Organizational hormone theory stresses the enormous effect hormones have earlier in life. Pup hormonal levels are known to shape sexual differentiation and social behaviour. Endogenous hormones, as well as maternal hormones and hormones that originate from siblings sharing the womb, can all affect the brains of pups (Adkins-Regan, 2005). Once I have the results from the relatedness genetic data, I can test maternal, paternal and co-siblings effects on pup sex, survival parameters, hormonal levels and social ranks.
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Fur coverage Fur coverage, among other things, seems to be a morphological indicator of androgen levels in female rock hyraxes. In adult males, T levels are weakly correlated with fur coverage. In adult females, A4 levels are negatively correlated with fur coverage. The immuno-competence-handicap hypothesis (ICHH) may apply to male rock hyraxes. If quality males have high T levels and maintain full coats, despite of immunological costs inflicted by the high T levels, than an honest signal of male quality is communicated. If T is an immunoenhancer, and not an immunosuppressor (Schoech et al., 1999; Evans et al., 2000), than males with high T levels would have healthier looking furs. Fur coverage is an immunocompetence 'bulletin board', needing maintenance for a healthy look. Another option is that T enhances the immune system behaviourally (i.e. dominance may increase access to food), which results in a better body condition and greater immunocompetence (Evans et al ., 2000).
Androgens are costly and females may pay more than males for elevated levels, since it not only survival is challenged, but reproduction is compromised as well. The negative correlation between androgen levels and fur coverage in females, implicate androgens as immunosuppressants in the rock hyrax, although C may be indirectly involved as well.
A key assumption in the ICHH is that there is a tight dose-dependant relationship between small variations in T levels and the degree of trait expression, which produces variations in signal expression and maintain the honesty of the signal by effecting immunocompetence (Folstad and Karter, 1992). The problem is that a dose-dependant relationship between the hormonal levels and the possible effects does not necessarily exist. On the contrary, most experiments to date have hinted that the hormonal mechanism is often more similar to a threshold response, with individual variation in thresholds, and not dose-dependant at all (Hews and Moore, 1997). Another possibility that Hews and Moore (1997) raise involve the uncoupling of trait expression and androgen levels. They suspect that the ICHH is open to 'cheaters' and is thus not evolutionarily stable, if a mutant arises that can exhibit a high degree of a trait elaborately without having high androgen levels (Hews and Moore, 1997). Examples for uncoupling that already occur in nature are species that breed using environmental rather than hormonal cues or species with sex role reversals (Eens and Pinxten, 2000). In black coucals ( Centropus grillii ) for example,
62 females are more aggressive yet males have higher T levels (Goymann and Wingfield, 2004b).
Social hierarchy In a preliminary report that included 10 males and 14 female hyraxes, I reported that male T levels were highly correlated with BBS ranks (Jameson et al ., 1999) and that there was no correlation between social ranks and T levels in females (Koren et al ., 2006). Increasing the sample size to 28 males and 26 females has resulted in the following opposite relationships: In males, no relationship is found between social status and hormonal levels. On the other hand, in females, social status is negatively correlated with androgen levels (both T and A4). Low ranking females have higher T levels and higher A4 levels than high ranking females. Overall, subordinate females have higher androgen levels than dominants, possibly causing them to act in a manner typical of mammalian males, such as a lack of choosiness in the mating season (McGlothlin et al ., 2004). Subordinate females may therefore mate with lower quality males. Experimentally elevated T levels have been shown in different bird systems to effect female fecundity (Rutkowska et al ., 2005) and delay the onset of female reproduction (Ketterson et al ., 2005). In fur seals, androgens regulate embryonic delay (Browne et al ., 2006). Since T can be costly to female reproduction, it will be interesting to test, whether in the hyrax system, low ranking-high T females have lower reproductive success than high ranking-low T females. Despite the fact that all adult females reproduce every year, regardless of their rank (LK, personal observation), subordinates may be producing less viable, lower-quality offspring, mediated by high T levels. Operational sex ratios may also be influenced by hormone-rank interactions (see general discussion). Significant correlations between dominance rank and T levels were documented in both sexes of the naked mole-rat (Clarke and Faulkes, 1997). In that eusocial mammal, queens are at the top of the female hierarchical ladder and have the highest level of T among females. In female hyraxes, the relation between androgen levels and social status is inverse, which makes the interpretation, as is the case with human and most other mammalian females, more obscure (Staub and DeBeer, 1997; Mazur and Booth, 1998; Christiansen, 2001).
In most social mammals, dominant males have higher T levels than subordinate males (Ketterson and Nolan, 1992; Creel et al., 1997; Wingfield, 2005), but there are exceptions. In stable groups, for example, members may attain a social 63 rank and hold on to it (i.e. 'social inertia'), regardless of T levels (Adkins-Regan, 2005). Another exception are spotted hyena males, where natal males are dominant to dispersing males, despite having significantly lower T levels (Holekamp and Smale, 1998). C levels in male hyaenas are not related to rank because of strict queuing rules (Goymann et al ., 2003). Animals with high T levels are expected to take risks, which would lead them to high stress levels (Schoech et al ., 1999), mediated through high C levels, which is useful for them since they need the fight or flight mechanism continuously operating (Creel et al ., 1996). Because of the trade-off with the immune system, social stress (i.e. high glucocorticoids levels) may be a cost (or a handicap) that offsets the benefits of high ranks (Creel et al., 1996; Creel, 2001). Schoech et al .(1999) showed that T is probably an immunocompetence enhancer, but by inducing C levels, the effect is minimized. Generally, dominants have elevated glucocorticoids levels when a hierarchy is unstable (see Muller and Wrangham (2004b) for an exception related to energy factors) or in systems where dominants fight more often than subordinates (Sapolsky, 1992; Creel, 2001; Abbott et al., 2003). Despite my results that dominant female rock hyraxes have low androgen levels, and that T and C are highly positively correlated, I also found that dominant females have marginally higher C levels than subordinates. High C levels are associated with high ranks in several cooperative breeders with a high reproductive skew (Creel, 2001; Creel, 2005). In ring-tailed lemurs, where females are dominant and reproductive skew is low, C levels are highest in the most dominant female group members (Cavigelli, 1999; Cavigelli et al ., 2003). In male lemurs, C is not associated with rank (Gould et al ., 2005). In female golden lion tamarins ( Leontopithecus rosalia ), there are no differences in C levels between dominants and subordinates (Bales et al ., 2005). This may be due to the low levels of aggression and the high level of social support available to subordinate females (Abbott et al ., 2003; Bales et al ., 2005). Hyrax females live in stable groups for extended periods (some their whole lives) and are likely to be genetically related. Reproductive skew is low and several chores, such as babysitting, are shared among group members. Despite that, dominants have higher C levels, possibly as a result of the high reproductive efforts that may be attached to their potentially higher reproductive success. Their lower T levels interfere less with reproduction, providing dominants with a strong fitness advantage over subordinates.
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Physiological metabolic costs vary between individuals, relating to body size, group size, food availability and many other biotic and abiotic factors, which can also mediate the relationship between social status and HPA axis response (Muller and Wrangham, 2004b; Lindstrom et al., 2005). Relationships involving hormones are very complex, influenced by almost every possible endo- and exogenous factor. Patterns are often masked due to vast individual differences (Guimont and Wynne- Edwards, 2006), social contexts (Wingfield et al ., 1990) and species peculiarities. To further complicate matters, when a behavioural trend is not reflected by circulating steroid levels, it may be due to changes in steroid receptor numbers in the brain, which changed due to social interactions that mediated gene expression (Fernandez- Guasti et al ., 2003).
Singing Hyrax males that sing are 'typical mammalian males' in the hormonal definition. Singers have higher T levels than non-singers. Singers often reply to other singers, countersinging in synchrony, or alternating songs, as a form of a contest. Singers may very well be involved in an ongoing competition, a situation which is known to elevate T levels (Mazur and Booth, 1998). Singers may be characterized as a 'hawk' personality type. They expose themselves to predation and to rival males, causing their C levels to rise ('fight or flight' mode is 'turned on'). In male ring- necked pheasants ( Phasianus colchicus ), dominant and high displaying males have, in the mating season, higher levels of both T and corticosterone, which are positively correlated (Mateos, 2005). Hyrax singers have lower E2 levels than non singers; possibly indicating that less T is aromatized (most remaining in the potent 'masculine state'). Also, among males that sing, C levels positively correlate with social ranks. High ranking singers have higher C levels than low ranking singers, a known trend in most studied socially breeding mammals (Creel, 2001), which may be explained by high aggression levels and activity attached to high rank. This trend is not present in the general adult male hyrax population.
Because of the high cost attached to high T and C levels, not singing, for some hyrax, might be a better strategy than singing. If males that do not sing live longer (because they are involved in less fights and suffer less injuries) and lose less energy (on fighting, as well as on singing), it might be an equally successful strategy. Even if yearly reproductive success is much lower in non-singers than in singers, life-time reproductive success (given longer, healthier lives), may be greater than that of 65 singers. It will be interesting to compare both singing and silent morphs in terms of total fitness.
Singing, after all is a signaling system. Other ways to signal information that cannot be seen, such as hormonal profile, in the rock hyrax, is using scent marking. Females should be assessing male quality using steroidal odours since these are likely to honestly reflect individual quality. Male hyraxes, both singers and non-singers mark rocks on popular paths by dribbling a white liquid from their penis (which is retracted during the spring). Another possible way to mark could be sneezing. Hyraxes sneeze a lot. In squirrel monkeys, sneezing rate is positively correlated with male T levels (Hennessy et al ., 1980). Hyraxes too may spray around information bearing molecules by sneezing. Another possible way for hyraxes to distribute chemical information is through their unique secretory dorsal gland. Both sexes have it, and hyraxes often lie on their backs on common paths. The purpose of this behaviour has been assumed to dust off ectoparasites, but it might alternatively or additionally serve as marking behaviour. Hyraxes also have cutaneous glands on their foot pads, which are supposed to improve the traction between their feet and the steep rock formations they move on. Despite their classification as eccrine (thermoregulation) and not as apocrine (scent) glands, they have several similarities to human sweat glands: secreting large amounts of fluids, and containing antibacterial activity (Stumpf and Welsch, 2002). These skin glands are good candidates as messengers, emitting information containing molecules about individual condition and quality, to the environment.
Hormones are involved in complex biochemical interactions, are influenced by and influence a vast array of fields. In the study of hormones, their impact and their interactions, the unknown, and possibly immeasurable, is, unfortunately, enormous. The hormone story is a black box. Given the complexity and their role in communication within and between animals, including hormones in behavioural ecology studies is still in the primary stage. Preconceptions hinder progress. Once more species are tested and examined, perhaps we will find out that hyraxes are not at all unusual, and accepted conventions will be altogether changed.
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Chapter 3: Why Sing? Introduction Singing discloses information about the identity of the singer and communicates information to conspecifics. Singers may advertise their virtues and prove their merits by providing information pertaining to a close by predator (Riede and Zuberbuhler, 2003), a rival or a food source, in their songs. Voices change with time throughout life, reflecting conditions of age and health (Titze, 2000). Vocalizations are useful for adversaries in aggressive interactions to assess the sizes of their opponents (Zahavi, 1975; Bee et al., 1999; Davidson and Wilkinson, 2004), and potential mates are expected to choose partners based on signals, such as time and energy consuming song repertoires, that reliably indicate individual quality (Lambrechts and Dhondt, 1986). Conspecifics extract information from solo vocalizations as well as from vocal interactions, and use that information in subsequent encounters (Peake et al., 2001; Leboucher and Pallot, 2004).
Vocal recognition Vocal recognition is an important part of group living. The ability to identify the sender of messages, and to react according to its reputation and reliability, has important fitness implications. Individual differences are likely to be found in all acoustically communicating species, and have been revealed in mice (Holy and Guo, 2005), harbour seals Phoca vitulina (Hanggi and Schusterman, 1994), mouse lemurs Microcebus murinus (Zimmermann and Lerch, 1993), chacma baboons Papio cynocephalus ursinus (Fischer et al ., 2001; Fischer et al ., 2002), Fallow deer Dama dama (Reby et al ., 1998), bowhead whales Balaena mysticetus (Potter et al., 1994; Mellinger and Clark, 2000), wolves (Tooze et al ., 1990), and in several penguin systems (Jouventin and Aubin, 2002; Searby et al ., 2004). Male common loons (Gavia immer ) produce individually characteristic yodels to differentiate themselves from other males. When they change their territories, they change their vocalizations so that they would be clearly differentiated from the last resident of the new territory (Walcott et al ., 2006). In several systems, vocalizations also provide information on sex (e.g. Kloss' gibbons; Tenaza, 1976) and group affiliation (e.g. African elephants (McComb et al ., 2003; Soltis et al., 2005b), Mangabeys Cercocebus albigena (Waser, 1977b), Arctic foxes Alopex lagopus (Frommolt et al ., 2003), and Rhesus macaques Macaca mulatto ; Ghazanfar et al ., 2001). Playback experiments showed that red deer
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(Cervus elaphus ) hinds are able to discriminate between the roars of their harem- holder stag and those of other neighbouring stags (Reby et al ., 2001). Juvenile Richardson's ground squirrels ( Spermophilus richardsonii ) showed greater vigilance in response to experimental neighbours' alarm calls relative to strangers' alarm calls (Hare, 1998). Male bullfrogs ( Rana catesbeiana ) also possess a capacity for individual voice recognition, on the basis of pitch. In a playback study, territorial males discriminated between the voice of a familiar neighbour and the calls of strangers, towards whom they were more aggressive (i.e. "dear enemy effect"; Bee and Gerhardt, 2002). Parent-offspring recognition is important in gregarious species (e.g. in herds of domestic sheep Ovis aries ; Searby and Jouventin, 2003). In the subantarctic fur seal ( Arctocephalus tropicalis ), only once the pups learn to recognize their mothers' voices, at 2 days old, do mothers depart to feed (Charrier et al ., 2001). In the migratory northern fur seal ( Callorhinus ursinus ), mothers and offspring retain recognition for at least 4 years (Insley, 2000). In several penguin systems parents also leave chicks for extended periods of time and then use acoustic call characteristics to locate them (Aubin and Jouventin, 2002; Jouventin and Aubin, 2002).
Signaling information honestly Songs are composed of various elements. In water pipits ( Anthus spinoletta ), a specific song element, the 'snarr', is key to intraspecific male interactions. Males with high 'snarr scores' are more dominant, mate more often, and have territories with less overlaps than males with low scores (Rehsteiner et al ., 1998). In male barn swallows ( Hirundo rustica ), a similar element, the 'rattle', is positively related to plasma T levels and inversely varied with body mass and condition. Other song features in male barn swallows vary according to their social environment, reflecting competitive potential (Galeotti et al ., 1997). Both the 'rattle' and the 'snarr' are emphasized in competitive situations. Stereotypic repetitive elements are often used in male-male communication (McElligott and Hayden, 1999). Such displays may permit individuals to assess their opponents, allowing them to avoid fighting when they are unlikely to win (Zahavi, 1975; Zahavi, 1977). Male song is a sexually selected trait shown to be related to reproductive success in many species (Andersson, 1994). Song may be used to communicate individual quality to potential female mates (Tenaza, 1976) as well as to potential male competitors (Bee et al ., 1999; McElligott and Hayden, 1999). Song production and quality have been shown to depend on and reflect parasite levels (Moller, 1991;
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Buchanan et al., 1999; Garamszegi, 2005), immunological condition (Saino et al ., 1997; Simmons et al ., 2005), food availability (Spencer et al ., 2003), hormonal levels (Ketterson and Nolan, 1992), as well as stamina (Fischer et al ., 2004). Females may choose males that sing more because they are likely to be in a better condition or more strongly motivated than others (Galeotti et al ., 1997). One of the first signs that vocalization is a source of information in mammals came from Clutton-Brock and Albon (1979) that showed in red deer, that roaring rates are highly correlated with fighting ability, providing an accurate indication of males' abilities to repel intruders (Clutton-Brock and Albon, 1979). McComb (1991) used playback experiments to show that hinds are more likely to look and move toward stags roaring at higher rates and toward stags initiating roaring bouts (McComb, 1991). In fallow deer, high vocalization rate is linked to mating success (McElligott et al ., 1999). In the greater white-lined bat ( Saccopteryx bilineata ), different male vocalizations are directed at males (noisy broadband calls) than at females (tonal calls), and song repertoires are linked to reproductive success (Davidson and Wilkinson, 2004). Several song parameters relate to winter dominance position, survival and lifetime reproductive success in the great tit ( Parus major ), reflecting male quality (Lambrechts and Dhondt, 1986). In sedge warblers (Acrocephalus schoenobaenus ), males with more complex songs, pair successfully (Airey et al ., 2000). In different bird systems, complexity of calls are also indications of developmental history, representing individual quality (Buchanan et al ., 2003b; Spencer et al ., 2003; Spencer et al ., 2004; Spencer et al ., 2005a; Spencer et al ., 2005b). Large song repertoire sizes have been shown to indicate in birds ultimate benefits (i.e. good genes for offspring viability), as well as proximate benefits (i.e. bigger higher vocal centers (HVC) in the brain; Airey et al ., 2000; Catchpole, 1996). Older male sedge warblers tend to have larger song repertoires and testes, but not greater sperm numbers (Birkhead et al ., 1997). Male singing may also serve to enhance female reproduction. Roaring by male red deer advances female ovulation, and harem-holding males improve their mating success by calling regularly (McComb, 1987). In canaries and in song sparrows, male song (both con-and heterospecific) enhances follicular development; females exposed to song lay more eggs, sooner (Bentley et al ., 2000). In captive canaries, egg size increases with exposure to playbacks of male songs, especially to 'sexy' experimentally synthesized syllables (Leitner et al ., 2006).
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The source-filter theory The main source of sound in mammals is the glottal airflow produced by vocal fold vibration (Titze, 2000). Air from the lungs is converted into acoustic energy in the larynx. Sound can also be produced in other places along the vocal tract by turbulences in the air stream or by the release of air pockets (Titze, 2000). Soft tissues (i.e. lips or tongue) can also oscillate to produce sound (Titze, 2000).
Fundamental frequency (F 0) is the rate at which the vocal folds (i.e. source) vibrate (Fitch and Hauser, 2002). It is connected to their length and stress and has traditionally been linked to body size, since the length of the vocal folds is assumed to increase with body size (Titze, 2000). On the basis of this assumption, it has often been predicted that larger animals should produce lower pitched vocalizations (Morton, 1977). In female hamadryas baboons ( Papio hamadryas ), body size is closely related to F 0 (Pfefferle and Fischer, 2006), yet in males, in a few systems that were examined, no relation was found between F 0 and physical measurements within age classes (Reby and McComb, 2003a; Reby and McComb, 2003b). When males become sexually mature F 0 systematically drops (Fischer et al ., 2002). Since F 0 is sensitive to glottal pressure and to fold stress it can reflect the motivation of callers.
As male baboons age, rank, as well as F 0 drop and calls shorten (Fischer et al ., 2004).
Calls produced by dominant male baboons have higher F 0 than calls produced by subordinate males (Fischer et al ., 2004). F 0 also has a strong heritable component; with sons inheriting the F 0 of their fathers (Reby and McComb, 2003b). The sound generated from the glottis includes many frequencies, which are filtered by the vocal tract (i.e. pharyngeal, oral and nasal cavities) before radiated out from the mouth and the nose. Since the air column in the vocal tract has elasticity and mass properties, it vibrates as the sound energy passes through it, filtering the source signal. The shape and the length of the vocal tract controls air flow and density, which discriminately filters certain frequencies out and amplifies others (i.e. formant frequencies; Titze, 2000). Formant frequencies are independent of the source, are not correlated to pitch (or F 0), and are inversely related to vocal tract length. They are supposed to provide an honest indication of body size since the vocal tract is firmly bounded by the bones of the skull, whose size is closely tied to overall body size (Fitch, 2000b). Formant dispersion, or the average spacing between formants, decreases with increased body size or mass in male dogs, red deer, rhesus macaques, black and white colobus monkeys, and in men (Fitch, 1997; Riede and Fitch, 1999; Reby and McComb, 2003a; Bruckert et al., 2006; Harris et al., 2006) and appears to 70 be an example of cheap, honest communication (Fitch and Hauser, 2002). As well as evidence of body size, formant frequencies can also provide reliable clues of identity, age, gender, social rank or reproductive success, as shown in baboons, red deer stags, and in men (Reby and McComb, 2003a; Fischer et al., 2004; Bruckert et al., 2006). In mammals, the vocal tract extends from the larynx to the lips or nostrils. It can therefore be elongated by extending the nose, protruding the lips, or by lowering the larynx in the throat during vocalization (Fitch, 2000a). In a few species the larynx has descended (Fitch and Reby, 2001), functioning to exaggerate the impression of body size (Fitch, 1997), and potentially dissociating the relationship between vocal tract and body size, as maybe seen in human males (Gonzalez, 2004), conflicting the above (Bruckert et al ., 2006). In tonal calls, formants are detectable only if they coincide with harmonics. Formants particularly stand out in harsh (aperiodic) sounds, where the sound source has a broad frequency spectrum and no harmonics which could be confused with the formants (Fitch and Hauser, 2002).
Hormones and song Androgens affect signaling frequencies. In general, males communicate at lower frequencies than females, even in electric fish (orders Mormyriformes and Gymnotiformes) with an electric organ discharge (EOD; Staub and DeBeer, 1997). In men (and not in women), higher T levels are significantly associated with low pitch voices (Dabbs and Mallinger, 1999), low formant frequencies and small formant dispersions (Bruckert et al ., 2006). Song features, such as acoustic structure or duration, that reflect muscle state, are also probably androgen mediated, since muscles are sensitive to steroids (Adkins-Regan, 2005). The larynx and the vocal folds can also be influenced by androgens, growing independently of the rest of the body and showing sexual dimorphism. T may physiologically change the bulk, length or tension of the vocal cords, or it may psychologically affect the motivation of an individual to alter its pitch in its social interactions (Dabbs and Mallinger, 1999). In human males at puberty, for example, androgen receptors in the laryngeal cartilages respond to increased circulating T with a profound growth spurt (Adkins-Regan, 2005). The mechanisms are still poorly understood despite centuries-old common knowledge, which led to the castration of choir boys in order to preserve their high pitch and avoid their voices from lowering at puberty.
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Singing in animals involves interactions of endocrine states and social circumstances. In male Thomas langurs ( Presbytis thomasi ), social changes as well as the increase in T levels underlie the loud call differences between males that live in all-male bands and males living in mixed-sex groups (Wich et al ., 2003). Bird song, in both sexes, is influenced by androgens. In young male songbirds, the development of song is mediated through T, secreted by the testicles. Castration leads to incomplete or unstable songs, and hormone replacement leads to mature songs. Females also sing complex male-like repertoires when induced by T treatment (Adkins-Regan, 2005). Castration of adults gradually abolishes song (Kroodsma et al , 1982). In several bird species, it has been shown that androgen levels, aggressiveness and singing are positively correlated (reviewed in Ketterson and Nolan, 1992). In song birds, T regulates the enlargement of "song control nuclei" (HVC, which are dedicated to song and to song perception), at great oxygen consumption costs (Wennstrom et al ., 2001). In canaries, only T has that effect, and only on the HVC (Sartor et al ., 2005). Experiments show that in song sparrows, not only T, but also the androgen DHT, and E 2 increase the volumes of three song nuclei (HVC, archistriatum and basal ganglia homolog), compared to castrated birds (Tramontin et al ., 2003). When the androgen precursor DHEA was added experimentally at physiological levels to non-breeding males, singing increased along with the volume of the HVC (Soma et al ., 2002), probably due to aromatization of DHEA to E 2 (Soma et al ., 2004). Aromatase, found in large amounts in the brains of male songbirds, converts androgens to E2, which binds to unique estrogen receptors, found only in the brains of songbirds (Fusani and Gahr, 2006). In canaries, male song complexity affects both the hormonal state and brain functions of female audience. Certain 'sexy' syllables experimentally played to females provoked greater sexual displays, higher T levels, and increased HVC and song discrimination abilities, relative to control females (Marshall et al ., 2005). Stress also effects singing performance. Stressed male zebra finches ( Taeniopygia guttata ) sing shorter, simpler songs than non- stressed males (Spencer et al ., 2003), whose songs are clearly preferred by females (Spencer et al ., 2005b). In light of the scarce body of data available on vocalization behaviour in social mammals, I wanted to examine the information that male hyraxes communicate through singing, pertaining to individual quality. The rock hyraxes in Ein Gedi sing
72 in individually distinctive voices (Koren, 2000), most of the year. For a few months following the mating period, singing stops, implicating male singing as a sexual advertisement. In this chapter, I attempt to connect specific vocal components to age, body size and condition, social status and hormonal parameters.
Methods Singing males were tape recorded during three field seasons (2002-2004). I recorded during the morning session (first light out to noon), when sound propagation is at its peak. Strong winds interfere with sound recordings in the afternoons.
Equipment All recordings were made with a Sennheiser ME 67 shotgun microphone (Frequency response 50-20000 Hz ±2.5 dB) with a Sennheiser K6 powering module, covered with a Sennheiser MZW70-1 blimp windscreen. Microphone was hand held or placed on a tripod, using a MZS20-1 shock mount with a pistol grip. Recordings were captured using a battery operated, portable Marantz PMD-222 cassette recorder
(Frequency response for CrO 2 Tape 40-14000 Hz; signal to noise ratio: 57 dB) on Maxell XLII-60 analogue tapes. The time, singer and social situation during which it was recorded were noted using the tape recorder's counter as a time reference. Vocalizations were digitized using Avisoft SAS Lab Pro (Specht, 2002) at a sampling frequency of 44 kHz, with 16 bits. After visual inspection, tracks were downsized to 22.05 kHz or 32 kHz using an appropriate antialiasing filter. Spectrograms were measured at 256 FFT lengths, 100% frame, using a Hamming window, which gave a frequency resolution of 86 Hz with a 112Hz bandwidth and a temporal resolution of 5.8ms at a 50% overlap.
Song analysis For each hyrax, up to 3 songs were analyzed per year. Songs were picked on merit of best signal to noise ratio. Noise pollution in the deep David gorge is greater than in Arugot due to its funnel shape. Three main song characters were identified and analyzed: wails, chucks and snorts (Fig. 43). Songs are composed of several bouts. Each bout usually begins with a wail. All hyrax songs from Ein Gedi contain wails. Song bouts are rarely composed purely of wails. Most wails are followed by one or by numerous chucks. Chucks are short, harmonic elements that are sometimes
73 interrupted by snorts. Snorts are not present in all hyrax songs, and vary in the number of repeats. They may also follow, precede or replace a wail or a chuck.
kHz kHz 10 kHz 14 8 10 12 10 6 8 8 6 4 6 4 2 4 2 2 0.5 1.0 1.5 s 0.1 s 0.2 s Figure 41: Song elements in hyrax song: wail, chuck and snort (from left to right). Fundamental frequency and equally spaced harmonics are seen in the wail and in the chucks. The heavier bands are the formant frequencies, easily distinguished in the snort's broad frequency band.
The different elements used in each song, their duration, number of repetitions, rates and temporal variation were measured from the sonograms using the Avisoft SASLabPro cursors, and transferred to Excel (Microsoft, 2002) for analyses. Fundamental and frequency of the harmonics were extracted in the same way. Measurements were averaged per hyrax per year. Measurements and abbreviations used are summarized in Table 6.
Filter estimation Measurements of the vocal anatomy of hyraxes were conducted with the help of Dr. Yuval Zohar (a nose, ear, and throat specialist), by detailed surgery of hyraxes that died from a car collision. These measurements were used to predict the number of formants expected (N) below the Nyquist frequency of the sampled signal ( fc ; roughly half the sampling rate). N= (2L/c) fc ;where L is vocal tract length and c is the speed of sound (350m/s in the moist 37 0C air of a mammalian vocal tract). In order to remain conservative and avoid measuring possible 'pseudo-formants', formant frequencies extracted from the wail element were not included in the analyses. First formant characteristics (minimum, average and maximum frequencies) were therefore considered only for snorting hyraxes, using the Praat software (Boersma and Weenink, 2004). Linear predictive coding (LPC) is a spectral modeling technique used to estimate formant frequencies in human speech. I used model order 6-8 and number of poles 3-9, visually selecting the optimum number of coefficients for each slice by superimposing LPCs on spectrographs. Formant analysis parameters were: 0.01-0.05 sec time step; 2-5 maximum number of formants; 5000-11000 Hz maximum formants. Formant frequency values were transferred to Excel and
74 averaged over the duration of the snort. Formant dispersion could not be calculated because the number of formants extracted for each hyrax varied (range 1-4).
Table 6: Summary of variables extracted from hyrax songs. Some variables are general, describing the performance of hyraxes during a given year and others are specific measurements of song variables and of elements. Abbreviations are used in calculations of PCA analysis. Element Measurement Abbreviation per hyrax overall song duration recorded duration per year number of files songs were recorded on files number of songs recorded nsongs average song length tsong average per number of bouts nbouts song bout length tbout bout rate (number of bouts per song) rbout total bout length divided by song length singing wail average number in a song nwails number per song length rwail total length twail average length ave.wail maximum length max.wail chuck average number in a song nchucks average length tchuck number per song length rchuck average number per bout chuck.bout maximum number per bout max.chuck snort average number in a song nsnorts total number of snorts recorded snorts maximum number of snorts in a row sno.row harmonics minimum fundamental frequency min.Fo maximum fundamental frequency max. Fo average fundamental frequency ave. Fo minimum first harmonic frequency min.2 maximum first harmonic frequency max.2 average first harmonic frequency ave.2 minimum second harmonic frequency min.3 maximum second harmonic frequency max.3 average second harmonic frequency ave.3 formants minimum first formant frequency min.first maximum first formant frequency max.first average first formant frequency ave.first
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Statistical Analysis Vocal recognition was analyzed using stepwise discriminate function analysis (STATISTICA; Stat soft, 71). Principal Component Analysis (SPSS; v. 13.0) was used to group the 21 measurements extracted for hyrax song into 6 components, the 3 measurements extracted from the formant frequency into 1 component and the 9 measurements for the harmonic frequencies into a single component as well. One of the 6 components (Rates) was correlated to the harmonics component and was therefore removed from the analyses. In order to test which song variables (harmonics, formant, snorts, wails, extremes, counts, and chucks; 7 independent variables) were used in hyrax communication, I set age, weight, morphometric component, fur coverage, hormonal levels and social status as dependant variables in a stepwise multiple regression (through randomization, 1000 permutations; Permute; v. 3.4). Other than weight (r=0.823; n=11; p=0.002), none of the dependant variables were related to age in singers. The formant component was limiting and not available for all singers (since not all singers snort and formants were extracted only from the snort element). Multiple regressions were therefore often calculated twice, once with all of the independent variables, and again without the formant component, when it did not contribute, in order to allow for more individuals to participate, increasing sample size.
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Results Only 17 out of 54 adult males sang during 2002-2004 (a third of the potential singers). The mean ±SD age of singers, at the time of singing, is 4.1±1.3 years. Eleven males were recorded singing only one year, one male was recorded singing two years and five males were recorded singing all three years. Table 7 shows the number singers and the number of songs that were recorded per year. On average, 15.9 songs were recorded per hyrax (range 1-86). Mean ±SD song duration is 85.7±50 seconds, with the longest recorded song lasting 214 seconds. Mean ±SD number of bouts per song was 16.7±10.9, with the maximum being 43. All songs contained wails, and except for one, all songs contained chucks, with the maximum number of chucks recorded in a row being 51 (average ±SD chucks in a row=12.8±10). Only 11 out of the 17 singers (i.e. 65%) snorted, with a maximum number of snorts recorded per song being 63 (average ±SD snorts per song=14.3±14.7).
Table 7: Summary of the number of male hyraxes singing and the number of songs that were recorded per year in Ein Gedi.
2002 2003 2004 Number of singers 10 6 12 Number of songs 103 106 61
Vocal recognition Discriminate function analyses (DFA) separated singers that sang at least 2 songs (6 in Arugot and 9 in David), on the basis of their song parameters (for Arugot and David, Wilks' Lambda=0.000001; p<0.00001). Step-wise DFA entered 18 significant song variables for Arugot and 13 for the David analyses. Discriminate analysis separated singers effectively in most cases. Squared Mahalanobis Distances (Sq. MD) test for differences among centroids of different singers. In Arugot most dyads were significantly separable (R3 and O1 are an exception; Table 8), while in David, five dyads could not be significantly separated, meaning that their song features were similar (for example M9 and X1; Table 9). The first two roots in the stepwise analysis were significant for both locations ( χ2; p<0.05; Table 10 for Arugot and Table 11 for David). Tables 12 and 13 (for Arugot and David, respectively) show the unstandardized canonical scores for the separate songs analyzed for the discriminate analysis. At the top of Tables 14 and 15 are the a priori classification probabilities which were calculated for each hyrax from the number of songs analyzed. These are
77 the probabilities that songs belong to a male at random. Below are the posterior probabilities calculated for Arugot and David hyrax (respectively), which note the success rate of discrimination. All songs were assigned to the appropriate singer. A graphical representation of the vocal recognition shows discrimination based on a plot of the first and second canonized score roots for the analyzed songs (Fig. 44 for Arugot and Fig. 45 for David).
Table 8: Summary of discriminant function analysis (DFA) in Arugot. Squared Mahalanobis Distances (Sq.MD), F-values and significance is shown for each individual centroid dyad. Singer R3 T6 M2 O1 T5 Q7 Sq.MD=1895 Sq.MD=177635 Sq.MD=42834 Sq.MD=1772 Sq.MD=19729 F(18,4) =68.9 F(18,4) =4699 F(18,4) =648 F(18,4) =27 F(18,4) =298 P=0.0005 P<0.0000001 P=000005 P=0.003 P=0.00003 R3 Sq.MD=215974 Sq.MD=62511 Sq.MD=201 Sq.MD=9471 F(18,4) =7856 F(18,4) =1119 F(18,4) =4 F(18,4) =170 P<0.0000001 P=0.000002 P=0.1 P=0.00008 T6 Sq.MD=46515 Sq.MD=212433 Sq.MD=315695 F(18,4) =703 F(18,4) =3211 F(18,4) =4772 P=0.000004 P<0.0000001 P<0.0000001 M2 Sq.MD=60485 Sq.MD=120408 F(18,4) =640 F(18,4) =1274 P=0.000005 P=0.000001 O1 Sq.MD=10451 F(18,4) =111 P=0.0002
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Table 9: Summary of discriminant function analysis (DFA) in David. Squared Mahalanobis Distances (Sq.MD), F-values and significance is shown for each individual centroid dyad. Singer Q1 X2 A67 X1 A12 T3 T1 O7 M9 Sq.MD=180.8 Sq.MD=406.7 Sq.MD=94.3 Sq.MD=33.8 Sq.MD=49.3 Sq.MD=287.2 Sq.MD=184.4 Sq.MD=639.8 F(13,8) =8.3 F(13,8) =21.45 F(13,8) =4.35 F(13,8) =1.9 F(13,8) =1.8 F(13,8) =13.3 F(13,8) =6.8 F(13,8) =33.7 P=0.003 P=0.00009 P=0.02 P=0.17 P=0.2 P=0.0005 P=0.005 P=0.00006 Q1 Sq.MD=846.6 Sq.MD=55.5 Sq.MD=86.7 Sq.MD=165.6 Sq.MD=790.1 Sq.MD=673.5 Sq.MD=1345.7 F(13,8) =44.65 F(13,8) =2.56 F(13,8) =5 F(13,8) =6.1 F(13,8) =36.5 F(13,8) =24.9 F(13,8) =71 P=0.000005 P=0.09 P=0.01 P=0.008 P=0.00001 P=0.00005 P=0.000001 X2 Sq.MD=809 Sq.MD=546.3 Sq.MD=312.4 Sq.MD=80.2 Sq.MD=211.6 Sq.MD=159.3 F(13,8) =42.7 F(13,8) =37.4 F(13,8) =12.8 F(13,8) =4.2 F(13,8) =8.7 F(13,8) =9.8 P=0.000006 P=0.000011 P=0.0006 P=0.02 P=0.002 P=0.001 A67 Sq.MD=38.7 Sq.MD=140 Sq.MD=654.7 Sq.MD=509.2 Sq.MD=1180.8 F(13,8) =2.2 F(13,8) =5.2 F(13,8) =30.2 F(13,8) =18.8 F(13,8) =62.3 P=0.13 P=0.01 P=0.00002 P=0.0001 P=0.000001 X1 Sq.MD=44.3 Sq.MD=416.7 Sq.MD=320 Sq.MD=847.1 F(13,8) =1.9 F(13,8) =24 F(13,8) =14.1 F(13,8) =57.9 P=0.17 P=0.00006 P=0.0004 P=0.000002 A12 Sq.MD=255.5 Sq.MD=223.2 Sq.MD=595.7 F(13,8) =9.4 F(13,8) =6.9 F(13,8) =24.4 P=0.002 P=0.005 P=0.00005 T3 Sq.MD=74.4 Sq.MD=120.3 F(13,8) =2.7 F(13,8) =6.3 P=0.078 P=0.0067 T1 Sq.MD=206.8 F(13,8) =8.5 P=0.0025
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Table 10: χ2 Tests with successive roots removed for Arugot. The first two roots are significant. Roots Eigen Canonicle Wilks' Chi-Sq. df p-level Removed value R Lambda 0 44344.6 0.999 0 259.7 90 0 1 27 0.982 0.0004 109.9 68 0.001 2 9 0.948 0.01 63.2 48 0.07 3 4.3 0.9 0.1 31.1 30 0.4 4 0.73 0.65 0.58 7.7 14 0.9
Table 11: χ2 Tests with successive roots removed for David. The first two roots are significant. Roots Eigen Canonicle Wilks' Chi-Sq. df p-level Removed value R Lambda 0 229.3 0.998 0.000001 228.7 104 0 1 19.8 0.976 0.000331 136.2 84 0.0003 2 4.1 0.896 0.006889 84.6 66 0.06 3 3.0 0.867 0.035 56.9 50 0.2 4 1.6 0.785 0.14 33.3 36 0.6 5 0.7 0.631 0.37 17.1 24 0.8 6 0.5 0.562 0.6 8.4 14 0.9 7 0.1 0.330 0.89 2 6 0.9
Table 12: Unstandardized canonical scores for analyzed songs performed by male singers in Arugot. Song Singer Root 1 Root 2 Root 3 Root 4 Root 5 1 R3 103.28 -1.06 -0.60 -2.15 0.69 2 R3 107.13 -2.35 -1.18 -1.49 -0.35 3 R3 107.28 -0.46 -0.33 -0.17 -0.94 4 R3 105.50 -2.12 1.41 -2.91 -0.60 5 R3 105.22 -2.74 0.22 -4.07 0.82 6 R3 105.37 0.48 0.98 -1.18 1.54 7 R3 104.93 -1.83 -0.43 -1.36 -0.14 8 R3 105.82 -0.73 0.36 -1.42 0.26 9 R3 104.91 -1.66 -2.02 -1.62 -0.40 10 R3 106.15 -0.28 0.82 -1.88 -0.61 11 R3 106.74 -1.15 2.32 -2.35 0.36 12 Q7 63.14 -3.39 -1.59 2.90 1.20 13 Q7 61.92 -2.85 -0.42 2.16 -3.45 14 Q7 63.39 -2.82 -0.89 0.41 -1.56 15 Q7 61.45 -3.63 0.31 3.09 -2.28 16 Q7 62.09 -3.28 -0.26 2.69 0.45 17 T6 -357.27 -2.22 0.61 -1.14 0.72 18 T6 -359.24 -2.61 0.98 1.16 0.15 19 T6 -359.58 -2.36 1.37 0.58 0.37 20 T6 -359.13 -1.76 0.30 0.13 0.11 21 T6 -360.07 -2.55 2.09 0.14 0.49 22 M2 -143.72 11.76 -5.58 -0.17 -0.06 23 M2 -144.17 11.35 -6.33 -0.92 -0.85 24 O1 101.96 8.33 6.79 1.81 0.11 25 O1 101.33 11.78 6.86 1.23 0.01 26 T5 202.50 0.56 -3.79 2.09 1.84 27 T5 203.07 -2.43 -1.99 4.42 2.13
80
Table 13: Unstandardized canonical scores for analyzed songs performed by male singers in David. Song Singer Root 1 Root 2 Root 3 Root 4 Root 5 Root 6 Root 7 Root 8 1 M9 5.31 -2.20 -0.14 1.64 0.74 0.93 -0.01 0.77 2 M9 6.04 -2.81 1.39 1.47 -3.16 -1.62 -0.75 0.03 3 M9 5.67 -2.63 1.42 2.58 -1.76 -1.26 2.07 -2.24 4 Q1 16.36 4.31 -0.69 -0.32 1.81 -1.42 -1.29 0.80 5 Q1 16.47 4.62 -2.82 0.72 0.56 -0.87 0.48 -0.66 6 Q1 17.49 3.32 -0.57 2.28 0.43 0.72 -2.19 -0.44 7 X2 -13.41 6.40 0.57 1.46 -0.64 0.19 0.73 0.73 8 X2 -12.26 6.32 0.65 1.37 -1.48 0.43 0.50 0.72 9 X2 -12.11 7.37 1.86 0.99 -0.36 -0.61 0.56 0.98 10 X2 -10.24 7.86 1.32 -0.26 1.20 0.39 -0.45 -1.62 11 A67 15.92 -3.41 1.59 -1.50 -1.93 1.92 -0.11 0.46 12 A67 14.19 -0.40 -0.05 0.24 -1.69 0.22 -0.51 0.25 13 A67 14.59 -2.18 0.83 0.85 -0.88 1.41 -0.74 0.14 14 X1 11.06 -1.37 -0.82 -0.97 0.55 -1.48 0.28 0.06 15 X1 10.46 -1.45 -1.08 0.60 0.73 -1.63 -0.24 -0.05 16 X1 7.59 -0.93 0.63 -1.08 0.62 0.92 -0.19 0.15 17 X1 9.65 -0.95 -1.22 -2.63 -0.42 0.39 1.90 -0.54 18 X1 9.69 -1.28 -0.35 -2.40 0.09 -1.30 1.51 1.78 19 A12 4.43 0.46 -2.26 -1.21 2.44 1.14 1.43 -0.72 20 A12 4.61 3.80 -0.56 -1.25 -0.16 1.78 0.01 -0.47 21 T3 -10.32 -0.81 2.04 -2.08 0.71 -1.54 -0.99 0.49 22 T3 -9.23 -0.81 1.83 -3.13 -0.73 0.96 -0.85 0.33 23 T3 -11.54 -0.90 4.38 -2.46 0.63 -0.47 -0.01 -1.50 24 T1 -6.72 -4.46 1.42 2.45 2.47 0.46 -0.13 0.14 25 T1 -6.73 -7.85 2.10 2.62 2.43 0.38 0.16 0.25 26 O7 -19.57 -2.84 -3.94 -0.19 -1.06 0.00 -1.20 0.17 27 O7 -17.86 -1.64 -1.44 1.33 0.01 0.57 1.65 1.51 28 O7 -19.60 -2.57 -3.93 0.21 -1.07 0.70 -0.44 -1.13 29 O7 -19.89 -2.96 -2.13 -1.35 -0.08 -1.31 -1.17 -0.42
81 Table 14: Posterior probabilities for Arugot singers. All hyrax songs were assigned, with the highest probability (p=1, a full match), to the appropriate male singer. A priori classification probabilities, based on the number of songs analyzed for each singer are presented in the top row. Observed Q7 R3 T6 M2 O1 T5 Classification p=0.185 p=0.407 p=0.185 p=0.074 p=0.074 p=0.074 1 R3 0 1 0 0 0 0 2 R3 0 1 0 0 0 0 3 R3 0 1 0 0 0 0 4 R3 0 1 0 0 0 0 5 R3 0 1 0 0 0 0 6 R3 0 1 0 0 0 0 7 R3 0 1 0 0 0 0 8 R3 0 1 0 0 0 0 9 R3 0 1 0 0 0 0 10 R3 0 1 0 0 0 0 11 R3 0 1 0 0 0 0 12 Q7 1 0 0 0 0 0 13 Q7 1 0 0 0 0 0 14 Q7 1 0 0 0 0 0 15 Q7 1 0 0 0 0 0 16 Q7 1 0 0 0 0 0 17 T6 0 0 1 0 0 0 18 T6 0 0 1 0 0 0 19 T6 0 0 1 0 0 0 20 T6 0 0 1 0 0 0 21 T6 0 0 1 0 0 0 22 M2 0 0 0 1 0 0 23 M2 0 0 0 1 0 0 24 O1 0 0 0 0 1 0 25 O1 0 0 0 0 1 0 26 T5 0 0 0 0 0 1 27 T5 0 0 0 0 0 1
82 Table 15: Posterior probabilities for David singers. All hyrax songs were assigned, with the highest probability (p=1, a full match), to the appropriate male singer. A priori classification probabilities, based on the number of songs analyzed for each singer are presented in the top row. Observed M9 Q1 X2 A67 X1 A12 T3 T1 O7 Classification p=0.103 p=0.103 p=0.138 p=0.103 p=0.172 p=0.069 p=0.103 p=0.069 p=0.138 1 M9 1 0 0 0 0 0 0 0 0 2 M9 1 0 0 0 0 0 0 0 0 3 M9 1 0 0 0 0 0 0 0 0 4 Q1 0 1 0 0 0 0 0 0 0 5 Q1 0 1 0 0 0 0 0 0 0 6 Q1 0 1 0 0 0 0 0 0 0 7 X2 0 0 1 0 0 0 0 0 0 8 X2 0 0 1 0 0 0 0 0 0 9 X2 0 0 1 0 0 0 0 0 0 10 X2 0 0 1 0 0 0 0 0 0 11 A67 0 0 0 1 0 0 0 0 0 12 A67 0 0 0 1 0 0 0 0 0 13 A67 0 0 0 1 0 0 0 0 0 14 X1 0 0 0 0 1 0 0 0 0 15 X1 0 0 0 0 1 0 0 0 0 16 X1 0 0 0 0 1 0 0 0 0 17 X1 0 0 0 0 1 0 0 0 0 18 X1 0 0 0 0 1 0 0 0 0 19 A12 0 0 0 0 0 1 0 0 0 20 A12 0 0 0 0 0 1 0 0 0 21 T3 0 0 0 0 0 0 1 0 0 22 T3 0 0 0 0 0 0 1 0 0 23 T3 0 0 0 0 0 0 1 0 0 24 T1 0 0 0 0 0 0 0 1 0 25 T1 0 0 0 0 0 0 0 1 0 26 O7 0 0 0 0 0 0 0 0 1 27 O7 0 0 0 0 0 0 0 0 1 28 O7 0 0 0 0 0 0 0 0 1 29 O7 0 0 0 0 0 0 0 0 1
83
Figure 42: First and second canonized score roots for Arugot singers. Discrimination is significant (Wilks' Lambda=0.000001; F (90,23) =11.893; p<0.00001). Ellipses represent 95% confidence limits around individual centroids.
84
Figure 43: First and second canonized score roots for David singers. Discrimination is significant (Wilks' Lambda= 0.000001; F (104,66) =3.8451; p<0.00001). Ellipses represent 95% confidence limits around individual centroids.
85 Factor structure Principle component analysis (PCA) united 21 song variables (described in the methods) into 6 factors or components (see Table 16). The first factor compressed all countable variables into one component ('Counts'), which explained 35.5% of the variance (i.e. the most). The second factor contained the chuck element variables ('Chucks'), and explained 16.45% of the variance. The third factor contained the rates measured ('Rates'), explaining 13.2% of the variance. The 4 th factor has 2 snort variables ('Snorts') and explained 9.4% of the variance. The 5 th ('Extremes') included the shortest element (chuck time) and the maximum wail measured and explained 9.3% of the variance. The 6 th factor is a measurement of the average wail ('Wail'), explaining 5.5% of the variance. The six factors combined explained 89% of the song variance (Table 17).
Table 16: Variables measured and their division into factors by Principal Component Analysis. Variables were rotated using the Varimax method with Kaiser normalization. The rotation converged in 5 iterations. See methods section for full explanation of measurements and abbreviations. Factors Components Variables 1 2 3 4 5 6 Counts nwails 0.972 -0.113 -0.021 0.068 -0.063 -0.071 nbouts 0.968 -0.119 -0.013 0.095 -0.077 -0.065 duration 0.939 -0.064 -0.172 0.060 0.003 -0.109 tsong 0.928 0.152 -0.033 0.229 0.143 -0.062 twail 0.897 -0.150 0.003 0.045 0.288 0.061 nchucks 0.878 0.256 -0.079 0.140 -0.170 -0.118 files 0.868 0.028 -0.052 0.238 0.241 -0.018 nsongs 0.742 0.115 0.069 -0.024 0.001 0.364 nsnorts 0.699 0.129 0.015 0.329 0.060 0.228 Chucks chuck.bout -0.110 0.939 -0.200 0.107 -0.063 0.018 rchuck -0.044 0.842 0.173 0.030 -0.338 0.042 tbout -0.036 0.840 0.006 0.218 0.461 0.068 max.chuck 0.274 0.833 -0.099 -0.046 -0.070 -0.206 Rates rbout -0.032 -0.182 0.974 -0.048 -0.042 0.064 rwail -0.078 -0.223 0.902 -0.054 0.276 0.097 singing -0.071 0.400 0.884 0.117 0.164 0.072 Snorts sno.row 0.202 0.097 -0.034 0.904 -0.106 -0.028 snorts 0.312 0.089 0.028 0.861 0.078 -0.087 Extremes tchuck -0.010 -0.051 0.198 -0.147 0.927 -0.045 max.wail 0.471 -0.151 0.201 0.228 0.660 0.184 Wail ave.wail -0.013 -0.065 0.145 -0.085 0.030 0.902
86 Table 17: Summary of significant results from principal component analysis on 21 measured song parameters. Six factors (or 'components') had Eigenvalues >1. Below 1 contribution becomes steeply irrelevant. For each factor a name was assigned reflecting the variables it represents. Factors are listed by their contribution to the overall variance. The counts factor explains most of the variance, the wail explains the least. All together, 89% of the song variance is explained by the 6 factors. Factor Name Unrotated Varimax Rotation Eigenvalues % of Cumulative Eigenvalues % of Cumulative % Variance % Variance 1 Counts 7.96 37.90 37.90 7.453 35.49 35.49 2 Chucks 3.72 17.73 55.63 3.454 16.45 51.94 3 Rates 3.1 14.76 70.39 2.771 13.20 65.14 4 Snorts 1.52 7.23 77.62 1.979 9.42 74.56 5 Extremes 1.46 6.94 84.56 1.958 9.32 83.88 6 Wail 1.01 4.81 89.37 1.152 5.49 89.37
PCA analysis united 9 measurements that described the harmonic frequencies into a single factor (termed 'harmonics'), separated from the above analysis. The 'rates' component (3 rd from the structure analysis) was correlated with the 'harmonics' component, and was therefore not used in the subsequent analyses.
87 Anatomical measurements Two adult hyraxes, a male and a female, which were run over by a car in Ein Gedi, were dissected by Dr. Yuval Zohar (a nose-ear-throat specialist). No anatomical differences were noted between the male and the female vocal tracts. The length of the male's vocal tract, from the epiglottis to the tusks, in the front of the mouth (Fig.46), is approximately 4cm. The cricothyroid muscle has freedom to move, allowing the larynx and the thyroid cartilage to retract a further 0.8 cm (i.e. 20% longer). The epiglottis is rigid, but the trachea is flexible, permitting the lengthening of the vocal tract. The formant dispersion, calculated from the anatomical measurements, is 3.5 kHz, with approximately 3 formants below the Nyquist frequency of 11 kHz, when the sampling rate is 22 kHz. The 3 extracted formant variables (average, minimum and maximum first formant), were unified into a single component (termed 'formants'), using PCA analysis. The 'formants' component is not related with the other components. The term component was used rather than factor since factors resulting from 3 different PCAs (from structural elements, harmonic and formant frequencies) were used.
larynx - מקור הקול vocal tract
- -
Figure 44: Dissection of an adult male Ein Gedi hyrax. Vocal tract, measured from the epiglotis to the open air, is approximately 4cm. Thyroid cartilage encasing larynx is shown as well.
88 Signaling information
Elements reflecting age, size and fur coverage Age is related only to the snort component (R 2=0.287; n=17; p=0.026; Fig. 47). Older, more experienced singers snort more than younger, new singers.
3.5
3
2.5
2
1.5
1
0.5
0 Snorts component -0.5
-1
-1.5 1 2 3 4 5 6 Age
Figure 45: Snort component as a function of age in male singers. Older, more experienced singers snort more than younger, inexperienced singers (R2=0.287; n=17; p=0.026).
Only the counts component was significantly correlated with weight (R 2=0.54; n=10; p=0.009; Fig. 48), with heavier hyraxes singing more. Body size (i.e. morphometric component) is related only to the chucks component (R 2=0.377; n=11; p=0.036; Fig. 49). Bigger hyraxes chuck more.
89 4 3.5 3 2.5 2 1.5 1 0.5 0 Counts Counts component -0.5 -1 -1.5 2.2 2.4 2.6 2.8 3 3.2 3.4 Weight
Figure 46: Counts component as a function of body weight (Kg; R 2=0.54; n=10; p=0.009). Singers that weigh more, sing more.
3
2.5
2
1.5
1
0.5
0
Chucks component Chucks -0.5
-1
-1.5 0.6 0.8 1 1.2 1.4 1.6 Morphometric component
Figure 47: Chucks component as a function of morphometrics component (R2=0.377; n=11; p=0.036). Bigger males use more chucks in their singing.
90 Fur coverage is associated only with the formant component (R 2=0.724; n=7; p=0.012; Fig. 50). Hyraxes that have fuller fur coverage also have lower formant frequencies.
2.5
2
1.5
1
0.5
0
-0.5 Formants component Formants
-1
-1.5 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 Fur coverage
Figure 48: Formants component as a function of transformed fur coverage (R2=0.724; n=7; p=0.012). Males with fuller fur coverage have a lower formant frequency.
Elements reflecting hormones Testosterone levels are not significantly explained by any song variables, but are marginally related to the snorts component (R 2=0.2397; n=14; p=0.077). If sample size was larger, ties would probably be more significant. Androstenedione (A4) levels are related to both the snorts and the harmonics components (R 2=0.765; n=14; p<0.001). Most (55%) of the variance of A4 levels could be explained by the snort component (R 2=0.553; n=14; p<0.001; Fig. 51). The harmonics component contributes only 21% to explanation of A4 levels (R 2=0.212; n=14; p=0.014; Fig. 52).
91 3
2.5
2
1.5
1
0.5
0
Snort component -0.5
corrected for harmonics component harmonics for corrected -1
-1.5 -2.5 -1.5 -0.5 0.5 1.5 Standardized Androstenedione corrected for harmonics component
Figure 49: Partial residual plot of the snort component as a function of standardized androstenedione (A4) levels in singing male hyraxes (R2=0.553; n=14; p<0.001). Both variables are corrected for the harmonics component. A4 levels are transformed.
3.5
3
2.5
2
1.5
1
0.5
0 corrected for snort component snort for corrected
Harmonics component Harmonics -0.5
-1
-1.5 -1.5 -1 -0.5 0 0.5 1 Standardized Androstenedione corrected for snort component
Figure 50: Partial residual plot of the harmonics component as a function of standardized androstenedione (A4) levels in singing male hyraxes (R2=0.212; n=14; p=0.014). Both variables are corrected for the snort component. A4 levels are transformed.
92
Estradiol (E2) levels are related to both the harmonics and to the extremes components (R 2=0.579; n=14; p=0.009). Contribution of the harmonics component 2 amounts to 25% of the variance of E 2 levels (R =0.255; n=14; p=0.019), while the extremes component accounts for 32% of the variance (R 2=0.324; n=14; p=0.028; Fig. 53).
1.5
1
0.5
0
-0.5
Extremes component -1 corrected for harmonics component harmonics for corrected
-1.5 -1 -0.5 0 0.5 1 Estradiol levels corrected for harmonics component
Figure 51: Partial residual plot of the extremes component as a function of standardized estradiol 2 (E2) levels in singing male hyraxes (R =0.324; n=14; p=0.028). Both variables are corrected for the harmonics component. Males with long chucks and wails have lower E2 levels. E2 levels are transformed.
Cortisol (C) levels were associated only with the chucks component (R 2=0.359; n=14; p=0.032; Fig. 54). The relationship was negative so that males that had high C levels chucked less than males with low C levels.
93 3
2.5
2
1.5
1
0.5
0
Chucks component Chucks -0.5
-1
-1.5 -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 Standardized Cortisol
Figure 52: Chucks component as a function of standardized cortisol (C) levels in singing male hyraxes (R 2=0.359; n=14; p=0.032). Males that use the chuck element more extensively have lower C levels.
Elements reflecting social status Males also relay their social status through their songs. Snorts explained nearly 67% of the variance in David's score among singing males (R 2=0.667; n=9; p=0.006; Fig. 55). Higher ranking males snort more than lower ranking singers. Rank derived using David's score is related to the formants components (R 2=0.862; n=7; p=0.002; 56). More dominant males, which rank in the first places in the social scale, have lower formant frequencies, which are 'richer' sounding voices (with more timbre).
94 0.4
0.2
0
-0.2
-0.4
-0.6 Snort component -0.8
-1
-1.2 3 3.5 4 4.5 5 5.5 David's score
Figure 53: Snort component and David's score (R2=0.667; n=9; p=0.006). Dominant animals, which have higher David's score, snort more.
2
1.5
1
0.5
0
-0.5 Formants component -1
-1.5 0 1 2 3 4 5 6 7 Rank (David's score)
Figure 54: Formant component as a function of rank, derived using David's score (R2=0.862; n=7; p=0.002). The most dominant animals rank 1 st and have lower formant frequencies.
95 Discussion Why sing? Male hyraxes probably sing in order to advertise their quality, as implied by old age (survival), social dominance and a favourable physiological state. Hyraxes use distinctive voices to sing unique songs, which contain components that are related to attributes of individual singers. In fallow deer, males vocalize primarily to threaten other males (McElligott and Hayden, 1999), while in Kloss' gibbons it is mainly for mate attraction (Tenaza, 1976). It is likely that hyrax song is mostly performed for female audience, since hyraxes stop singing for a few months following the mating periods (LK, personal observation), although certain song elements are also likely to convey information to nearby males. If vocalizations are an indicator of male quality, they should be expensive to produce, in terms of time, energy or risk. One indication of a social cost may be that young hyraxes, which would probably be challenged by older males if they sing, are silent. Repertoire size increases with age, from juvenile-hood to adulthood, and old singers that stop singing lose their lives (LK, personal observation).
Vocal recognition Based on the different song elements and harmonic frequencies, all singers were significantly separated and all songs were properly identified by discriminate function analysis. Like in male loons, males need to differentiate themselves from other potential competitors (Walcott et al ., 2006). Although it may be beneficial for less valuable males to imitate the songs of higher quality males, the males whose voices were more alike resided in more distant groups. In penguins, recognition is purely acoustic, based on vocal signature, without visual recognition what so ever (Jouventin and Aubin, 2002). Hyraxes in Ein Gedi use chemical (odour), as well as visual and behavioural cues to communicate and to recognize each other; yet, their environment consists of complex topography, for which acoustic recognition between group members is vital. Male hyraxes sing, at lower rates, many months before the mating period, maybe as part of intrasexual competition and hierarchy formation. In fallow deer, males who start vocalization activity earlier in the season gain more mating success, possibly because it is familiarizing females to its voice (McElligott et al ., 1999). In the red deer, hinds discriminate in a playback experiment, between the roars of their harem-holder stag and those of other stags (Reby et al ., 2001). It is also possible, that like in fallow deer, males that start vocalizing early have an intersexual advantage. It
96 will be interesting to see whether females prefer males who sing more and earlier in the season.
Signaling information honestly I found evidence that males signal their states honestly by singing. Essential information can probably be extracted from male songs via multiple signals that can be pooled together in order to provide a good indication of male attributes. In the hyrax, using stepwise multiple regressions, I found specific song components that are reliable indicators of physical, hormonal and social attributes.
Elements reflecting age, size and fur coverage Male hyraxes may advertise their age by the snort component. Snorts are broadband sounds, not performed by younger, inexperienced males. Some males add the snort component to their singing repertoires after a year or two of vocalizing and others never do. Snorts are carried out by only 65% of singers (11 out of 17), perhaps because they are low frequency sounds, whose production and radiation by small animals is a physical constraint (Fitch and Hauser, 2002), despite the fact that animals often perform singing to the limit of their physical constraints (Podos, 1997). Higher weight in male hyraxes was conveyed through higher vocalization rates, which included longer song, bout and element durations, and the usage of numerous repeated elements (i.e. the counts component). Weight is positively related to reproductive success in many mammals (e.g. the gray mouse lemur; Eberle and Kappeler, 2004). Physical attributes, such as height and weight, as well as age can be reliably inferred from human voices (Krauss et al., 2002; Bruckert et al., 2006). Sound provides information on body size by default. Interactions between sound and body do not need to invoke biological advantage or selection at all (Fitch and Hauser, 2002). Hyraxes that are physically bigger (i.e. bigger morphometric component) chuck more. Chucks require both concentration and muscular control to perform precise rhythmic pulses, with exact lengths and spaces in between. Males that are bigger produce more chucks in a row. Rows of chucks seem to be produced in one breath, which may indicate bigger lungs, which are related to bigger body size (Fitch and Hauser, 2002). Loud calls should also indicate a large caller. I did not measure the amplitude of hyrax songs, yet louder sounds should travel further, although the distortion caused by the structure of the gorges in Ein Gedi, detract from its effectiveness.
97 In a few invertebrate systems, such as in the cricket (Teleogryllus commodus ), secondary sexual traits, such as song, showed correlations with male immune function and female preferences (Simmons et al ., 2005). I used fur coverage as an indicator of general body condition. In my study system, the formant component was related to fur coverage, so that hyraxes with fuller furs (i.e. better body condition) also had lower formant frequencies, which indicate, using the source-filter theory, larger bodies (Fitch, 1997), and in my study, higher social status. Fuller furs, in male hyraxes were also marginally correlated with higher T levels, which possibly reflects higher quality (i.e. ICHH; see chapter 2 discussion). Although T levels and social status were not found to be directly related in this study, their relationship may be mediated by fur coverage, represented in hyrax song by the formant component.
Elements reflecting hormones Snorts are low, noisy sounds, which satisfy the definition of sounds that accompany aggression (Fitch, 1997), which is often associated with androgen levels. Both of the androgens that I essayed were associated with the snort component. T levels were marginally positively correlated with snorts while A4 levels were negatively related to snorts and to harmonics. Males with high T levels snort more while males that snort more have lower A4 levels and higher pitched voices. The opposite trends that T and A4 showed pertaining to the snort element are suggestive of a biochemical balance between the two androgens in singers. Since the hormonal synthesis pathway between T and A4 is bidirectional, and since hyraxes are generally considered to have high androgen levels, it is possible that in order to prevent an androgen over dose, there is negative feedback between the two hormones, whereas the level of one increases, the level of the other decreases. Since T and A4 are not correlated in singers, their relationship may be mediated through additional factors or conditions.
E2 has an important role in bird song. In male hyraxes, E 2 levels are lower in singers, compared with non singers. Despite that, E 2 levels have a role in signals that are conveyed through hyrax songs since two elements: the harmonics and the extreme components are negatively related to it. The harmonics component represents the fundamental frequency (F 0), along with its first and second harmonics. Males that had high E 2 levels had lower pitched voices, which intuitively implicate bigger body size.
High E 2 levels also related to shorter wails and chucks, which logically indicate
98 smaller sized males, contrasting the impression above. If in hyraxes, females prefer males with lower E 2 levels (i.e. singers) then longer wails and chucks should perhaps be preferred as well. Long average wail, however, represented by the wail component, was not found to be significantly correlated with any of the individual variables I measured.
The harmonic component related negatively to both E 2 and A4 levels suggesting that in hyrax, as in other mammalian males, F 0 may not be a reliable indicator of body size or dominance ranks (Fitch and Hauser, 2002; Fischer et al., 2004), despite the widespread assumption that a deep voice indicates a large, assertive body (Morton, 1977; Pfefferle and Fischer, 2006). The size of the vocal folds can be independent of body size, and can experience a hormone-dependent growth spurt, further decoupling body size and F 0, particularly in males (Fitch, 1997). Also, since F 0 is dependent on vocal fold length and stress, motivation and social situation is likely to change it, raising F 0 in exciting or stressful situations (Fischer et al., 2004).
In song birds, stress levels negatively effect song development (Spencer et al ., 2003) and females prefer songs of 'relaxed' males (Spencer et al ., 2005b). In hyrax, C levels were higher in singers, compared to non singers, and C levels negatively correlated with singers' social status, as observed in other social mammals (Creel, 2005). C levels were expressed in hyrax song by the chucks component. Males that chucked more had lower C levels. Chucks are likely to necessitate relaxed muscles and high concentration levels. Since high chuckers had low C levels, which were associated in singers with low social status, chucking in singers may be used to signal submission. Among male hyraxes, no connection was found between size and social ranks (see chapter 1), yet increased chuck rates and long rows of chucks convey large body size. I am suggesting that big hyraxes that wish to avoid aggression, chuck to signal it. Repetitive elements, such as chucks, are often used in male-male communication (McElligott and Hayden, 1999). Such sounds may permit individuals to assess their opponents (Zahavi, 1975; Zahavi, 1977) since information can be extracted from their rate and variance over time, signaling motivation and concentration. Chucks were found to be related to body size on one hand, and to C levels on the other, making them an interesting element in hyrax male-male communication.
99 Elements reflecting social status David's score was used to rank group members. Males with higher David's scores (i.e. that were more dominant) snorted more. The snort was the only element that was not used by all singers. It was positively related, in singing hyraxes, to age, T levels, and to dominance, despite them being unrelated. In the literature, plenty of examples linked age, T levels and dominance ranks (Creel et al., 1997; Kraus et al., 1999; Eberle and Kappeler, 2004; Muller and Wrangham, 2004a). It is interesting to find the snort element, which is potentially a highly honest signal, connecting these parameters. Another indicator of rank in hyrax singers is the formant component. Formant frequencies have been linked in a few species to age and to dominance ranks (Reby and McComb, 2003a; Fischer et al., 2004; Bruckert et al., 2006). Snort in itself, other than maybe difficult to produce is a broadband sound that extenuates formants. In order for the formant frequencies to be efficiently advertised, they need to be coupled, in the hyrax, to the snort component. Dominant animals, which have higher David's scores, ranking first on the social scale had lower formant frequencies, which honestly reflect body size in several male mammals (Fitch, 1997; Riede and Fitch, 1999; Reby and McComb, 2003a; Bruckert et al., 2006; Harris et al., 2006).
Male hyrax morphology is only slightly different from females'. One of the only visual differences between the sexes, that I could detect, is the wider male nose, which is related to head diameter (a sexually dimorphic trait), and may serve an acoustic amplifying function. The male larynx may also be bigger and rest lower in the throat than in females. In the dissection of the male hyrax, we saw that there is a possibility for an approximately 0.8 cm retraction of the larynx (i.e. elongating the vocal tract by 20%). When the vocal tract is elongated, format frequencies and spacing decrease. In red deer, the larynx can be pulled far down to the sternum while calling, significantly lowering the formant frequencies (Fitch and Reby, 2001; Reby and McComb, 2003a), and exaggerating body size (Fitch, 1997). In dogs, cotton-top tamarins, pigs and goats, the larynx is also lowered during vocalization into the oral cavity which closes the velum, sealing off the nasal cavity and avoiding nasal anti- resonances (Fitch, 2000a). Male hyraxes may also elongate their vocal tract by extending out their necks as they sing; possibly in order to exaggerate their body size. This extension along with the retraction of the larynx may weaken formants as an honest signal (as seen in human males; Gonzalez, 2004), unless all singers extend their necks and larynxes to the maximum that is anatomically possible (Reby and
100 McComb, 2003a), or that neck extension reflects the motivation of the caller (Reby and McComb, 2003b). In red deer, formant frequencies decrease with age, which may be a consequence of growth (neck and vocal tract lengthen) but may also be a result of increased elasticity in the thyro-hyoid ligaments, which allows older deer more efficiency at fully extending their vocal tracts (Reby and McComb, 2003b). Other means to possibly exaggerate body size include subhyoid air sacs, which may also function in roars of black and white colobus monkeys ( Colobus guereza ; Harris et al ., 2006). Neck extension and possession of full furs can also be a mean to visually exaggerate body size.
Nonlinearities Oscillations of the vocal folds are never perfectly periodic. Deviations or nonlinearities can potentially serve as acoustic indicators of developmental stability, which may assist in mate choice (Fitch et al ., 2002). Period doubling, biphonation (the presence of two independent frequencies) and deterministic chaos are generated by irregular opening and closing of the glottis, which causes nonlinearities in the dynamics of the vocal folds (Fitch and Hauser, 2002). Subharmonics are caused by period doubling and may be produced by small animals attempting to sound large, especially during aggressive interactions (Fitch et al ., 2002), since perception is that lower pitch designates a large body (Morton, 1977). Nonlinearities are important in acoustic call morphology. They may provide a way to generate highly complex and unpredictable vocalizations without requiring complex mechanisms (Fitch et al ., 2002). They may also aid in individual recognition and in the estimation of size, by producing loud broadband sounds that accentuate formant information and project it over significant distances (Fitch et al ., 2002). Nonlinearities are likely to be adaptive since listeners cannot habituate to them, making them difficult to ignore. Examples are a crying baby, or a scream, which are unpleasant because they are unpredictable (Fitch and Hauser, 2002). Escape behaviour is the visual parallel of acoustic nonlinearities (Fitch et al ., 2002). Asymmetrical vocal folds can increase the probability of nonlinearities in the voice. These may be judged as unattractive by potential mates since they deviate from physical symmetry, providing an indication of developmental (in) stability (Moller and Thornhill, 1998). In hyraxes, however, nonlinearities are prevalent. Most songs (belonging to 16 out of 17 singers) contain nonlinearities, with variances
101 between singers and songs (see Fig. 57 for examples). Strictly harmonic songs are rare; most contain subharmonics and a few show biphonation. Deterministic chaos is very common in hyrax songs and is difficult to ignore. It is manifested as broadband noise, often superimposed on the harmonic structure. One example of informative deterministic chaos is the snort element, which highlights and projects the formant frequencies. Since nonlinearities are often linked to high sub-glottal pressure, they may also communicate the singers' motivational state (Reby and McComb, 2003b). I could not find a proper way to describe nonlinearities in a quantitative way that would allow me to compare the degree of such disturbances between songs and hyraxes.
kHz kHz 10 10
8 8
6 6
4 4
2 2
0.5 s 0.5 1.0 1.5 s
kHz kHz kHz 10 10 14 8 8 12 10 6 6 8 4 4 6 4 2 2 2
0.5 s 0.5 1.0 s 0.2 s
Figure 55: Examples of nonlinearities: subharmonics, biphonation, and deterministic chaos in hyrax sonograms. The top left sonogram, representing 5 chucks, and the top right sonogram, of a wail, both show the three types of nonlinearities. The two wails on the bottom row (left and middle sonograms) show subharmonics. The middle bottom wail has deterministic chaos as well as possible biphonation. The snort, on the bottom right is a smear of broadband frequencies, typical of deterministic chaos. The formant frequencies are highlighted at approximately 1.5kHz and at 4.5kHz (typical 3n formant frequency pattern).
The costs involved with producing the snort element are not obvious. It may be, like I have already suggested, the exposure of the formants that keeps the snort signal honest. Males that cannot advertise lower formant frequencies (or dispersion) may simply choose not to snort. Also, the cost might be testosterone mediated, since snorts are associated with T levels, and the immunocompetence handicap hypothesis might mediate the cost of snorts as an honest signal (see chapter 2). Snorts may also
102 serve as status badges (e.g. in birds). They seem to be effective in male-male competitions since snort is a good predictor of social rank. I tried running various playback experiments to test this hypothesis, however, no reaction was attained to snorts or to any other artificial or full song broadcasted. Another approach is to attempt to determine whether snorts carry an energetic cost. If snorts are expensive to produce, in terms of energy expenditure or a social risk, then lower quality hyraxes will have a higher burden to shoulder and snorts can be considered a handicap (Zahavi, 1975), although Silk et al .(2000) showed that honest signs do not have to be expensive. Signals can be cheap if individuals are familiar with each other, interacting repeatedly, even when there are conflicts of interest (Silk et al ., 2000).
Another question is whether females find snorts attractive. I would expect females to choose males who snort with lower formant frequencies, advertising dominance and fitness. Snorts are composed of oscillations, which can cause females to miss appropriate indicators (Van Doorn and Weissing, 2004), but at least in humans, evidence shows that women do extract the proper parameters from male voices (Bruckert et al ., 2006). Fore coming results of the molecular analysis of reproductive success, will shed some light on female preferences of such specific, selective, song elements, as the snort.
More to choose from… Choruses and countersinging in male hyraxes may be a stage for males to compare performances, allowing them to assess opponents and avoid fighting when they are unlikely to win (Zahavi, 1975; Zahavi, 1977). Hyraxes often disrupt each other, chorusing in synchrony. Countersinging may either camouflage or keep the singers honest, bringing out individual differences. For instance, male American toads ( Bufo americanus ) dropped the dominant frequency of their call when they vocally interacted with other males, improving its correlation with their body size (Howard and Young, 1998). In red deer, playback experiments showed that adult harem holders were less likely to answer young stags' roars than older stags (Clutton- Brock and Albon, 1979). In hyraxes, when new males appeared, resident males increased their vocalizations and chased them away, forcing immigrants to sneak around (LK personal observation). In gibbons, mostly females solo sing. Males sing longer songs, but only in choruses with other males (Geissmann and Nijman, 2006). Tenaza (1976) suggested
103 that they do it primarily as an adaptation, reducing predation risk to singers. In tree frogs ( Smilisca sila ) as well, males lowered the cost of calling by synchronizing their calls with neighbours. This anti-predatory behaviour effectively reduced bat predation (Tuttle and Ryan, 1982). Since predation is a significant factor for hyraxes in Ein Gedi as well, countersinging may have evolved in order to protect singers from predators focusing and locating them. Countersinging may also be advantageous for females, who can sample at once all of the males singing from different parts of the gorge, compare them and choose appropriate mates. In the domestic canary, females listen to playbacks of interacting males and prefer the males that overlap opponents' songs (Leboucher and Pallot, 2004). Public male-male competitions are useful for female choice (Kokko, 1997b). Females across species eavesdrop on male contests and collect information, for used in mate choice. Finally, male singing may also enhance female reproduction in various ways (McComb, 1987; Bentley et al., 2000). In song birds, attractive male songs increased female T levels, HVC and song discrimination abilities, allowing females to select higher quality males (Marshall et al ., 2005). If male hyrax song has a similar stimulating effect on female T levels, female discrimination is likely to decrease (McGlothlin et al ., 2004), maybe explaining the motivation of non resident males to sing. In this chapter I showed that hyrax voices are highly individual and that songs contain specific information that is linked to individual quality. Analyses of specific song parameters show that hyrax songs can reliably communicate information pertaining to age, body size and condition, hormonal levels, and social status. I hypothesize that the chucks component is important in male-male interactions, while the snort element advertises qualities valuable to females in mate choice. Altogether, songs contain a vast amount of information pertaining to different aspects of hyrax life. Further research is necessary to find out how male and female audiences use this information.
104 General Discussion Now that I have addressed the questions: who sings? what is its hormonal profile? and why sing? I can draw a profile for singers. Singers, in the rock hyrax, are mostly males that are older and are more dominant, on average, than most sexually mature males. Singers are typical mammalian males in the hormonal sense, in their elevated T levels and low E 2 levels. They also have higher C levels than non singing males. Singer C levels are also associated with their social ranks, as in most cooperative breeders, where dominant animals have the highest C levels, and subordinate animals the lowest. In addition, hyrax voices are individually distinct, and songs are possibly used to advertise their age, body size, body condition, hormonal levels and social status. Combining all of these results and the different singer attributes points to the conclusion that indeed, hyrax song does contain in it information indicating individual quality. The question that remains is whether this information is translated into choice. Females can exercise mate choice either directly, through preference or resistance, or indirectly, through such acts as estrous advertisement that increase male-male competition. Females can also choose for direct or indirect benefits. Direct benefits include paternal care, food, protection, a good territory, or any advantage that a male may provide for a female or her young. Indirect benefits are based on what a male may inherit to his offspring, to help them survive and breed, such as good looks or good genes. Sexy sons benefits (Kokko, 2005) can occur where a male trait, such as full fur coverage, is inherited (Kokko, 2001; McNamara et al ., 2003). If females find full fur coverage, for example, attractive, males with that phenotype would gain reproductive success, passing that trait on to their sons. Fisher (1930) described this preference as the reason that some frivolous traits are exaggerated through sexual selection, not actually indicating any advantageous quality. Yet, if a trait, as useless as it may be adds reproductive benefits to its attractive bearer, than it becomes valuable (Kokko, 2001; McNamara et al ., 2003). The handicap hypotheses asserts that the more useless and expensive a trait is, the more honest it will be as a signal for quality, being too deleterious to lower-quality individuals (Zahavi, 1975; Zahavi, 2003)
105 There is plenty of evidence that females choose males based on their attractiveness (i.e. displays and ornaments; Andersson, 1994). Genetic quality, though, cannot be inferred from just a few fitness enhancing parameters, but rather from reproductive success. "Good genes" are supposed to improve reproductive success. Olsson et al . (2005) looked at potential good genes effects in adult male sand lizards ( Lacerta agilis ). They isolated, using molecular tools, a specific Major Histocompatability Complex (MHC) genotype whose carriers have less ectoparasites under increasing physiological stress, are not constrained by parasites at production of status coloration, and have a higher reproductive success. Results I present in this study suggest that fur coverage is sexually selected. Over the past eight years I have also been searching for polymorphic microsatellites that will be sufficient to screen the study population. Incorporating genetic analysis of paternity and relatedness, in order to assess reproductive success can provide the appropriate framework for discussion of ultimate-level mechanisms adjacent to my morphological, behavioural and hormonal analysis.
Female choice Rock hyrax females mate polyandrously. In Arugot, all adult females (ages 2- 8) gave birth to 1-4 pups, every year. In David, not all adult females were directly observed suckling pups, yet in general, female hyraxes reproduce throughout life, even in old age (Millar, 1971). Females' receptivity, in Ein Gedi, is modulated (i.e. unsynchronized yet seasoned). Females may overlap and each is receptive for a few days in a row. Females advertise estrus by specific vocalizations and possibly by urine marking, increasing the potential for female choice (Buesching et al ., 1998). Males, which have been fighting for months, respond to female's receptivity advertisement by 'patiently' lining up and taking turns mounting the female. This behaviour literally constitutes cooperative breeders (Watts, 1998); perhaps sharing breeding resources among egalitarian males, or simply for a lack of choice. Female hyraxes may be choosing to mate promiscuously (rather than hiding after the first mating) in order to ensure fertilization or to perhaps increase offspring genetic diversity. In baboons, females mate polyandrously to avoid infanticide (Clarke, 2006). Female hyraxes may also mate with multiple males in order to mask paternity (i.e. protecting their offspring from possible harm or even infanticide), gain multiple paternity (Zeh and Zeh, 2003) or to avoid sexual harassment (Wolff and Macdonald,
106 2004). In the hyrax though, rape is unlikely, since like in the elephants, female genital morphology requires feminine cooperation for successful copulation (Glickman et al ., 2005). Outside the receptive period, female hyraxes respond aggressively to male mounting attempts.
It is possible that females control paternity through mating with preferred males at more fertile times. Also, perhaps the order of the mating is important (first or last in line). Other opportunities for female choice may be before mating (Eberle and Kappeler, 2004), and at conception, through sperm selection and other cryptic choices (Birkhead et al ., 1997; Setchell and Kappeler, 2003; Simmons, 2003), although female hyrax are not likely able to remove mating plugs. Even after mating females may selectively abort a selected sex or an embryo of a certain father, and later choose which pups to care for. Zeh and Zeh (2003) discuss polyandry and suggest that it may be used either to create the setting for sperm competition, increasing the probability for fertilization by a high quality sperm (and male), or to increase offspring diversity. Its purpose may also be inbreeding avoidance, in its 'genetic incompatibility avoidance' version, by allowing the reproductive tract to 'decide' what is compatible (Zeh and Zeh, 2003).
Ovulation in baboons is concealed by a long estrus, and can be at any point, unrelated to the anal swelling (Clarke, 2006). In the gray mouse lemur, Eberle and Kappeler (2004) showed that males can discriminate between different periods in the estrus cycle. In hyraxes, only limited data is available on ovulation (Millar, 1971), and no information was found on its variance or whether males possess any means with which to determine its occurrence. Also, to the best of my knowledge, no infanticide was reported in this system in the wild, although I observed male aggression (biting and chasing) directed at pups, and males have been observed attacking and killing pups in captivity (Mendelssohn, 1965). I suggest that females actively choose fathers for their offspring, and that males invest energy in singing behaviour in order to advertise their stamina, as a handicap, to improve their chances of siring offspring. Another option though, is that the apparent lack of choosiness, perpetrated by females mating polyandrously with all males in the area, reflects a real lack of choosiness, a by-product of high T levels (McGlothlin et al ., 2004). If males forfeit their ability to choose mates for higher T levels, it is possible that female hyraxes, with their species- inherent high T levels,
107 lost that benefit as well. Under this assumption, dominant females have an advantage. Results from the hormonal analysis of Ein Gedi females showed that T levels were negatively associated with social ranks. Perhaps dominant females, that have lower T levels, can and do choose higher quality males, while subordinate females, with higher T levels, have no mate choice due to both their biology and sociology. Future study of mating order and offspring paternity, for high and low ranking females, will shed light on this hypothesis.
In fallow deer, groaning rates increase after copulation, possibly to reduce extra-pair matings (McElligott and Hayden, 1999). In hyraxes, once all reproduction activity for the season ceased, vocalization did as well, hinting that one of its purposes may have been to elicit female reproduction behaviour. 'Sexual conflict theory' suggests that females evolve counter-adaptations to male attempts to manipulate their reproduction (Zeh and Zeh, 2003). One possible adaptation, in the hyrax (as well as in the spotted hyena and in the lemurs), is female dominance, which can inhibit male copulatory behaviour. Subordination can decrease male reproductive success because of slower ejaculation, as seen in macaques (Caldecott, 1986).
More hyrax thoughts and further research In hyraxes, like in sifakas (Lemuridae), there is male dispersal, yet an even pup and adult sex ratio (Kappeler and Schaffler, 2006). No paternal care is provided, yet females, contrary to expected (Emlen and Oring, 1977), are not monopolized by males, but are actually, on average, dominant to them. Hyraxes and sifakas, both have a short mating season, during which females mate polyandrously. Kappeler and Schaffler (2006) reported that in sifakas, more than 90% of the offspring are sired by the resident male, who is older and more dominant than the natal males, who do not reproduce. Reproduction is highly skewed among males, yet natal males stay with the group, enjoying group living. In sifakas it was shown that as the number of males present in the group increased, reproductive success in the group increased as well, due to less infanticide and takeovers (Kappeler and Schaffler, 2006). In hyraxes, social groups are composed of females, pups, late dispersers (males up to 2yrs old) and an adult male, whose presence is not obligatory. Hyraxes are cooperative breeders, sharing babysitting and area guarding amongst group members. Juvenile males are evicted from their natal group before the age of two by the older resident males. They live on their own or in bachelor groups (Koren, 2000),
108 fending for themselves. Many migrate, vulnerable to disease and predators. They are wounded by other males and by the ragged environment. Their dwindling energy is invested in finding a receptive group of females to join. The advantages to living among a mixed sexed group may be increased reproduction opportunities, favourable nesting sites or the security of extra vigilant eye numbers. All resident males are immigrants. Males native to the area are all bachelors, although they mate with receptive local females. Results of reproductive success (molecular paternity analysis) will shed some light on alternative male strategies; their choice to either disperse or stay as bachelors around their natal groups.
Fur coverage and body condition Fur is connected to body maintenance and to the ability to advertise. Females probably prefer mates (from the available male variation) that are strong and can withstand ectoparasites and different pathogens. Fur coverage might also give evidence of genetic quality since the immune system is expensive to keep up. Females have been shown to prefer males with a Major Histocompatability Complex (MHC) different from their own, which may indicate a preference for maximizing offspring MHC polymorphism in order to increase disease and pathogen resistance (Olsson et al ., 2005). Since I have DNA from all hyraxes in the study, MHC region can be studied and included in the growing body of information collected for each individual and tested with relation to reproductive success. Full fur coverage, as well as a light fur colour, can be used to exaggerate body size. Hyrax pups possess very dark furs, which become lighter with age. Dark furs may serve to camouflage inexperienced pups from predators, or to stress small body size, age class and subordination to conspecifics. Older hyraxes should theoretically have lighter furs, although colouration is individual, with an entire spectrum of colours and shades (from blonde, to reddish, brown, and dark). Experimental work in vertebrates showed that different forms of colouration, particularly carotenoid and melanin based, are differentially sensitive to stressors such as diet manipulations and parasitic infections (Bortolotti et al ., 2006; Griffith et al ., 2006; Stoehr, 2006). Fur colour is an additional parameter that can be tested in the future, to see if it is indicative of individual quality. It may provide interesting information as to the role of fur in the hyrax catalogue of sexual signals.
109 Rank and hormones Hyrax age is apparently a key indicator of rank, in both sexes. In the spotted hyenas, rank is maternally inherited (Jenk et al ., 1995), while in the lemurs, rank is independently achieved through agonistic interactions (von Engelhardt et al ., 2000). In the hyrax, I am awaiting molecular maternity results, to link to social status, as well as to pup sex ratio. Sex ratios at birth should theoretically favour females if the mother is older, stressed, and in a poor physical condition (Love et al ., 2005). Male offspring, which are more 'expensive' to produce in mammals, are more likely if the mother is dominant, has higher T levels, and is generally in a good condition (Veiga et al., 2004; Pike and Petrie, 2005; Rutkowska and Cichon, 2006). In hyraxes, females are more dominant, have higher T and C levels, but are generally in a worse body condition than males, and pup sex ratio is even. This is an interesting system to test such evolutionary strategies, focusing on individual female hormone levels, ranks and resulting offspring sex ratios. Organizational hormone theory talks about the enormous effect hormones have earlier in life. Pup hormonal levels shape sexual differentiation and social behaviour. Endogenous hormones as well as maternal and sibling hormones can effect pups brains. Once I have the maternal and sibling relatedness data I can shed light on that, as well as on the growth rate of offspring. One can also look at the heritability of hormonal levels, in both sexes. Female hyraxes' elevated androgen levels are unusual for mammals, but are not as rare in other vertebrates; female teleost fish and monogamous birds have elevated androgen levels as well (Ketterson et al ., 2005). The high androgen levels seen in female rock hyraxes may be a by-product of selection for another trait, linked with androgens, or a remnant of early development; as seen in the African elephant, a sister taxon to the rock hyrax, where its unique uro-genital development starts in the masculine form. Opposite to the 'normal' mammalian development, female elephants (and spotted hyenas) first develop male genitalia. As a result, adult females have angled genitalia that require their co-operation for mating (Glickman et al ., 2005). Experimental manipulation of T levels using implants can be used to investigate the relationship between T and C in male hyraxes and the costs associated with elevated T (and perhaps C as a by-product) levels on male singers and non- singers and on female hyraxes. The hyrax model is ideal for checking the ICHH, challenge hypothesis and other decision making strategy models. Implants can also
110 be used to investigate the relationship between hormones and song production in male hyraxes. T implants can be used, in females, to investigate T's effect on choosiness and mate preferences, in relation to advertised male quality, as well as to pup sex ratios. A complementary approach to implants is using receptor blockers. It is possible, for example, to inhibit T's binding to androgen receptors by administering the androgen receptor antagonist flutamide (Flut). Aromatase inhibitor 1-4-6 androstatrien-3,17-dione (ATD) blocks T's conversion into E 2 (Hau et al ., 2000).
Vocalization: songs, survival and females On two occasions I witnessed active resident singers becoming silent. Within two weeks of their silencing, they disappeared. Disappearance of ex-singers can be either due to eviction (by group females or by immigrant males), related to a loss of social status, or due to an illness related death, both of which can be reflected and preceded by a singing cessation. Either way, it may support my hypothesis that song is an indication of fitness in the rock hyrax. The discriminate function analysis shows that hyraxes sing in distinctive voices, and the canonical scores also show that no two songs are identical. Hyraxes create individual songs, using temporally and harmonically unique element combinations. Other than in countersinging, where hyraxes often adopt similar ping- ponging patterns (data is not yet analyzed), I saw no evidence for mimicry. The fact that every song is different may indicate that hyrax song is context specific, used to communicate complex messages, and not mere binary information. The failures of all of my various playback experiments strengthen this hypothesis. In hyraxes, since voices are individually recognizable and since songs seem to contain honest signals of individual quality, it is interesting to see whether males grouped with known vs. strange males sing differently, and whether the reliability of the messages they give out changes (Howard and Young, 1998). Other types of calls probably also contain valuable information. Alarm calls can be interesting to investigate in the context of the identity of the caller (i.e. who takes risks by sounding them), as well as the reaction of the group to them (i.e. reliability: who elicits a response and who is ignored). Countersinging is interesting in both the context of female audiences, examining whether there is a precedence effect (McElligott et al ., 1999), and in the context of males, and what they gain from displaying together (Kokko, 1997b). Hyrax song displays may also resemble performance arenas (Emlen
111 and Oring, 1977; Kokko et al ., 1999), yet polyandrous mating is paradoxical to it by definition. Over the years, I have recorded two females singing high pitched songs. A rise in female androgen levels (A4 and T) can produce male traits, such as singing (Staub and DeBeer, 1997). Female song may signal receptiveness, like female alpine accentors ( Prunella collaris ) that sing only when fertile (Langmore et al ., 1996). Male Barbary macaques ( Macaca sylvanus ) discriminate between different stages of the estrus cycle, based on females' copulatory calls, responding the strongest to calls given when the female is most fertile (Semple and McComb, 2000). Ultrasound and infrasound are both likely in the hyrax. A team of bat researchers (headed by Prof. Schnitzler) recorded ultrasonic hyrax song at around 40 kHz using bat detectors, and described it as 'a symphony' (Yossi Yovel, personal communication). Hyraxes also use very low frequencies in their snorts, and are related to elephants, which are notorious for using low frequency sounds. Infrasound is more likely in large animals, with large singing and projecting apparatus (e.g. elephants) that live in open spaces, where sound can travel to great distances without disturbances. With the right equipment, an entire range of sounds is probably waiting, outside of the human hearing range. The most important verification of this study is its synthesis with reproductive success, whose relationship to age, body size and condition, social status, hormonal levels, singing behaviour and song features, will provide precious clues as to what traits are viewed by females as valuable. Many models rationalize how female preferences and male traits coevolve (Fisher, 1930; McNamara et al ., 2003). Females' preferences are likely to be as varied as males' qualities (Howard and Young, 1998), which is actually a pre-requisite to natural selection. Choice may also be context dependant (Coleman et al ., 2004; Kirkpatrick et al ., 2006), and therefore needs to be tested in various female ages, belonging to different social classes.
112 Postlude Singing in the rock hyrax seems to be an honest signal, potentially advertising age, body size and condition, hormonal levels and social ranks. Its multiple quality and motivation related messages may provide both males and females valuable information about the singer. The most important next step is to identify the relevant targets of the different messages (intended and unintended audience), and to assess their interpretation of the information. The ultimate benefits that singers can gain, in terms of reproductive success, have yet to be assessed, but they must be considered in order to properly appreciate the costs of singing, balancing it with alternative strategies.
113 Appendix Further validations for hair-testing protocol determining hormonal levels Before a new method can be widely used for a species and a substrate it needs to be validated using an established method. A concern may be, for example, with respect to methanol extraction, and the possibility that it may introduce substances that would interfere with the detection and lead to false detection of hormone levels. I wish to provide some of the preliminary work that led to our essay. The cortisol immunoassay was linear between 1ng/ml to 80ng/ml (r2=0.998). The coefficient of variation of the test was 7.5% at the lower end and 3.5% at the upper end. The limit of detection of the essay was 0.04ng/mg when 5mg of hair was used in the assay. The coefficient of variation for six replicates performed in the same assay (intra-assay) was 4.6% and when one sample was analyzed in six consecutive days (inter-assay) the coefficient of variation was 8.3%. The specifications for the testosterone immunoassay were similar. The test was linear between 0.2ng/ml and 16ng/ml (r2= 0.998) with similar coefficients of variation. The detection limit was 0.01ng/mg when 5mg of hair was used. The recovery of testosterone spiked hair was stable and the analytical process was not associated with breakdown of the molecule. The results detected by the immunoassay were further validated by the use of liquid chromatography-mass spectrometry (HPLC/MS/MS). 20 mg of hair, from 10 hyraxes, was minced into 2-3 mm pieces and thoroughly mixed, in order to overcome potential differences in levels along the hair shaft. The mixture was divided into 12 glass vials and incubated overnight at 40°C on a shaker with 1 ml Soerensen buffer
(38.8 ml KH 2PO 4 (9.07 g/L) and 61.2 mL NA 2HPO 4 (11.87 g/L)) at pH 7.6. Standards were obtained by adding 50 µl of 0.25-20 ng/ml testosterone in methanol solution to 20 mg of blank (human female) hair. To each sample, 50 µl of deuterated testosterone (5ng/ml) as an internal standard was added. After incubation, the solutions were transferred into clean tubes and centrifuged. The supernatant was placed on C-18 bound-elute columns (activated with 3 ml of methanol and rinsed with
3ml deionized H 20). Columns were rinsed with 1ml deionized water followed by 1 ml deionized water: methanol (90:10, v/v) and than dried for 30 minutes. Testosterone was eluted three times with 0.5 ml methanol, which was than evaporated to dryness under nitrogen. The residue was reconstituted in 50 µl mobile phase (75% methanol:
114 acetonitrile (60:40): 25% ammonium acetate 10 mM). 10 µl of each sample was injected on a column (Phenomenex Luna; 5µM; C18; 100A; 150 x 4.6 mm) using an HPLC system consisting of a LC binary pump and a thermo auto sampler (Agilent 1100). Flow rate was 0.6 ml/min, using a linear gradient of 10 mM ammonium acetate from 5 to 25% and a methanol: acetonitrile (60:40) solution from 95 to75%. The run time was 10 minutes. The retention time of testosterone was 5:45 minutes. The HPLC/MS/MS method detected testosterone in the hair. There was significant correlation between the results of the HPLC/MS/MS and the ELISA (r 2=0.71, P<0.01). A ligand free media, in this case a testosterone or a cortisol-free hair sample is impossible since hormones are transported to any growing hair from the plasma. Similar to other naturally occurring molecules and xenobiotics, hormones are incorporated into the hair during its formation. Because all plasma has hormones present, every hair does as well. The hormonal levels derived from every set of analyses are used on a comparative basis to test among different experimental animals and/or conditions. When the results are to be applied in this way, the absolute values of the hormonal levels are of little importance. Unlike serum or saliva, testosterone from hair needs to be extracted by solvents, sonicator, and other manual steps. Slight variations in the extraction protocol between sets of samples may influence the final concentration detected. In other words, concentrations of testosterone obtained in two different assays are not fully comparable. Therefore, testosterone in hair is a relative measure and comparable samples are only those obtained from the same microplate. To enhance hair testing as a comparative tool, I compared testosterone levels in plucked versus cut hair, different segments of hair from the same animal and washed versus unwashed hair samples. The extraction and detection protocols are detailed in Koren et al , 2002.
Plucked versus cut hair For five animals, hair was both plucked and cut close to the skin from the same area in the body (average ±SD was 10.5±1.8 mg). A t-test between testosterone levels detected for the two different treatments showed no significant difference (t4=0.04, P=0.97; Power =0.028). Hair can therefore be cut with scissors instead of plucked.
115 Light versus dark hair segments For 5 males and 5 females, whose hair was dark close to the skin and light away from it, hair was separated (samples cut) into light and dark segments. Segments weighed an average (±SD) of 21.4±3.1 mg with a maximum of 10% difference between pairs. No significant differences were seen between testosterone levels detected in the different segments. ANOVA showed no difference between sex
(F(1,16 )=0.01; P=0.9), segment (F(1,16 )=0.29; P=0.6) and interaction between sex and segment (F(1,16) =0.84; P=0.4). Despite the lack of statistical difference, the dark portion of the hair had three times more hormones than the light segments (dark = 1355.13±2033.85 pg/mg hair; light = 370.94±620.64 pg/mg hair). Because of the difference in means and the possible effect of melanin on the incorporation of xenobiotics (Raul et al. , 2004), it should be stressed that hair should be consistently sampled from the same area in the body, as well as the same colour of fur.
Washed versus unwashed hair For 6 males and 14 females, duplicate samples were weighed. Each hair sample averaged (±SD) 20.4±0.4 mg, with a maximum of 4% difference between pairs. For each animal, one hair sample was not treated, and one hair sample was washed with 10% SDS, vortexed, and then washed 10 times with warm distilled water until all suds were out. Higher levels of hormones were detected in the washed hair than in the unwashed hair (washed 67.4±25.2 pg/mg hair; unwashed 40.7±41.6 pg/mg). This result emphasizes the internal source of the hormones essayed and the marginal influence of additional sources, such as other animals rubbing against or marking each other. The lower testosterone levels detected in the untreated hair samples must be due to the masking of hormones by dust and other debris from the field. Despite the fact that testosterone levels in the washed hair were significantly higher then in the untreated (paired t-test: t16 =3.78; P=0.0016), they were highly correlated (r=0.724; Z=3.42; P=0.0006; N=17). The high correlation between the two shows that the untreated hair collected from the field represents the animals' testosterone level. Consequently, it permits skipping the washing stage in our study. Use of hair has tremendous yet unexplored potential in wild animal behavioral research as a non invasive hormone collection method. This method evaluates long- term hormonal status, reflecting chronic exposures, rather than a momentary one that
116 is reflected in blood levels, which are heavily confounded by the stress of capture and handling of wild animals (Koren et al , 2002). This new comparative tool can provide wild animal researchers the unbiased hormonal profiles at the basis of many behavioural phenomena observed.
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143 תודות
למנחים: דר " אלי גפן ודר " עופר מוקדי , שמצד אחד החזיקו יד ומצד שני נתנו לי חופש פעולה מלא להתפתח לכיוונים שונים משלהם . . לוועדה המלווה שתמכה : פרופ ' עוזי מוטרו , פרופ ' יורם יום- טוב , ופרופ ' ארנון לוטם . . לפרויקטנטים שהזיעו : ליאת , גיל , נתי , מודי ולאה . . לטכנאים שהזיעו עוד יותר : 'בת , איתמר , אורן , יותם וענבל . . לענת , תמר ואופיר שהחזיקו בגבורה שפנים צפוניים . . לש" שנים : ינון , דפנה , תאיר , נועם , שימי , ובני מחזור 2005 . . למדריכי ומנהלי ביס" ש ע" ג -2005 1999 . . לרשות הטבע והגנים שאפשרה לי לעבוד בתחום שמורה . . למיכאל בלכר , לאבי ד ויד עוד כמנהל , ולפקחי שמורת ע" ג -2005 1999 . . ליחידת חילוץ ע -ג" ערן חופש , אלון שחל ויעקוב , על הציוד והעזרה . . לחברי בע ג" : יעל , מיכל ודורון , נשות החוג למחול מזרחי ( במיוחד לרוחיק , חיה , שרה ואורנה .) .) ל חניתה ויעל שטיפלו באוצר שלי . . לאלו שמכירים את השפנים מולקולארית : צליל , מיכל , רונית , אדר , תמר והגר . . לחברותי ממועדון הסלט : סיגל , שרון , ורד , תמר וענבר . . לרחל ( המכשפה המתוקה ) פז ולחברי המכון לשמירת טבע ( במיוחד לפרופ ' דיני איזיקוביץ ' שקיבל אותי ולדר " שריג גפני עבור המקרר והתמיכה ). ). לדר" יובל זוהר שניתח שפנים דרוסים . . לדר" נועם לידר, "דר עופר אמיר ו "דר נועם אמיר, שעזרו עם ההתחלה האקוסטית . . לאבא על הכניסה לארץ האפשרויות האינסופית של שיער והורמונים ולמעבדה בקנדה על האירוח . לאימא שחיזקה את תחושות הבטן שלי . . לטל אחותי הנפלאה שתמיד נמצאת לידי , ו לבן זוגה יובל . . לאמיר שהושיע יותר מפעם אחת . . למשפחה המורחבת: לסבתא שעזרה לי למצוא מטפלת בעין גדי , ולחברות שהן חלק ממני , במיוחד לפיפי מורתי וחברתי א'ול, חיותי י ' ענת ורונית . . לאבי , בן זוגי המסור . . לנור שסייעה ותמכה , נתנה והטעינה וכל כך לא הפריעה . ש אפשרה וזרמה עוד לפני שנולדה , ואחר כך התעניינה ושאלה . . לש פנים , תודה על הקולות שמעלים בליבי חיוך , עדיין ...... וגם לנמרים , לזאבים , לצבועים ולשיטפונות , שעזרו לי לקום ולצאת כל בוקר ב- 3:30 מהמיטה ...... תודה ! !
אלפי סליחות אם שכחתי מישהו שעזר עם כל הלוגיסטיקה המורכבת של מחקר זה . . ולסיום , שיר שאבי כתב עבורי בעודי תינוקת בת מספ ר חודשים ... " לי שלי גוזלי עוללי ... מה הם אומרים לך השפנים נים לא נים? ומה את עונה לשפנים נים לא נים? יבוא יום תדעי לדבר , תדברי אבל אז כבר לא תזכרי " " ... גורלי נקשר עם המובייל שמעל למיטתי ( מן הסתם הם היו ארנבונים , אבל לא נורא )... ומאז אני בחיפוש אחרי מה הם א ומרים , השפנים ...... תקציר תקשורת קולית ( ווקאלית ) קיימת ב מיני שוני של בעלי חיי, למטרות קיומיות שונות כמו חיזור ואזהרה . תקשורת באמצעות שירה נדירה ביונקי יחסית לעופות . תיאוריות רבות נוסחו על מנת להסביר ולחזות תהליכי קבלת החלטות , לקיחת סיכוני , יצירת מערכו ת חברתיות ושרידות בבעלי חיי חברתיי . בהקשר זה מסקרנת במיוחד השירה שכרוכה בהשקעת זמ , אנרגיה וסיכו טריפה . סביר להניח שבצד ה' מחיר ' קיימת ג תועלת אשר מצדיקה את ההשקעה . על ידי בחינת שירת ה זכרי , באמצעות כלי מתחו הפיזיולוגיה וה התנהגות , ניסיתי במחקר הנוכ חי לתת תשובות לשאלות בעלות היבט אבולוציוני סביב שאלת המחקר העיקרית הא שירה מעידה על איכות הפרט בשפני סלע? במיני שוני של עופות , דו חיי , חרקי ויונקי נמצא קשר בי אלמנטי שוני של שירה לבי מצב גופני , מדרג חברתי, רמות הורמונאליו ת והצלחה רביתית . כמו כ נמצא שבבעלי חיי שוני, זכרי מקשיבי לקולות שכניה ומשני את התנהגות בהתא למעמדו החברתי של משמיע הקול והיכרות עמו . שפני הסלע מהווי מודל אידיאלי לחקר נושאי אלה, כיוו שה בעלי חיי חברתיי , פעילי יו , מתקשרי בצורה קולית הנשמעת למרחקי ונחקרי בסביב ת הטבעית הכוללת טורפי וסכנות טבעיות אחרות . בעוד שזכרי צעירי משמיעי קולות לטווחי קצרי בלבד וזכרי מתבגרי משמיעי לעיתי קולות פעייה צרודי , חלק מה זכרי ה בוגרי מתאפייני בשירה של ממש מז. רי שוני נבדלי זה מזה ב זמ שה משקיעי בשירה ב, אור! השיר ,י וב מרכיבי השיר . מטרת המחקר הנוכחי הייתה בח ינת הקשר בי מרכיבי שירת הזכרי , המדרג החברתי , ה רמות של מספר הורמוני , מצב הגופני ו שרידות ה. מחקר התבצע בשמורת עי גדי , וכלל תצפיות שדה וניסויי בשדה ובמעבדה במש! חמש עונות . אוכלוסיית שפני המחקר בנחל ערוגות ובנ חל דויד, המונה מעל מא תיי ושלושי פרטי, סומנה סימו אינדיווידואלי על ידי קולרי , תגי אוז ושבבי תת עוריי . בנוס" לסימו , כל פרט נשקל , נמדד וצול . כמו כ , נלקחו מכל שפ דגימו ת שיער לצור! אנליזה הורמונאלית . הסימו האינדיווידואלי אפשר ער יכת תצפיות התנהגותיות , וריוד ג הפרטי ב חמש ה קבוצ ות החברתי תו . באופ מפתיע התברר שהפרטי הדומיננטיי ביותר בקבוצות השונות ה נקבות . בכ! מצטר" שפ הסלע לקבוצה אקסקלוסיבית ב עלת מבנה חברתי מטריארכאל ,י הכוללת את הפיל האפריקאי ( הקרוב פילוגנטית לשפ הסלע ) , ) הצבוע הנק וד והלמורי . בעזרת רגרסיה של גילאי ידועי של שפני מול נתוני מורפומטריי שלה , יכולתי לקבוע את גיל של כל השפני . הגיל הממוצע של הנקבות הבוגרות עלה על גיל של הזכרי . מצאתי כי מספר נקבות הגיעו לגיל 10 בעוד שזכר בודד באוכלוסיית המחקר הגיע לגיל 7 . זכרי , אשר נאלצי להיפרד מקבוצת הא שלה בהגיע לבגרות מינית , חשופי יותר למחלות וטריפה ונאלצי להתחרות ע זכרי רבי . למרות זאת , ה גדולי יותר מנקבות , ומצב גופ , אשר מיוצג על ידי מצב פרווה ומשקל מתוק לאור! גו" , טוב יותר מאשר זה של הנקבות . פרטי דומיננטיי , בשני הזוויגי , הינ מבוגרי וגדולי יותר מפרטי בעלי מעמד חברתי נמו! . . ב דגימות שיער נית למדוד רמות הורמוני שוני . בכל הפרטי מדד תי את ההורמוני טסטוסטרו ו אנדר וסטנדיו ( שמסווגי כאנדרוגני הורמוני ' זכריי )' , אסטרדיול ( אשר נחשב להורמו ' נקבי )' וקורטיז (ול ממשפחת הורמוני ה stress) . באופ שונה מהדג היונקי המקובל , רמות ה אנדרוגני אשר נמדדו בנקבות בוגרות דמו ל רמות שנמדדו בזכרי בוגרי . תוצאה זו היא ראשונה מסוגה . במעט המחקרי שפורסמו עד כה ביונקי , בה נמדד טסטוסטרו בשני הזוויגי, נמצא שרמתו בנקבות בוגרות נמוכה משמעותית מזו של זכרי בוגרי . . לנקבות בוגרות ג רמות קורטיזול גבוהות יותר מאשל לזכרי , יתכ שזו תוצאה של המאמ& וההשקעה הקשורי לרבייה . מעמד חברתי בנקבות נמצא בקשר ישיר ע רמות קורטיזול ובקשר הפו! ג ע מצב גופני וג ע רמות אנדרוגני . מצאתי שהשונ י באסטרטגיות ההורמונאליות בי הזוויגי מתחיל בגורי . תמותת הגורי גבוהה בשני הזוויגי , א! גורי זכרי ששרדו את שנת חייה הראשונה מאופייני ברמות קורטיזול נמוכות , בעוד שגורות ששרדו מאופיינות ברמות גבוהות ה של קורטיזול וה של טסטוסטרו . צירופי הורמונאליי אלה ה ה' מתכו ' לנחיתות חברתית בבוגרי , בשני הזוויגי . . שליש מהזכרי הבוגרי שרי . זכרי ששרי ה מבוגרי יותר ודומיננטיי יותר מזכרי שלא שרי . רמות הטסטוסטרו של זמרי גבוהות יותר ורמות האסטרדיול נמוכות יותר מזכרי שלא שרי . רמות הקורטיזול של זמרי ג בוהות יותר מזכרי שלא שרי וה עומדות בקשר ישיר ע המעמד החברתי שלה , בדומה לקשר בנקבות . פרשנות אפשרית לכ! היא שזמרי דומיננטיי מצויי במצב ' לחו& ' יותר מאשר זמרי נחותי יותר , יתכ בגלל שה מעורבי ביותר אינטראקציות אנטגוניסטיות , ובכ! מסתכני יותר . במרכ ז עבודת השדה , הקלטתי שיר ה של 17 זכרי . ווקליזציה זו עב רה קידוד דיגיטאל י י ואנליזה שאפשרה זיהוי אינדיווידואלי לכל זכר ששר . באמצעות תכנת מחשב ייעודית פורק ה ה השירה למרכיביה . כימתתי שלושה מרכיבי מרכזיי בשירה יללה (wail), גיהוק (chuck) , ונחירה (snort) - וב חנתי את החשיבות היחסית של כל אחד בהעברת מסרי ואמינות . מצאתי שכל מדד שיכול להעיד על איכות הפרט של השפ עומד במתא ע מרכיב אחר של השירה . למשל , גיל השפ , רמות האנדרוגני והמעמד החברתי שלו ניתני לקביעה על פי אלמנט הנחירות . משקלו של השפ עומד במתא ע מש! ה זמ שהוא משקיע בשירה , וגודלו ורמות הקורטיזול שלו נמצאי במתא ע אלמנט הגיהוקי . תדר הפורמנטי , אשר קשור לצינור העברת הקול , נמצא במתא ע מצב הפרווה וע המעמד החברתי . יתכ וקשרי אלו מעידי שאלמנט הגיהוק מעביר מסרי שנועדו לזכרי אחרי , בעוד שאלמנט הנחיר ות אולי נועד בעיקר לפרסו בפני נקבות . . שפני סלע זכרי מבוגרי אינ נפוצי בעי גדי . זכרי ששרדו והגיעו לגודל הסופי מפרסמי את גיל , גודל , מצב הגופני , מצב ההורמונאלי ומעמד החברתי באמצעות שירה . . שירה כרוכה בהשקעת זמ , אנרגיה וסיכו טריפה . בנוס" , גורמי נו ספי המאפשרי שירה מפותחת , כגו רמות הורמונאליות גבוהות , כרוכי א" ה בסיכוני רבי . איכות הפרט צריכה להימדד ג בהצלחה רביתית , והיתרו הסלקטיבי של שירה צרי! לכלול ג את העדפת הנקבות . תוצאות המחקר שלי מרמזות שאכ שירה מכילה מסרי אמיני לגבי מדדי רבי אשר יכולי לפרס את איכות הפרט בשפני סלע . .
עבודה זו נעשתה בהדרכת דר " אלי גפ ודר " עופר מוקדי
הא שירה מעידה על איכות הפרט בשפני סלע???
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