BEHAVIORAL AND GENETIC CORRELATES OF REPRODUCTIVE SUCCESS IN MALE (ADDAX NASOMACULATUS, DE BLAINVILLE 1816)

BY

EDWARD M. SPEVAK

DISSERTATION

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology with a concentration in Ecology, Ethology, and Evolution in the Graduate College of the University of Illinois at Urbana-Champaign, 2015

Urbana, Illinois

Doctoral Committee:

Professor Kenneth Paige, Chair, Director of Research Professor Andrew Suarez Professor C.-H. Christina Cheng Associate Professor Alfred Roca

ABSTRACT

The addax (Addax nasomaculatus) is a critically endangered whose range is now restricted to a few small populations in the and thus the focus of national and international conservation efforts. Very little is known about their general behavior and social structure, either in the wild or in captivity. This study examined the behavior of semi-free ranging herds of addax to establish the mating strategies male addax pursue to increase their reproductive success. This study demonstrates that addax have the ability to become territorial under certain conditions, possibly due to the high population density and constant resource base as seen in related species. Two mating strategies were evident among male addax in this population: territorial and following or non-territorial. Resources and the behavior and density of females influenced the presence of these different mating strategies with male age and rank and available territory determining the strategy. Male reproductive success of addax was assessed using both behavioral observations and genetic paternity analysis using microsatellites and the second exon of Adna DR3 of the major histocompatibility complex (MHC). There was a significant relationship between the age of the male and the number of offspring sired (rs =.46, N = 66, p = .0002) and a male's rank and his reproductive success (rs = -.88, p = .0007). There was no difference between the total number of calves sired by territorial versus non-territorial males. However, individual territorial males sired on average significantly more calves than non-territorial males. Possession 2 of a territory was a significant component of reproductive success (R = .27, F.05= 11.77, d.f. = 2, 63, p < .05). There was a significant difference between behavioral and genetic estimates with behavioral data overestimating the reproductive success of younger age classes (1-2 year olds and 2-3 year olds) and underestimated the 3 year olds and above. In this study, comparisons between genetic and behavioral estimates of reproductive success showed that behavioral estimates consistently underestimated the absolute reproductive success of successful males and overestimated the success of many unsuccessful males. However, in the absence of known paternity behavioral methods give an adequate estimate of male reproductive success for multi- male herds. This is the first study of addax in which territorial behavior was observed and where its benefits, in terms of reproductive success, were assessed. This study also demonstrates the usefulness of microsatellites and DRB3 of the MHC in helping to establish paternity and

ii therefore reproductive success in addax. Additionally, a further examination of Adna DRB3 of the MHC found low levels of allelic diversity in addax. Despite low levels of allelic diversity, as measured by DR measures of heterozygosity within and among addax populations were found to be informative, correlating highly with infant survivorship and average heterozygosity across the genome as measured using microsatellite markers.

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ACKNOWLEDGMENTS

So many people contributed in so many ways towards the development, execution, and final completion of this project. I would especially like to thank my third and final graduate advisor, Dr. Ken Paige, who took me on as his first graduate student and has put up with me these many years hoping that I would complete my dissertation. He has been the best advisor a student could ever ask for. He has been a good friend and colleague, without whose support, enthusiasm, and humor I could never have finished.

This research could never have been done without the support, staff, facilities, and addax of the Fossil Rim Wildlife Center and Fossil Rim Wildlife Foundation. To the late Jim Jackson and Christine Jurzykowski who gave me support, a home, and family in one of the most beautiful spots on earth. Also to Dr. Evan Blumer, a good friend and colleague, who was instrumental in getting me to Fossil Rim and getting me started and who worked closely with me on the setup of this research project.

To Adam Eyres, Rodney Marsh, Kelly Snodgrass and Bruce Williams who supplied me with more than ample amounts of friendship and good times and who cared about the addax as much as I did. To Drs. Evan Blumer, Tom Demar and Steve Osofsky for drawing countless tubes of blood and Cathy Thurman for packing and shipping blood and tissue samples to me in the lab. Also to Rebecca Christie and Peg Gronemeyer for keeping an eye on my addax while I was away from Fossil Rim and keeping me informed about their daily lives.

I would like to thank the late Dr. Tom Foose for his initial faith in me and for recommending me to the Addax Species Survival Plan (SSP) that I be the one to undertake and complete this research. Also to Terrie Correll who helped me piece together the history of the captive addax population and Dr. Robert Lacy for guidance.

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To Dr. Harris Lewin and the members of his lab Drs. John Beever, Michiel van Eijk, and Runlin Ma for introducing me to the major histocompatibility complex (MHC) and the beauty of its uses and information contained therein and how to run the lab tests to bring out the information.

I would like to thank the , Cheyenne Mountain Zoo, Henry Doorly Zoo, Fossil Rim Wildlife Center, Louisville Zoo, Miami Metro Zoo, Oklahoma City Zoo, , San Diego Wild Park, and the for supplying me with the blood and tissue samples to complete the project. To the Denver Zoological Society for providing me with additional financial support for lab equipment and supplies.

I would like to thank my first advisor, Dr. Michael Lynch, for giving me my first “home” at the University of Illinois- Urbana Champaign and to my second advisor, the late Dr. M. Raymond Lee, who gave me another “home”, support, and laughter when this project began.

To Drs. Christine Spolsky and Tom Uzzell for allowing me to use their lab in the startup of this project and to Dr. Chris Phillips for his humor and his initial guidance in my lab work. To Drs. Jim Jacobson and John Patton for collecting many of the initial blood samples and introducing me to new methods of DNA extraction and to the potential benefits of PCR.

I would also especially like to thank one woman who was instrumental in helping me start and complete this research. To my wife and best friend Mary Brong for encouraging me to go back to grad school, for always being there in the beginning and knowing that I could do it, and for being there at the end.

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TABLE OF CONTENTS

CHAPTER 1: INTRODUCTION ...... 1

CHAPTER 2: MATING STRATEGIES OF MALE ADDAX (Addax nasomaculatus): DOMINANCE AND TERRITORIALITY ...... 4

CHAPTER 3: A COMPARISON OF GENETIC AND BEHAVIORAL ESTIMATES OF REPRODUCTIVE SUCCESS IN MALE ADDAX ...... 23

CHAPTER 4: LOW GENETIC VARIABILITY AT MHC LOCUS Adna DR3 AND FITNESS IN ADDAX (Addax nasomaculatus) ...... 47

CHAPTER 5: CONCLUSION ...... 57

FIGURES AND TABLES ...... 60

BIBLIOGRAPHY ...... 90

APPENDIX A ...... 124

APPENDIX B ...... 127

APPENDIX C ...... 129

APPENDIX D ...... 134

APPENDIX E ...... 138

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CHAPTER 1: INTRODUCTION

The addax (Addax nasomaculatus) is a critically endangered antelope whose range is now restricted to a few populations in the Sahara. Addax are probably the most -adapted of all the and now one of the most endangered. They formerly ranged across the Sahara from in the east to the Rio de Oro and Senegambia in the west (Dolan, 1966; Haltenorth and Diller, 1980; Harper, 1945). However, they have been exterminated over most of their range (Harper, 1945; Newby, 1980, 1984; UNEP/CMS 2006; Durant et al., 2014) due predominantly to hunting and poaching (Newby 1980, 1990), along with habitat loss, and (Newby 1980). Addax are currently listed as critically endangered on the IUCN Red List of Threatened Species (2015).

Very little is known about their general behavior and social structure, either in the wild or in captivity, let alone the reproductive and mating strategies males pursue. Early explorers and observers had seen addax in the wild in herds ranging from about 10 to 12 individuals to over 1000 when migrating between different grazing areas (Dolan, 1966, Gillet, 1965, Harper, 1945, Newby, 1980, 1984). It is assumed that small herds are usually groups of females with one male (Newby, 1980, Newby, 1984) similar to related oryx species (Estes, 1991, Estes, 1993). However, nothing is known of their behavior in larger aggregations. Where large, multi-male addax populations do exist, different males may pursue alternative mating strategies depending on age, status, body size, habitat and area limitations effecting territory numbers and size, and the number of potential competitors. A few studies of addax have been undertaken on small captive or semi-free ranging populations (Gordon, 1987, 1989; Gordon and Bertram, 1987; Gordon, et al., 1988; Gordon and Wacher, 1986, Mackler, 1984; Manski, 1979, 1982, 1991; Rice, Unpublished). However, only one study observed animals for longer than three consecutive months (Manski, 1979, 1982, 1991), and no study has dealt with individual reproductive strategies.

It is presently very difficult to study addax in the wild because of their low numbers and the logistics of observing them within the large desert environment of the Sahara (Wacher et al.,

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2004; Wacher et al., 2007; Wacher et al, 2010b; Spevak pers. obs.). Census information on wild populations indicates an estimated 250 to 300 individuals left in small isolated populations across Southern and Central Sahara in , , and , (UNEP/CMS, 2006) with the last major population of around 200 individuals in the Tin Toumma desert of eastern Niger (UNEP/CMS 2006; Wacher et al. 2004; Wacher et al., 2008).

Addax are well established in captivity in North America. Based upon the International Addax Studbook (Correll, 1989a) current records of addax in North America began with a pair received by the Brookfield Zoo from the Khartoum Game Commission in the in 1935. In 1955 another pair of addax from the Hannover Zoo of Khartoum Zoo origin arrived at the Brookfield Zoo. In the early 1960s more addax (3 males and 8 females) arrived in North America. Most of these animals were wild caught from Fort Lamy, Chad or zoo stock from the Hannover and Khartoum Zoos. It is reasonable to assume that the Fort Lamy animals were related and the Hannover and Khartoum Zoo were certainly related. The only addax received in this period that were not related to the Fort Lamy, Hannover and Khartoum animals were a pair received by the San Diego Zoo from NNW of Obeche, Chad. Since this time lineages have been mixed and animals moved around North America. An analysis of the studbook data finds only 14 identifiable founders to the North American captive population. However, due to lack of certain pedigrees within various captive populations there is the possibility of an additional six founders.

The addax herd at the Fossil Rim Wildlife Center in Glen Rose, Texas (the population under study here) was started in late 1974, with the first calf born in 1975. Additional animals were acquired through the 1970s. All animals were acquired from other zoos and animal dealers. Records are very poor on numbers acquired and from which institutions and dealers. As the last imports of addax into the U.S. occurred in the 1960’s all animals are derived from captive stock and would be related to other captive populations.

Observations of captive or semi-free ranging animals are presently the only way to learn about the reproductive biology of addax as well as many other . Examinations of the behavior of animals in captivity compared to the wild suggest that large changes or differences in

2 behavior are not necessarily expected (Ricker, et al., 1987). Changes in behavior that do occur in captivity are in many cases qualitative changes in the expression of behaviors, not changes in the patterns of the behavior themselves (Price, 1984).

Addax are a critically endangered species (IUCN, 2015) and thus the focus of national and international conservation efforts (Grettenberg, 1989; Mallon et al, 2001; Newby and Grettenberg, 1986; Spevak et al., 1993; UNEP/CMS, 2006; Wacher et al. 2004, 2008). These data on the behavior and social structure and genetic variability of this species will be instrumental for successful conservation and reintroduction efforts (Wielebnowski, 1998).

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CHAPTER 2: MATING STRATEGIES OF MALE ADDAX (Addax nasomaculatus): DOMINANCE AND TERRITORIALITY

Introduction

Most of the 75 species of African bovids, which includes the antelope, are polgynous and male fitness is determined by the number of mating opportunities they can monopolize. Only members of the dwarf antelope (), i.e., dik-diks (Madoqua sp.), ( campestris), (Oreotragus oreotragus), (Ourebia ourebi), and other dwarf antelope ( sp.), along with the common (Redunca arundinum) (Reduncini) and some of the (Cephalophini) exhibit monogamy (Estes, 1991). Among antelope, male mating strategies are hypothesized to have evolved to maximize access to receptive females (Gosling 1986). Males have two main options to maximize their number of matings. Males can follow females over all or part of their range, and maintain dominance over other males that are also following. This mating strategy is termed female or harem defense polygyny (Emlen and Oring, 1977). Alternately, they can sit and wait in part of the females' range and defend it against other males. The sit-and-wait strategy is usually termed resource defense polygyny (Emlen and Oring, 1977), as the male defends sedentary females or a resource that females require (e.g., food, cover, calving sites, etc.).

Gosling (1986) describes the following conditions that would favor following and resource defense strategies in antelope. Conditions that favor a following strategy are: 1) low density and unpredictable food resources causing female numbers to be low and their presence unpredictable, 2) low degree of breeding synchronicity with females receptive during a large part of the year, 3) females having a tendency to form groups that a male could monopolize by following, and 4) potential mates and competitors being too concentrated in space and time for a sit and wait strategy to be economic. Resource defense territoriality would be favored when: 1) there is a high quality and clumped food resource, or other required resources, so that the number of females and their tendency to form groups is high, 2) there is a heterogeneous or seasonal food supply that would increase the amount of time females remained in one area, 3) there is a high degree of

4 breeding synchronicity, 4) males familiar with a particular area are less likely to be killed by predators, and/or 5) the costs of resource defense territoriality are lower when males have an owner advantage over males that adopt a following strategy. However, in any system there are often males that adopt either a following or sit-and-wait strategy because of age, status or space availability or limitations for territories and movement.

Because resources may have a major impact on social structure and mating strategies, a number of studies have examined the implications of an antelope's ecology upon its social structure (Estes, 1974; Geist, 1974; Gosling, 1986; Jarman, 1974; Owen-Smith, 1977; Rubenstein, 1989; Western, 1979). In general, these studies have found that across species, social systems and reproductive strategies vary with ecology and population density. For example, ( lunatus) males use different reproductive strategies in different populations, from lekking to resource defense territories to the following of large mobile female herds (Gosling, 1986; Jewell, 1972). These differences were due to female density and movement patterns. Variations in reproductive strategies have also been found in other antelope species including, (Connochaetes taurinus) (Attwell, 1982, Estes, 1969; Talbot and Talbot, 1963), ( kob) (Floody and Arnold, 1975), (Oryx gazella) (Estes, 1991; Gosling, 1986) and dorcas (Gazella dorcas) (Baharav, 1989). However, it is not clear how variations in mating strategies and social structure within populations affect an individual’s reproductive success.

In addition to the effect of differing social structures and reproductive strategies on reproductive success, an individual male’s dominance status and territorial behavior can also affect the reproductive success of that individual male. Dominance and high social status may either exclude many potential breeding individuals from reproducing and/or provide for exclusive access to potential mates and in territorial species affect an individual's ability to establish and maintain a breeding territory (Clutton-Brock, et al., 1982; Duncan, et al., 1984; Estes, 1969; Floody and Arnold, 1975; Fryxell, 1987; Leuthold, 1977; Lott, 1981; Murray, 1982; Owen- Smith, 1984; Poole, 1989; Schaller, 1972; Sinclair, 1977).

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The addax (Addax nasomaculatus) is an antelope whose range is now restricted to a few populations in the Sahara. Very little is known about their general behavior and social structure, either in the wild or in captivity, let alone the reproductive and mating strategies males pursue. Early explorers and observers had seen addax in the wild in herds ranging from about 10 to 12 individuals to over 1000 animals when migrating between different grazing areas (Dolan, 1966, Gillet, 1965, Harper, 1945, Newby, 1980, 1984). It is assumed that small herds are usually groups of females with one male (Newby, 1980, Newby, 1984) similar to related oryx species (Estes, 1991, Estes, 1993). However, nothing is known of their behavior in larger aggregations. Where large, multi-male addax populations do exist, different males may pursue alternative mating strategies depending on age, status, body size, habitat and area limitations effecting territory numbers and size, and the number of potential competitors. A few studies of addax have been undertaken on small captive or semi-free ranging populations (Gordon, 1987, 1989; Gordon and Bertram, 1987; Gordon, et al., 1988; Gordon and Wacher, 1986, Mackler, 1984; Manski, 1979, 1982, 1991; Rice, Unpublished). However, only one study observed animals for longer than three consecutive months (Manski, 1979, 1982, 1991), and no study has dealt with individual reproductive strategies.

This study examined the behavior of semi-free ranging herds of addax to establish the mating strategies male addax pursue. Specifically, I used behavioral observations of semi-free ranging animals to address the following questions: Do addax exhibit territorial behavior or do they only pursue a following strategy? What are the possible benefits and costs of differing strategies? Finally, how do age, dominance hierarchy, the behavior and distribution of females, and space limitations effect the choice of male reproductive strategies? Addax are a critically endangered species (IUCN, 2015) and thus the focus of national and international conservation efforts (Grettenberg, 1989; Mallon et al, 2001; Newby and Grettenberg, 1986; Spevak et al., 1993; UNEP/CMS, 2006; Wacher et al. 2004, 2008). This data on the behavior and social structure of this species will be instrumental for successful conservation and reintroduction efforts (Wielebnowski, 1998).

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Materials and Methods

Species Information Addax are medium-sized antelopes from belonging to the tribe Hippotragini of the subfamily Hippotraginae of the (Artiodactyla). Other related extant members of this tribe include five species of oryx, i.e., (Oryx leucoryx), gemsbok (Oryx gazella), scimitar-horned oryx (O. dammah), beisa oryx (O. beisa beisa) and fringe-eared oryx (O. b. callotis) and the sable ( niger) and roan antelopes (H. equinus) (Dixon and Jones, 1988; East, 1999; Haltenorth and Diller, 1980).

Addax exhibit relatively little , females are 95 to 110 cm at the shoulder and weigh up to 90 kg, while males are about one fifth heavier, up to 150 kg, and 105 to 115 cm at the shoulder (Haltenorth and Diller, 1980). Both sexes possess spiraled horns that may reach a length of 110 cm with male's horns being thicker.

Study Herd and Area Behavioral observations were conducted at the Fossil Rim Wildlife Center (FRWC) in Glen Rose, Texas, from September through December 1989, and from June 1990 through June 1992. The study herd of addax lived in an enclosure covering approximately 420 acres. The enclosure is part of a drive-through facility and open to the public. However, only approximately fifty acres of this enclosure is accessible to the general public. As in other facilities and national parks animal behavior appeared little affected by presence unless people left their vehicles (Schultz and Bailey, 1978).

All individuals in the study were identifiable by ear tags and differences in shape, body size, and coat pattern. The herd was composed of 48 individuals (12 males, 36 females) at the beginning of the study, with individuals ranging from 0 months to 15+ years of age. The size of the herd at the end of the study was 67 (23 males, 44 females) ranging from 0 months to 18+ years of age. Other species of present in the enclosure included ,

7 gemsbok, (Kobus ellipsiprymnus), fallow ( dama), (Antelope cervicapra), axis deer or (Axis axis) and whitetail deer ( virginianus).

Vegetative cover was primarily open grassland or oak (Quercus sp.) savanna with some denser stands of oak or juniper (Juniperus sp.). Topography was hilly with elevations ranging between 290m and 380m. Water is continuously available from a stream and four man-made ponds. Animals forage naturally, but are supplemented once a day with a 20% protein pelleted herbivore mix in the public areas of the pasture. Animal forage was supplemented during fall and winter with bales of alfalfa or lucerne (Medicago sativa), and round bales composed of coastal Bermuda grass (Cynodon dactylon) and Kleingrass ( coloratum) placed throughout the enclosure. Mineral and salt blocks were also present. Shelters were available for inclement weather.

Sampling Over 1200 hours of observations were made of the study population. Observations were performed from a jeep or pickup using 10 x 50 Bushnell binoculars. To facilitate data recording an ethogram, i.e., a comprehensive list of behaviors along with their descriptions and characteristics, as developed for addax was used during the study (Lehner, 1996) (Appendix 2). Scan samples (Lehner, 1979) of the herd were taken near dawn, mid-day, and dusk. For each scan sample date, time, weather conditions, i.e., sunny, overcast, rain, etc., temperature, group composition, and individual behavior and location were recorded. Sender and receiver were indicated for any interactive behaviors (e.g., mounting, horn-wrestling, etc.). The entire enclosure was traveled during observations and an attempt was made to find every individual for each observation period. Because the habitat in parts of the pasture was either heavily wooded or inaccessible by vehicle not every individual was observed every day during the study. All occurrence data on agonistic behavior were recorded as well as ad lib notes on general and reproductive behavior.

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Variables and Definitions

Dominance Rank Dominance ranks were established by two methods. Animals were ordered in a win/loss matrix, with winners listed in rows and losers in columns, to minimize the number of reversals (i.e., the number of interactions listed below the diagonal) (Lehner, 1979) (Appendix D). Ranks were also determined by weighing an individual’s rank by the rank of its opponents (Clutton- Brock, et al., 1982). This rank was determined by the number of males an individual defeated (B), plus the total number of males they defeated excluding the subject (b) divided by the number of males he lost to (L), plus the total number they lost to excluding the subject (l). This index of fighting success was:

(B + b + 1) / (L + l + 1)

Adding one to each side of the ratio reduced the chances of an anomalous result in instances where an individual was never observed to defeat another or never observed to be defeated. In both methods of estimating ranks it was assumed that an individual that defeated another could also defeat all those individuals that the latter defeated. The two methods for assigning dominance ranks, win/loss matrix (Lehner, 1979) and weighting an individual’s rank by the rank of its opponents (Clutton-Brock et al., 1982), produced very similar results (R2 = .932, p <.0001). Based upon observed movements and behavior, the win/loss matrix was used in the analyses of reproductive success. Only males that were capable of siring offspring were examined in the dominance hierarchy, i.e., ten months and older.

Territorial Behavior A territory can been defined in a number of ways including: "any defended area" (Noble, 1939), "a spatially fixed area within which a given animal consistently prevents certain other individuals from engaging in certain activities." (Leuthold, 1977), or a “system of mutually exclusive areas occupied by males” (Owen-Smith, 1975). Each of these definitions has limitations when applied to observed behavior in addax, e.g., not allowing for distinctions between territorial and non-

9 territorial defense of an area (Noble, 1939; Walther et al. 1983), implying defensive behaviors in regard to a territorial male when dominant offensive behaviors may be more relevant (Leuthold, 1977; Walther et al, 1983), and that territories have precise boundaries and other males are not tolerated (Leuthold, 1977, Owen-Smith, 1975; Walther et al, 1983) The definition by Walther et al (1983) of a territory as "a place in which an animal lives for a variable period of time and around which that animal has established a subjective boundary" addresses previous limitations in definitions of territories (Walther et al., 1983) and works well for and other ungulates.

I used the criteria by Walther et al. (1983) for establishing the existence of territoriality in male addax. Walther et al. (1983) describe several behaviors associated with his definition of territory: 1) intolerance or dominance over conspecifics of the same sex within its boundaries, 2) higher thresholds for flight inside the territory, 3) efforts to herd back conspecifics of the opposite sex when attempting to leave the territory, 4) sudden halting of the territorial animal when arriving at the boundary, 5) occasional occurrence of agonistic behavior by other males outside owners territory when the owner temporarily leaves his territory, and 6) the establishment of a scent- marking system related to the structure of the territory. In regard to scent-marking systems, certain behaviors were included that would be indicative of territorial males.

When not accompanying possibly receptive females, presumed territorial males continually patrolled their territories. Constant patrolling may deposit scent from interdigital glands (Estes, 1991). Territorial males also performed vegetation-horning (Estes, 1991), i.e., thrashing bushes and sapling trees, which could serve as a visual marker as well as a possible olfactory sign (Estes, 1991). Territorial males also defecated regularly in a ritualized squat posture (Estes, 1991, Mackler, 1984, Manski, 1979, 1991). Additional data were recorded on the movements of males and their behavior toward other males and females.

Age Classes Maximum life span for an addax is approximately 30 years (Correll, 1989a). The maximum known age of an individual at death in the study population was a female at 18 years and 8 months. Approximate ages were established for most individuals born prior to 1989 and were

10 based upon previous breeding history, physical appearance, and available records. Most individuals born after 1988 had known birthdates. Four age classes were used for the purpose of this study: 0 to 1 year, 1 to 2 years, 2 to 3 years, and 3 years and above. These designations were based upon behavioral and physical development.

0 to 1 year: Males are able to sire their first offspring. Age at sexual maturity for males had been described in the literature as approximately 2 3/4 years (Densmore and Kraemer, 1986; Haltenorth and Diller, 1980). However, paternity analysis identified three males siring offspring before one year of age (Spevak, unpublished). Two males had known birth dates and based on a gestation period average of 260 days (range 257-264 days) sired offspring at 319 and 325 days of age respectively (Spevak, unpublished).

1 to 2 Years: Females are able to conceive and calve their first offspring. Several females gave birth to their first calf at 2 years of age. Assuming a gestation period of approximately 260 days, females reached sexual maturity between 15 and 16 months.

2 to 3 years: Males and females reach adult size and males begin performing secondary sexual behaviors, e.g., squat defecating (Estes, 1991; Mackler, 1984; Manski, 1979, 1991).

3 years+: Males may establish territories.

Calving In the wild, addax calves are born between the cold and wet seasons, i.e., winter or early spring (Newby, 1984) but can be born throughout the year over a wide range of months, both hot season and cool season (Wacher et al., 2010b). In captivity females calved and conceived throughout the year with no defined rut (Densmore and Kraemer, 1986) (Figure 1.2). Although calves are born throughout of the year, there is a peak with over half of all births in the North American population occurring from December through June (Densmore and Kraemer, 1986). The apparent uneven distribution in calving may be due to a partial seasonal anovulatory period noted in addax from November through June (Asa et al., 1996). This anovulatory period is similar to that

11 exhibited by the related scimitar-horned oryx (O. dammah) also exhibiting a seasonal birth peak (Morrow et al., 1999)

Females separate themselves from the rest of the herd prior to giving birth (Manski, 1979, 1991). This separation by the female is found in most species of antelope and is an anti-predator response (Estes, 1991). Addax females usually remain in one area (i.e., territory) for the first one to two weeks after calving then move between different male territories.

Females come into a post-partum estrus shortly after calving (Densmore and Kraemer, 1986) followed by another approximately three weeks later (Wacher, 1988). There appears to be a 20 to 21 day estrus cycle similar to the related scimitar-horned oryx (O. dammah) (Gill and Cave- Browne, 1988). These estrus periods were evident from observations of both male and female behavior. During these times a territorial male was usually in consort. Frequent conceptions during the post-partum estrus leads to an average inter-birth interval of approximately nine months (Densmore and Kraemer, 1986).

Statistical Analyses Although individual fitness (sensu Darwin) is not necessarily easily defined and its definition is highly context dependent, most authors agree that fitness equates to some measure of genetic contribution to future generations (Brommer et al. 2004), and empirical studies conventionally use LRS (Lifetime Reproductive Success, usually defined as the number of offspring surviving to breeding age sired by a parent) as a valid single-generation proxy of long-term genetic contribution (see Clutton-Brock 1988; Brommer et al. 2004). In this study, because calves were caught as neonates and the duration of the study precluded many of these calves reaching reproductive age, reproductive success was defined as the number of born offspring sired by a male, as a proxy of individual male fitness. Although this measure does not integrate a juvenile survival component, calf survival should be almost exclusively affected by maternal rather than by paternal influences (Gaillard et al. 2000). The number of calves/offspring sired was the dependent variable in most analyses. Independent variables include age, dominance rank, number of other males present, whether males were territorial or non-territorial, and individual

12 identification. Territoriality was analyzed as both a dependent and independent variable in analyses when identifying factors affecting territorial behavior and male reproductive success, respectively. Statistical analyses, including ANOVA, stepwise regression, Spearman’s rank correlation were completed using the SYSTAT statistical package, version 5.2 (SYSTAT, 1992).

Results

Territorial Behavior Using the criteria of Walther et al (1983), addax in this population exhibited territorial behavior. Males in this population did not establish territories until they were at least three years of age. At the beginning of the study only two territories were identifiable (Figure 2.1a). However, there were only two males over three years of age in the herd. By the end of the study five territories had been established with seven different males over three years of age (Figure 2.1c).

As a male reached 3 years of age he might isolate himself from the rest of the herd in an area and not move with the female groups moving throughout the pasture, becoming more sedentary in an apparent attempt to carve out a territory or usurp a territory from an established territorial male. Agonistic encounters between territorial males often occurred at a territorial boundary and would often either reinforce or alter a territory's boundaries. Territorial males occasionally left their territories for food or to attempt to usurp females from an adjacent territory. When a male was ousted from his territory he did not rejoin the herd as a following non-territorial male. He found another area of the pasture within which he established another territory. By this method territory boundaries changed and territory sizes decreased until a maximum of five territories were established. One territory comprising half of the pasture at the beginning study was at various times held by four different males and divided into three territories (Figure 2.1b). Most territories also had some obvious topographic features that acted as boundaries, e.g., streams and/or roads.

Territorial males continually attempted to herd females, (i. e., those of breeding age or with calves) away from territorial boundaries and towards the center of territories. Females were often successful at getting past these males. Two territorial males were observed herding calves within

13 their territories. By this method they were successful in attracting the calves' mothers back into the territory. Among small groups of addax and within territories, movement was usually led by a dominant female with the dominant male following and establishing the direction of movement. Males established direction for a group by herding females in the direction of travel and cutting off and threatening those females that moved off in a different direction. Males that did not possess territories moved with female groups as they moved between territories. These males had little influence on a group's movement.

Males over two years of age defecated in a ritualized squatting posture that appears to be a secondary sexual behavior that appears with age and demonstrates status. Males defecate in a standing or squatting posture. A squat defecation was usually performed by a territorial male or the most dominant male in an area (Estes, 1991; Mackler, 1984) but was occasionally observed during agonistic encounters. For example, a lower ranking male occasionally performed a squat defecation in the presence of a higher-ranking male after defeating another male in a horn- wrestling bout. Outside of this context a higher-ranking male would challenge if he observed a subordinate squat defecating. The posture itself is a major display component as males were occasionally seen squatting without defecating.

In territorial males this defecation did not appear to mark the boundaries of territories (R. D. Estes pers. comm.) but was usually placed along routes and in areas frequented by other addax. No specific defecation areas or routes were noticeable in non-territorial males that squat defecated.

Non-territorial or Following Behavior The number of non-territorial or following males was always greater than the number of territorial males. At the beginning of the study there were 10 following non-territorial males compared to 2 territorial males. At the end of the study there were 15 non-territorial males and 5 territorial males. There are three probable reasons for an individual male to exhibit following behavior. The age of the individual male was a factor as only males 3+ years of age attempted to hold territories. Also, only the highest ranked individuals possessed territories. There are always

14 subordinate individuals. Finally, there was an apparent lack of space for additional territories to be established as there were two additional 3+ year old males at the end of the study that did not possess territories. All these factors led some males to pursue an alternate mating strategy of following.

Dominance Hierarchy Addax established and maintained dominance status through a variety of agonistic behaviors, e.g., overt aggression, ritualized displays (See Appendix B), and displacement of individuals. Occasionally, a dominant individual displaced a subordinate without physical contact or display when a subordinate moved away at the approach of a dominant addax. This form of displacement was more apparent in interactions between males and females and males and calves. Displacement was also more apparent in interactions between females and between calves. Males appeared to use their horns more readily and engage in physical contact in contests. Closely ranked males took part in horn wrestling bouts, inter-locking horns in an apparent test of fitness. Individuals would also jab a subordinate with the tips of the horns or strike with the shaft. Displays of horns without contact occur in various ritualized postures where horn position, e.g., forward, upright, laid along the back, indicate status and a willingness to escalate an encounter (Estes, 1991). Compared to other antelope, addax appear to exhibit great variation in horn configuration ranging from a wide "v" with tight spirals to narrow set and open spirals. Occasionally individuals broke off all or part of one or both horns. However, individuals appeared to adapt to these differences and modified their behavior accordingly in horn wrestling bouts.

Four different dominance hierarchies where observed during the course of the study for the time periods 9/89 to 12/89, 6/90 to 2/91, 3/91 to 11/91, and 12/91 to 6/92 (Table 2.1) (Appendix D). The number of males in each hierarchy ranged from 12 at the beginning of the study to 20 at the end. Only potentially breeding males were ranked, i.e., ten months and older (see Age Classes). These four hierarchies represented periods when there were major changes in the population (e.g., males reaching sexual maturity, more territories established, males removed from herd,

15 etc.) and were predominantly linear. Each hierarchy was treated as an independent event for analysis (Cowlishaw and Dunbar, 1991).

Males were usually lost to the hierarchies by being sent to other institutions. Only one male during the study was lost due to death. On February 12, 1991 a fight occurred between two males over access to a receptive female. During the encounter the alpha male's horns (Male 61) pierced the abdomen of a three year old male (Male 107) severing a renal artery and causing severe internal hemorrhaging. He died within minutes. Most male-male interactions resulted in no injuries or only minor cuts and scratches.

Calving During the study at Fossil Rim, 63 of 89 or 70.8% of calves were born from December through June. In addition, 7 of 13 (53%) first-calf births by females during this study occurred during February, March and April. Other primiparous calves were born throughout the year.

Females calved in various parts of the enclosure usually away from the public. However, females did not calve in all areas equally but showed a preference for an area furthest away from the public drive with over 44% of calves born there (2= 68.057, d.f. = 7, p<.0001) (Figures 2.3 and 2.4). The territory holder of this calving site changed several times with three different males occupying it at various times. These males were often not the sires of the calves (Spevak, Chapter 2). This area was also usually frequented at the end of the day by most of the addax, excluding other territorial males.

Twenty-nine of 57 (51%) calves to iteroparous females at FRWC were conceived during the first and second post-partum estrus periods. One female in the study herd produced four calves in succession nine months apart. With a nine month interbirth interval no discernable rut was evident and therefore sexually receptive females were present throughout the year.

Correlation and stepwise multiple regression analyses were conducted to examine the relationship between territoriality in male addax and various potential predictors including a

16 male’s age, dominance rank, individual identification, and number of breeding males in the population. Male rank correlated with territoriality, as higher-ranking males were territorial. However, age was a greater predictor of territoriality as only males three years of age and above attempted to possess territories. Although age and rank on territoriality were significantly correlated (R2 = .553, F = 37.719, p < .0001), only age was significant in the relationship (stepwise multiple regression coeff. = -.49, p = .0008). The negative value of the coefficient indicates the presence of multicollinearity (Neter et al., 1996), especially as indicated between age and rank on territoriality, as older males tend to be higher ranked as well as territorial.

Discussion

Addax Territoriality Addax have never been described as territorial most likely because previous studies of addax were of small and/or low-density populations and no territorial behavior was noted (Gordon, 1987, 1989; Gordon and Bertram, 1987; Gordon, et al., 1988; Gordon and Wacher, 1986; Mackler, 1984; Manski, 1979, 1982, 1991). This study demonstrates that addax have the ability to become territorial under certain conditions. The existence of territoriality in the herd at the Fossil Rim Wildlife Center (FRWC) is probably due to the high population density and constant resource base (Gosling, 1986). Addax herd density during the study ranged from one addax per 3.54 hectares at the beginning to one addax per 2.53 hectares at the end. Strong territorial behavior was exhibited throughout the study. These high densities are an artifact of captivity and most likely much higher than may have occurred in the wild. At what densities addax become territorial is presently unknown and needs to be established. Additionally, it is currently unknown whether density or resource availability is the stronger determinant of territorial behavior or a combination of the two. However, in the wild, two related oryx species, gemsbok (O. gazella) and fringe-eared oryx (O. beisa. callotis), also become territorial when population densities are high and/or resources are constant (Estes, 1974, 1991, 1993; Wacher, 1988). Territorial behavior under conditions of high density or constant resources has been observed in other species as well, including topi (Gosling, 1986; Jewell, 1972), wildebeest (Attwell, 1982: Estes, 1969; Talbot and Talbot, 1963), kob (Floody and Arnold, 1975), (Baharav, 1989), and ( nippon) (Balmford et al, 1993). This of behavioral plasticity and

17 variation in social structure appears to correlate with environmental influences of diet and habitat preferences (Brashares et al. 2000). Additionally, variation in mating strategies within populations of individual species “is most likely maintained as a conditional strategy influenced by multiple internal factors (especially age, health, body size) and external factors (particularly density at small, local scales)” (Isvaran, 2005b).

Addax are a specialist of sandy desert regions and are the characteristic occupant of Saharan dunes, adapted to very dispersed pastures (Newby, 1984; Grettenberger and Newby, 1990). Early explorers and observers in the Sahara had seen addax in the wild in herds ranging from about 10 to 12 (Dolan, 1966; Gillet, 1965; Harper, 1945; Newby, 1980, 1984). A recent survey of the last viable population of addax in the wild in the Tin Toumma region of Niger found group sizes ranging from 1 to 20 individuals (Wacher et al., 2008) with mean and typical observed group sizes in this same region from 2007 to 2009 ranging from 4 to 7 and from 7 to 16, respectively (Wacher et al, 2010b). These small herds are usually groups of females and young with one dominant male and additional subordinate males (Newby, 1980, 1984; Wacher et al, 2008). Due to the ephemeral and widely distributed nature of forage addax travel many miles between feeding areas (Wacher et al., 2008) but the actual distances traveled by groups are still unknown. Historically, there have also been observations of groups of several hundred individuals presumed to be animals migrating between different grazing areas (Dolan, 1966, Gillet, 1965, Harper, 1945, Newby, 1980, 1984) but were probably also a result of many smaller herds congregating seasonally and temporarily in areas of exceptional grazing (Newby, 1987; reviewed in UNEP/CMS 2006). However, surveys of the Ouadi Rimé-Ouadi Achim Game Reserve in Chad, a grassland and open savanna habitat, found numerous historical signs of prior occupation by addax, i.e., old horn fragments (Wacher et al, 2010a), indicating a possible more permanent residence and in area of more abundant resources. Finally, the Sahara desert though estimated to be between seven and eleven millions years old (Zhang et al., 2014) went through at least three “greening” episodes over the past 120,000 years when much of the Sahara would have been covered with extensive grasslands (Tjallingii et al., 2008). The behavioral plasticity of male addax in regard to reproductive strategies would have allowed them to adapt and exploit these changing conditions and diverse habitats.

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Among Walther's (1983) criteria for establishing the existence of territoriality, which was not directly verifiable in addax, was the establishment of a scent marking system related to the structure of the territory. However, there were indications that a scent marking system may exist. Males continually patrolled their territories which may have deposited scent from interdigital or pedal glands (Dolan, 1966; Estes, 1991). Additionally, the ritualized squat defecation by territorial males leaves very conspicuous piles of fecal pellets along routes and in areas frequented by other addax. This may serve to advertise to others of a territorial male's presence and act as both visual and olfactory cues. Males were often observed sniffing at these dung piles and depositing their own nearby or directly on top of an older pile or that of another male’s.

Territorial males also performed vegetation-horning (Estes, 1991), i.e., thrashing bushes and sapling trees, outside of agonistic encounters. For example, when male 108 was returned to his territory following minor medical treatment he frequently squat defecated and horned vegetation. No other addax were present during the performance of behaviors. Whether this behavior serves as an auditory and visual advertisement of a territorial males presence, as it does in sable antelope (Estes, 1991), or an olfactory advertisement by laying down sent from glandular skin on the forehead, as in (Aepyceros melampus) (Estes, 1991), needs to be examined.

Male Mating Strategies and Reproductive Success Two mating strategies were evident among male addax in this population: territorial and following or non-territorial. Resources and the behavior and density of females influenced the presence of these different mating strategies. In addition, age and rank and available habitat for territories determined the strategy that a male undertook. A male's subsequent reproductive success could be affected by his mating strategy.

The observation that females separate themselves from the rest of the herd at calving would also have a strong influence on male mating strategies. When females isolate they are often followed or visited by the territorial or dominant male. Following is due, in part, to the fact that prior to parturition, females display body postures and hormonal changes similar to estrus (Manski, 1979, 1982, 1991). In small non-territorial mobile herds the dominant male often isolates himself with

19 the female, as in the related Arabian oryx (O. leucoryx) (Stanley-Price, 1989). These subgroups may last two weeks or more. In these cases a male might easily monopolize a female during the post-partum estrus period. However, with no defined rut, with females calving and cycling throughout the year, and the female's tendency to isolate when calving, the location of receptive females becomes less predictable. As female numbers increase territorial behavior would become advantageous. Resident males would tend to encounter females on a more or less regular basis (Wacher, 1988). However, not all territorial males would be equally successful in monopolizing a post-partum female. Females calved in various locations (Figure 2.4), yet all areas of the pasture were part of one male’s territory. In addition, individual territorial males may be at a disadvantage as females moved among territories. Several times territorial males were observed making forays into another territory in an attempt to herd possibly receptive females back into their territories.

Calves hide for the first two to six weeks of life after which they spend most of their time with their mother and the herd. Prior to the female's introduction of her calf to the rest of the herd most of her associations are with other females with similar aged calves (i.e., nursery or peer group), territorial males, or non-territorial males. These nursery/peer groups, which may remain stable for up to six months, also formed a temporary crèche of calves, with or without an adult present. Mothers returned periodically after foraging to nurse their offspring. Non-territorial males often only had access to a post-partum female when the territorial male left or the female was apparently not in estrus.

A large portion of females (over 50%) calve during late winter and early spring. This would increase the frequency with which a territorial male encountered post-partum estrus females. However, females are asynchronous in their calving and receptive post-partum females could be found throughout the year. With over half of all conceptions for iteroparous females occurring shortly after calving, when females are isolated from the rest of the herd, there is an advantage for males to become territorial. Additionally, as females with similar aged calves form nursery groups this would increase the total number of females a territorial male has access to during the post-partum estrus periods.

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Non-territorial males have one advantage over territorial males in that they are able to follow females throughout their movements and would be in a better position to detect non-post-partum estrus cycles. In theory there could be an upper and lower threshold when following or non- territorial behavior could be more successful. This might happen when female numbers are so low that a territorial male might rarely encounter them or when female numbers are so large that an individual territorial male could not control all of them or might spend more time trying to defend them from other males than breed with them.

Increased reproductive success for territorial males may also be related to the location of territories. For example, topi males on leks have the highest reproductive success if they possess the central territories regardless of which male possessed the territory (Gosling and Petrie, 1990). Additionally, Deutsch and Nefdt (1992) found that female Kafue (Kobus leche kafuensis) and Uganda kob (K. kob thomasi) frequented successful lek territories not successful lek males. This selection may be related to olfactory cues in the territory, i.e., the scent from urine of previous female occupation (Deutsch and Nefdt, 1992). Addax females did show an apparent preference for particular territories or rather locations within territories in which to calve. The use of a territory by a female to calve may encourage other females to use the territory. Therefore, males should compete for those areas that females frequent most and potentially increase their reproductive success due to female choice of the territory. Since over 44% of calves were born in one area (area 1 see Figure 2.4), the male possessing this area would have an increased opportunity to breed during the post-partum estrous cycles. However, in this study no apparent increase in reproductive success for the territorial male was evident.

At Fossil Rim the number of territories and, therefore, territorial males was probably limited by the available space in the enclosure. At the beginning of the study only two territorial males were evident. These two males were also the only males over three years of age. At the end of the study five males held territories. An indication that space may affect the total number of territories is the fact that there were two additional males over 3 years of age that did not possess territories. The two 3-year-old males which did not possess territories were also lower ranked in the dominance hierarchy than the territorial males.

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However, once a male became territorial, he did not revert to being a follower moving with the females. For example, during the time of this study male 108 dropped in the dominance hierarchy from second to fourth. He remained a territorial bull but probably due to his status was only able to establish a territory in an area relatively little used by females for foraging or calving. Interestingly, prior to male 108's establishment of his new territory no female had been observed calving in this area; afterwards five different females calved in this area. Whether females felt the area now was secure for calving with 108 present or there was some female choice for breeding with 108 during the post-partum estrus cycle is unclear.

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CHAPTER 3: A COMPARISON OF GENETIC AND BEHAVIORAL ESTIMATES OF REPRODUCTIVE SUCCESS IN MALE ADDAX

Introduction

Male mating strategies involving dominance and territoriality are thought to have evolved to maximize access to receptive females. Many estimates of reproductive success, however, have been based upon behavioral observations of copulations and/or associations between individuals (Poole 1989; Le Bouef 1974; Clutton-Brock et al. 1982; Clutton-Brock et al. 1988; Clutton- Brock 1988; Le Boeuf and Reiter 1988; Struthsaker and Pope 1991; Cheney et al. 1988; Thirgood 1990; Berger and Cunningham 1987). Such observations might tend to assign a higher or lower proportion of offspring to various males for a number of reasons. First, it is often difficult to determine estrus in females when males would have a higher probability of siring offspring. Second, the use of alternate reproductive tactics by males may not be taken into account where covert reproductive behavior may not assign offspring. Third, males that mate guard would tend to be assigned a higher proportion of offspring than other males even though no copulations were observed. Fourth, behavioral observations often assume that all breeding males have the same reproductive potential.

Accurate measures of paternity are required to better understand the costs and benefits of different reproductive strategies, e.g., territoriality, lekking, cuckoldry, etc. Additionally, understanding reproductive success is fundamental to understanding social structure, population genetics, and conservation biology (Struthsaker and Pope 1991). Over the years various genetic techniques from allozymes (blood and serum proteins) to nuclear and extra-nuclear DNA have been developed to examine and answer questions of evolution and systematics, speciation and hybridization, population and individual levels of genetic diversity and fitness, and population social structure (Avise, 2004; Lowe et al., 2004). These techniques have also been used to assign paternity in order to verify and corroborate behavioral observations and estimates of reproductive success (Cain et al., 2014; Coltman et al., 1999; Coltman et al., 2002; DeWoody, 2005; DeYoung et al., 2009; Hynes et al., 2005; Mooring and Penedo, 2014; Rasmussen et al., 2008; Willisch et

23 al., 2012; Wroblewskia et al., 2009). Both allozymes and nuclear DNA have been used to assess paternity (Burke, 1989; Hill, 1987; Juneja et al. 1987; Lacy et al., 1988; Payne and Westneat, 1988; Foltz 1981; Bowling and Touchberry, 1990; Goodloe et al., 1991). Allozymes have the advantage of speed in analyses, routine nature of the procedure, and ease of scoring many individuals (Avise, 2004; Lowe et al., 2004). There are, however, some species and populations that are relatively monomorphic and lack genetic variability as revealed by allozymes (e.g., Northern elephant seal (Mirounga angustirostris) Bonnell and Selander, 1974; endangered felids, Newman et al., 1985; (Acinonyx jubatus) O'Brien et al., 1985; addax (Addax nasomaculatus), Spevak et al., 1993). This increases the number of allozyme systems that need to be examined in order to find enough polymorphic regions to confidently assign paternity (Lacy et al., 1988).

DNA fingerprinting has been used in establishing paternity (Hill, 1987; Jeffreys and Morton, 1987) and in some cases genetic analyses of behavioral estimates of reproductive success have been found to be accurate (e.g., (Cervus elaphus), Pemberton et al., 1992; (Panthera leo), Packer et al., 1991). However, in heavily inbred captive populations DNA fingerprinting has shown to lack the variability necessary for determining paternity (Bruford and Altmann, 1993). Additional disadvantages including the time it takes to produce and develop a "fingerprint" (Burke, 1989), it is DNA "expensive", requiring large amounts of high molecular weight DNA (Russell et al., 1993), and there are problems associated with the interpretation of gels with small sample sizes that lack information on familial relationships to determine segregation patterns (Burke, 1989).

A group of genetic markers that possess fewer limitations and have successfully been used in paternity analyses and population genetics are microsatellites. Microsatellites, Simple Sequence Repeats (SSR), or Simple Tandem Repeats (STR) are synonymous for stretches of nucleotide repeats ranging from di- to penta- nucleotides (Weber and May, 1989; Stallings et al., 1991; Ashley and Dow, 1994; Schlotterer and Pemberton, 1994; Avise, 2004; Lowe et al., 2004). The most commonly used microsatellites have been dinucleotides (Moore et al., 1991; Ostrander et al., 1993). These tandemly repeated (GT/AC)n dinucleotide DNA sequences, can be highly

24 polymorphic and randomly distributed across the genome. These characteristics make microsatellites useful genetic markers for examining paternity (Ellegren, 1992; Ellegren et al., 1992; Stallings et al., 1991; Weber and May, 1989). Microsatellites have additional advantages over allozymes and DNA fingerprinting. It is unknown whether allozymes are selectively neutral as they perform various physiological and biochemical functions. Therefore due to selection allozymes may show little or no variability where microsatellites and other DNA techniques have demonstrated appreciable variability (Yuhki and O'Brien, 1990). Unlike DNA fingerprinting microsatellites are not as "DNA expensive" (Russell et al., 1993) and require smaller amounts of DNA for analysis. Additionally, PCR can readily amplify enough DNA from small hair, fecal, blood, and tissue samples (Waits and Paetkau, 2005). In addition the costs in terms of time and money for microsatellites are usually considerably less than traditional DNA fingerprinting (Burke, 1989).

An additional class of useful genetic markers in examining population as well as individual level questions is the Class I and II genes of the Major Histocompatibility Complex (MHC). The MHC is central to the vertebrate immune system (Klein, 1986). It is a multigene family that encodes key receptor molecules that recognize and bind foreign peptides for presentation to specialist immune cells and subsequent initiation of an immune response (Klein, 1986). These genes are the most polymorphic loci yet detected in vertebrates (Klein, 1986), representing a useful region of the genome for estimating levels of allelic diversity and heterozygosity, particularly in populations lacking allozyme variation (Klein, 1986; Aguilar et al. 2004; Castro-Pieto et al., 2011).

In this study, I examined male reproductive success of addax (Addax nasomaculatus) using both behavioral observations and genetic paternity analysis. Because addax have no observable rut, the effectiveness of behavioral estimates of reproductive success was assessed using molecular genetic techniques (microsatellites and MHC loci) to directly determine male reproductive success in addax. I also correlated inferred and realized reproductive success with male characteristics including age, dominance and territorial behavior. Together these data help

25 elucidate the costs and benefits of differing reproductive strategies and behaviors, furthering our knowledge of a little studied arid land antelope.

Materials and Methods

Species Information Addax are medium-sized antelopes from North Africa belonging to the tribe Hippotragini of the subfamily Hippotraginae of the Bovidae (Artiodactyla). Behavioral observations were conducted at the Fossil Rim Wildlife Center (FRWC) in Glen Rose, Texas, from September through December 1989, and from June 1990 through June 1992.

All individuals were identifiable by ear tags, differences in horn shape, size, and coat patterns. The herd was composed of 48 individuals (12 males, 36 females) at the beginning of the study, with ages of individuals ranging from 0 months to 15+ years of age. Size of the herd at the end of the study was 67 (23 males, 44 females) ranging in age from 0 months to 18+ years of age. Approximate ages were established for most individuals born prior to 1989 and were based upon previous breeding history, appearance, and available records. Most individuals born after 1988 had known birthdates. Four age classes were used for this study: 0 to 1 year, 1 to 2 years, 2 to 3 years, and 3 years and above. These designations were based upon behavioral and physical development: 0 to 1 year: males are able to sire their first offspring, 1 to 2 Years: females are able to conceive and calve their first offspring, 2 to 3 years: males and females reach adult size and males begin performing secondary sexual behaviors, 3 years+: males may establish territories.

Over 1200 hours of observations were made of the study population. To facilitate data recording an ethogram was developed for addax that was used during the study (Appendix B) (Lehner, 1979). Scan samples (Lehner, 1979) of the herd were taken near dawn, mid-day, and dusk. For each scan sample date, time, weather conditions, i.e., sunny, overcast, rain, etc., temperature, group composition, and individual behavior and location were recorded. Sender and receiver were indicated for any interactive behaviors, e.g., mounting, horn-wrestling, etc. All occurrence

26 data on agonistic behavior were recorded as well as ad lib notes on general and reproductive behavior.

Blood and tissue samples were collected from all addax maintained at the FRWC for paternity testing and genetic analyses. All new calves were caught shortly after birth for neonate and health checks. Blood was collected for health assessments and DNA testing. All other addax in the population were caught and blood samples were collected for this study. Blood was collected in either heparin or EDTA and stored whole in 700 l aliquots in an ultra-cold freezer at -70 to -80 °C. Tissue samples were stored in 90% ethanol or placed in an ultra-cold freezer.

Microsatellites/SSR (Simple Sequence Repeats) Genomic DNA was prepared from whole blood, lymphocytes, or tissue, i.e., liver or heart, using a modified protocol of Kirby (Kirby, 1990). Seven hundred l of fresh, whole blood was aliquoted into microcentrifuge tubes and frozen at -70 to -80°C. Samples were then thawed and 800 l of 1X SSC was added, mixed and centrifuged in a microfuge for 1 minute (~12,500x g RCF). The supernatant was removed and the pellet was re-suspended in 1 ml 1X SSC and centrifuged again for 1 minute. The supernatant was removed and the pellet re-suspended in 375 l 0.2 M NaAc (pH 5.2) + 25 l 10% SDS. 10 l of 20 mg/ml Proteinase-K was added and mixed gently. Samples were incubated at 55°C for at least two hours to overnight. Samples were then extracted once with phenol:chloroform (1:1), twice with chloroform and precipitated with ethanol. The precipitate was centifuged for 15 min. and the pellet was washed in 70% ethanol, dried briefly under vacuum and re-suspended in TE, pH 8.0.

Tissue samples were first powdered in liquid nitrogen using a mortar & pestle and 100 mg of the powder was placed into 500 l 0.1M NaCl TE, 100 l 20% SDS and 50 l Proteinase-K

(10mg/ml), then incubated for 1-2 hours at 55°C. 200 l of 7.5 M NH4Ac was added and incubated on ice for one hour and then centrifuged. The supernatant was then transferred to a clean labeled tube. Samples were then extracted and precipitated as per whole blood. DNA samples were diluted in distilled water to a concentration of 10 ng/l.

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Initial reactions were carried out with 100 ng of genomic DNA in a 50 l final volume containing

48 l PCR buffer (final concentration 50 mM KCl, 10 mM Tris-HCl, 2.5 mM MgC12, 0.01% gelatin), 50 M dNTPs, 0.15 M of each of the microsatellite primers and 1 unit of Taq polymerase (Perkin Elmer Cetus, Norwalk, Connecticut). The thermal cycling profile for the amplification was: 4 min. at 94 °C, followed by 33 cycles of 1 min. at 94 °C, 2 min. at 55 °C and 5 min. at 72 °C. Initial examination of amplified fragments were by 6% polyacrylamide gel electrophoresis (PAGE), (Hoefer model SE200, Hoefer Scientific Instruments, San Francisco, California) at 122v for 45 min. stained with ethidium bromide and photographed under UV light. Due to close phylogenetic relationship to and sheep, 30 microsatellite/SSR primer sets from published and unpublished sequences for cattle and sheep were screened for use in addax (Moore at al., 1991). Four primer sets were selected which exhibited polymorphism in a screening of nine unrelated animals from Fossil Rim. The four primer sequences were:

Microsatellite Primer Sequence URB008 (Ma et al., 1996) 5’-GGAGCAGCACCTGTTAGAAGAGA-3’ 5’-ACTTTACTAGGGAAGGAGGGTGG-3’

BM 43 (Bishop et al., 5’-AGGGAGGGTCACCTCTGC-3’ 1994) 5’-CTTGTACTCGTAGGGCAGGC-3’

MAF 70 (Buchanan and 5’-CACGGAGTCACAAAGAGTCAGACC-3’ (GT strand) Crawford, 1992) 5’-GCAGGACTCTACGGGGCCTTTGC-3’ (CA strand)

MAF 209 (Buchanan and 5’-TCATGCACTTAAGTATGTAGGATGCTG-3’ (GT strand) Crawford, 1992) 5’-GATCACAAAAAGTTGGATACAACCGTGG-3’ (CA strand)

Samples were then amplified with these four primer sets under the same conditions except for the addition of P32dCTP at a final concentration 1 Ci per reaction. Samples were denatured and 10 l of formamide stop buffer was added (0.3% xylene cyanol FF, 0.3% bromophenol blue,

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10mM EDTA pH 7.5, 97.5% formamide). A total of 2.25 l of each sample was loaded in a .4mm 7% polyacrylamide denaturing gel. Microsatellite reactions were double loaded per well. An uncut M13 control sequencing reaction was run adjacent to the samples providing an absolute size marker. Samples were run at 70 watts in a 1x TBE buffer for approximately 3 hours until the dye front runs off the gel. Gels were rinsed, fixed, dried, and autoradiographed at room temperature for at least eight hours.

MHC Adna DR Genomic DNA was prepared as described above from whole blood or tissue and diluted in distilled water to 10 ngl. Oligonucleotide primers based on previously published sequences of Bola DR alleles (Groenen et al., 1990; Sigurdardóttir et al., 1991) were used for amplification of the second exon of Adna DR Amplification of DR alleles were performed in two stages. First round amplification primers were: HL030; 5'-ATCCTCTCTCTGCAGCACATTTCC-3', and HL031; 5'-TTTAAATTCGCGCTCACCTCGCCGCT -3'. The primers HL030 and HL031 contain seven and eight nucleotides of the 5' and 3' ends of exon 2 respectively, plus intron sequences. Heminesting (Li et al., 1990) was used to increase yield and specificity of the PCR product in the second round of amplification, employing primers HL030 (as above) and HL032; 5'-TCGCCGCTGCACAGTGAAACTCTC-3'. HL032 consists entirely of nucleotides at the 3' end of exon 2 and has an eight basepair overlap with the 3' end of HL031.

Reactions were carried out in a 50 l final volume containing PCR buffer (final concentration 50 mM KCI, 10 mM Tris-HCI, 0.01% gelatin), 1 l (10 ng) DNA, 100 M dNTPs, 0.5 M of each of the DR primers, 2.5 mM MgC12, and 1 unit of Taq polymerase. The thermal cycling profile for the first round of amplification was: 4 min. at 94 °C followed by 10 cycles of 1 min. at 94 °C, 2 min. at 60 °C and 1 min. at 72 °C. Final extension was for 5 min. at 72 °C. Subsequently, 2 l of first round product were transferred to another 500 l tube containing the mix described above, with the exception of DNA, for a second round of PCR according to the following thermal cycling profile: 27 cycles of 1 min. at 94 °C and 30 seconds at 65 °C, followed by a final extension for 5 min. at 72 °C.

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Ten 1 of second round PCR product were digested with 5 units of either RsaI, HaeIII (Bethesda Research Laboratories, Gaithersburg, Maryland) or BstYI (New England Biolabs Inc., Beverly, Massachusetts) in a total volume of 15 1, according to the manufacturers' recommendations. Digestions with BstYI were done in 500 l microfuge tubes, using a thermal cycler. After incubation of 1 hour and 15 minutes, BstYI digested product was heated to 85 °C for 4 minutes to denature the enzyme in order to obtain clear resolution of the restriction fragments. Restriction fragments were resolved by 6% polyacrylamide gel electrophoresis at 122v for 45 min. An Mspl digest of pBR322 (New England Biolabs Inc., Beverly, Massachusetts) was used as a standard for determination of restriction fragment size. Fragments were visualized after staining with ethidium bromide (1 mg/ml) for 5 min., and photographed using high speed black and white film (Polaroid 667, Polaroid Corp., Cambridge, Massachusetts) under UV illumination.

Digestion of Adna DR products with RsaI resulted in the identification of three distinct fragment patterns. Similarly, two BstYI patterns and two HaeIII patterns were identified (Figure 3.5). The combination of restriction patterns obtained with the three enzymes lead to the identification of seven DRB alleles in this population. Alleles of Adna DR are described using a three letter code following the convention of van Eijk et al (1992) (e.g., MAE, where M is the RsaI, A is the BstYI, and E is the HaeIII pattern).

Behavioral Estimates of Reproductive Success From behavioral observations reproductive success of a potentially breeding male was estimated using the number of days he was seen in consortship with a female during the time she could have conceived. Males were assigned a portion of the calf for every day they were seen with a female during the period when the calf was conceived (Clutton-Brock et al., 1988; Clutton-Brock et al., 1982). A constant probability that a female conceived on each day was assumed. Due to the size of the herd and the habitat in parts of the pasture being either heavily wooded or inaccessible by vehicle very few copulations were ever observed so that methods using this technique were not applicable (Cowlishaw and Dunbar, 1991; Struthsaker and Pope, 1991).

30

Conception was determined by back dating from the date of birth the gestation period of 257 to 264 days (Dittrich, 1972). Lacking any standard deviation on this range, an additional five days was added to each end to potentially include the entire period that a female conceived. Reproductive success was estimated for the eight-day period of conception (i.e., gestation) and the 18-day period (i.e., gestation plus ten days). These will be referred to as the 8-day and 18-day methods respectively.

All potential breeding males from each age class were included in the estimates (see age classes). The number of potential breeding males present at any one time ranged from 12 to 20 during the study.

Genetic Based Paternity Assignment Genetic based paternity assignments were obtained by comparing allele patterns for the four microsatellites and Adna DR between known dams and calves and all possible sires. The four microsatellites displayed two to seven alleles with Adna DRB3 exhibiting an additional seven allelic patterns (Table 3.1). Paternity was assigned only on shared allelic patterns (Figure 3.1, 3.2, Appendix A). Males were assigned paternity only if they shared the allelic pattern with a putative offspring. If two or more males possessed the same allelic pattern with a potential calf no certain paternity was assigned for analyses. Along with allelic patterns additional data on the possible sires was also used for paternity assignment. These included the ages of possible sires, i.e., when an individual was physiologically able to sire offspring, 10 months and older (Spevak unpublished), and the addax herd's composition at the time of a calf’s conception, i.e., which potential sires were present or absent. Only calves with known dams were examined for paternity assignment. To assess the power of Adna DRB3 and this suite of microsatellite loci in assessing paternity a combined probability of exclusion (PE) in CERVUS 3.0 (Marshall et al., 1998; Kalinowski et al., 2007) was calculated.

31

Results

Behavioral and Genetic Estimates of Reproductive Success in Males Data were available for behavioral estimates of paternity for 28 calves for the 8-day method and 31 calves for the 18-day method. The difference was due to the lack of observations of breeding males with some females during the 8-day conception period. This usually occurred with females that had recently calved, had isolated themselves from the rest of the herd, and could not be observed. Except in the proportion of males siring different numbers of calves no significant difference was found between the 8-day and 18-day method.

Male reproductive success ranged from 0 to 3.169 calves for the 8-day method and 0 to 2.952 calves for the 18-day method. The distribution of the proportion of males siring different numbers of calves differed significantly between the two conception intervals 2 = 39.5, d.f. = 3, p < .001) (Figure 3.3). The proportion of males siring between 0 to 1 calves was identical for the 8-day and 18-day methods. The 18-day method found no males siring more than 3 offspring. Additionally, differences appeared between the proportion of males siring between 1 to 2 calves and 2 to 3 calves. The 8-day method assessed a higher proportion of males siring fewer numbers of calves than did the 18-day method.

Sires for 41 of the 77 calves examined with known dams that conceived between February 1989 and July 1992 were identified by allele patterns and knowledge of the age of potential sires and whether they were present during the time of conception. Sires were also established for three additional individuals conceived prior to the study. Without knowledge of whether possible sires were present during conception the use of Adna DR3 and the suite of four microsatellites for paternity testing offers a probability of exclusion (PE) of 0.6677, suggesting that approximately one in three males will falsely appear to be a father of an offspring with unknown paternity. Future investigations should include additional or more polymorphic loci to increase likelihood of assigning paternity.

Genetic analysis established that males were able to sire offspring before they were one year of age. In this study, three males less than one year of age sired offspring. Two males had known

32 birthdates and based on an average gestation period of 260 days (range 257 - 264 days) (Dittrich 1972) sired calves when they were 319 and 325 days old.

Age and Estimates of Reproductive Success From behavioral estimates males over 1 year of age sired the most calves (Figure 3.4). Age classes 1 to 2, 2 to 3, and over 3 years sired approximately the same number of calves with no significant correlation between age class and number of offspring per age class for the 8-day and

18-day methods (rs = .80, p = .1659). However, there were fewer males in the 3+ age class and therefore, fewer offspring per individual males in the 1 to 2 and 2 to 3 year age class than in the 3+ age class (.401 (.459), .719 (.876) vs. 1.269 (1.452) offspring/male for 1-2, 2-3 vs. 3+ year old males, 8-day method and 18 day method, respectively). There was a significant correlation between the age class of the individual male and the number of offspring sired for both the 8-day

(rs = .569, p < .0001) and 18-day method (rs = .539, p = .0003) indicating older males were more successful breeders.

Based on genetic analysis significantly more calves were sired by older males than expected if females bred with males randomly based on the frequency of potential mates 2 = 16.875, d.f. = 3, p = .0007) (Figure 3.5). The age of a female had no relationship to the age of the sire of her offspring (rs = .417, N = 44, p = .6765). Females did not preferentially mate with similar aged males. Both young and old females bred predominantly with males three years and older.

There was a significant relationship between the age of the male and the number of offspring sired (rs =.46, N = 66, p = .0002). Of the 44 calves genetically assigned sires, 3 (6.8%) were sired by males under 1 year of age, 4 (9.1%) were sired by males between 1 and 2 years of age, 10 (22.7%) by males between 2 and 3, and the remaining 27 (61.4%), by males 3 and over.

Behavioral estimates of the reproductive success of different age classes found all classes except 0 to 1 year having equal success. There was, however, a significant difference between behavioral and genetic estimates (ANOVA: F = 3.279, d.f. = 2, 27, p <.0001). Compared to the distribution determined genetically, behavioral data over-estimated the reproductive

33 success of younger age classes (1-2 year olds and 2-3 year olds) and under-estimated the 3 year olds and above (Figure 3.6).

Dominance Rank and Estimates of Reproductive Success Compared to other ranked males the alpha and beta males sired more offspring than any other males: 3.345 and 3.538 (8-day method), and 3.094 and 3.517 (18-day method) for alpha and beta males respectively (Figure 3.7). The alpha and beta males together accounted for 23% (8-day method) and 22.7% (18-day method) of all calves. There was a significant correlation between the rank of a male and his reproductive success for both the 8-day (rs = -.75, p = .0027) and 18- day method (rs = -.791, p = .0016). However, most males appeared to have sired only between 1 and 2 offspring.

There were 12 males in the dominance hierarchy at the beginning of the study. During the study up to 20 potential breeding males were included in the hierarchy. Of these 20 males only 11 sired offspring. The dominance rank of the sires for 38 calves was identifiable. Seventeen of 38 (44.7%) of the calves were sired by the alpha and beta males (Figure 3.8). Other males that sired offspring averaged 2.3 offspring per an individual's dominance rank. There was a significant correlation between a male's rank and his reproductive success (rs = -.884, p = .0007).

Both the 8-day and the 18-day methods assigned reproductive success to 19 males in the dominance hierarchy, whereas only 11 actually sired offspring (Figure 3.9). There was a significant difference between the 8-day method and the genetic data 2 = 31.577, d.f. = 18, p = .0247). However, no significant difference was found between the 18-day method and the genetic data 2 = 27.917, d.f. = 18, p = .0633) though a trend was found. Both behavioral methods underestimate the reproductive success of males that did reproduce and overestimate the reproductive success of unsuccessful males.

Territoriality and Estimates of Reproductive Success The mean number of calves sired by territorial males for the 8-day method (x=1.675, s.e.= .413) was significantly higher than the mean number of calves sired by non-territorial males (x=.805,

34 s.e.= .122)(t = 2.72, d.f. = 26, p = .0115) (Figure 3.10). The mean number of calves sired by territorial males for the 18-day method (x=1.702, s.e.= .424) though higher than the mean for non-territorial males (x=.948, s.e.= .151) was marginally significant and in the predicted direction (t = 2.002, d.f. = 26, p = .0558). The lower overall mean number of calves per non- territorial male may in part be due to the increased competition among non-territorial males, i.e., more non-territorial than territorial males.

The sires of 36 calves could be identified as being either territorial or non-territorial using the criteria of Walther et al (1983). There was no difference between the total number of calves sired by territorial (N=18) versus non-territorial males (N=18). However, the number of territorial sires was less than half the number of non-territorial sires (N=5 and N=12, respectively), with individual territorial males siring on average significantly more calves (3.8 +/- .917) than non- territorial males (1.583 +/- .288) (t = 3.064, d.f. = 15, p = .0079).

Reproductive success of territorial males was underestimated by the 8-day method 2 = 42.591, d.f. = 27, p = .0288). The 18-day method also underestimated the success of territorial males; though not significant, a trend existed 2 = 38.874, d.f. = 27, p = .0651) (Figure 3.11). However, both behavioral and genetic methods found higher reproductive success per individual territorial male than for non-territorial males (number of calves: genetic 3.8 +/- .917 vs. 1.583 +/- .288, 8-day 1.675 +/- .413 vs. .805 +/- .122, 18-day 1.702 +/- .424 vs. .948 +/- .151).

Female Calving, Post-partum Estrus and Male Reproductive Success Females separate themselves from the rest of the herd prior to giving birth (Manski 1979, 1991). They then usually remain in one area (territory) with their calf for the first one to two weeks after calving then move among different territories. Prior to the female introducing her calf to the rest of the herd, most associations are with other females with similar age calves (nursery group), territorial males, or non-territorial males. Non-territorial males usually only have access to the female when the territorial male leaves.

35

Female addax come into estrus throughout the year and appear to have a post-partum estrus shortly after calving (Densmore and Kraemer, 1986; Asa et al., 1996) followed by another approximately three weeks later, similar to oryx (Oryx sp.) (Wacher, 1988). During these times a territorial male is usually in consort. The conception period for 29 calves could be established as occurring during the first or second post-partum (N = 17) or the non-postpartum estrus periods (N = 12) by back dating the gestation period (Dittrich, 1972) from parturition. Other calves were either primiparous (N = 6) or the female's previous calving date was unknown (N = 3).

Of those calves conceived during the first and second postpartum estrus periods, territorial males accounted for 9 of 17 (53%) calves (Figure 3.12) but only 4 of 12 (33%) calves conceived during non-postpartum estrus periods. Therefore, of the calves sired by territorial males 9 of 13 (69.2%) were conceived during the postpartum estrus periods. Of calves sired by non-territorial males conception occurred equally between postpartum (8 of 16, 50%) and non-postpartum (8 of 16, 50%) estrus periods. A territorial male’s greatest reproductive success occurred during the postpartum estrus periods when females tend to be sedentary. Non-territorial males achieved higher reproductive success outside these periods when females move between areas and territories.

Mate Choice and Individual Preference There were a total of 26 males that were of breeding age during the study. However, at most only 20 possible breeding males were present at any one time. The number of breeding males varied as individuals were sent to other institutions, became injured, or died. Of the 26 potential breeding males examined only 14 (53.8%) sired offspring (Figure 3.13). Of the 38 calves sired by these males, 68.4% (26 of 38) were sired by only five males. All five males ranked high in the dominance hierarchy and became territorial after the age of 3 years.

However, if females exhibit individual male preference one might expect consecutive offspring to be sired by the same male (Amos at al., 1995). Using paternity assignment data, sires for 11 pairs of consecutive offspring from 10 females were identified through paternity analysis. Of these 11 pairs only one pair of consecutive offspring had the same sire. In all other cases different

36 males sired different calves of a pair. In total, 11 different males were involved in these conceptions.

Factors Affecting Male Reproductive Success A step-wise multiple regression analysis compared “genetic” reproductive success to the variables territoriality, rank, male age, individual male identification, and male competition, i.e., the number of other breeding males present. Possession of a territory was a significant 2 component of reproductive success (R = .272, F.05= 11.771, d.f. = 2, 63, p < .05). However, examining individual components separately, step-wise regression found rank to also be 2 significant in predicting reproductive success (R = .226, F.05= 18.707, d.f. = 1, 64, p <.05). Neither the number of breeding males, age, nor individual male identification were significant components of male reproductive success. A previous analysis found a significant correlation between a male's rank and his reproductive success (rs = -.884, p = .0007). Among non-territorial males rank is a good estimator of reproductive success.

Behavioral Versus Genetic Estimates of Paternity Figure 3.14 shows the proportion of males siring different numbers of calves. Genetic analysis revealed three males that sired six offspring apiece. Behavioral data underestimated absolute reproductive success of males. Neither the 8-day nor 18-day methods revealed males that sired more than 4 or 3 calves, respectively. There was a significant difference between the 18-day method and the genetic data 2 = 45.253, d.f. = 6, p < .001). However, there was no significant difference between the 8-day method and the genetic data 2=7.75, d.f. = 6, NS). The 18-day method assessed a large proportion of males siring 2 to 3 calves which was not seen in either the 8-day or genetic methods. However, though behavioral estimates under- or over-estimated individual reproductive success they did correlate significantly with genetic assignments of paternity (8-day: R2 =.572, p < .0001, and 18-day: R2=.532, p < .0001) indicating that in lieu of genetic data behavioral observations may be useful estimators.

37

Discussion

Territoriality had a greater payoff in terms of individual reproductive success than did following or being non-territorial. Although, as a group, non-territorial males sired more offspring than territorial males, territorial males sired significantly more offspring per individual (1.675 +/- .413) than did non-territorial males (.805 +/- .122). Throughout the study there were always fewer territorial males than non-territorial. At the end of the study there were five territorial males compared to 15 non-territorial. Only males 3+ years of age attempted to hold a territory, due to dominance, experience, or some physiological change associated with age. Both age and rank were significantly correlated with territoriality (R2 = .553, F = 37.719, p < .0001), though only age was significant in the relationship (stepwise multiple regression coeff. = -.49, p = .0008). Even though males in every age class reproduced, males over 3 years of age sired proportionally more offspring per individual. As males over 3 years of age acquired and maintained territories, territoriality increased their access to females. However, the reproductive success of males over three years may be an artifact of territoriality in this population; throughout most of the study all three-year-old males possessed territories.

The dominance hierarchy also affected the reproductive success of individual males. Dominance hierarchies may not remain stable from season-to-season or year-to-year or even during the breeding season (Carlini et al., 2002). Miller (1981) found that mating systems in horses (Equus caballus) changed with changes in the dominance hierarchy. Dominance hierarchies may preclude many potential breeding individuals from reproducing (Clutton-Brock, et al., 1982; Duncan, et al., 1984; Estes, 1969; Floody and Arnold, 1975; Leuthold, 1977; Schaller, 1972; Sinclair, 1977). However, as population size or density increases, males lower in the dominance hierarchy may be gaining more copulations because dominant males are unable to control or accompany all receptive females allowing additional individuals to breed (Le Bouef, 1974; Le Bouef and Reiter, 1988; Røed et al., 2002). Work on wild horses indicates that the most dominant animal in a group may be getting less than 50% of all copulations (Berger, 1987). In this study, based on behavioral observations, the alpha and beta addax males together sired 23% of all offspring. Though the alpha and beta males sired the largest proportion of offspring there

38 was still a significant relationship between a male’s rank and the number of offspring sired. Higher ranking males were able to sire more offspring through competition with other males and maintaining access to receptive females. However, dominant status does not impart exclusivity to receptive females nor preclude lower ranked males from breeding through alternative mating strategies (Wrobewskia et al. 2009).

Paternity assignment versus behavioral estimates of reproductive success In this study, comparisons between genetic and behavioral estimates of reproductive success showed that behavioral estimates consistently underestimated the absolute reproductive success of successful males and overestimated the success of many unsuccessful males. In a study of tammar wallabies (Macropus eugenii) in a captive situation, Hynes et al (2005) found that the dominant  male sired only 50% of offspring, though accounting for 60% of first copulations observed in receptive females. A similar result was found in American (Bison bison) where 60% of observed copulations by dominant tending males that did produce a calf did not accurately predict the sire (Mooring and Penedo, 2014).

Even though behavioral methods under- or over-estimated reproductive success they did demonstrate a general trend for higher ranked and territorial males to sire more offspring; no age related success was noted using behavioral methods. In other studies comparing behavioral and genetic assessments of reproductive success, behavioral estimates accurately identified the relative success of individual males but were poor predictors of absolute individual reproductive success (e.g., , Mooring and Penedo, 2014; red deer, Pemberton et al., 1992). This was also true in this study where general population trends of male reproductive success were revealed through behavioral observations. Behavioral methods underestimate the true success of successful addax males and overestimate the success of many addax males who, in fact, fail to father any calves. Although, behavioral estimates were not useful in determining sires of calves from particular females, they did provide insights into patterns of reproductive success that the use of genetic data alone could not provide, such as the role of alternate mating strategies (Mooring and Penedo, 2014).

39

Behavioral estimates from the 18-day method were not a significant improvement over the 8-day method. When compared with genetic estimates the 8-day method, which included only the reported gestation period for addax, was a more useful estimator of relative reproductive success than the 18-day method, although both were limited. In the absence of known paternity and where time for observations is limited the 8-day method gives an adequate estimate of male reproductive success for multi-male herds. However, estimates of individual reproductive success based upon associations between male and female addax is limited.

Comparing behavioral and genetic data certain points are evident. In the absence of genetic data behavioral parameters related to reproductive success, i.e., age, rank, territoriality, etc., may themselves be better indicators of a male’s reproductive success along with estimates from associations. Male reproductive success in addax increased with age, rank, and territoriality. Behavioral observations indicated that males in each age class above the age of one year have equal fitness in regard to siring offspring. However, paternity analysis found that over 50% of all calves were sired by males three years and above. The most successful males in this population were older, i.e., over three years of age, territorial, and high ranking. However, many of these parameters are interrelated. For example, males did not attempt to hold a territory until they were at least three years of age. Step-wise regression analysis showed that there was a significant relationship between rank, territoriality, and reproductive success, however, the higher ranking males were usually the older, territorial males in this study. As there was a strong correlation between age, rank, and territory possession (R2 = .553, F = 37.719, p < .0001), territoriality would appear to be a greater predictor of reproductive success. Where addax are not territorial, e.g., low density populations, age and rank would likely be useful indicators of a male’s potential reproductive success.

This is the first study of addax in which territorial behavior was observed and where its benefits, in terms of reproductive success, were assessed. Two related oryx species, gemsbok (O. gazella) and fringe-eared oryx (O. beisa callotis), have also been found to be territorial when population densities are high and resources are constant (Estes, 1974, 1991, 1993; Wacher, 1988) however, reproductive success was never assessed. As addax and oryx densities become higher males

40 appear to become more sedentary. However, females and young males still move between territories. What is the benefit of becoming territorial for a nomadic antelope?

In this study individual territorial males had higher overall and individual reproductive success than non-territorial males. A male's reproductive success is due to female behavior. The social structure of addax appears to be comprised of groups of females and young males with a single dominant male (Newby 1980, 1984). In small harem groups the number of females cycling at any one time would be low and a single dominant male or herd bull would be able to stay in consort. If a female left a group to calf, a single male would be able to stay with her until she cycled. As females with similar aged calves often associate (Spevak pers. obs.) a single male would also be able to protect them until they cycled. All of these scenarios presume low numbers of male competitors and low numbers of breeding females. In this type of a situation a single male has the potential of breeding with all estrous females.

Addax, historically, had been observed in large herds of up to a thousand individuals (Dolan, 1966; Gillet, 1965; Harper, 1945; Newby, 1980, 1984) and in certain situations would have been found at higher densities. As population densities increased the number of competitors would also increase, as would the number of breeding females. Under these conditions males might adopt one of two reproductive strategies: sit and wait or following (Gosling, 1986). A major advantage for territorial males is a female's tendency to become sedentary for the first one to two weeks after calving and remaining separate from the rest of the herd for the calf's first month. During this period females come into two estrous cycles (Densmore and Kraemer, 1986; Wacher, 1988). Territorial males are then in a good position to breed females during these two post- partum estrus cycles. In this study territorial males accounted for 53% of all calves conceived during this period. In addition 69.2% of calves sired by territorial males were conceived during this same period. A territorial male’s highest reproductive success occurs shortly after a female gives birth during post-partum estrus cycles. A non-territorial male's highest reproductive success occurs during those estrus periods when females are nomadic or moving with the herd. Additionally, female movements and grouping may also determine the reproductive strategy of males (Hogg, 1987; Isvaran, 2005a).

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In addax, the advantages of territoriality cannot be completely explained through behavioral observations. Paternity exclusion data did find overall that territorial males sired more than twice as many offspring per individual (3.8 +/- .917) than did non-territorial males (1.583 +/- .288). However, how this additional reproductive success manifests itself is not apparent. Female addax have been observed mating with more than one male during an estimated time of conception and behavioral observations may assign offspring conceived during an estrus period to more than one male. Male are able to identify estrus periods in females by testing their urine (Hart et al., 1989; Muller-Schwarze, 1991). Male addax periodically test a female's state of estrus by placing their nose into a female's stream of urine, lifting their head, and performing flehmen or lip curl, allowing the urine to flow across the palate into the vomero-nasal organ (Hart et al., 1989; Muller-Schwarze, 1991). Therefore, male addax are in a position to identify when a female will most likely conceive. Behavioral observations of addax estrus cycles are not as precise. Only genetic data are able to identify actual paternity during different estrus periods (i.e., postpartum and non-postpartum).

Paternity Analysis Allozymes, serum and blood proteins, have been successfully used in assessing levels of genetic variability and paternity testing in a wide variety of natural and captive populations (e.g., Bowling and Touchberry 1990; Burke 1989; Cohn 1990; Foltz 1981; Goodloe et al, 1991; Hedrick et al 1986; Hill 1987; Juneja et al. 1987; Lacy et al. 1988; Lee et al. 1989; Payne and Westneat, 1988). Although many have appreciable diversity, there are some populations that are relatively monomorphic (Bonnell and Selander, 1974; Newman et al., 1985; O'Brien et al., 1985). For example, allozyme studies on several captive herds of addax by Robert Lacy at the Brookfield Zoo (Illinois) demonstrated an overall heterozygosity level of only 0.3% (Spevak et al. 1993). With such low levels of variability allozymes may not always be informative (Lowe et al., 2004).

DNA fingerprinting has been used successfully for paternity testing and corroborating or refuting behavioral estimates in mammals (e.g., tammar wallabies, Ewen et al., 1993; red deer, Pemberton et al., 1992; lions, Packer et al., 1991) but it has associated problems such as the time it takes to

42 produce a "fingerprint" [Burke 1989] and the large amounts of DNA required (Russell et al., 1993).

This study demonstrates the usefulness of microsatellites and DRB3 in helping to establish paternity and therefore reproductive success in addax. One of the major limitations of microsatellites are suitable primer sets for specific loci. For related taxa the same primer sets may be used, e.g., cattle primers for wild cattle and antelope (Engel et al., 1996; Marshall et al., 1999; Schnabel et al., 2000; Udina et al, 1999), dog primers for wolves (Canis lupus) and other wild canids (Moore, 1991). However, roughly only half of these primer sets will work with approximately half showing polymorphism (Stallings et al., 1991). Species-specific microsatellite primers will yield the greatest amount of information, with higher levels of polymorphism. One additional concern with microsatellites, as it is with allozymes and other nuclear DNA analyses, is a possible lack of genetic variability due to genetic drift, founder effect or a population bottleneck. The North American addax population is derived from 14 to 20 (Correll, 1989a, 1989b) and like many species maintained in captivity, management practices have allowed extensive inbreeding, over-representation of individual founders, and often little exchange of animals. This has reduced the overall predicted genetic diversity of the captive population (Correll, 1898b) and resulted in a predicted Founder Genome Equivalent (FGE) of approximately 4 (Correll, 1989b). The number of founder genome equivalents is similar to the effective founder number, but the former has been devalued based on the proportion of its genome that has probably been lost to drift (Lacy, 1989). Other examinations of addax microsatellites have also found very low numbers of polymorphic loci (Armstrong et al., 2011; Heim et al., 2012) probably due to this captive history.

In this study DR3 added another potentially highly polymorphic locus for paternity assignment. MHC genes are the most polymorphic loci yet detected in vertebrates (Klein, 1986), with as many as 100 alleles per locus and heterozygosity levels as high as 100% in mammals (Klein, 1986). Even in species that have seen severe historical population bottlenecks in the wild substantial MHC variability has still been observed (e.g., (Syncerus caffer), Wenink et al., 1998; American bison (Bison bison), Mikko et al., 1997). However, this is not

43 always the case as other species that have gone through severe population declines or bottlenecks have seen a loss in MHC diversity (e.g., Arabian oryx (Oryx leucoryx), Hedrick et al. 2000; (Damliscus pygargus pygargus), Van der Walt et al., 2001, cheetah (Acinonyx jubatus), Castro-Prieto et al., 2011, (Bison bonasus), Udina et al., 1998; Northern elephant seal (Mirounga angustirostris), Weber et al, 2004; Spanish ibex ( pyrenaica), Amills et al., 2004) In addax only eight alleles were identified across the North American population compared to over 46 alleles (i.e., DRB RFLP haplotypes) reported for lines of domestic cattle ( taurus and B. indicus) (Davies et al., 1994). This is to be expected from the low number of founders to the captive population and disproportionate breeding of individuals (Correll, 1989b). However, it is still a useful region of the genome for examining paternity estimating levels of allelic diversity and heterozygosity where populations lack variation at other loci and systems (Klein, 1986).

DNA fingerprinting may have been able to identify more sires than the microsatellites and MHC analyses but with greater difficulties. In heavily inbred captive populations, like addax, DNA fingerprinting has proved almost impossible due to a lack of variability (Bruford and Altmann, 1993; John Patton unpublished data). Additionally, due to the nature of DNA fingerprinting it is often difficult scoring and comparing bands across gels (Burke, 1989). For accurate estimates every potential father would have to be run in lanes adjacent to every mother-calf pair under study (Pemberton et al., 1991), which is both time consuming (Burke, 1989) and DNA “expensive” (Russell et al., 1993). The microsatellites and DRB3 of the MHC alleviate these problems, though the use of only five separate loci limited the resolution capabilities compared to traditional DNA fingerprints. In future studies more identified loci could be used for a more complete analysis (Armstrong et al., 2011; Heim et al., 2012).

The study of reproductive success is fundamental in examining how species evolve and adapt. In addition, studies of reproductive success can have an immediate impact on conservation programs for endangered species. The social structure, mating strategies, and reproductive success of few large polygynous species have been examined in depth, exceptions include red deer (Cervus elephus) (Albon and Guinness, 1988; Clutton-Brock et al., 1982;

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Carranza et al., 1990), northern elephant seal (Mirounga angustirostris) (Le Bouef, 1974; Le Boeuf and Reiter, 1988; Carlini et al, 2002), African bush elephant (Loxodonta africana) (Poole, 1989; Poole, 1989; Moss, 1988; Rasmussen et al., 2008), horse (Equus caballus) (Berger 1987; Berger and Cunningham, 1987; Bowling and Touchberry, 1990; Miller, 1981), American bison (Bison bison) (Berger and Cunningham, 1994; Komers et al., 1992; Lott, 1981; Lott and Minta, 1983; Maher and Byers, 1987; Shull and Tipton, 1987; Mooring and Penedo, 2014), and (Panthera leo) (Packer et al., 1988; Packer et al., 1991; Schaller, 1972; Wildt et al., 1987). Research examining the correlation of reproductive success and dominance, age, mating strategy, and kinship avoidance looking at primates (e.g., Bauers and Heran, 1994; Berard et al., 1994; Cowlishaw and Dunbar, 1991; de Ruiter et al., 1992; Smith, 1995), ungulates (e.g., Byers and Kitchen, 1988; Coltman et al. 2002; DeYoung et al., 2009; Komers et al., 1992; Lott, 1981) and marsupials (e.g., Ewen et al., 1993; Hynes et al., 2005) illustrates that reproductive success in different species based on certain criteria, e.g., age, dominance, and territoriality, may vary seasonally, yearly and between populations.

Genetic examination of reproductive success helps to confirm or refute behavioral estimates. More accurate measures of reproductive success allow for fine-tuning behavioral estimators (Coltman et al., 1999; DeYoung et al., 2009; Pemberton et al., 1992) and identify those characters that are most important in establishing an individual’s reproductive success, e.g., territoriality. Additionally, genetic analysis uncovers aspects that are not observable. In the case of addax, the difference in post-partum conceptions between territorial and non-territorial males could not be obtained through behavioral observation. Therefore, the additional selective advantage of becoming territorial would remain unknown if behavioral observations alone were used.

However, genetic analysis through microsatellites or DNA fingerprinting are not always much more informative than behavioral observations. Pemberton et al. (1992) used DNA fingerprinting to test previous behavioral estimates of reproductive success of red deer (Albon and Guinness, 1988; Clutton-Brock et al., 1982) and found a significant correlation between the two. However, in red deer there is a defined breeding season or rut, females move very little between harems and

45 males, and in general, mate only once (Albon and Guinness, 1988; Clutton-Brock et al., 1982). These factors would tend to allow for good behavioral estimates. Addax, in comparison, have no defined rut, females move often between male territories, and may mate more than once during an . Some females were observed mating with up to five different males in one estrus period. These factors tend to limit the effectiveness of behavioral estimators (Pemberton et al. 1992). Therefore, a general knowledge of a species social structure and mating system are essential in determining whether genetic analyses need to be undertaken.

An understanding of a species mating system and individual reproductive success is beneficial in furthering our understanding of and the selective pressures leading to evolutionary change. A number of studies of antelope have examined the implications of a species' ecology upon its social structure (Brashares et al., 2000; Estes, 1974; Geist, 1974; Gosling, 1986; Isvaran, 2005b; Jarman, 1974; Owen-Smith, 1977; Western, 1979). For example, sex biased dispersal, the formation of long term matrilineal relationships, agonistic interactions, territoriality, dominance hierarchies, reproductive behavior, population density, group size, sex ratios, age composition, etc., all have an impact on the social structure of a population. The social structure of the population in turn determines the reproductive strategies of males and females which ultimately establishes the population's genetic structure. The mating strategy and reproductive success of an individual can have a direct bearing on the amounts and rates of gene flow within and between populations which can effect evolutionary potential and/or change (Chepko-Sade and Halpin, 1987). Therefore, although evolutionary change, e.g., changes in allele frequencies, may take a number of generations to detect, initial examinations of factors that may be responsible for future evolution in a species, i.e., reproductive success, are essential in determining the causes and results of evolution.

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CHAPTER 4: LOW GENETIC VARIABILITY AT MHC LOCUS Adna DR3 AND FITNESS IN ADDAX (Addax nasomaculatus)

Introduction

Allozymes have been successfully used in assessing levels of genetic variability in a wide variety of organisms in both natural and captive populations. Although most populations have appreciable diversity, there are some populations that appear monomorphic over large sections of their genomes (Bonnell and Selander, 1974; Newman, et al., 1985; O'Brien, et al., 1985). For example, recent allozyme studies on several captive herds of endangered addax (Addax nasomaculatus) demonstrated an overall heterozygosity level of only 0.3% (Lacy, pers. comm.) (Spevak, et al., 1993). Such low levels of genetic variability are likely due to a history of inbreeding and/or a lack of diversity among founders or a limited founder population (Correll 1989a, 1989b). With such low levels of variability allozymes are not always informative. Class I and II genes of the major histocompatibility complex (MHC) are the most polymorphic loci yet detected in vertebrates (Klein, 1986), representing a useful region of the genome for estimating levels of allelic diversity and heterozygosity, particularly in populations lacking allozyme variation. Here I present the use of a PCR-RFLP technique for rapid typing of one of the more polymorphic regions of the bovine MHC, the second exon of the BoLA DR gene (van Eijk et al., 1992). Using this technique, we report exceedingly low levels of allelic diversity in addax, levels lower than those reported for breeds of domestic cattle. Despite these low levels of allelic diversity, as measured by DR measures of heterozygosity within and among addax populations were found to be informative, correlating highly with infant survivorship and average heterozygosity across the genome as measured using microsatellite markers.

Materials and Methods

Study Sites and Organism Addax are probably the most desert-adapted of all the antelopes and now one of the most endangered. They formerly ranged across the Sahara from Egypt in the east to the Rio de Oro and

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Senegambia in the west (Dolan, 1966, Haltenorth and Diller, 1980, Harper, 1945). However, they have been exterminated over most of their range (Harper, 1945; Newby, 1980, 1984; UNEP/CMS 2006) due predominantly to hunting and poaching (Newby 1980, 1990), along with habitat loss, and drought (Newby 1980). Addax are currently listed as critically endangered on the IUCN Red List of Threatened Species (2015).

From initial imports of addax to the United States in the 1930s and 1960s there are now over 700 in North America; around 300 are in managed programs in zoos. This North American herd is derived from only 14 to 20 founders (Correll, 1989a; 1989b) and like many species maintained in captivity, management practices have allowed extensive inbreeding, over-representation of individual founders, and often little exchange of animals. This has resulted in a number of inbred, related herds. Minimizing potential detrimental effects associated with the consequent loss of genetic variability requires an assessment of genetic diversity and an understanding of how such variability affects survival both in wild and captive populations. Such information is essential in the design of appropriate breeding schemes and in reintroduction programs (Woodruff, 1989; Frankham et al., 2010).

Genetics To assess genetic diversity within and among addax populations a PCR-RFLP technique for typing the second exon of the Bola DR gene was used (van Eijk, et al., 1992). The MHC genes encode highly polymorphic cell-surface molecules that present foreign peptides to T-cells, triggering appropriate immune responses to infectious agents. Blood and tissue samples were collected from 175 addax from 11 captive facilities in North America. Four institutions supplied 10 or more samples: Fossil Rim Wildlife Center (Glen Rose, Texas; N=114), Henry Doorly Zoo (Omaha; Nebraska; N=19), St. Louis Zoo (St. Louis, Missouri; N=11), and Brookfield Zoo (Brookfield, Illinois; N=17).

Genomic DNA was prepared from whole blood, lymphocytes, or tissue using a modified protocol of Kirby (Kirby, 1990). Seven hundred l of fresh, whole blood was aliquoted into microcentrifuge tubes and frozen at -70 to -80°C. Samples were then thawed and 800 l of 1X

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SSC was added, mixed and centrifuged in a microfuge for 1 minute (~12,500x g RCF). The supernatant was removed and the pellet was re-suspended in 1 ml 1X SSC and centrifuged again for 1 minute. The supernatant was removed and the pellet re-suspended in 375 l 0.2 M NaAc (pH 5.2) + 25 l 10% SDS. Following suspension 10 l of 20 mg/ml Proteinase-K was added and gently mixed. Samples were incubated at 55°C for at least two hours to overnight. Samples were then extracted once with phenol:chloroform (1:1), twice with chloroform and precipitated with ethanol. The precipitate was centrifuged for 15 min. and the pellet was washed in 70% ethanol, dried briefly under vacuum and re-suspended in TE, pH 8.0.

Tissue samples were first powdered in liquid nitrogen using a mortar & pestle and 100mg of the powder was placed into 500 l 0.1M NaCl TE, 100 l 20% SDS and 50 l Proteinase-K

(10mg/ml), then incubated for 1-2 hours at 55°C. Two hundred l of 7.5 M NH4Ac was added and incubated on ice for one hour and then centrifuged. The supernatant was then transferred to a clean, labeled tube. Samples were then extracted and precipitated as per whole blood. DNA samples were diluted in distilled water to a concentration of 10 ng/ml.

Oligonucleotide primers based on previously published sequences of Bola DR3 alleles (Groenen et al., 1990; Sigurdardóttir et al., 1991) were used for amplification of the second exon of Adna DRB3. Amplification of DR alleles was performed in two stages. First round amplification primers were:

HL030; 5'-ATCCTCTCTCTGCAGCACATTTCC-3', HL031; 5'-TTTAAATTCGCGCTCACCTCGCCGCT -3'.

The primers HL030 and HL031 contain seven and eight nucleotides of the 5' and 3' ends of exon 2 respectively, plus intron sequences. Heminesting [Li et al. 1990] was used to increase yield and specificity of the PCR product in the second round of amplification, employing primers HL030 (as above) and HL032; 5'-TCGCCGCTGCACAGTGAAACTCTC-3'. HL032 consists entirely of nucleotides at the 3' end of exon 2 and has an eight basepair overlap with the 3' end of HL031.

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Reactions were carried out in a 50 l final volume containing PCR buffer (final concentration 50 mM KCI, 10 mM Tris-HCI, 0.01% gelatin), 1 l (10 ng) DNA, 100 M dNTPs, 0.5 M of each of the DR3 primers, 2.5 mM MgC12, and 1 unit of Taq polymerase (PerkinElmer, Cetus, Norwalk, Connecticut). The thermal cycling profile for the first round of amplification was: 4 min. at 94 °C followed by 10 cycles of 1 min. at 94 °C, 2 min. at 60 °C and 1 min. at 72 °C. Final extension was for 5 min. at 72 °C. Subsequently, 2 l of first round product were transferred to another 500 l tube containing 48 1 PCR buffer containing the mix described above, with the exception of DNA, for a second round of PCR according to the following thermal cycling profile: 27 cycles of 1 min. at 94 °C and 30 seconds at 65 °C, followed by a final extension for 5 min. at 72 °C.

Selection of restriction endonucleases was based on analysis of 14 sequenced DR3 alleles in cattle; three enzymes were chosen which could distinguish all sequenced alleles (van Eijk, et al., 1992). Ten 1 of second round PCR product were digested with 5 units of either RsaI, HaeIII (Bethesda Research Laboratories, Gaithersburg, Maryland) or BstYI (New England Biolabs Inc., Beverly, Massachusetts) in a total volume of 15 1, according to the manufacturers' recommendations. Digestions with BstYI were done in 500 l microfuge tubes, using a thermal cycler. After incubation of 1 hour and 15 minutes, BstYI digested product was heated to 85 °C for 4 minutes to denature the enzyme in order to obtain clear resolution of the restriction fragments. Restriction fragments were resolved by 6% polyacrylamide gel electrophoresis (PAGE), (Hoefer model SE200, Hoefer Scientific Instruments, San Francisco, California) at 122v for 45 min. An Mspl digest of pBR322 (New England Biolabs Inc., Beverly, Massachusetts) was used as a standard for determination of restriction fragment size. Alleles of the addax orthologue of DR3 were visualized after staining with ethidium bromide (1 mg/ml) for 5 min., and photographed using high speed black and white film (Polaroid 667, Polaroid Corp., Cambridge, Massachusetts) under UV illumination (Figure 3.1). Alleles of Adna DR3 are described using a three letter code following the convention of van Eijk et al (1992) (e.g., MAE, where M is the RsaI, A is the BstYI, and E is the HaeIII pattern) (Figure 4.1).

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Observed heterozygosity for each population was calculated by summing the total number of heterozygotes and dividing by the sample size. Expected heterozygosity was derived by using the 2 following formula: H = 1 - pi , where pi represents the frequency of a given allele.

Infant Mortality Infant mortality was assessed 30 days following birth. Data on infant mortality was derived from individual institution records and the International Addax Studbook (Correll, 1989a). Although infant mortality is usually estimated through six months of age (Ballou and Ralls, 1982; Ralls, et al., 1979), previous research on inbreeding depression indicates that the majority of genetic deaths occur early in life (within the first 30 days), with no significant differences when longer time periods are used, e.g., six months (Lacy, et al., 1993; Templeton and Read, 1983; Spevak unpublished data). Infant mortality was examined from January 1, 1989 to June 1, 1992 for all herds (few records exist for the Fossil Rim herd prior to 1989) based upon data from the Addax International Studbook (Correll, 1989a).

Results and Discussion

Results of the genetic analysis demonstrate low levels of variability in addax (Table 4.1). Class I and II genes of the MHC are the most polymorphic loci yet reported in mammals, with as many as 100 alleles per locus and heterozygosity levels as high as 100% (Klein, 1986). Digestion of Adna DR3 products with RsaI resulted in the identification of three distinct fragment patterns. Similarly, two BstYI patterns and two HaeIII patterns were identified. The combination of restriction patterns obtained with the three enzymes lead to the identification of eight Adna DR3 alleles across the North American addax herd. All allele patterns were identified from segregation patterns among individuals of known relationship. Allele survivorship among herds ranged from 3 to 7 alleles, with no herd possessing all eight alleles (Table 4.1). One allele was identified in only a single animal from a sample of two animals from a herd not used in the analysis. These levels are lower than those reported for breeds of domestic cattle where over 46 alleles (i.e., DRB RFLP haplotypes) have been reported in both Bos taurus and B. indicus (Davies et al., 1994).

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Because MHC molecules encoded by different alleles differ in their peptide-binding capacities (Amills et al, 1998; Behl et al. 2012; Buus, et al., 1987; Doherty and Zinkernagel, 1975; Lewin et al., 1999), higher levels of allelic variation in MHC loci may better protect organisms against a wider variety of infectious agents than those with lower levels of variation. MHC diversity has been interpreted as an adaptive strategy for countering rapidly evolving pathogens that afflict natural populations (Borghans et al, 2004; Ditchkoff et al., 2005; Hedrick and Kim, 2000; Hill, et al., 1991; McClelland et al., 2003; Oliver et al., 2009; Schad et al., 2005; Sommer, 2005; Yuhki and O'Brien, 1990). There is both direct (Gavora, et al., 1986; Hill, et al., 1991, Ilmonen et al., 2007; McCleland et al., 2003; Oliver et al., 2009; Schad et al., 2005) and indirect (Ditchkoff et al., 2005; O'Brien, et al., 1985; Watkins, et al., 1990; Watkins, et al., 1990) evidence supporting this proposition.

The results of the Adna DR3 analysis suggest a relationship between heterozygosity within addax populations and infant mortality lending support to the idea that heterozygosity, as measured by Adna DR, positively affects fitness. Observed heterozygosity levels for Adna DR ranged from 52.9% to 73.7% between herds (Table 4.1). These levels were within the range recorded for various breeds of domestic cattle, ranging from 12% to 94%, though substantially lower than the high of 94% within a breed (Nassiry et al., 2008; Ruzina et al., 2010; Udina et al., 1998; Takeshima et al., 2003). It should be noted that even cattle breeds which showed some of the lowest levels of observed heterozygosity had higher allelic diversity than addax, i.e., 19 versus 8 alleles for DR3 (Nassiry et al., 2008). Infant mortality ranged from a high of 67% at Brookfield Zoo to a low of 6.25% at the Henry Doorly Zoo; Brookfield Zoo (67%, 4 out of 6), Fossil Rim Wildlife Center (14.29%, 11 out of 77), Henry Doorly Zoo (6.25%, 1 out of 16), and St. Louis Zoo (25%, 3 out of 12). Regression analysis revealed a significant negative correlation between 30-day infant mortality and observed heterozygosity at Adna DR3 (R2=-0.957, F = 39.169, d.f. = 1,3, p = 0.0216) (Figure 3.2). No significant relationship was found between infant mortality and the number of alleles in the herd (R2=-0.615, F = 3.191, d.f. = 1,3, p = 0.216). Similar results were also observed within the Fossil Rim herd. At Fossil Rim, for example 2.8 times as many homozygous offspring died as heterozygous offspring (19.1% of all homozygous offspring died while only 6.8% of all heterozygous offspring died; N = 21 and 44

52 homozygotes and heterozygotes, respectively). Although these results are correlational, they lend support to the idea that there is generally a positive relationship between MHC diversity as evidenced by Adna DR3 heterozygosity with fitness and infant survival (Ober et al., 1998; Wedekind et al., 1996). High infant mortality and low litter survivorship in cheetah (Acinonyx jubatus) has also been interpreted as related to the ancestral loss of genetic variability (Marker and O’Brien, 1989) as evidenced by low levels of diversity at the MHC (O’Brien et al., 1985; O’Brien et al., 1987).

In order to test the applicability of using DR polymorphism to estimate genome wide diversity, observed heterozygosity was compared to average heterozygosity measured across the genome using microsatellites. Tandemly repeated (GT/AC)n dinucleotide DNA sequences (microsatellites) have been shown to be highly polymorphic (Tautz, 1989, Weber and May, 1989; Lowe et al., 2004) and randomly distributed across the genome (Ostrander, et al., 1993). These characteristics make microsatellites useful genetic markers for examining genetic diversity. Microsatellite primers designed for cattle and sheep were used with the addax due to their close phylogenetic relationship (Moore et al., 1991; Engel et al., 1996). Three polymorphic microsatellites were chosen for estimating average heterozygosity (Table 4.2). Observed heterozygosity of Adna DRB3 correlated with average heterozygosity of microsatellites (R = .864) though it was not significant. Using more microsatellites would probably increase the correlation and significance. Note, there was no significant correlation between individual microsatellite heterozygosities and observed levels of DR heterozygosity or infant mortality. Studies by Yukhi and O'Brien (1990) have shown concordance between MHC variability and genome-wide diversity, as measured with allozymes, further supporting the use of the MHC in estimating the extent of genetic diversity in natural and captive populations.

MHC and Mate Choice Females may prefer individual males as breeding partners. These decisions may be based on such things as genotypic similarities or differences (Brown, 1997; Kempenaers, 2007; Tregenza and Wedell, 2000), horn symmetry, body conformation or ornamentation (Arcese, 1994) or even territory location and quality (Balmford et al., 1992; Bro-Jørgensen, 2002). Females may also be

53 selecting mates based upon their genetic identity at the MHC locus, affecting male reproductive success. A number of studies have elucidated the potential role of the MHC in mate choice (Brown and Eklund, 1994; Jordan and Bruford, 1998; Hedrick, 1992; Grob et al., 1998; Penn and Potts, 1999; Penn, 2002; Zelano et al., 2002; Bonneaud et al., 2006; Setchell et al., 2010; Wedekind et al., 1995). MHC molecules encoded by different alleles differ in their peptide- binding capacities (Amills et al, 1998; Buus, et al., 1987; Doherty and Zinkernagel, 1975; Lewin et al., 1999; Behl et al. 2012). Higher levels of allelic variation in MHC loci and heterozygosity may better protect organisms against a wide variety of infectious agents and parasites and be an adaptive strategy for countering rapidly evolving pathogens that afflict natural populations (Borghans et al, 2004; Ditchkoff et al., 2005; Hedrick and Kim, 2000; Hill, et al., 1991; McClelland et al., 2003; Oliver et al., 2009; Schad et al., 2005; Sommer, 2005; Yuhki and O'Brien, 1990). Studies of mate choice and the MHC with house mice (Mus musculus) and other species (Brown and Eklund, 1994) indicate that individuals may be choosing mates based on how similar or dissimilar at the MHC they are to each other as detected in urine (Coopersmith and Lenington, 1990; Egid and Brown, 1989; Eklund et al., 1991; Potts, et al., 1991; Singer et al., 1997; Carrol et al., 2002). Choosing mates based on genetic non-identity may maintain heterozygosity levels in a population (Winternitz et al., 2013).

An indication that addax may be choosing mates based upon MHC genotypes comes from behavioral observations and observed heterozygosity levels. Male mammals identify estrus periods in females by testing their urine through flehmen, as noted earlier (Hart et al., 1989; Muller-Schwarze, 1991). If the major function of this behavior is to establish estrus then one would not expect females to perform this behavior towards males. Females were observed occasionally performing this behavior to males. Males do not go through any known hormonal cycles but if addax were able to identify MHC genotypes as observed in mice and other species (Brown and Eklund, 1994; Coopersmith and Lenington, 1990; Egid and Brown, 1989; Eklund et al., 1991; Potts, et al., 1991; Singer et al., 1997; Carrol et al., 2002) this behavior might be expected of females toward males. Additionally, as females appeared to select territories of particular males in which to calve there may be additional phenotypic characteristics, such as horn length or shape, body conformation, etc. that was indicative of “good genes” (Ditchkoff et

54 al., 2001). Another indication that mate choice might be influenced by the MHC comes from observed levels of heterozygosity. Heterozygosity at Adna DR is higher than would be expected from Hardy-Weinberg equilibrium (observed = .675, expected = .602). In addition,

Wright's F statistic Fis with a value of -.1213 also indicates an excess of heterozygotes. Excess heterozygotes may indicate negative assortative mating within the population (Lowe et al., 2004). However, the excess of heterozygotes may indicate previous migration into the population through the acquisition of new individuals. This is less likely as this population has remained closed since the original acquisitions in the late 1970’s (Correll, 1989a).

To examine whether heterozygosity may be maintained by negative assortative mating a coefficient of relatedness or coancestry (Lowe at al., 2004) was estimated for each sire and dam of a calf from Adna DR data. This value is the probability that any two randomly chosen alleles in a population are identical by descent (Hartl and Clark, 1989). Values for the coefficient of coancestry range from 0, when no alleles are shared between parents, to 1, when all alleles are shared (Table 4.3). There was no significant difference between the number of offspring sired by males of different coefficients of coancestry (kinships) to females than if females bred at random 2 = 1.034, d.f. = 3, p = .7937) (Figure 4.3). Previous investigation found that consecutive mates tend to be different (see Mate Choice and Individual Preference). However, there was no significant difference between the coefficient of consanguinity between consecutive males 2 = 2.033, d.f. = 3, p = .5655) (Figure 4.4). Additionally, no allelic preference in mates compared to population frequencies was evident 2 = 4.448, d.f. = 6, p = .6163) (Figure 4.5). Negative assortative mating based upon MHC haplotypes or alleles has been observed (Coopersmith and Lenington, 1990; Bernatchez and Landry, 2003; Huchard et al., 2013; Potts et al., 1991) but is certainly not universal (Paterson and Pemberton, 1997). It should be noted that although not observed intrasexual competition by females for preferred males can reduce observable MHC dissimilarities (Garner et al., 2010). To further examine whether there was an heterozygote advantage and possible balancing selection maintaining or increasing heterozygosity at Adna DRB3, Ewens-Watterson Test for neutrality (Ewens, 1972; Watterson 1978a; 1978b; 1986) was performed using the program Arlequin ver. 3.5.2.2 (Excoffier et al., 2005). Examination of Adna

DRB3 observed Hardy-Weinberg homozygosity (ƒe = 0.3953) found allelic frequencies slightly

55 more even than expected but did not differ significantly from equilibrium homozygosity (ƒeq = 0.4261, Slatkin’s Exact test P-value = 0.2217) (Slatkins, 1994;1996). The higher than expected level of heterozygosity may be maintained by negative assortative mating but Adna DR of the MHC may not be the region to examine.

Although arguments exist as to the merits of using only MHC loci in developing breeding and conservation programs (Gilpin and Wills, 1991; Hughes, 1991; Hedrick, 2003; Hedrick et al., 2000; Miller and Hedrik, 1991; Ujvari and Belov, 2011; Vrijenhoek and Leberg, 1991), when little else is available or when individuals are similar in other respects (e.g., a lack of allozyme diversity) the MHC provides a sensitive alternative tool for assessing genetic diversity. We have also recently evaluated the PCR-RFLP method on a number of related species, with primers amplifying orthologous genes from ten different species (Lewin et al., 1993) including American bison (Bison bison), (Bubalis arnee), Arabian oryx (Oryx leucoryx), scimitar- horned oryx (O. dammah), (Gazella dama), blesbok (Damaliscus pygargus phlippsi), wildebeest (Connachaetes taurinus), and (Giraffa camelopardalis) demonstrating the widespread utility of this technique.

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CHAPTER 5: CONCLUSION

Conservation Implications An understanding of male and female mating strategies and consequent reproductive success is fundamental to understanding a species social and population genetic structure (Struthsaker and Pope, 1991). The population and social structure of a species can have a direct bearing on the amounts and rates of gene flow within and between populations (Chepko-Sade and Halpin, 1987). The reproductive system of an organism may also have an enormous impact on gene frequencies and therefore evolution. Examinations of the levels of genetic variability found in wild populations have led to controversies as to whether an organism's population size or its social structure is more important in determining overall levels of genetic variability (Chepko- Sade and Halpin, 1987; Grieg, 1979; Lacy, 1987). It is ultimately the behavior and social structure of an organism that affects individual reproductive success and therefore gene transmission that determines how genetic variability is structured within a species or population.

In 1974, Bonnell and Selander (1974) discovered that the northern elephant seal (Mirounga angustirostris) had no variability at 24 electrophoretic loci examined. They attributed this to the fact that the northern elephant seal went through a severe bottleneck of approximately 20 individuals in the 1890's. What complicated the elephant seal's problem was its social structure. Males develop a dominance hierarchy and establish harems of females (LeBouef, 1974). With low population numbers it is possible for one male to control all reproductive females thereby altering the breeding sex ratio. After a short time, all individuals would effectively be related and inbreeding would reduce the overall levels of genetic variability (Charlesworth and Charlesworth, 1987; Frankham et al, 2010)). In addition, variations in reproductive success, due to dominance hierarchies or territoriality, or the exclusion of possible breeding individuals from closed social groups would reduce the effective population size (Chepko-Sade et al. 1987; Frankham et al. 2010), i.e., the size of an ideal population having the same rate of decrease in heterozygosity as the observed actual or non-ideal population (Hartl, 1981).

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Management of Captive and Wild Populations Wallach and Boever (1983) believed that herd management was one of the most universally neglected facets of ungulate management. In order to manage species more effectively, the Association of Zoos and Aquariums (AZA) developed the Species Survival Program (SSP) (Foose, 1986; Foose et al. 1986). The SSP concept along with other regional zoo breeding programs is to manage species in captivity as a metapopulation, with captive populations interacting through the processes of immigration and emigration (Gusset et al., 2014; Lacy, 2012; WAZA, 2005). Eventually the captive populations will interact more and more with the remaining wild populations, through the exchange of individual animals, embryos, or gametes, to bolster each (Foose, 1986; Foose et al., 1986; Foose et al., 1995, Lacy, 2012; WAZA, 2005).

Zoo or conservation breeding populations are often managed with animals of known lineage and are manipulated in such a way as to minimize inbreeding and maintain genetic diversity and simplify management. Much of this is accomplished through the selection of breeding partners (Frankham et al., 2010; Rodriguez-Clark, 1999). Additionally, “suitable” breeding males are often determined by such behavioral criteria as (a) engaging in minimal conflict, (b) more likely to assess than fight or flee, and (c) actively tending females by retaining proximity and assessing their readiness for copulation (Packard et al., 2014). This “suitability” may inadvertently select for certain phenotypic traits, e.g., behavior, more suited to a captive than a wild situation (Snyder et al., 1996). If animals are to be returned to the wild they must be able to function as wild populations are functioning (Kleiman, 1980, 1989; Stanley-Price, 1989). Additionally, “Released animals should exhibit behaviors essential for survival and reproduction, and for compatibility with any conspecifics in the release area” (IUCN/SSC, 2013). Maintaining various social structures and reproductive strategies, including multi-male multi-female breeding groups, gives rise to a host of management problems (Wielebnowski, 1998), not least of which is the maintenance of genetic variability (Fa et al, 2011; Frankham et al, 2010). For example, many antelopes are kept in harem situations in captivity with one male and many females. This does not allow for any male-male competition. In the wild a "harem" may actually contain more than one male, as is the case with oryx species (Oryx sp.) (Wacher, 1988; Walther, 1972, 1978). Captive animals not kept in similar situations may be at a competitive disadvantage when put

58 back into the wild. The maintenance of long term genetic diversity of a population is of critical importance (Frankham et al, 2010), however, allowing competition between males and mate choice (Grahn et al., 1998) is becoming an important component and consideration for the long- term maintenance of genetic diversity (Quader, 2005; Wedekind, 2002). Maintaining variability in social structure and behavior is as important as maintaining genetic variability for successful future reintroductions (Fa et al., 2011; Kleiman, 1980, 1989; Stanley-Price, 1989).

National parks are also becoming "megazoos", with wild populations requiring more intensive management in order to maintain an ecological balance (Fosse et al., 1995; Luard, 1985). However, national parks and reserves do not always have the luxury of moving single animals or deciding who should breed with whom. The species must be managed as a population, with all the associated factors of population and social structure, and the lack of knowledge of relationships between individuals. Understanding the dynamics of reproductive success and population structure in a controlled semi-free ranging system with known individuals, e.g., addax, will assist in the development of effective long-term management techniques and models for wild populations with unknown individuals (Wielebnowski, 1998). In addition, the more that is known of a species behavior, i.e., social structure and reproductive strategy, and its variability the greater our potential to understand the intricacies of evolution and our ability to conserve endangered species (Schaller, 2000).

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FIGURES AND TABLES

Figure 2.1. Maps of the Main Pasture at the Fossil Rim Wildlife Center illustrating male territories observed during the study during different time observation periods. Numbers indicate the male that possessed the territory. Dashed lines indicate approximate boundaries of male territories. Solid lines are fences bordering the pasture.

Figure 2.1a. Male territories at the beginning of the study (9/1989) showing only two males in possession of territories.

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Figure 2.1b. Male territories during the middle of the study (2/1991) showing three males in possession of territories.

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Figure 2.1c. Male territories at the end of the study (6/1992) showing five males in possession of territories.

62

Figure 2.2. Distribution of addax births and conceptions by month at the Fossil Rim Wildlife Center (FRWC) from 1989 through 1992.

63

Figure 2.3. Distribution of addax births by location at the Fossil Rim Wildlife Center.

1-Lodge, 2- Stream Pasture, 3-Water Buck Shed, 4 – Adam Lane, 5-Addax Shed, 6-Front, 7- Rodney Road/Old Coyote Den, 8-Cheetah Hill

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Figure 2.4. Map of FRWC Main Pasture with general locations of addax female calving areas.

3 7

2

6

1 5

4

8

65

Figure 3.1. Restriction patterns of Adna DR3 exon 2 alleles obtained by digestion with RsaI, HaeIII, and BstYI. Allelic patterns for RsaI (lanes 2-5), HaeIII (6-8), and BstYI (lanes 9-10) are indicated at the bottom.

66

Figure 3.2. Polyacrylamide (7%) gel electrophoresis of PCR amplifications of the bovid microsatellite locus BM43 using addax DNA. Six animals are shown for paternity exclusion analysis alongside an uncut M13 control sequencing reaction providing an absolute size marker. 124 and 115 are breeding males. They are shown alongside breeding females 111 and 105 along with their respective offspring 0011 and 202. Banding patterns suggest that male 115 sired both 0011 and 202. Additional loci examined revealed that 115 sired neither calf. (Extra bands surrounding major bands are likely to be PCR artifacts due to slippage in the DNA synthesis).

67

Figure 3.3. The distribution of the proportion of males siring different numbers of calves, comparison between 8-day and 18-day methods.

68

Figure 3.4. Reproductive Success of Males by Age Class, comparison between 8-day and 18-day methods.

69

Figure 3.5. Observed and Expected Number of Offspring Sired by Males in Different Age Classes as determined by examination of allelic patterns in Adna DRB3 and four microsatellites.

70

Figure 3.6. Behavioral versus Genetic Estimates of Male Reproductive Success by Age Class.

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Figure 3.7. Number of Calves Sired by Different Ranked Males, comparison between 8-day and 18-day methods.

72

Figure 3.8. Number of Calves Sired by Males of Different Ranks as determined by examination of allelic patterns in Adna DRB3 and four microsatellites.

73

Figure 3.9. A comparison between genetic and behavioral methods estimating the number of calves sired by different ranked males.

74

Figure 3.10. Number of Calves Sired by Territorial and Non-territorial Males, comparison between 8-day and 18-day methods.

75

Figure 3.11. A comparison between genetic and behavioral methods of the percent of calves sired by territorial and non-territorial males.

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Figure 3.12. Postpartum and Non-postpartum Conceptions by Territorial and Non-territorial males as determined by examination of allelic patterns in Adna DRB3 and four microsatellites.

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Figure 3.13. Number of Calves sired by Individual Addax Males.

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Figure 3.14. A comparison between genetic and behavioral methods of the proportion of males siring different over-all number of calves sired.

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Figure 4.1. Restriction patterns of Adna DR3 exon 2 alleles obtained by digestion with RsaI, HaeIII, and BstYI. Allelic patterns for RsaI (lanes 2-5), HaeIII (6-8), and BstYI (lanes 9-10) are indicated at the bottom.

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Figure 4.2. Correlation between Adna DR3 Observed Heterozygosity and 30-Day Infant Mortality across four captive Addax populations.

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Figure 4.3. Observed and expected number of offspring sired by males of different Coefficients of Coancestry to females.

82

Figure 4.4. Comparison of Coefficient of Coancestry between males from consecutive matings.

83

Figure 4.5. Frequency of Adna DRB3 alleles within the population compared to the frequency in known breeding males.

84

Table 2.1. Male addax dominance hierarchy at FRWC for the four observational time periods of the study.

Rank 9/1989-12/1989 6/1990-2/1991 3/1991-11/1991 12/119-6/1992 1 61* 61 61 109, 115 2 108 115 109 108 3 115 109 108, 115, 125 125 4 133 107, 108** 51 61 5 109 51 124 124 6 107 130 137 159 7 125 137 159 0011, 137 8 51 182 161 176 9 130 124 179 187 10 137 159, 179 0011 183, 186 11 124 161 176, 177, 183 177 12 182 186 179 13 187 188 14 188 196 15 193 193 16 197 17 202

*Numbers indicate male ID based on ear tag numbers ** Non-linear relationship where more than one male ranked equally

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Table 3.1. Alleles and allele frequencies for MHC locus Adna DR3 and 4 microsatellites identified in the Fossil Rim Wildlife Center addax herd.

Alleles* and Frequencies

MHC Locus Microsatellite Loci Adna DR URB008 BM 43 MAF 70 MAF 209 MAE - .5746 165 - .2191 178 - .1207 126 - .01 115 - .3957 LAE - .0526 163 - .0281 176 - .0402 122 - .79 113 - .6043 OAE - .0175 161 - .4719 174 - .1609 114 - .19 LAG - .1184 157 - .2135 172 - .1437 LBE - .2018 155 - .0674 170 - .4598 LBG - .0307 168 - .0747 MAG - .0044

Obs. H = .675 Obs. H = .786 Obs. H = .805 Obs. H = .542 Obs. H = .451

* Allele designations for Adna DR are restriction patterns following the convention of van Eijk et al (1992). Allele designations for the microsatellites are allele sizes in base pairs.

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Table 4.1. Adna DR3 Allele Frequencies and Observed and Expected Heterozygosities in Captive Addax (Addax nasomaculatus) Populations.

Population Alleles and Frequencies H H exp. obs. 1 2 3 4 5 6 7 8 St Louis .5000 .0455 .1364 .1364 .1364 .0455 .0000 .0000 .636 .69 Zoo (N=11) Brookfield .4120 .0000 .0000 .5290 .0590 .0000 .0000 .0000 .529 .547 Zoo (N=17) Henry .1316 .0263 .4737 .1053 .2632 .0000 .0000 .0000 .737 .677 Doorly Zoo (Omaha) (N=19) Fossil Rim .5746 .0526 .0175 .1184 .2018 .0307 .0044 .0000 .675 .602 (FRWC) (N=111) N. .2778 .0159 .1984 .2778 .2143 .0079 .0000 .0079 .603 .760 American Pop. (excluding FRWC) (N = 63)††

† Expected Heterozygosity is based upon Hardy-Weinberg Equilibrium †† Samples from Brookfield Zoo, St. Louis Zoo, Henry Doorly Zoo, San Diego Zoo, Cheyenne Mountain Zoo, Louisville Zoo, Miami Metrozoo, San Antonio Zoo, Oklahoma City Zoo, Horizon Ranch, and Camp Cooley Ranch

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Table 4.2. Adna DRB3 and Microsatellite or Simple Sequence Repeats (SSR) Allele Frequencies and Observed Heterozygosities in Captive Addax (Addax nasomaculatus) Populations.

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Table 4.3. Coefficients of relationship or consanguinity (r) between males and females based on their genotype at a selected locus. The value r is the probability that alleles are identical by descent.

Dam’s Genotype Sire’s Genotype r AA AA 1 AB AA 1/2 AB AB 1/4 AB AC 1/4 AB CD 0

89

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APPENDIX A

Appendix A.1. MHC and SSR genotype patterns of addax sampled at FRWC. For Adna DRB3 allelic RFLP patterns are listed after the convention of van Eijk et al. (1992). For each SSR/Microsatellite the presence of a band/allele is indicated by “I.” Where only one allel was identified the individual is presumed to be homozygous at that locus and is indicated by “II.” Greyed cells indicate that no gene product was able to be amplified for that locus.

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APPENDIX A.1 (cont’d)

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APPENDIX A.1 (cont’d)

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APPENDIX B

Appendix B.1. Animal species mentioned in text

Addax (Addax nasomaculatus) African elephant (Loxodonta africana) American Bison (Bison bison) Arabian oryx (Oryx leucoryx) Axis deer or chital (Axis axis) Beisa oryx (O. beisa beisa) Blackbuck (Antelope cervicapra) Blesbok (Damaliscus pygargus philippsi) Bontebok (D. p. pygargus) Common Reedbuck (Redunca arundinum) Dama gazelle (Gazella dama) Dik-dik (Madoqua sp.) Domestic cattle (Bos taurus and B. indicus) Dorcas gazelle (G. dorcas) Duikers (Cephalophini) Dwarf Antelope (Neotragus sp.) Fallow deer (Dama dama) Fringe-eared oryx (O. beisa callotis) Gemsbok (O. gazella) Giraffe (Giraffa camelopardalis) Horse (Equus caballus) House mouse (Mus musculus) Impala (Aepyceros melampus) Kafue lechwe (Kobus leche kafuensis) Klipspringer (Oreotragus oreotragus) Kob (Kobus kob) Lion (Panthera leo)

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Northern elephant seal (Mirounga angustirostris) Oribi (Ourebia ourebi), Red deer (Cervus elaphus) (Hippotragus equinus) Sable Antelope (H. niger) Scimitar-horned oryx (O. dammah) Sika deer (Cervus nippon) Spanish ibex (Capra pyrenaica) Steenbok (Raphicerus campestris) Tammar wallaby (Macropus eugenii) Topi (Dammaliscus lunatus) Uganda kob (Kobus kob thomasi) Waterbuck (Kobus ellipsiprymnus) Water buffalo (Bubalis arnee) White-tail deer (Odocoileus virginianus) Wildebeest (Connochaetes taurinus) Wolf (Canis lupus)

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APPENDIX C

Appendix C.1: Ethogram of addax used in this study.

Postural or Locomotory Alert Posture: Standing or occasionally lying, staring in direction of disturbance or object of interest with both ears forward. Charge: Quick movement towards another individual. Chase/Pursue: Following in which both animals are galloping/running. Crawling: Observed once in a calf attempting to move away from an aggressive adult male. Body was held close to ground and calf walked on while maintaining its belly a couple inches above the ground. Following: Movement where one animal attempts to approach while the other attempts to depart. Gallop: In addax, appears as a rather stiff-kneed gate with little flexing of the joints of the hind legs Jumping/Cavorting: Jumping, bucking, spinning, usually displayed by calves or young addax, occasionally seen in adults during high intensity interactions. Observed in adult addax and oryx, especially with a change in weather. Kneeling: As a prelude to or part of fighting or horning the ground though may be symbolic and continue with grazing or lying. Lying: Often with one or more legs extended. Lying Out: Best observed in calves where calf lies with head low on the ground. Also observed in adults, especially females that are trying to avoid advances by males or a dominant female. Scraping or Pawing: Scraping or pawing of ground with forefoot to clear area of debris before lying down or as a prelude to urination or defecation. Stand: Self-explanatory Stare: Looking in one particular direction for a prolonged period, usually caused by a disturbance or at a subordinate. Trot: Stylized movement with alternate legs moving in unison, head is held high and moved synchronously from side to side. Also observed in Oryx sp.

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Walking: Self-explanatory

Input/Output Behaviors Bunting: The striking of the udder of a cow by the calf to stimulate the flow of milk. Defecate: Elimination of fecal material. Individuals usually hold tail strait out behind them. Two variations are the Tail Curl and the Squat, both so far only observed in males. Defecate/Squat: Defecation posture performed only by males. Individual adopts a low crouch, supporting weight on forelegs and tips of hind hooves. Tail is curled up and out of the way. Squat defecation or defecation crouch is usually performed by the most dominant male in a group or immediately by the winner of a horn wrestling bout. Defecate/Tail Curl: Only observed in males. Animal defecates in standing position but curls tail up so that tip is higher than rump. The tail curl position is similar to that seen in the squat defecation. Observed in dominant males where they were apparently too preoccupied to squat. Feeding: Intake and mastication of foodstuffs. Can be divided into grazing and feeding of pelleted diets and hay. Ruminating: Regurgitation and re-mastication of foodstuffs. Common when individuals are lying. Suckling: Nursing by calves. Urinate: Excretion of metabolic wastes. Vocalizations: Addax have a number of vocalizations. Terms used to describe sounds are very subjective. Grunt: A vocalization where the mouth appears to be closed or open only slightly. A sound that appears to remain in the throat of the animal similar to a hum in humans. Function? Contact? Moaning/Lowing: A repeated call with mouth open and tongue curled up. In calves, the call is higher pitched and time between moans is of a much shorter duration. This is usually an alarm call in calves. Any female with a young calf will respond to a human mimicked calf call, but a calf that actually vocalizes appears to be recognized only by its mother. This same call in adult females may be a sign of nervousness or as a contact with a calf or the rest of the group.

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Roar/Bellow: A single loud low call often performed by an individual after being struck by another. Snort: A sneeze-like sound indicating alarm or nervousness

Interactive Behaviors Allo-grooming: Addax will usually only groom a calf or themselves. Allo-grooming of calves usually takes the form of licking or combing with the incisors. Auto-grooming: Individuals groom themselves with four parts of their body depending upon the location of the irritation. Addax usually groom with their tongue, lower incisors, horn tips and hind feet. Ano-Genital Licking: Usually performed by mothers to young calves while suckling, in order to stimulate defecation and remove feces. Ano-Genital Scenting: Sniffing of genitalia of another individual. Usually male to female.

Agonistic Behaviors: Kick (Hindleg): A backward kick of the hindleg towards a pursuer. Rare Erect Posture: Neck raised above horizontal Lateral Presentation: Standing sideways to opponent often with erect posture and angle-horn. Very obvious display in addax when complete. Individual usually stands perpendicular or parallel to opponent with erect posture (head held high), horns angled at opponent and the facing ear pointed toward opponent. Very stylized. Clash Fighting: While standing with heads high, individuals quickly deliver powerful forward and downward blows with their horns. Over The Shoulder Stabbing: While standing in parallel or reverse-parallel, individuals will try to deliver a stabbing blow over their shoulder to their opponent. Also observed when not in parallel when it is an individual’s only recourse for a retaliatory blow. Angle-Horn: Horns angled in direction of opponent with chin turned away. High-Horn Presentation: Neck raised, nose level or angled down. Low-Horn Presentation: Head lowered, chin in, horns pointing at opponent. Medial Horn Presentation: Neck at about shoulder level, horns pointing upward.

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Horn Butt: The striking of another individual with the shaft or base of the horns Horning: Striking inanimate objects with horns, e.g., bushes. Horn Jab: The striking of another animal with the tip of the horns Horn Sweeping: Abrupt sideways movement of the horns to shoulder or flank. Offensive threat and displacement. Horn Wrestling or Horn Pressing: Pushing, twisting and grappling performed with interlocking horns. Head-Low/Chin Out: Horns pointed backward while chin is lifted. Submissive behavior. Head-Low Posture: Chin drawn in with horns usually pointing upward. Defensive threat or leads to offensive action Head Shaking or Horn Wag: A side to side motion of the horns. Head Turn: Sideward turn of head usually in frontal orientation, Nodding: A forward and downward movement of the horns, symbolic butting. Supplanting: Displacing a subordinate by walking towards it. Usually with little or no obvious agonistic displays.

Reproductive Behaviors: Chin Resting: Resting of chin on cow’s rump prior to mounting by males Circling/Mating whirl-around: Described by various terms, e.g., reverse parallel, courtship circling, paarungskreisen, etc. Circling reverse parallel orientation of male and female during mating ritual. Female usually in head-low/chin out posture. Also occurs in agonistic encounters between males. May remain stationary for short amounts of time. Ejaculation: Usually indicated during mounting and intromission with a slight jump with a forward thrust and the throwing back of the head. Flehmen: Also called lip curl or urine testing. Usually an individual places his nose in a stream of urine voided by another individual. The individual then raises his chin and curls its upper lip to allow urine to pass across the palate and the vomero-nasal organ. Performed by both males and females although usually by males in order to test the reproductive status of a female. Intromission: The observed penetration of the penis into the vagina.

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Laufschlag/Foreleg Kick: A very pronounced lifting of the bent foreleg by a male towards a female. Usually directed at the flank or rump. Occasionally seen performed stiff legged but then usually directed at the chest of a female, apparently to stop her forward progress. Mounting: Resting of the forequarters of an individual, usually male, on the rump of another individual, usually female. Nuzzle: Nudging with the muzzle Tail Raise: Tail held away from body when not defecating or urinating. May indicate imminent parturition or estrus of females

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APPENDIX D

Appendix D.1. Dominance hierarchy win/loss matrix for male addax at FRWC from 9/1989 to 12/1989

61 108 115 133 109 107 125 51 130 137 124 182 W

61 3 3 4 2 1 13 108 5 2 2 9 115 21 3 2 2 1 29 133 6 5 4 2 1 1 19 109 1 11 5 6 2 2 27 107 1 6 6 2 1 16 125 12 2 3 1 1 19 51 8 1 1 10 130 1 1 2 137 2 2 4 124 1 1 182 0

L 0 3 4 31 13 21 17 27 15 6 6 6 149

W = Wins, L = Losses

Dominance matrix for male addax in the Main Pasture herd from October through December 1989. Agonistic encounters won (W) are in the upper part of the matrix, losses (L) are in the lower portion. Animals are ranked by the number of individuals defeated versus the number of individuals that defeated them. Values below the diagonal are reversals.

(* 115 did not move after encounter but retaliated immediately.)

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Appendix D.2. Dominance hierarchy win/loss matrix for male addax at FRWC from 6/1990 to 2/1991

61 115 109 108 107 125 51 130 137 182 124 159 179 161 W 61 28 26 1 13 10 25 8 7 4 3 125 115 21 9 11 11 14 3 2 4 2 77 109 2 20 19 23 14 1 9 3 2 93 108 3 2 1 6 107 6 5 5 1 2 19 125 14 10 2 7 3 36 51 1 22 3 5 1 32 130 1 17 11 3 32 137 11 6 17 182 15 1 16 124 7 5 3 15 159 1 3 4 179 1 1 161

L 28 47 12 44 50 83 62 21 60 51 5 5 5 473

W = Wins. L = Losses

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Appendix D.3. Dominance hierarchy win/loss matrix for male addax at FRWC from 3/1991 to 11/1991

W = Wins, L = Losses

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Appendix D.4. Dominance hierarchy win/loss matrix for male addax at FRWC from 12/1991 to 6/1992

W = Wins, L = Losses

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APPENDIX E

Appendix E.1: List of reagents used in analyses.

1X SSC ~12,500x g RCF 10% SDS TE

0.1M NaCl TE 10 mM Tris-HCl

10mM EDTA 1x TBE

Tris-HCI

NaCl TE pH 7.5 .1 M NaCl .584g/100mls (2mls 5M NaCl/100mls) .05 M Tris .605g/100mls (5mls 1M Tris pH 7.5/100mls) .001 M EDTA .037g/100mls (.2mls .5M EDTA pH 8.0/100mls)

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