Inbred Prairie Voles (Microtus Ochrogaster) Under Captive and Semi-Natural Conditions

SURVIVAL AND REPRODUCTIVE SUCCESS OF INBRED AND NON- INBRED PRAIRIE VOLES (MICROTUS OCHROGASTER) UNDER CAPTIVE AND SEMI-NATURAL CONDITIONS

A Thesis

Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Master of Science Department of Zoology by Kathryn Lynn Williams Miami University Oxford, OH 2008

Advisor______(Dr. Brian Keane)

Reader ______(Dr. Nancy G. Solomon)

Reader______(Dr. Thomas O. Crist)

ABSTRACT

SURVIVAL AND REPRODUCTIVE SUCCESS OF INBRED AND NON- INBRED PRAIRIE VOLES (MICROTUS OCHROGASTER) UNDER CAPTIVE AND SEMI-NATURAL CONDITONS

by Kathryn Lynn Williams

Inbreeding’s harmful consequences have been well documented under artificial conditions; however, studies under natural conditions are limited. I examined the effects of inbreeding on the fitness of prairie voles (Microtus ochrogaster) in captivity and the field. In captivity, sibling and non-sibling pairs did not differ with regard to time to the first litter, litter size, or offspring weight. Another laboratory experiment examined these same variables in the following un-related pairs: non-inbred female/ male, inbred female/ male, non-inbred female/ inbred male, and inbred female/ non-inbred male. The only significant result was that the weaning weight of offspring born to non-inbred pairs was greater than offspring born to non-inbred female/ inbred male pairs. There also was no significant difference in the survival and reproduction of unrelated inbred and non-inbred voles released into semi-natural enclosures. This study did not find any evidence that inbred adults have lower fitness than non-inbred adults.

Table of Contents

List of Tables iii List of Figures iv Dedication v Acknowledgements vi Chapter One: Introduction 1 Methods 4 Results 6 Discussion 8 Chapter Two: Introduction 12 Methods 14 Results 19 Discussion 20 Tables and Figures 25 Literature Cited 34

List of Tables

Table Page

Chapter One

Table 1.1 25 Table 1.2 26

Chapter Two

Table 2.1 27 Table 2.2 28 Table 2.3 29

iii

List of Figures

Figure Page

Chapter Two

Figure 2.1 30 Figure 2.2 31 Figure 2.3 32 Figure 2.4 33

Dedication For my Dad William Randolph Williams

Acknowledgements

I would like to thank my advisor, Dr. Brian Keane, and the members of my committee, Dr. Nancy Solomon, and Dr. Thomas Crist. I would also like to thank the Solomon Lab and the Meikle Lab. In addition, I would like to thank Dr. Douglas Meikle, Loren Hayes, Lana Knoch, Mark Spritzer, Charity Crowe, Chrissy Anderson, Chris Wood, and Bob Davis.

I could not have completed this thesis without the friendship and support of Sheri and Chris Barton, Aaron Roberts, Jennifer Hoffman, Carrie Smith, and Sarah Harvey.

Thanks to my family ~ My brother, Andrew.

My Mom deserves special thanks for all of her love, support, friendship, and guidance throughout my life.

And finally, to my husband and muse, Jeremy Shuck. “A riddle, wrapped in a mystery, inside an enigma” —Winston Churchill

Thank you for the motivation to finish this degree and to finally go to law school. Most of all, thank you for putting up with me throughout this tenuous process.

Chapter One INTRODUCTION

“Nature abhors perpetual self-fertilization” −Charles Darwin

The consequences of inbreeding have been thoroughly documented since the writings of Charles Darwin (1868). Inbreeding depression is the reduction in reproductive success and survival due to mating between relatives. Studies have documented inbreeding depression in a wide variety of taxa including the following: plants (Darwin 1868; Charlesworth and Charlesworth 1987), insects (Saccheri et al. 1998; Armbruster et al. 2000), fish (Waldman and McKinnon 1984), reptiles (Waldman and McKinnon 1984), birds (Keller et al. 1994), and mammals (Jimenez et al. 1994; Meagher et al. 2000; Slate et al. 2000). Fitness components such as inter-birth-interval, offspring survival, parental behavior, mating ability, sperm quality, plant height, and offspring weight may be negatively affected by inbreeding (Crnokrak and Roff 1999; Frankham et al. 2002). Inbreeding increases the chance an individual will inherit the same alleles at a particular locus, thus being homozygous at that locus (Frankham et al. 2002). Increased homozygosity may decrease survival or reproductive success (Frankham et al. 2002). The importance of two non-mutually exclusive hypotheses, overdominance and partial dominance, are debated as genetic mechanisms of inbreeding depression (Roff 2002). The overdominance hypothesis suggests that heterozygotes will have greater fitness compared to homozygotes (Mitton 1993). Because inbreeding may lead to an increase in the homozygous state, fitness may be reduced if heterozygous individuals have an advantage in the population (Charlesworth and Charlesworth 1987). The partial dominance hypothesis states that inbreeding could increase the chance that harmful recessive alleles may be expressed in offspring (Mitton 1993). Because inbreeding increases the chance of the homozygous condition, deleterious recessive alleles may be exposed resulting in deleterious consequences for an individual (Frankham et al. 2002). In a unique study designed to separate the genetic mechanisms at work, Roff (2002) crossed several different lines of 14 generations of brother-sister matings in sand crickets (Gryllus firmus). Following out-crossing, if the partial dominance hypothesis was operating, trait means were expected to exceed original non-inbred levels (hybrid vigor), while if the overdominance hypothesis was operating,

trait means would return to the original non-inbred levels (Roff 2002). The partial dominance hypothesis was supported by the results because trait means exceeded original levels suggesting deleterious recessive alleles were causing a decline in fitness (Roff 2002). The fitness decline was alleviated after crossing with a population that did not have the same deleterious recessive alleles (Roff 2002). Additional indirect support for the deleterious effects of inbreeding comes from the behaviors that may have evolved in many species as a means to reduce the opportunity for mating between close relatives. In mammals, behavioral mechanisms such as sex-biased dispersal and kin recognition may have evolved to limit inbreeding (Charlesworth and Charlesworth 1987; Pusey and Wolf 1996; Berger et al. 1997). Although inbreeding avoidance may not be the only reason for sex-biased dispersal, if either female or male offspring disperse, the chance of breeding with relatives is reduced via geographic separation. A review by Pusey and Wolf (1996) outlined two vole species (Townsend vole- Microtus townsendii and meadow vole- Microtus pennsylvanicus) that were more likely to disperse in the presence of relatives. The authors also looked at one species of mouse (white-footed mice- Peromyscus leucopus noveboracensis) that was less likely to disperse when the opposite-sex parent was not present (Pusey and Wolf 1996). If the sexes do not differentially disperse, individuals may recognize kin via association or phenotype matching to actively avoid inbreeding (Fadao et al. 2000). An experiment on the root vole (Microtus mandarinus) showed a breeding preference for unfamiliar individuals, regardless of actual relatedness (Fadao et al. 2000). This demonstrates that individuals avoid breeding with the animals with which they were reared, such as littermates (Fadao et al. 2000). A cross-fostering study on the montane vole (Microtus montanus) found that males caused sexual maturation in unfamiliar females and unfamiliar biological daughters, but not in daughters or foster daughters when housed together (Berger et al. 1997). Most evidence for inbreeding depression in mammals comes from data in laboratory and zoological populations (Lacy et al. 1993). Captive inbreeding studies have mainly focused on only juvenile mortality as one fitness component (Shields 1993). A review, of the costs of inbreeding among captive populations of 38 species of mammals, estimated juvenile mortality was an average of 33% higher among inbred offspring compared to non-inbred offspring (Ralls et al. 1988). However, a focus on only juvenile mortality may not accurately reflect the effects of inbreeding on reproductive success because inbred offspring could be more competitive, parental, or fertile (Shields 1993). Thus, a trade- off of “quantity for quality” would go undetected if inbred offspring that survived were more successful

at reproduction despite having a higher chance of mortality as juveniles (Shields 1993). Alternatively, looking solely at juvenile mortality may underestimate the deleterious effects of inbreeding because inbred individuals may also have lower reproductive success. The objective of my study was to determine if inbreeding reduced total reproductive success by affecting several components of fitness. A socially monogamous rodent, the prairie vole (Microtus ochrogaster), was used as a model organism to examine the effects of inbreeding. The prairie vole was used in this study because they are abundant and socially monogamous with bi-parental care (Carter and Getz 1993; Getz and Carter 1996). Additionally, inbreeding avoidance can be overcome in prairie voles by separating animals for short periods of time (Gavish et al. 1984). Prairie voles also have relatively short gestation and weaning periods, making them an ideal model organism (Nadeau 1985; Carter and Getz 1993; Getz and Carter 1996). First, I measured the reproduction of sibling pairs and non-sibling pairs in which none of the parents were inbred. The survival of offspring from these pairs was also examined. This is the “traditional” inbreeding test to compare the affects of close inbreeding on the offspring’s birth weight, weaning weight, and survival until weaning. A full-sibling mating was predicted to result in fewer offspring produced, lower birth or weaning weights, or longer time to first reproduction relative to non-sibling matings. Next, to determine how inbreeding might affect an individual’s reproductive success in adulthood I examined four types of matings: inbred female/ inbred male, non-inbred female/ non-inbred male, inbred female/ non-inbred male, and non-inbred female/ inbred male pairs. This experimental design was unique because the paired individuals were not related; so the offspring produced were not inbred. Since pairs were not related, the direct genetic effects of inbreeding (deleterious recessive alleles and/ or loss of heterosis) were not expected to be observed in offspring. Instead, this experiment was designed to determine if behavioral or physiological traits of the inbred but un-related parents would manifest a difference in reproductive success even though the offspring produced by these pairs were not inbred. The non-inbred female/ non-inbred male pairs served as the control group. The inbred female/ inbred male, inbred female/ non-inbred male, and non-inbred female/ inbred male pairs were expected to experience lower reproductive success (lower offspring weight, fewer offspring, longer time to first reproduction) compared to the control (non-inbred female/ non-inbred male pairs).

METHODS Experiments took place from June 2003 to June 2004 at the Miami University Ecology Research Center in Butler County, Ohio (39° 30′N, 84° 44′W). Individuals used in this project originated from a prairie vole breeding colony maintained at Miami University. Colony animals were derived from founders originally caught in southern Illinois during the summer of 2000. Animals used in this research were collected from colony pairs living in captivity between four to seven generations. Attempts were made to limit inbreeding in this stock with the periodic addition of wild caught animals; however, inbreeding coefficients for this population could not accurately be determined.

Experiment One: Sibling Inbreeding Two treatments were established to examine the effect of full-sibling breeding on reproductive success and offspring survival. Experimental parings were created by separating prairie vole pups at weaning (21 days of age) and housing them with same-sex siblings for ≥ 8 days to overcome inbreeding avoidance (Gavish et. al. 1994). Animals between 35-45 days were randomly assigned to pairings of either full-sibling (inbred; n = 16) or non-related individuals (non-inbred; n = 18). Under laboratory conditions, prairie voles do not breed if they are paired prior to 31 days of age (Solomon 1991), so all pairs were old enough to initiate breeding. Pairs were selected to limit relatedness to the extent possible. Thus, the last common ancestor for each pair was at least four generations in the past. Paired animals were housed in standard plastic shoebox cages (28 x 18 x 13 cm) until parturition. After parturition, pairs and offspring were placed in 36 x 30 x 18cm plastic cages. Animals were held under climate controlled conditions (14:10 light/dark cycle (lights on at 0600h), 25 + 3°C) at Miami University’s animal facilities. All cages contained processed paper bedding (Cell Sorb Plus, A & W Products, Inc., New Philadelphia, OH), dried alfalfa, and cotton Nestlet (Ancare Corp., North Bellmore, NY) for nesting material. Animals were given ad-lib Rodent Chow food (Purina Mills LLC, St. Louis, Missouri) and water. Time from pairing to the birth of the first litter (days), litter size at birth, litter size at weaning (day 21), and offspring weight were recorded for each pair. Cages were checked for births each day. To estimate growth rate, all pups were weighed with an electronic scale (Explorer Pro, Ohaus Co., Pine Brook, NJ) to the nearest hundredth of a gram one day after birth and every three days thereafter until weaning (21 days). Litter weight at birth and weaning

was averaged because individual pups were not marked. Pairs were given 60 days to reproduce, after which time non-producing pairs were deemed to have failed to reproduce and separated.

Experiment Two: Inbred v. Non-inbred Parents Four treatments were established to test the effects of inbreeding on survival of offspring and reproduction with regard to the sex of the inbred individual. Experimental animals were weanlings produced by inbred and non-inbred pairs from Experiment One. Individuals ranging from 35 to 60 days of age were randomly assigned to treatments. Four treatment groups were established: inbred female/ inbred male (I/I; n = 14), non-inbred female/ non-inbred male (N/N; n = 15), inbred female/ non-inbred male (If/Nm; n = 15), non-inbred female/ inbred male (Nf/Im; n = 16). The sex of the inbred animal for inbred/ non-inbred pairs varied to account for any difference in inbreeding due to sex. Pairs were selected to limit relatedness between family lines so that the last common ancestor for each pair was at least three generations in the past. Paired animals were housed in standard plastic shoebox cages (28 x 18 x 13 cm) until parturition, after which they were placed in 36 x 30 x 18cm plastic cages. Animals were held under controlled conditions (14:10 light/dark cycle (lights on at 0600h), 25 + 3°C) at Miami University’s animal facilities. All cages contained processed paper bedding (Cell Sorb Plus, A & W Products, Inc., New Philadelphia, OH), dried alfalfa, and cotton Nestlet (Ancare Corp., North Bellmore, NY) for nesting material. Animals were given ad-lib Rodent Chow food (Purina Mills LLC, St. Louis, Missouri) and water. Time from pairing to first reproduction (days), litter size at birth, litter size at weaning, and offspring weight were recorded for each pair. All pups produced were weighed with an electronic scale (Explorer Pro, Ohaus Co., Pine Brook, NJ) to the nearest hundredth of a gram one day after birth and every three days thereafter until weaning (21 days). Individual pups were not marked so the litter weight was averaged for birth and weaning weights respectively. Pairs were given 60 days to reproduce, after which time non-producing pairs were considered to have failed to reproduce and separated.

Statistical Analysis Offspring weight at weaning was averaged per litter to take into consideration large litters with light-weight pups versus small litters with heavy pups. For experiment one, the mean pup weight at weaning from full sibling pairs was compared with that of non-sibling pairs using a

two-tailed Student’s t-test (analyzed using Excel - Microsoft Office 2003). The mean number of offspring produced per litter by full sibling pairs and non-sibling pairs was compared at birth and at weaning using a two-tailed t-test. Litters in which all offspring died before weaning were included in the “litter size at weaning” analysis as zeros. The number of days from pairing until first reproduction was recorded to determine if sibling pairs were slower or less likely to reproduce compared to non-sibling pairs. Each pair was given 60 days to reproduce, after which they were considered a non-breeding pair and assigned a “60” in the analysis. A two-tailed t-test was performed to compare the number of days from pairing until birth of the first litter for sibling and non-sibling pairs. For experiment two, the mean weight at weaning of offspring from pairings of inbred female/ inbred male (I/I), non-inbred female/ non-inbred female (N/N), inbred female/ non- inbred male (If/Nm), and non-inbred female/ inbred male (Nf/Im) was compared using an ANOVA and Bonferroni post hoc tests (analyzed using STATVIEW 5.0.1). Litters in which all pups died before weaning were not included in the analysis. In experiment two, mean litter sizes for each treatment (I/I, N/N, If/Nm, Nf/Im) were compared at birth and weaning using an ANOVA and Bonferroni post hoc tests. Litters in which all offspring died before weaning were included in the “litter size at weaning” analysis as zeros. An ANOVA and Bonferroni post hoc tests were performed to compare the number of days from pairing until birth of the first litter for each of the four treatments (I/I, N/N, If/Nm, Nf/Im).

RESULTS

Experiment One: Sibling Inbreeding Sibling pairs averaged fewer days (33.69 ± 3.7) from pairing to birth of the first litter than non-sibling pairs (37.78 ± 3.9) but this was not significantly different (t-statistic = 0.75, df = 32, p = 0.46; Table 1.1). Thirteen of the 16 inbred pairs successfully bred (81% success rate), while 12 of the 18 non-inbred pairs produced offspring (66% success rate). Overall, 73.5% of the paired voles produced litters. Two of the inbred pairs produced litters in which zero pups survived to weaning (one pair produced 2 pups; the second pair produced 3 pups). None of the non-inbred pairs lost an entire litter. Litter size ranged from 0 to 6 pups per inbred pair (n = 16) and 0 to 5 pups per non-inbred (n = 18) pair. Mean litter size at birth was not significant

between treatments (t-statistic = 0.337, df = 32, p = 0.74). Sibling pairs had a mean of 2.3 pups (n = 36) per litter and non-sibling pairs had a mean of 2.1 pups (n = 37) per litter (Table 1.1). Birth weight averages were not analyzed because some litters were found on day one while others were found on day two resulting in a disparity of weights. Non-siblings weaned an average of 1.78 pups (n = 32) per litter which was not significantly different from sibling pairs that averaged 1.75 pups (n = 28) per litter (t-statistic = 0.047, df = 32, p = 0.96; Table 1.1). The mean pup weight at weaning for inbred pairs ranged from 15.9 to 23.6 (g). The mean pup weight at weaning for non-inbred pairs ranged from 16.3 to 24 (g). Non-sibling pairs averaged slightly heavier pups (20.1 g ± 0.8) than sibling pairs (19.3 g ± 0.6), however, the difference in offspring weight at weaning was not statistically significant (t-statistic = 0.82, df = 20 p = 0.42; Table 1.1).

Experiment Two: Inbred v. Non-inbred Parent The number of days from pairing until the first litter born was not significantly different among treatments (F = 0.92, df = 3,56, p = 0.43; Table 1.2). Inbred female/ inbred male pairs averaged the fewest days to first reproduction while non-inbred pairs, non-inbred female/ inbred male pairs, and inbred female/ non-inbred male pairs took longer to produce pups. Overall, 78% of the pairs produced litters, with 92% of the inbred/ inbred pairs, 81% of the non-inbred female/ inbred male pairs, 73% of the non-inbred/ non-inbred pairs, and 66% of the inbred female/ non- inbred male pairs producing litters. Of the pairs that produced litters, the number of pups born ranged from 1 to 6 for non-inbred/ non-inbred pairs, 2 to 5 for inbred/ inbred pairs, 1 to 6 for inbred female/ non-inbred male pairs, and 2 to 6 for non-inbred female/ inbred male pairs (n = 60 pairs). The range of the number of pups weaned was the same for each group. Only two pairs produced litters in which none of the pups survived to weaning (non-inbred female/ inbred male: all 3 pups died; inbred female/ non-inbred male: all 4 pups died). The mean number of pups born in each litter (F = 0.70, df = 3,56, p = 0.56) and the size of the litter at weaning (F = 0.83, df = 3,56, p = 0.49) were not significantly different between treatments (Table 1.2). The mean offspring weight at weaning was significantly different between the non-inbred/ non-inbred pair and the non-inbred female/ inbred male pair (F = 3.06, df = 3, 149, p = 0.03; Table 1.2). All other comparisons of offspring weight at weaning were not significantly different from each other.

DISCUSSION Inbreeding depression has been well established in captive studies. Reproduction between full siblings results in the greatest loss of heterozygosity and should be the most severe form of inbreeding in out-crossing species (Mitton 1994). When testing the negative effects of inbreeding, many studies use an inbreeding coefficient of F = 0.25, or full-sibling breeding, and find significant results (Jimenez et al. 1994; Crnokrak and Roff 1999; Armbruster et al. 2000; Meagher et al. 2000). In general, a 15-25% reduction in fitness is expected among offspring from mating between first-generation relatives (Keller and Waller 2002). If genetic factors associated with inbreeding influenced the fitness of offspring, then I would have expected that litters of full-sibling pairs would weigh less or have fewer offspring compared to unrelated pairs. However, I did not find any evidence of inbreeding depression at F = 0.25 in this study. There was no reproductive difference between inbred and non-inbred pairs in the days from pairing to birth of the first litter, litter size at birth, litter size at weaning, or offspring weight at weaning. Similarly, a study on the captive breeding programs of Red (Canis rufus) and Mexican (Canis lupus baileyi) wolves did not find an effect of inbreeding on juvenile survival or litter size (Kalinowski et al. 1999). However, this study only looked at two aspects of fitness (juvenile survival and litter size) and litter size actually declined over the years. One possible explanation for my results is that the animals in my experiment experienced a bottleneck when brought into captivity and bred for several generations. After several generations, even the non-inbred individuals possibly had inbreeding coefficients greater than zero (more related than non-relatives). The bottleneck hypothesis is supported by comparing my litter size results with other studies on prairie vole litter size. A study comparing first time mothers with experienced mothers reported prairie vole mean litter size at birth to be 4 pups for primiparous litters and 5.2 pups for multiparous litters (Wang and Novak 1994). In my study, mean litter size at birth was 2 pups for non-sibling pairs and 2.3 pups for sibling pairs. At weaning, my study found litter size was 1.8 pups for both sibling and non-sibling pairs. Wang and Novak (1994) report 2.7 pups as the mean litter size at weaning for primiparous litters. Because inbreeding coefficients could not be calculated for the founding population, the degree of inbreeding prior to this study is unknown. The bottleneck and continued breeding in captivity may have purged deleterious recessive alleles thereby allowing subsequent generations to forgo

the measurable, deleterious effects of further inbreeding with both “inbred” and “non-inbred” litters having similar fitness (Hedrick and Miller 1992; Hedrick and Kalinowski 2000). Another explanation is that the artificial condition of captivity, lacking competition, predation, and harsh weather were too benign to detect a difference between the fitness of inbred and non-inbred offspring. Several authors note that inbreeding depression is more severe under natural conditions (Jimenez et al. 1994; Crnokrak and Roff 1999; Hedrick and Kalinowski 2000; Meagher et al. 2000). Most studies on inbreeding measure fitness with traits such as offspring survival, litter size, and weight. Other traits, however, such as adult survival, mating ability, or competitive ability may be affected by inbreeding but were not measured in my study (Crnokrak and Roff 1999; Meagher et al. 2000). In the second experiment, pairs of males and females: non-inbred female/ non-inbred male, inbred female/ inbred male, inbred female/ non-inbred male, non-inbred female/ inbred male, were not related so I did not expect to see direct genetic evidence of inbreeding depression in the offspring produced. However, the “inbred” members of these pairs were the result of mating between full-sibling pairs, and therefore were more likely to have reduced fitness as a result of loss of heterosis or recessive deleterious alleles than less related individuals. Physiological or behavioral differences among the inbred parents, such as poor parental care, infertility, reduced milk production, or infanticide, were traits that could have resulted in lower offspring survival or growth. However, a significant difference was not found for the any of the measures of reproductive success (birth to first litter, litter size at birth and weaning, and offspring weight) between three categories, non-inbred pairs, inbred pairs, and inbred female/ non-inbred male pairs. Several factors may have contributed to a lack of a significant difference being detected. First, while juvenile survival and weight are typical measures of fitness, other traits such as foraging behavior, offspring defense, or nest building by parents could have been affected by inbreeding but were not detected. Thus, if these differences did exist, they did not result in fewer offspring weaned or lower offspring weight. Second, inbred and non-inbred groups were comprised of individuals from shared ancestry that experienced a bottleneck after being captured four to seven generations prior to this study. The largest mean for litter size at birth was 3.3 pups for the inbred-inbred pair. This was still smaller than the mean for other primiparous litters at birth of 4 pups (Wang and Novak

1994). My study found the highest mean litter size at weaning was 3.1 for inbred female/ inbred male pairs. This was actually higher than the mean reported for primiparous litters at weaning by Wang and Novak (2.7 pups; 1994). However, in my study the smallest mean litter size at weaning was 2.1 pups, for the inbred female/ non-inbred male pair, which is less than what Wang and Novak report. Therefore, in my study, perhaps even the “non-inbred” individuals were related enough to result in similar fitness to the more “inbred” individuals. The conditions of captivity, which lack natural stressors such as disease, food shortage, and parasites, were possibly too benign to detect a difference between the reproductive success of inbred and non-inbred animals. In the wild, prairie voles are territorial and defend a home range as male-female pairs, communal groups, or single individuals (Getz and Carter 1996) in order to defend offspring from wandering voles or other male residents (Hodges et al. 2002). Because my study of solitary male-female pairs prevented the measurement of aggressive interactions with other adults during offspring protection, I could not detect if inbred parents could be poorer at territory and offspring defense. A study of another rodent species, house mice (Mus domesticus), showed that inbred males had lower reproductive success than non-inbred males because they were poorer competitors for territories (Meagher et al. 2000). Therefore, the measure of reproductive success in captivity may be abnormally high for inbred animals if they are poor at winning aggressive interactions. A captive prairie vole study, similar to my experiment, tested inbreeding depression between non-inbred/ siblings, non-inbred/ non-siblings, inbred/ non-siblings, and inbred/ siblings (Bixler and Tang-Martinez 2006). In this study, the non-inbred/ sibling pairs and the non-inbred/ sibling pairs were the same as my Experiment One. The non-inbred/ non-siblings and inbred/ non-siblings resembled my Experiment Two non-inbred/ non-inbred, inbred/ inbred pairings. Bixler and Tang-Martinez (2006) reported an effect of inbreeding on the likelihood of reproducing, number of litters, number of young produced, inter-birth interval, offspring weight at birth and weaning, and growth rates. Further, the authors found that the parents’ level of inbreeding (inbred or non-inbred) and parents’ level of relatedness (siblings or non-siblings) did not affect latency to birth of the first litter (Bixler and Tang-Martinez 2006). This finding is similar to both my results in the first and second experiments in which I did not find a difference in pairs’ time to first reproduction. Because distinctions in parental behavior were not found, Bixler and Tang-Martinez (2006) attributed the results to physiological differences between

inbred and non-inbred animals. They alternatively stated that a behavioral difference was not detected due to the small sample size (Bixler and Tang-Martinez 2006). Direct behavioral differences were also not measured in my study but may have an effect in a natural environment when animals are not confined to a small space or when conditions are harsher. Additionally, the Bixler and Tang-Martinez study used animals taken directly from the wild, which may explain why they found an effect while I did not. Again, the bottleneck and successive generations in captivity may explain the similar fitness for “inbred” and “non-inbred” voles in my study. One unexpected result was a significant difference in offspring weight at weaning between non-inbred female/ non-inbred male pairs and non-inbred female/ inbred male pairs. Offspring with two non-inbred parents were significantly heavier than offspring with a non- inbred mother and an inbred father (see Table 1.2). Average litter size was the same for both groups so the differences did not arise from number of offspring produced. One explanation for this result is suggested by limited studies on the effect of inbreeding on sex (Robert et al. 2005). One such study that examined inbreeding in two subspecies of monogamous Oldfield mice (Peromyscus polionotus) found paternal behavior of inbred males was significantly reduced in terms of nest-building and time in contact with young; although this did not affect offspring survival (Margulis 1998). However, inbreeding did not negatively affect female parental care (Margulis 1998). Since the monogamous prairie vole males also share in parental care, males could influence offspring weight by inadequately building a nest or neglecting to huddle with young, causing the offspring to spend more energy on thermoregulation. If a “poor-inbred- father” existed, I would have expected to see an effect in the inbred female/ inbred male pair as well. This, however, was not observed. Interestingly, the study using the Oldfield mouse (Peromyscus polionotus), found inbred females showed higher levels of parental care than non- inbred females while inbred males showed lower levels of parental care compared to non-inbred males (Margulis 1998). A modeling study on inbreeding suggested that Margulis’s results may be due to the genetically reduced parental ability in one individual being compensated by the other parent (Robert et al. 2005). Robert et al. (2005) went on to state that differences in parental behavior may be due to deleterious mutations at sex-linked loci. Lighter offspring from non-inbred female/ inbred male pairs can be explained if inbred males were inadequate fathers but inbred females were superior mothers. However, the inbred female/ non-inbred male pair did not produce more or heavier offspring as would be expected if

non-inbred males provided adequate care while inbred females produced superior care. This should be further examined to determine if there is an actual sex-biased link between parental care and inbreeding in prairie voles. Overall, it appears that in a socially monogamous species, one generation of sibling mating does not result in lower reproductive success in captivity as a result of adult parental behavior or physiological defects.

Chapter 2 INTRODUCTION

Research on inbreeding depression is primarily based on studying captive populations (Lacy et al. 1993). Because natural conditions can be harsher than laboratory settings, it is expected that the effects of inbreeding depression could be more severe under natural conditions. As a result of natural condition stressors, including predation, competition, and resource limitation, presumably genetically inferior inbred individuals may be less apt to survive and reproduce (Meager et al. 2000; Keller and Waller 2002). Weather, disease, and parasites may also affect the reproduction or survival of inbred individuals (Keller and Waller 2002). A review of data on mammal populations by Crnokrak and Roff (1999) found that inbreeding depression was greater under natural conditions compared to captive conditions. Only a handful of studies examining inbreeding depression look beyond juvenile mortality to the entire lifetime of an organism, as is necessary to determine the detrimental effects of inbreeding (Shields 1993). The limited examination of traits over time may over- or under-represent the significance of inbreeding (Shields 1993). For example, inbred offspring could have lower survival rates but be more “vigorous or fertile” than non-inbred offspring resulting in a greater lifetime reproductive success (Shields 1993). Conversely, only looking at juvenile mortality may under represent the harmful effects of inbreeding if inbred animals are poorer competitors, mates, or parents later in life. To determine the extent that inbreeding is detrimental, several components of fitness need to be examined under natural conditions to better estimate the lifetime reproductive success of both inbred and non-inbred individuals (Shields 1993). Studies of lifetime survival and reproduction under natural or semi-natural conditions find that inbred adult animals have lower reproduction and survival rates (Meagher et al. 2000;

Slate et al. 2000; Jimenez et al. 1994). Meagher et al. (2000) found that inbred wild house mice (Mus domesticus), living in predator-proof barns, exhibited decreased survival and reproduction relative to non-inbred mice. This study also found male-male competition accentuated the detrimental effect of inbreeding, resulting in inbred males producing one-fifth as many offspring as non-inbred males (Meagher et al. 2000). In red deer (Cervus elaphus), lifetime reproductive success was greater in non-inbred deer compared to relatively more inbred deer (Slate et al. 2000). Keller et al. (1994) detected inbred song sparrows (Melospiza melodia) were selected against in natural population crashes due to inclement winter weather. Jimenez et al. (1994) found that inbred white-footed mice (Peromyscus leucopus) had lower survival than non-inbred mice simultaneously released loose inside a zoo. Few studies examine inbreeding depression under natural conditions, and still fewer compare the effects of inbreeding in captive and natural conditions within a species (Crnokrak and Roff 1999). These comparisons are vital to determine the differences between artificial versus natural conditions so that the results may be extrapolated to other species in which such experiments are not feasible (e.g., endangered species). Gundersen et al. (2001) tested inbreeding depression in a field population-growth experiment using a vole species (root vole- Microtus oeconomus) that was previously tested in captivity. In the field experiment, inbreeding did not affect rates of population growth (Gundersen et al. 2001). Previous experiments, however, showed lower individual reproductive rates for inbred voles in captivity (Gundersen et al. 2001). It is important to note that the sample size in Gundersen’s study was low and competition between inbred/ non-inbred animals did not occur (Gundersen et al. 2001). A captive and field comparison study on the replacement rates of a tree-hole-breeding mosquito (Aedes geniculatus) showed that natural conditions resulted in overall lower fitness for both inbred and non-inbred mosquitoes (Armbruster et al. 2000). Fitness was similarly reduced in captivity and natural conditions when inbreeding levels increased, but inbreeding depression was not more severe in the field than in captivity (Armbruster et al. 2000). Because inbreeding depression studies usually examine only juvenile survival rather than total lifetime fitness (Shields 1993), the aim of my study was to compare how competitive interactions in semi-natural conditions between un-related inbred and non-inbred individuals affected survival and reproduction. The prairie vole (Microtus ochrogaster), a socially monogamous rodent with bi-parental care, was used as a model organism because they are

highly trappable (Cochran and Solomon 2000) and short lived (Getz et al. 1997). In addition, there is an existing technique to establish parentage with microsatellite markers in voles (Getz et al. 1997; Solomon et al. 2004). Prairie voles were released into natural enclosures so that survival and reproductive success could be monitored when competition between and among both sexes of un-related, inbred and non-inbred animals could occur. Even though half of the individuals placed together were inbred, none of the animals were related. Thus, the direct genetic effects of inbreeding depression (deleterious recessive alleles or loss of heterozygosity) were not expected to appear in their offspring. Instead, reproductive success may vary between inbred and non-inbred (more or less heterozygous) adults due to different behavioral qualities (e.g., parental care, competitive ability, or ability to acquire resources) or physiological differences (e.g., gamete viability). First, I wanted to determine if non-inbred animals would have higher reproductive success and survival than the inbred animals. I would have expected to see the non-inbred animals produce more offspring and survive longer than the inbred animals because the harsher conditions may accentuate difference in parental care, competition, or predator avoidance between the two groups. The second objective of this study was to determine if the costs of inbreeding differed under more stressful natural conditions compared to the artificial environment of captivity. I would have expected to see greater survival and reproductive differences between inbred and non-inbred animals in the enclosures than in captivity because of the harsher environmental conditions in the enclosures.

METHODS Experiments took place June-November 2003 at the Miami University Ecology Research Center in Butler County, Ohio (39° 30′N, 84° 44′W). Founding individuals used in this project originated from a prairie vole breeding colony maintained at Miami University. Colony animals were derived from founders originally caught in southern Illinois during summer 2000 and were in captivity for four to seven generations. Attempts were made to limit inbreeding in this stock with the periodic addition of wild caught animals, however, inbreeding coefficients could not accurately be determined for this population. To derive the animals for this study, pups were separated at weaning (21 days of age) and housed with same-sex siblings for a period of time no less than 8 days to overcome inbreeding avoidance (Gavish et. al. 1994). Animals between 35-45 days were randomly assigned to

pairings of either full-siblings (inbred; n = 16) or un-related individuals (non-inbred; n = 18). Animals were selected to limit relatedness between family lines to at least four generations. Pups produced from these pairs were randomly assigned to the enclosures described below. The study area was an old field with vegetation dominated by perennial grasses and forbs, and consisted of four 0.1 ha small mammal enclosures. Each 0.1 ha (30m x 30m) enclosure was delineated by a galvanized steel fence. The sheeting stood 0.75-m above ground and 0.45-m below ground to restrict movements of rodents in either direction. A 1-m strip was mowed on the inside perimeter of each enclosure to discourage digging by prairie voles. A line of electric fencing surrounded the perimeter of the set of 4 enclosures to exclude most mammalian predators (primarily raccoons). Avian and reptilian predators were not actively excluded. Live trapping for removal of all Microtus species living in the enclosures took place one week prior to release of the study animals. Animals were released into the four semi-natural enclosures after reaching sexual maturity (35-40 days). Four females (2 inbred, 2 non-inbred) and four males (2 inbred, 2 non- inbred) were released into each enclosure; for a total of 32 founders. Individuals sharing enclosures were not closely related. That is, while 4 of the 8 animals in each enclosure were inbred, none of the 8 were closely related (i.e., shared a common relative within 4 generations). This allowed for the examination of the effects of inbreeding on survival and reproduction, but negated any possible effect of kinship. Densities in this experiment were representative of low to moderate population densities found in Illinois (Getz et al. 1987). Prior to release, all animals were uniquely marked with Passive Intergraded Transponders (“PIT tags;” AVID Microchip ID System, Inc., Folsom, LA). Voles were anesthetized with Isoflurane during subcutaneous injection of PIT tags posterior to the cranium for individual identification in the field (Fagerstone and Johns 1987). These microchips are a minimally invasive method used to permanently mark individuals for identification in multiple recapture studies (Schooley 1993). PIT tags were checked before and after insertion to assure they worked properly. PIT tags were read in the field with a portable tag reader (AVID Microchip ID System, Inc., Folsom, LA). An ear clip from each individual was taken while the animal was anesthetized as a source of DNA for later microsatellite analysis of parentage. Live trapping was started two weeks after release to monitor individual survival and check for offspring produced in the field. Ugglan multiple-capture live traps were baited with

cracked corn, a low quality food (Desy and Batzli 1989), and placed in a 5x5-m grid pattern in each enclosure. This method has yielded trapping success of greater than 80% of the population in prior experiments (Cochran and Solomon 2000). Live traps were set between 1800-2000h three times a week and then checked the following morning at 0700-0900h. The site of capture, body mass, sex, PIT tag number, and reproductive condition were recorded for each individual. Females were powdered with UV reflective power and released so that a trail could be followed to locate burrows (Lemen and Freemam 1985). Four additional traps were set around burrow entrances to increase the chance of catching offspring. These traps were set and checked on the same schedule as the grid traps (see above). Offspring weighing 13g or greater were marked with a unique PIT tag number, ear punched (for later DNA extraction), and weighed before release in the area in which they were caught. Offspring weighing less than 13g were not PIT tagged due to the inability to safely anesthetize and insert tags in the field. Untagged offspring were sampled for DNA, weighed, and released. PIT tag number (if assigned), area of capture, date, and body mass were recorded on each sample vial. Because the average prairie vole lifespan in one study was 66 days in the wild (Getz et al. 1997), live trapping continued for three months to attempt to collect entire lifetime history data of founding individuals. All founders and offspring were trapped out of the enclosures at the end of the experiment.

Genetic Analysis Tissue collected from ear punches was frozen at -70 ºC in eppendorff tubes for parental analysis. DNA was extracted by digesting tissue with 20µl proteinase K and 180 µl lysis buffer in a 65°C water bath overnight. This was followed by rinsing with phenol and chloroform washes following procedures in Keane et al. (1994). Samples were rinsed twice with 70 % Ethanol before being dissolved in 50 µl water. Primers for five microsatellite loci (AV13, MSCRB-6, MSMM-5, MSMM-6, and MOE 2) were used for parentage analysis because of previous success in amplifying prairie vole DNA (Keane et al. 2007; Table 2.1). Primers were selected based on degree of polymorphism and reliability of scoring. DNA was amplified using polyermase chain reaction for each locus in 15 µl reactions with 50 ng/ µl DNA, 10mM Tris-HCL, 0.2 mM dNTP’s, 1.0 mM MgCl2, 0.67 µM fluorescent- labeled primers (forward and reverse), and 0.5 U Taq DNA polymerase. Only the forward primer was fluorescently labeled with either the HEX, 6-FAM, or NED tag. After initial

denaturation at 95 °C for 3 minutes, DNA was amplified by cycling 35 times under the following conditions (following methods in Solomon et al. 2004): denature 90°C, 30 seconds; anneal 48- 62°C (depending on primer, Table 2.1), 20 seconds, extend 72°C, 20 seconds, final elongation 5 minutes. Amplified products were combined with the internal standard Rox (35-500 bp) and analyzed with an ABI 3100 (Applied Biosystems, Foster City, CA). Output was then evaluated with Genescan and Genotyper software to determine fragment size (Applied Biosystems, Foster City, CA). Genotyper defaults were used to assign alleles and molecular weights.

Parentage Analysis Parentage was determined for offspring using the genetic data with the program CERVUS 2.0 (Marshall et al. 1998). CERVUS calculates a likelihood ratio score for each candidate parent to identify the male and female that are most likely to be the true parents of a particular offspring. The statistical confidence of these parentage assignments is calculated using a simulation that takes into account population allele frequencies, an estimate of genotyping error, proportion of missing genotypes, total number of candidate parents sampled, and the proportion of candidate parents sampled (Marshall et al. 1998). Each enclosure was treated as a different population. A separate simulation was run to assess the level of statistical confidence in parentage assignments within each enclosure. All simulations were performed for 10,000 cycles with a 0.005 genotyping error rate. The 0.005 error rate used for each simulation was previously determined from the transmission of alleles between mother and in utero offspring (Solomon et al. 2004). The remaining input parameters for the each simulation were based on the actual data from the enclosures. Parentage of only the first litter born to females in the enclosures was examined so that only the founding male voles would be potential sires. Separate maternity and paternity analyses were conducted assuming neither parent of an offspring was known. Maternity or paternity was accepted for a particular offspring if the confidence level was at least 80%. I also performed two-parent analyses assuming an unknown mother and an unknown father. This analysis was done twice, once selecting a mother first and then a father first. These analyses allowed me to assign both parents for an offspring and once again parentage was accepted if the confidence level was at least 80% for the assignment of both parents.

To be conservative, the analysis for enclosure 4 contained males from both enclosures 4 and 3 due to holes under the fencing separating the enclosures. These holes were sealed upon discovery but it was not determined if animals traversed the enclosures. Therefore, the analysis in determining parentage for enclosure 4 was weaker because it contained 8 possible fathers instead of 4. Enclosure 3 had to be entirely replaced by enclosure 5 due to human error in trapping.

Statistical Analysis Survival I equated survivorship with the minimum number known alive (“MNKA”) each week until the conclusion of the experiment at week 12. MNKA was determined by the date of the last capture for each individual. For example, if a vole was captured in week 6 but not again until week 9, it was still recorded as being present in the experiment during weeks 7 and 8. Based on the capture data, a few voles avoided capture some weeks but were still present in the enclosures. Therefore, a confident distinction between mortality and trap-shyness could not be made unless carcasses were found. Any animals not trapped by the conclusion of the experiment were considered deceased. Survival was analyzed as 1) combining weeks 11 and 12 as the end of the experiment and 2) treating only week 12 as the end. Survival was analyzed each way because sometimes animals were not captured each week but were more often captured in a two week period. The minimum number, of inbred or non-inbred voles, know alive were compared using a chi-square analysis. MNKA was also analyzed according to sex with a chi-square test. All chi-square analyses were completed using GraphPad (GraphPad Software, Inc. 2002-2005). MNKA of the level of breeding and sex was analyzed with a Fisher’s Exact test because the expected values were less than five. The Fisher’s exact test was calculated using an on-line statistical analysis program (http://www.matforsk.no/ola/fisher.htm#INTRO).

Reproduction Following the parental assignments, the number of inbred versus non-inbred mothers and fathers over the 80% confidence level was analyzed with a chi-square test. All offspring for

which two parents could be assigned at the 80% confidence level were grouped according to parentage (I/I, N/N, If/Nm, Nf/Im) and were used in the analysis.

RESULTS

Survival During the final week of trapping 6 non-inbred and 8 inbred founders were captured (Figure 2.1). The minimum number of inbred versus non-inbred voles known alive during week 12 was not significantly different (χ² = 0.29; df = 1; p = 0.59; Table 2.2). In the final two weeks of the experiment, 10 non-inbred and 9 inbred animals were trapped (Figure 2.1). Survival between inbred and non-inbred animals was not significantly different (χ² = 0.053; df = 1; p = 0.82; Table 2.3). Nine females and 5 males were trapped during week 12 (Table 2.2) but this difference was not significant (χ² = 1.143; df = 1; p = 0.29; Table 2.2). There was also not a significant difference in survival between males and females during weeks 11 and 12 (χ² = 0.47; df = 1; p = 0.49; Table 2.3). Six inbred females, 3 non-inbred females, 2 inbred males, and 3 non-inbred males (Figure 2.3 and 2.4) were captured during the final week of trapping. No significant difference was found when analyzing inbred versus non-inbred voles by sex at week 12 (p = 0.58; Table 2.2). Six inbred females, 5 non-inbred females, 3 inbred males, and 5 non-inbred males (Figure 2.3 and 2.4) were caught during the last two weeks of the experiment. Weeks 11 and 12 were not significant when examined by sex and breeding type (p = 0.65; Table 2.3).

Reproductive Success Of 44 juveniles genotyped, 38 (86%) could be assigned a mother and 35 (80%) assigned a father at the 80% confidence level. When analyzing only the reproductive success of females, inbred individuals produced 20 offspring while non-inbred animals produced 18. There was not a significant difference (χ² = 0.105, df = 1, p = 0.75) between inbred or non-inbred females. When male reproductive success was examined separately, non-inbred animals produced 21 offspring while inbred animals produced 14 pups. This result was also not significant (χ² = 1.40, df = 1, p = 0.12).

Two parents could be assigned to 25 (57%) offspring at the 80% confidence level. Non- inbred/ non-inbred pairs produced 10 offspring while inbred/ inbred pairs produced 6, inbred female/ non-inbred male pairs produced 7, and non-inbred female/ inbred male pairs produced 2 offspring. There was not a significant difference in number of offspring produced among treatments (χ² = 2.49, df = 1, p = 0.11). For offspring in which two parents could be assigned, non-inbred males produced 17 offspring while inbred males produced 8. Again, this result is not significantly different but there is a tendency towards non-inbred males fathering more offspring than inbred males (χ² = 3.24, df = 1, p = 0.07). Non-inbred females produced 12 offspring while inbred females produced 13 offspring; there was no statistically significant difference between treatments (χ² = 0.04, df = 1, p = 0.84).

DISCUSSION Many captive studies have established the negative consequences of inbreeding, but the magnitude of inbreeding depression under natural conditions is less clear (Lande 1988; Saccheri et al.1998; Keller et al. 2002). Inbreeding was not found to have an effect on survival or reproduction under the semi-natural conditions of my study. Because founding animals were not related, the direct genetic effects of inbreeding were not expected in the offspring they produced. Instead, the physiological and behavioral characteristics of inbred parents were factors that could have led to reduced adult fitness. Behavioral effects were not directly measured in this study and the lab experiment (Chapter One) did not yield any results that would implicate significant physiological effects (e.g. gamete inviability) present under the controlled conditions of captivity. Prairie voles are a socially monogamous species in which bi-parental care for the young is observed (Getz and Carter 1996). Offspring could be negatively affected if inbred parents were poor nest builders, infrequently huddled with young, or did not retrieve wandering pups. One study on two subspecies of Oldfield mice (Peromyscus polionotus) found that male parental behavior, recorded in time spent huddling and nest building, was negatively affected by inbreeding (Margulis 1998). In addition, establishing a good territory is vital to prairie voles when acquiring resources or protecting young from infanticide (Hodges et al. 2002). In house mice (Mus domesticus), under semi-natural conditions, inbred males produced one-fifth as many offspring as non-inbred males, partly because they struggled to secure and defend territories

(Meagher et al. 2000). Jimenez et al. (1994) found that the survival of inbred white-footed mice (Peromyscus leucopus noveboracensis) was lower than non-inbred mice when released into a zoo habitat. Both inbred and non-inbred mice started with similar body mass at the time of release but only inbred male mice lost weight during the experiment (Jimenez et al. 1994). Therefore, inbreeding may compromise the competitive and aggressive interactions necessary to defend a specific territory. If inbred voles did not defend territory as well as non-inbred voles, offspring with an inbred father may be more susceptible to infanticide by wandering voles. As a result, inbred males would have lower reproductive success. While this study did not find a significant difference of the number of offspring produced between inbred and non-inbred males, a trend towards inbred males producing fewer offspring was observed. Non-inbred/ non-inbred pairs produced 10 pups while non-inbred females/ inbred male pairs produced 2 pups. Even though this is not a significant result, when paired with the results from Chapter One (non-inbred female/ inbred male pairs produced significantly lighter pups at weaning compared to non- inbred/ non-inbred pairs) it supports the hypothesis that inbred males are poor at parental care, specifically, or reproduction, in general. Inbreeding depression may not have been detected in the field because of suitable, if not ideal, environmental conditions. A study of an island population of finches (Geospiza fortis and Geospiza scandens) showed that the degree of inbreeding depression was closely related to environmental conditions (Keller et al. 2002). In these finches, inbreeding affected juvenile survival only when conditions were poor (low food; Keller et al. 2002). Although not the case for every species (see Keller and Waller 2002), Keller et al. (2002) found the negative effects of inbreeding on adult survival in an environment with less food and more competition. Another study on birds recorded a population crash with a significant loss of more inbred song sparrows (Melospiza melodia) when environmental conditions were severe (Keller et al. 1994). In my field experiment, corn, a low quality food, was available in baited traps every other day. Mammalian predation, primarily raccoon (Procyon lotor), was actively discouraged with electric fencing surrounding the enclosures. However, other predators, such as birds, snakes, and shrews, were not removed or specifically excluded from the enclosures. No significant weather disturbances were of note and the climate in southwestern Ohio is relatively mild in late summer to early autumn. Competition with other rodent species was minimal since all Microtus species

were trapped and excluded before the commencement of the experiment. Prairie vole densities were low to moderate relative to natural densities found in other studies (Getz et al. 1987; Getz and Carter 1996). Mate acquisition (a balanced sex ratio existed) and territory competition (low competition for space) appear to be the remaining stressors which could have affected released prairie voles in my study. Taking all of these factors into consideration, inbreeding may not have influenced reproductive success and survival given the low the environmental stress experienced by founding voles. The low number of alleles and heterozygosity in my study suggests a bottleneck occurred when the animals were brought into captivity and bred for several generations. For example, a study on prairie voles that used three of the same loci found AV 13 had 21 alleles, Moe-2 had 12, and MSMM-6 had 6 (Ophir et al. 2008 in press); whereas in my study AV 13 had 13, Moe-2 had 5, and MSMM-6 had 3 alleles. The Ophir et al. (2008 in press) study using, AV 13, Moe-2, and MSMM-6, had levels of heterozygosity at 0.83, 0.75, and 0.37 respectively, while my study with the same loci had heterozygosity levels at 0.68, 0.31, and 0.56 respectively. These comparisons suggest that bringing the voles into captivity, followed by successive generation of breeding led to decreased heterozygosity and loss of alleles. Thus, the captive population was to some extent inbreed. Further, deleterious alleles may have already been purged thereby allowing subsequent generations to forgo the possible harmful effects of further inbreeding (Hedrick and Miller 1992; Hedrick and Kalinowski 2000). Therefore, the effects of one full-sibling mating on top of the bottleneck may not have been detrimental enough to influence the characteristics I directly measured in the field (survival) or the characteristics measured indirectly through offspring production, such as parental care, mate acquisition, fertility, or territory defense. Life expectancy of prairie voles ranged from 34.3 to 76.0 days in a study by Getz et al. (1997). Nonetheless, the 84 day length of my experiment may not have been long enough to fully determine adult survival because some released animals were still alive during the final trapping week. A more complete picture of survival and reproduction would be produced if several cohorts were followed for a longer period and the date of death of each animal could easily be determined. Even though there was not a significant difference in male reproductive success, there was a trend towards non-inbred males fathering more offspring than inbred males. Because there was no significant difference among survival of inbred and non-inbred males, perhaps females

were choosing non-inbred, more heterozygous, mates. One study modeled the evolution of female choice under inbreeding conditions and found that females who chose non-inbred males, complementary males, or males with fewer detrimental recessive mutations could decrease inbreeding depression in their offspring (Reinhold 2002). Reinhold (2002) stated that each possible strategy for female choice results in the avoidance of inbred males as mates. The complementary choice hypothesis suggests that a female will select a male that has a complementary genotype to their own, resulting in offspring with increased heterozygosity (Tregenza and Wedell 2000). One review suggested a heterozygous advantage for male mating in butterflies (Colias eurytheme and Colias philodice eriphyle) (Mitton 1993). Heterozygous males represented only 46% of the population while they accounted for 69% of the matings (Mitton 1993). Because inbreeding may decrease the condition of males through decreased heterozygosity and the unmasking of recessive alleles, females may determine which males were inbred and avoid mating with these sub par males (Frankham et al. 2002). Although behavior was not measured in my study, inbred males may have behaved differently when searching, soliciting, or retaining mates which may explain the trend towards non-inbred males producing more offspring. To my knowledge, there are no published studies testing female choice in mammals of un-related, inbred or non-inbred males to support this hypothesis. As discussed previously, a study by Meagher et al. (2000) found that inbred male mice were poorer competitors for territory which contributed to inbred males producing fewer offspring than non- inbred males. Thus, another possible explanation for the trend of inbred males having lower reproductive success is that these males poorly defend territories. Evidence suggests inbreeding may decrease survival and reproduction for the individual but conservation biologists are divided on the magnitude inbreeding may have on population- and species-level extinction. As individuals lose heterozygosity through inbreeding populations may experience a loss of genetic diversity. Because of the loss of genetic diversity, populations may lose their ability to adapt to changing environmental conditions (Wilson 1975; Keller et al. 1994). Saccheri et al. (1998) found that inbreeding among a meta-population of the Glanville fritillary butterfly (Melitaea cinxia) contributed to an increased extinction risk. These findings were further supported with experimental evidence in a study by Nieminen et al. (2001) on the same species. However, an alternative hypothesis suggests that demographic or environmental

stochasticity will cause extinction of small populations before genetic factors reach an extinction threshold (Lande 1988). As in many experiments with mammals, the sample size of four replicate enclosures, with 32 individuals, may have been too low to be able to detect a significant difference between groups. For example, when analyzing the reproductive success of both parents, a trend emerged in which non-inbred males produced twice as many offspring as inbred males. With a larger sample size, the differences in male reproductive success between inbred and non-inbred animals may become statistically significant. In addition to a larger sample size, components of fitness other than the number of offspring produced should be examined to determine if inbreeding depression is in fact not present (Hedrick and Kalinowski 2000). Additional traits associated with the negative effects of parental inbreeding on offspring, such as parental care, foraging behavior, or predator avoidance could also be tested. In conclusion, these results suggest that inbred individuals do not appear to be negatively effected, even under competition, in semi-natural conditions by one generation of consanguineous mating. Furthermore, having full-sibling parents does not appear detrimental to an individual’s survival or success in producing offspring under good environmental conditions. These results suggest that animals may not suffer adverse effects one generation of close inbreeding after experiencing a bottleneck by being brought into captivity and breed for several generations. This is promising for captive breeding programs that have little choice but to breed somewhat related individuals for the preservation of endangered populations. Although inbreeding depression was not found in either the captive or the field components of this study, the voles did have lower levels of heterozygosity at some loci than other studies on prairie voles. Thus, caution should be taken in the interpretation of these results since evidence from other taxa suggests that severe inbreeding should be guarded against, particularly in rare species (Frankham and Ralls 1998) where stochastic events could lead to extinction.

Table 1.1— Mean (±SE) days from pairing to birth of the first litter, litter size at birth, litter size at weaning, and offspring weight at weaning for sibling and non-sibling pairs. T- tests were used to compare each measure of reproductive success. Days to first Litter size at Litter size at Offspring weight (g) at N litter birth weaning weaning Non- 37.8 2.1 1.8 20.2 Siblings 18 (± 3.9) (± 0.4) (± 0.4) (± 0.8)

33.7 2.3 1.8 19.3 Siblings 16 (± 3.7) (± 0.4) (± 0.4) (± 0.6)

P = 0.46 P = 0.74 P = 0.96 P = 0.42

Table 1.2— Mean (±SE) days from pairing to first litter, litter size at birth, litter size at weaning, and offspring weight at weaning for non-inbred (N/N) pairs, inbred (I/I) pairs, non-inbred female/ inbred male (Nf/Im) pairs, and inbred female/ non-inbred male (If/Nm) pairs. Analysis of variance (ANOVA) and Bonferroni post hoc tests were used to compare means of each treatment. Means with different superscripts are significantly different (P < 0.05). Days to first Litter size at Litter size at Offspring weight Female Male N litter birth weaning (g) at weaning N N 15 36.5 (± 4.0) 2.6 (± 0.1) 2.5 (± 0.1) 18.79 (± 0.5)ª

I I 14 28.6 (± 2.7) 3.3 (± 0.3) 3.1 (± 0.3) 17.55 (± 0.3)ab

N I 15 34.3 (± 3.8) 2.8 (± 0.4) 2.5 (± 0.5) 17.30 (± 0.3)b

I N 16 36.4 (± 4.2) 2.4 (± 0.5) 2.1 (± 0.5) 17.62 (± 0.5)ab

ANOVA P = 0.43 P = 0.56 P = 0.49 P = 0.03

Table 2.1— Microsatellite primer sets, annealing temperature (Ta), and number of alleles detected.

ο Locus Ta ( C) No. alleles Ho Reference for initial characterization AV13 55 13 0.68 Stewart et al. 1998 Moe 2 60.5-62 5 0.31 Van de Zande et al. 2000 MSCRB-6 47.6-51 4 0.27 Ishibashi et al. 1997 MSMM-5 52 11 0.66 Ishibashi et al. 1999 MSMM-6 52 3 0.56 Ishibashi et al. 1999

Table 2.2 — Minimum number known alive (MNKA) during the final week (12) of the experiment by level of inbreeding, sex, and a combination of level of inbreeding and sex. Minimum number known alive, chi-square, degrees of freedom, and P-values (for chi- square and Fisher’s exact test) are listed. MNKA χ² df p Inbred 8 0.29 1 0.59 Non-inbred 6

Female 9 1.14 1 0.29 Male 5

Inbred Female 6 Non-inbred Female 3 0.58 Inbred Male 2 Non-inbred Male 3

Table 2.3 — Minimum number known alive (MNKA) during the final two weeks (11 and 12) of the experiment broken down by level of inbreeding, sex, and a combination of level of inbreeding and sex. Minimum number known alive, chi-square, degrees of freedom, and P-values (for chi-square and Fisher’s exact test) are listed. MNKA χ² df p Inbred 9 0.05 1 0.82 Non-inbred 10

Female 11 0.47 1 0.49 Male 8

Inbred Female 6 Non-inbred Female 5 0.65 Inbred Male 3 Non-inbred Male 5

8 Number ofVoles

0 123456789101112 Weeks

Figure 2.1 — The minimum number of inbred (■) voles and non-inbred (▲) voles known alive per week, from weeks 1-12.

8 Number of Voles Number

0 123456789101112 Weeks

Figure 2.2 — Minimum number of females (●) and males (▲) known alive per week from week 1 to week 12.

4 Number of Voles of Number

0 123456789101112 Weeks

Figure 2.3 — Minimum number of inbred females (■), inbred males (▲), non-inbred females (X), and non-inbred males (O) known alive per week from week 1 to week 12.

2 Number of Voles

0 Inbred Males Inbred Females Non-inbred Non-inbred Males Females

Figure 2.4 — The minimum number of voles known alive during the final week (gray) and the final two weeks (black) broken down by level of inbreeding and sex.

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