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Title:
Reproductive characteristics, multiple paternity and mating system in a central florida population of the gopher tortoise, Gopherus polyphemus
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Book
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English
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Colson-Moon, Jamie Colleen
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University of South Florida
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Tampa, Fla.
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Subjects / Keywords:
promiscuous mating
polygyny
radiograph
reproduction
microsatellites
Dissertations, Academic -- Biology -- Masters -- USF   ( lcsh )
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government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Summary:
ABSTRACT: I studied the reproductive characteristics and mating systems of a central Florida population of gopher tortoises (Gopherus polyphemus). Using x-radiography, females were monitored for stage in egg-shelling and clutch size. Eggs began to appear on x-ray photographs in the first week of May in both 2001 and 2002; however, fully shelled eggs were not found before the end of May. In total 55% of the females x-rayed were gravid. Clutch sizes ranged from 4-12 with a mean of 7.29, with a mean clutch mass of 40.9 g. Clutch size increased with an increase in mean carapace length and mean plastron length. Mean clutch mass also increased with mean carapace length of females. Hatchlings began to emerge in late August, with incubation times ranging from 83 to 96 days. 50% of the eggs hatched, with 16.2% of the eggs showing no signs of development when opened. Hatchling mass averaged 30.7 g and was positively correlated with egg mass. DNA was extracted from blood samples obtained from females and their offspring, and from the sexually mature males in the population. Nine microsatellite loci were amplified and genotypes constructed for each individual. There is evidence for promiscuous mating in gopher tortoises. Multiple paternity was detected in two of the seven clutches (28.6 %). In the clutches with multiple fathers, fertilization was highly skewed to one male, with primary male fertilizing over 70% of the clutch. Females with multiple-sired clutches were significantly smaller than females with single-sired clutches. Among the clutches assayed only one male fertilized more than one clutch, indicating that insemination of females is evenly spread among males of similar sizes. However, males assigned as fathers were significantly larger than other sampled males which may mean that larger males have an advantage in fertilization of clutches. Conservation efforts should consider the impact of the mating system on reproduction in a population, and the possible impact of the relocation of larger males on recipient populations.
Thesis:
Thesis (M.S.)--University of South Florida, 2003.
Bibliography:
Includes bibliographical references.
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Statement of Responsibility:
by Jamie Colleen Colson-Moon.
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Title from PDF of title page.
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Document formatted into pages; contains 69 pages.

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aleph - 001432579
oclc - 53244331
notis - AJL6127
usfldc doi - E14-SFE0000115
usfldc handle - e14.115
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ABSTRACT: I studied the reproductive characteristics and mating systems of a central Florida population of gopher tortoises (Gopherus polyphemus). Using x-radiography, females were monitored for stage in egg-shelling and clutch size. Eggs began to appear on x-ray photographs in the first week of May in both 2001 and 2002; however, fully shelled eggs were not found before the end of May. In total 55% of the females x-rayed were gravid. Clutch sizes ranged from 4-12 with a mean of 7.29, with a mean clutch mass of 40.9 g. Clutch size increased with an increase in mean carapace length and mean plastron length. Mean clutch mass also increased with mean carapace length of females. Hatchlings began to emerge in late August, with incubation times ranging from 83 to 96 days. 50% of the eggs hatched, with 16.2% of the eggs showing no signs of development when opened. Hatchling mass averaged 30.7 g and was positively correlated with egg mass. DNA was extracted from blood samples obtained from females and their offspring, and from the sexually mature males in the population. Nine microsatellite loci were amplified and genotypes constructed for each individual. There is evidence for promiscuous mating in gopher tortoises. Multiple paternity was detected in two of the seven clutches (28.6 %). In the clutches with multiple fathers, fertilization was highly skewed to one male, with primary male fertilizing over 70% of the clutch. Females with multiple-sired clutches were significantly smaller than females with single-sired clutches. Among the clutches assayed only one male fertilized more than one clutch, indicating that insemination of females is evenly spread among males of similar sizes. However, males assigned as fathers were significantly larger than other sampled males which may mean that larger males have an advantage in fertilization of clutches. Conservation efforts should consider the impact of the mating system on reproduction in a population, and the possible impact of the relocation of larger males on recipient populations.
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Reproductive Characteristics, Multiple Paternity and Mating System in a Central Florida Population of the Gopher Tortoise, Gopherus polyphemus by Jamie Colleen Colson-Moon A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Biology College of Arts and Sciences University of South Florida Co-Major Professor: Henry R. Mushinsky, Ph.D. Co-Major Professor: Earl D. McCoy, Ph.D. Stephen A. Karl, Ph.D. Date of Approval: July 10, 2003 Keywords: microsatellites, reproduction, radiograph, polygyny, promiscuous mating Copyright 2003 Jamie Colson-Moon

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Dedication To Mom and Dad Without you, I would have never been introduced to the beauty of nature. Thanks to you, I learned to ask questions and find my own answers. To Daniel Words can not express the depth of gratitude I feel for all of the support and love you have given me. Thank you.

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Acknowledgments I would like to thank my committee, Henry Mushinsky, Earl McCoy and Stephen Karl for all of the guidance and input they have given me over this project. Special thanks to the Karl Lab, Tonia Schwartz, Anna Bass, Caitlyn Curtis, Ken Hayes and Cecila Puchulutegui for all of their patience in answering my questions about molecular methodology. I gratefully acknowledge the assistance of the Gopher Tortoise Council. This project was supported by the J. Landers Student Research Award provided by the Gopher Tortoise Council.

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Table of Contents List of Tablesii List of Figuresiii Abstract iv General Introduction References5 Chapter One: Reproduction in a Central Florida Population of Gopher Tortoises, Gopherus polyphemus 9 Introduction9 Methods11 Results13 Pre-oviposition Data13 Post-oviposition Data14 Discussion15 References19 Chapter Two: Multiple paternity and mating system in a Central Florida Population of Gopher Tortoises, Gopherus polyphemus 28 Introduction28 Methods31 Results36 Paternal Assignment36 Multiple Paternity38 Mating System and Relatedness40 Discussion41 References44 Appendices58 Appendix A: Genotyping results for 75 gopher tortoises from the USF Ecological Research Area59 i

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List of Tables Table 1.1Reproductive characteristics of the gopher tortoise. 24 Table 1.2Number of radiographs taken of 47 female G. polyphemus taken from 2001-2002, followed by percentage of total radiographs for the year in parentheses.25 Table 1.3Mean largest egg diameters for individual clutches taken from eggs and x-ray photographs in 2002. 26 Table 1.4Summary of linear regressions and Pearson Product Moment correlations of adult and clutch characteristics.27 Table 2.1 Allele frequencies, heterozygosities, probability of identity, and exclusion probabilities for nine microsatellites in Gopherus polyphemus .51 Table 2.2Maternal genotypes inferred paternal genotypes and not excluded male candidates per clutch. 52 Table 2.3.Mother, clutch and father characteristics for single-sired clutches.55 Table 2.4 Relatedness values of mother gopher tortoises and assigned fathers compared to the average relatedness of the mother and other males in the population.57 ii

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List of Figures Figure 1.1Ecological Research Area in Tampa, FL. 56 iii

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Reproductive Characteristics, Multiple Paternity and Mating System in a Central Florida Population of the Gopher Tortoise, Gopherus polyphemus Jamie C. Colson-Moon ABSTRACT I studied the reproductive characteristics and mating systems of a central Florida population of gopher tortoises ( Gopherus polyphemus ). Using x-radiography, females were monitored for stage in egg-shelling and clutch size. Eggs began to appear on x-ray photographs in the first week of May in both 2001 and 2002; however, fully shelled eggs were not found before the end of May. In total 55% of the females x-rayed were gravid. Clutch sizes ranged from 4-12 with a mean of 7.29, with a mean clutch mass of 40.9 g. Clutch size increased with an increase in mean carapace length and mean plastron length. Mean clutch mass also increased with mean carapace length of females. Hatchlings began to emerge in late August, with incubation times ranging from 83 to 96 days. 50% of the eggs hatched, with 16.2% of the eggs showing no signs of development when opened. Hatchling mass averaged 30.7 g and was positively correlated with egg mass. DNA was extracted from blood samples obtained from females and their offspring, and from the sexually mature males in the population. Nine microsatellite loci were amplified and genotypes constructed for each individual. There is evidence for promiscuous mating in gopher tortoises. Multiple paternity was detected in two of the iv

PAGE 8

seven clutches (28.6 %). In the clutches with multiple fathers, fertilization was highly skewed to one male, with primary male fertilizing over 70% of the clutch. Females with multiple-sired clutches were significantly smaller than females with single-sired clutches. Among the clutches assayed only one male fertilized more than one clutch, indicating that insemination of females is evenly spread among males of similar sizes. However, males assigned as fathers were significantly larger than other sampled males which may mean that larger males have an advantage in fertilization of clutches. Conservation efforts should consider the impact of the mating system on reproduction in a population, and the possible impact of the relocation of larger males on recipient populations. v

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1 General Introduction The gopher tortoise ( Gopherus polyphemus (Daudin)) is one of four native North American tortoise species. Its range extends along the southeastern coastal plain from Louisiana to South Carolina (Auffenberg and Franz 1982). It is associated with four main habitats in Florida: longleaf pine-oak uplands, xeric hammocks, sand pine-oak ridges, and ruderal areas (Auffenberg and Franz 1982). Within these habitats, two of the main factors affecting the density of tortoises are openness of canopy and soil type. Generally, the more open canopies with more light reaching the ground will have higher tortoise densities, as will well-drained, sandy soils (Auffenberg and Franz 1982). These well-drained sandy soils are especially important to gopher tortoises as substrates for burrows, which serve as refuges for both the gopher tortoise and a host of other organisms, including the gopher frog, the Florida mouse, and the eastern indigo snake (Diemer 1986 and included references). With the importance of the gopher tortoise’s burrows to so many organisms, any negative impacts on gopher tortoise populations may have far-reaching effects on the communities of which they are a part. Gopher tortoise populations are declining across the range of the species (Auffenberg and Franz 1982). Because populations are declining, the species has gained some form of state or federal protection in many parts of its range (Ernst et al. 1994). In Florida, the gopher tortoise is listed as a species of special concern (Myers 1990). Unfortunately for the gopher tortoise, their main habitats are prime candidates for real-

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2 estate development, and habitat loss has become one of the greatest threats to gopher tortoise numbers (Diemer 1986). Because of the degree of habitat loss in Florida, federal, state and local parks lands harbor much of the remaining gopher tortoise habitat. These lands, such as the study site used here, are relatively free from development, often are managed by prescribed burning, and sometimes may achieve reletively high densities of tortoises. Even populations on protected lands could be in decline, however, the wellbeing of populations must be monitored (McCoy and Mushinsky 1992). It has become crucial to understand the biology of tortoises under these conditions, as in the future, these will be the populations most likely to remain in the face of habitat degradation and loss (Auffenberg and Franz 1982). The gopher tortoise is a long lived species. The age at which individuals reach sexual maturity varies among populations, from 9-21 years (Diemer and Moore 1994). Mating occurs in the spring, and eggs are deposited between May and July with juveniles emerging from August to September (Diemer and Moore 1994, Butler and Hull 1996, Iverson 1980). Eggs, hatchlings, and juveniles face intensive predation. Loss of eggs and juveniles of the gopher tortoise occur from avian, mammalian (raccoons, foxes, and skunks), and ophidian predation (Butler and Sowell 1996, Landers et al. 1980). Because of predation, estimates of mortality of eggs and juveniles range from 41-94% (Diemer 1994). Mature female at sites in Georgia produced a successful clutch once in 9-10 years, because in most years all eggs and hatchlings are lost to predators (Landers et al.1980). Because of the gopher tortoise’s low fecundity, any factor that impacts the reproductive abilities of a population becomes an important component of any

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3 conservation attempt. Although some data on oviposition exist (see above), little is known about the reproductive behavior of gopher tortoises. Some field reports of courtship behavior exist; however, it is not known which males are successful in the insemination of females (Douglass 1976, McCrae et al. 1981). The use of molecular techniques, such as highly polymorphic markers (e.g. microsatellites), allows for the determination of which males are fertilizing clutches in the population. In this thesis, I used molecular techniques to assign fathers to the offspring in clutches and determine whether multiple fathers were present in the clutch. Then by assessing patterns of paternity, I was able to determine the mating system displayed in the study population. One mating system which may be observed in the gopher tortoise is polygyny. In particular, a form of polygyny, the harem system, has been suggested based on observations of tortoise behavior (Douglass 1976). During the spring, incidences of male gopher tortoises' aggressive behavior towards each other have often been noticed (Hailman and Layne 1991). A dominance hierarchy has been described in gopher tortoises, with larger males often proving the victor in aggressive interactions (Douglass 1976; McCrae et al. 1981). If the mating system of gopher tortoises is polygynous, it is possible that these aggressive displays may be a form of harem guarding, with larger males insuring their chance to fertilize females by defeating smaller males in aggressive interactions. It has been suggested that male tortoises may not be able to continuously guard a harem of females, thus allowing for the possibility of other males mating with females courted by the dominant male (McCrae et al 1981). In which case, gopher tortoises may exhibit a promiscuous mating system in which both males and females mate with

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4 multiple partners. In a promiscuous mating system, not only would males be mating with multiple females, females would show multiple paternity of clutches due to mating with multiple males. Many species of turtles have multiple paternity in clutches (review in Pearse and Avise 2001). While females turtles do not gain direct effects, such as food gifts or paternal care of the clutch, they may be acquiring indirect genetic benefits (Pearse and Avise 2001), such as gaining ‘good genes’ (Kempenaers et al. 1992, Otter and Radcliffe 1996, Watson 1998), avoiding genetic incompatibility (Zeh and Zeh 1996; Kempenaers et al. 1999, Tegenza and Wedell 2000), or increasing genetic diversity of offspring (Madsen et al. 1992, Byrne and Roberts 2000) by mating with multiple males. While these studies have been done on non-Testudines, it is possible that female tortoises may receive similar genetic benefits from multiple matings. Testing for multiple paternity is especially important in conservation plans because multiple paternity can increase the effective size of a population over that of a population with single paternity (Sugg and Chesser 1994). Regardless of mating system, dominant male gopher tortoises may fertilize a larger percentage of a population than smaller males, either by guarding and mating with a harem of females or by defeating smaller males in aggressive interactions thereby gaining more opportunities to court females. The movement of dominant males out of or into a population may prove disruptive to the current reproductive individuals and may increase or decrease fertilization opportunities for males. Changes in the numbers or status of dominant males could come from several sources, including relocation during conservation efforts or isolation due to habitat fragmentation. Gopher tortoises are often relocated during conservation efforts (Diemer 1986), but it is unknown if these

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5 movements disrupt the current mating structure of both the relocated and recipient populations. Fragmentation of habitat could also disrupt mating structure, particularly if the fragmentation leads to a loss or excess of dominant males. The mating configuration of the population could be totally restructured if mating opportunities previously utilized by large dominant males become available or lost due to fragmentation. By studying the genetic makeup of the offspring, it is possible to determine paternity of clutches. Paternity identification could be used to determine the mating system of the population, evaluate multiple paternity within clutches, and to discover if large males dominate the fertilization of eggs. Such information would allow for a greater understanding of reproductive behavior, as well as illustrate several conservation concerns, including the impact of relocation and fragmentation on social structure and reproductive behavior and its implications for effective population size evaluations. References Auffenberg, W. and R. Franz. 1982. The status and distribution of the gopher tortoise ( Gopherus polyphemus ). In R.B. Bury (ed.), North American Tortoises: Conservation and Ecology, pp. 95-126. U.S. Department of Interior, Fish and Wildlife Service, Wildlife Research Report 12. Butler, J.A. and T.W. Hull. 1996. Reproduction of the tortoise, Gopherus polyphemus in northeastern Florida. Journal of Herpetology 30:14-18. Butler, J.A. and S. Sowell. 1996. Survivorship and predation of hatchling and yearling gopher tortoises, Gopherus polyphemus Journal of Herpetology 30:455-458.

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6 Byrne, P.G. and J.D. Roberts. 2000. Does multiple paternity improve the fitness of the frog Crinia georgiana ? Evolution 54:968-973. Diemer, J.E. 1986. The ecology and management of the gopher tortoise in the southeastern United States. Herpetologica 42:125-133. Diemer, J.E. and C.T. Moore. 1994. Reproductive biology of gopher tortoises in northcentral Florida. In R.B. Bury and D.J. Germano (eds.), Biology of North American Tortoises, pp. 129-137. U.S. Fish and Wildlife Service, Fish and Wildlife Research Report 13. Douglass, J.F. 1976. The mating system of the gopher tortoise, Gopherus polyphemus in southern Florida. M.S. thesis, University of South Florida, Tampa, FL. 79 pp. Ernst, C.H., R.W. Barbour, and J.E. Lovich. 1994. Turtles of the United States and Canada. Smithsonian Institution Press, Washington, USA. Hailman, J.P. and J.N. Layne. 1991. Notes on aggressive behavior of the gopher tortoise. Herp Review 22:87-88. Iverson, J.B. 1980. The reproductive biology of Gopherus polyphemus (Chelonia:Testudinidae). The American Midland Naturalist 103:353-359. Kempenaers, B., G.R. Verheyen, M. Vandenbroeck, T. Burke, C. Vanbroeckhoven, and A.A. Dhondt. 1992. Extra-pair paternity results from female preference for highquality males in the blue tit. Nature 357:494-496. Kempenaers B., B. Congdon, P. Boag, and R.J. Robertson. 1999. Extra-pair paternity and egg hatchability in tree swallows: Evidence for the genetic compatibility hypothesis? Behavioural Ecology 10:304-311.

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7 Landers, J.L., J.A. Garner, and W.A. McRae. 1980. Reproduction of gopher tortoises ( Gopherus polyphemus ) in southwestern Georgia. Herpetologica 36:353-361. Madsen, T., R. Shine, J. Loman, and T. Hakansson. 1992. Why do female adders copulate so frequently? Nature 355:440-441. McCoy, E.D. and H.R. Mushinsky. 1992. Studying a species in decline: Changes in populations of the gopher tortoise on federal lands in Florida. Florida Scientist 55:116-125. McCrae, W.A., J.L. Landers, and J.A. Garner. 1981. Movement patterns and home range of the gopher tortoise. American Midland Naturalist 106:165-179. Myers, R.L. 1990. Scrub and high pine. In R.L. Myers and J.J. Ewel (eds.), Ecosystems of Florida, pp. 150-193. Univ. Central Florida Press, Orlando, USA. Otter, K. and L. Ratcliffe. 1996. Female initiated divorce in a monogamous songbird: Abandoning mates for males of higher quality. Proceedings of the Royal Society London B 263:351-354. Pearse, D.E. and J.C. Avise. 2001. Turtle mating systems: Behavior, sperm storage, and genetic paternity. Journal of Heredity 92:206-211. Sugg, D.W. and R.K. Chesser. 1994. Effective population sizes with multiple paternity. Genetics 137:1147-1155. Tregenza, T. and N. Wedell. 2000. Genetic compatibility, mate choice and patterns of parentage. Molecular Ecology 9:1013-1027.

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8 Watson, P.J. 1998. Multi-male mating and female choice increase offspring growth in the spider Neriene litigiosa (Linyphiidae). Animal Behaviour 55:387-403. Zeh, J.A. and D.W. Zeh. 1996. The evolution of polyandry I: Intragenomic conflict and genetic incompatibility. Proceedings of the Royal Society London 263:17111717.

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9 Chapter One: Reproduction in a Central Florida Population of Gopher Tortoises, Gopherus polyphemus Introduction Gopher tortoise ( Gopherus polyphemus (Daudin)) population sizes are declining across the range of the species (Auffenberg and Franz 1982), and, as such, have gained some form of state or federal protection throughout the southeastern US (Ernst et al. 1994). In Florida, the gopher tortoise is listed as a state species of special concern (Meyers 1990). Unfortunately for gopher tortoises, their main habitats are prime candidates for real-estate development. Habitat loss has become one of the greatest threats to gopher tortoise numbers (Diemer 1986). Because of the degree of habitat loss in Florida, federal, state and local parks have become major refuges for tortoise populations. These lands, like the study site reported in this paper, are free from development, often managed by prescribed burning, and may also achieve relatively high densities of tortoises. In the future, these will be the populations most likely to remain in the face of habitat degradation and loss (Auffenberg and Franz 1982). Thus, it becomes crucial to understand the biology of tortoises under these conditions when creating a conservation plan. The formulation of any conservation strategy should include knowledge of the biology and ecology of the species of question. One of the most important and obvious areas is reproduction. Gopher tortoises become sexually mature in 9-21 years (Diemer and Moore 1994, Mushinsky et al. 1994). Mating occurs in the spring, with eggs being

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10 deposited between May and July (Diemer and Moore 1994, Butler and Hull 1996). The incubation period for eggs in north Florida is 80-90 days, with the juveniles emerging from August to September (Iverson 1980). Various estimates of mortality among eggs and juveniles range from 41-94% (Diemer 1994). Mature females in Georgia were estimated to produce a successful clutch once in 9-10 years (Landers et al. 1980). Reproductive biology characteristics, such as nesting season, clutch size and egg mass, vary among populations of gopher tortoises (Table 1.1). Diemer and Moore (1994) suggested the creation of a statewide database of reproductive characteristics to compare variation in gopher tortoise reproductive biology. The availability of specific data on the reproductive characteristics of a population allow for the construction of a more complete conservation plan. Conservation plans will be most effective when formulated using the best available data on the population under consideration. Because reproductive characteristics vary among gopher tortoise populations, data should be gathered on the specific reproductive characteristics of as many populations as possible. Of particular interest are the populations found on protected and maintained lands, as these populations are likely to remain in the face of increasing habitat loss. During this study, the reproductive characteristics of a central Florida population of gopher tortoises, located in a protected and fire-maintained area, were examined and compared to findings from other populations. Specifically, I studied the period during which x-ray photography was most effective in determining the presence of eggs, the average clutch and hatchling characteristics in the population, and relationships between mother and offspring characteristics.

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11 Methods The study was conducted at the University of South Florida’s Ecological Research Area (ERA), a 200 ha reserve located in Hillsborough County in west-central Florida (28.05oN, 82.20oW). Approximately 20 ha of sandhill habitat within the ECA have been exposed to controlled burning since 1976 (Mushinsky 1992). The controlled burning area is separated into plots which are burned on frequencies of one year, two years, five years, or seven years, or are left as unburned “controls” (Mushinsky 1985). A thriving population of about 280 tortoises occupies the plots (Mushinsky et al. 1994). All plots were trapped for tortoises during the course of the study.From April to August of 2001 and 2002, all active and inactive burrows (classification based on Mushinsky and McCoy 1994) in each plot were located and marked. The width of each burrow was measured at a depth of 500 mm and used as an estimate of the carapace length (CL) of the resident tortoise (Wilson et al. 1991). Burrows greater in width than the minimum CL of sexually mature females in the population, 240mm (Mushinsky et al. 1994) were trapped. Pit traps, consisting of 9.5 L buckets camouflaged with brown fabric and sand, were placed in the ground with the opening level with the burrow entrance floor. When in place, the traps were checked every two hours during daytime. Individuals were also gathered by hand when encountered in and around the plots. Portable x-ray machines allow a researcher to gather information on whether or not females are carrying eggs and, if so, how many eggs. By using x-ray photography, estimates of reproductive output can be gathered from captive females. However, assessment of reproductive characteristics by x-ray will only be effective when shelled eggs are present in the female. Therefore, knowing the interval between the shelling of

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12 eggs and oviposition for the population is critical. The sex of captured tortoises was determined by measuring the plastral concavity (PC) of the tortoise. Females had a PC of less than 6 mm, and males had a PC of greater than 6 mm in this population (Mushinsky et al. 1994). Females thought to be sexually mature were x-radiographed to determine whether they were gravid. The radiographs were made using “The Inspector” x-ray source, Model 200 (Golden Engineering, Centerville, IN). The x-ray source has an output of 3 millirads per 60 ns pulse. The film was processed using the Polaroid 8 x 10 Radiographic Film Processor, Model 85-12 (Polaroid, Waltham, MA), set at a 45 second exposure time. The date of the x-ray, the presence or absence of eggs, and the shelling status of the eggs were noted for each female x-rayed. Gravid females with completely shelled eggs were given the hormone oxytocin by injection to stimulate oviposition (Ewert and Legler 1978). The amount of 3% oxytocin administered was determined by body mass: 0.15 ml per 100 g of body mass (J. Iverson, personal communication). Females were restrained during ovipositioning with a custommade Tortoise Restraint Device (TRD), to prevent accidental damage to the eggs. After eggs were oviposited, females were released at the location where they were captured. Egg and clutch characteristics were determined for each of the clutches after oviposition. Egg mass was determined to the nearest 0.01 g immediately after oviposition and diameter (maximum and minimum diameter) was measured to the nearest 0.01 mm. Eggs were incubated at 30oC in moist vermiculite (1:1 weight to volume ratio of vermiculite to water) (Burke et al 1996, Demuth 2001). Eggs were inspected daily after 75 days of incubation and hatchlings removed when discovered. Eggs that did not

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13 hatch after the 120 days of incubation were removed and opened to extract the embryo. Hatchling wet mass was determined within two days of hatching. Data on mother, clutch and hatchling characteristics were collected. From the xray photography, the presence or absence of eggs, number of eggs, and egg diameters from the x-rays were noted. Mass and CL were recorded for each gravid female. For collected clutches, the date of oviposition, clutch size, egg mass and diameter were measured. The date of hatchling and the mass of hatchlings were recorded for all hatchlings. Data were reported as means SD with sample size in parentheses. T-tests were used to compare clutch sizes and mean clutch mass between years; as well as differences between egg diameter measurements taken from the eggs and from the x-ray photographs. Linear regression and Pearson Product Moment correlations were used to assess relationships between variables such as mother and clutch characteristics and egg and hatchling characteristics. Results Pre-oviposition Data Forty-seven sexually mature females were x-rayed between April and August of 2001 and between April and August of 2002. Of these females, twenty-two showed signs of shelled eggs when x-rayed. Radiographed females first showed the presence of incompletely shelled eggs in the first week of May in both years studied (Table 1.2). Females had fully shelled eggs between the last week of May and the second week of June of both years. The highest percentage of x-rayed females with shelled eggs occurred between 5/15 and 5/31. No females x-rayed after the first eight days in June had shelled eggs.

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14 When x-rays were compared to actual oviposition data, the number of eggs observed on the x-ray matched the number of eggs deposited in all clutches except for one. In that clutch, six eggs appeared on the radiograph, however, after observing the female for 24 hours, only five eggs were oviposited. Comparisons of egg diameter measurements on x-rays compared to actual egg diameters showed a significant difference between the two sizes in each of the clutches sampled (Table 1.3). In all cases, the mean diameters taken from x-ray in each clutch were larger than the mean diameters of the eggs. Thus, direct measurements of diameter taken from x-rays are not reliable estimates of egg diameter. Post-oviposition Data Clutch sizes between years were not significantly different (t = -1.254, df = 22, P > 0.5), so both years were combined for further data analysis. Clutch size ranged from 3 to 12 eggs with a mean of 7.29 2.26 (N=24). Carapace lengths of gravid females ranged from 255 mm to 317 mm (N = 15). Clutch size increased significantly with an increase in female CL (Table 1.4). Increases of 16.3 mm in CL lead to an increase in clutch size of one egg (Table 1.4). Clutch size also significantly increased with PL, with a 14.2 mm increase in PL leading to an increase of one egg in clutch size (Table 1.4). In 2001 and 2002, the mean egg masses were 38.1 g 7.66 (N = 47) and 43.4 g 4.64 (N = 66) respectively. Mean egg mass was not significantly different (t = -1.644, d.f. = 13, P = 0.124) between years and the combined mean clutch mass was 40.7 g 6.71 (N = 113). Mean individual egg mass in 2002 was 43.9 4.6 g, with egg mass ranging from 34.4 g to 51.9 g (N=66). Maximum egg diameter in 2002 ranged from

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15 39.7 mm to 49.2 mm, with a mean diameter of 43.9 2.2 mm (N=67). Mean clutch egg mass was positively correlated with female CL (Table 1.4). All eggs from 2001 were incubated until the 120 days after oviposition. Because no eggs had hatched at that point, the eggs were opened and inspected. In one clutch, three eggs each contained a badly decayed, small embryo. All other eggs showed no signs of development. Hatchlings from 2002 emerged from 8/14/02 to 9/13/02, with 4 of the 6 clutches hatching between 8/28 and 9/2. Incubation times ranged from 82 to 95 days (N = 33). The longest period between the first and last hatchling in the clutch emerging was seven days. There was a 50% hatching success rate for all clutches in 2002, with 16.2% of the eggs showing no signs of development. Hatchling mass ranged from 24.5 g to 39.5 g with a mean of 30.7 g 3.01 (N = 33). Hatchling mass was positively correlated with individual egg mass (Table 1.4). Discussion Conservation plans for the gopher tortoise should take in to account the variation in reproductive characteristics that exists between populations. The gopher tortoise population examined in this study is located in an area maintained by prescribed burning. Areas maintained by prescribed burning will often have open canopies that allow light to reach the ground, producing habitat in which higher densities of gopher tortoises are often found (Auffenberg and Franz 1982). As populations found on managed lands are most likely to remain in the future, understanding the reproductive characteristics of these populations is important in creating any future conservation efforts for the gopher tortoise.

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16 This study found that eggs were detectable to x-ray photography from the first week of May through the first two weeks of June in the central Florida population of gopher tortoises studied. Those dates are similar to other studies of Florida gopher tortoise populations (Godley, 1989, Diemer and Moore 1994, Linley and Mushinsky, 1994). Thus, when attempting to efficiently identify gravid females by x-ray methods in a Florida population of gopher tortoises, the optimal time to census females would be from the beginning of May to the middle of June. Diemer and Moore (1994) found that between 85% and 89% of sexually mature females x-rayed in a north Florida population were gravid between May 12 and June 10. In this study, 46.8% of the females x-rayed where gravid which is similar to another central Florida population where 66% of the females x-rayed between May and June were gravid (Godley 1998). Examination of the percentage of gravid females between the two years reveals a large discrepancy between years. In 2001, only 26.9% of x-rayed females were gravid, while in 2002, 71.4% of females x-rayed were gravid. There are several possible explanations for the difference between the two years. Because radiographic techniques can only detect eggs once they begin shelling, it was possible for females to be gravid but for the eggs to be undetectable in x-rays. Females who did not appear gravid when x-rayed in 2001 may not have begun to shell eggs at the time of the radiograph. However, during the same time periods in the next year, many more females showed shelled eggs on radiographs (Table 1.2). This would indicate either a change in the period during which eggs were shelled during 2001 or that the females which did not show eggs on the radiogaphs were not gravid during 2001. Other studies of gopher tortoises, in which radiography was not used, have reported no signs of egg laying during

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17 one or more years by sexually mature females (Auffenberg and Iverson 1979, Landers et al. 1980). The use of x-ray photography for detecting the presence of eggs is a convenient method for determining clutch size; however, determination of egg diameters from x-rays is more problematic. Egg diameters measured from x-rays were consistently larger than the actual dimensions. In some radiographs taken in this study, the difference was amplified as the female moved closer to the x-ray source. The x-ray platform used for this study only limited horizontal, not vertical movement of the tortoise. While the females were placed directly on their plastron on the platform, some females managed to stand up, moving closer to the x-ray source. Therefore, females who were x-rayed more than once in a sampling year sometimes showed large differences in egg diameter measurements from radiographs. It might be possible to estimate the actual egg diameter from x-rays if the female was immobilized and the distance between the tortoise and the x-ray source accurately measured. The mean clutch size was 7.2 eggs, similar to other recorded clutch sizes in central Florida (Godley 1989, Linley 1994). Mean clutch sizes reported for central and southern Florida were on average 2.1 eggs larger than mean clutch sizes for populations in north Florida (Table 1.1). Whether this difference is due to environmental factors, genetic factors or differences in the size or age of the tortoises sampled is unclear. Many studies, including this one, have reported a positive relationship between female carapace length or plastron length and clutch size (Landers et al. 1980, Diemer and Moore 1994, Smith 1995). Thus, the mean clutch size reported in a study may vary with the sizes of tortoises captured. Landers et al. (1980) and Iverson (1980) reported markedly different

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18 PLs and mean clutch sizes, with Landers et al. reporting a mean clutch size of 7.0 eggs and a mean PL of 283 mm, compared to the mean clutch size 5.2 and mean PL of 261 mm found by Iverson. In this study, the mean clutch size of 7.2 was 2.9% and 38.5% larger than those reported by Landers et al. and Iverson, respectively. The 3.4% and 12.1% respective increase in PL in this study’s population over those found in the populations mentioned above could account for the difference seen in clutch sizes. The mean mass of 2001 clutches, 38.1 g 7.66, is identical to both a study completed sixteen years earlier on the same population (Linley and Mushinsky 1994) and a study on a population on the eastern coast of central Florida (Demuth 2001). The mean clutch mass in 2002, however, was 43.4 g 4.55. The difference in mean clutch mass of 5.9 g between the two years is fairly large, although not significantly different. Female CL was positively related to mean clutch mass, so differences in the CL of females between years might account for differences in clutch mass. However, there was no difference in the CL of females between years in this study. Thus, the difference between the two means reported in this study may be due to natural variation in resource availability or allocation of energy for eggs. During 2000 and the beginning of 2001, precipitation levels for almost all months were well below normal, while rainfall in 2002 was above normal for the year (NOAA Annual Climatological Summary). A positive correlation was found between egg mass and hatchling mass in this study. Experimental analysis of survivorship in hatchling Trachemys scripta found that hatchling body size had a significant impact on survivorship (Janzen et al. 2000). Differences in egg mass, whether due to environmental resource availability or maternal energy allocation, may impact survivorship of hatchlings.

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19 In 2001, hatching success of incubated eggs was 0%. Examination of the eggs revealed a total lack of embryonic development in all but three of the eggs. Because no methodological cause for the lack of development could be discovered, the same incubation regime was utilized the following year. In 2002, only 16.2% of the eggs incubated showed no signs of development, which is close to the 13% reported in a southwest Georgia population (Landers et al. 1980). Hatchlings emerged after a mean incubation period of 86.7 days, which falls between the 88.6 and 83.1 days for 29oC and 30oC incubation temperatures, respectively, found by Demuth (2002). The reproductive characteristics reported in this study are similar to those found in other studies of central Florida populations of gopher tortoises. However, gaps in knowledge still exist. Information needs to be obtained about the reproductive season, including the timing of nesting and hatching and interannual variations in reproductive characteristics, such as egg mass. For management purposes, it is also important to determine whether the females in these populations lay clutches on an annual basis or less frequently, as suggested by this study. As gopher tortoise habitat is lost to development across its range, areas maintained for gopher tortoise management (see Diemer 1986 for suggestions), such as the site used in this study, may become important refuges for tortoise populations. In such cases, understanding the reproductive capabilities of the population become vital in making long term plans for tortoise conservation. References Arata, A.A. 1958. Notes on the eggs and young of Gopherus polyphemus (Daudin). Quarterly Journal of the Florida Academy of Science 21:274-280.

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20 Auffenberg, W. and R. Franz. 1982. The status and distribution of the gopher tortoise ( Gopherus polyphemus ). In R.B. Bury (ed.), North American Tortoises: Conservation and Ecology, pp. 95-126. U.S. Department of Interior, Fish and Wildlife Service, Wildlife Research Report 12. Auffenberg, W. and J.B. Iverson. 1979. Demography of terrestrial turtles. In Turtles: Research and perspectives, pp. 541-569. Wiley-Interscience, New York. Burke, R.L., M.A. Ewert, J.B. McLemore, and D.R. Jackson. 1996. Temperature-dependent sex determination and hatching success in the gopher tortoise ( Gopherus polyphemus ). Chelonian Conservation and Biology 2:86-88. Butler, J.A. and T.W. Hull. 1996. Reproduction of the tortoise, Gopherus polyphemus in northeastern Florida. Journal of Herpetology 30:14-18. Butler, J.A. and S. Sowell. 1996. Survivorship and predation of hatchling and yearling gopher tortoises, Gopherus polyphemus Journal of Herpetology 30:455-458. Demuth, J.P. 2001. The effects of constant and fluctuating incubation temperature on sex determination, growth, and performance in the tortoise Gopherus polyphemus Canadian Journal of Zoology 79:1609-1620. Diemer, J.E. 1986. The ecology and management of the gopher tortoise in the southeastern United States. Herpetologica 42:125-133.

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21 Diemer, J.E. and C.T. Moore. 1994. Reproductive biology of gopher tortoises in northcentral Florida. In R.B. Bury and D.J. Germano (eds.), Biology of North American Tortoises, pp. 129-137. U.S. Fish and Wildlife Service, Fish and Wildlife Research Report 13. Ernst, C.H., R.W. Barbour, and J.E. Lovich. 1994. Turtles of the United States and Canada. Smithsonian Institution Press, Washington, USA. Ewert, M.A. and J.M. Legler. 1978. Hormonal induction of oviposition in turtles. Herpetologica 34:314-318. Godley, J.S. 1989. A comparison of gopher tortoise populations relocated onto reclaimed phosphate-mined sites in Florida. In J.E. Diemer, D.R. Jackson, J.L. Landers, J.N. Layne and D.A. Wood (eds.). Gopher Tortoise Relocation Symposium, pp 43-58. Florida Game and Fresh Water Fish Commission, Nongame Wildlife Program Technical Report 5. Hallinan, T. 1923 Observations made in Duval County, northern Florida, on the gopher tortoise ( Gopherus polyphemus ). Copeia 1923:11-20. Iverson, J.B. 1980. The reproductive biology of Gopherus polyphemus (Chelonia:Testudinidae). The American Midland Naturalist 103:353-359. Janzen, F.J., J.K. Tucker, and G.L. Paukstis. 2000. Experimental analysis of an early life-history stage: selection on size of hatchling turtles. Ecology 81:2290-2304. Landers, J.L., J.A. Garner, and W.A. McRae. 1980. Reproduction of gopher tortoises ( Gopherus polyphemus ) in southwestern Georgia. Herpetologica 36:353-361.

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22 Linley, R.T. 1994. Tortoise density, age/size class distribution and reproductive parameters of a central Florida population of Gopherus polyphemus In D.R. Jackson and R.J. Bryant (eds.), The Gopher Tortoise and Its Community, pp. 2132. Proceedings of the 5th Annual Meeting of the Gopher Tortoise Council. Linley, T.A. and H.R. Mushinsky. 1994. Organic composition and energy content of eggs and hatchlings of the gopher tortoise. In R. B. Bury and D. J. Germano (eds.), Biology of North American Tortoises, pp. 113-128. U.S. Fish and Wildlife Service, Fish and Wildlife Research Report 13. McLaughlin, G.S. 1990. Ecology of gopher tortoises ( Gopherus polyphemus ) on Sanibel Island, Florida. M.S. Thesis, Iowa State University, Ames. 115 pp. Mushinsky, H.R. 1985. Fire and the Florida sandhill herpetofaunal community: with special attention to responses of Cnemidophorus sexlineatus Herpetologica 41:333-342. Mushinsky, H.R. 1992. Natural history and abundance of southeastern five-lined skinks, Eumeces inexpectatus, on a periodically burned sandhill in Florida. Herpetologica 48:307-312. Mushinsky, H.R. and E.D. McCoy. 1994. Comparison of gopher tortoise populations on islands and on the mainland in Florida. In R.B. Bury and D.J. Germano (eds.), Biology of North American Tortoises, pp. 39-47. U.S. Fish and Wildlife Service, Fish and Wildlife Research Report 13. Mushinsky, H.R., D.S. Wilson, and E.D. McCoy. 1994. Growth and sexual dimorphism of Gopherus polyphemus in central Florida. Herpetologica 50:119-128.

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23 Myers, R.L. 1990. Scrub and high pine. In R.L. Myers and J.J. Ewel (eds.), Ecosystems of Florida, pp. 150-193. Univ. Central Florida Press, Orlando, USA. Smith, L.L. 1995. Nesting ecology, female home range and activity, and population size-class structure of the gopher tortoise, Gopherus polyphemus on the Katherine Ordway Preserve, Putnam County, Florida. Bull. Florida Mus. Nat. Hist. 38, Pt.I (4):97-126. Wilson, D.S., H.R. Mushinsky, and E.D. McCoy. 1991. Relationship between gopher tortoise body size and burrow width. Herp Review 22:122-124. Wright, J.S. 1982. The distribution and population biology of the gopher tortoise ( Gopherus polyphemus ) in South Carolina. M.S. thesis, Clemson University, S.C. 70 pp.

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24 Table1.1. Reproductive characteristics of the gopher tortoise. NR = Not reported. *Eggs visible on x-ray as completely shelled.LocationNesting Season Hatching Date Mean Clutch Size Mean Egg Mass(g) or Range (if mean not reported) Mean Maximum Egg Diameter (mm) Citation South Carolina 5/27-7/1NR3.839.443.3Wright 1982 South Carolina NRNR6.538.0NRBurke et al. 1996 Southwest Georgia 5/18-6/278/29-10/97.044.544.8Landers et al. 1980 North Florida 5/18NR5.0NR41.6Hallinan 1923 North Florida NR8/20-9/295.240.943.3Iverson 1980 North Florida 6/8-6/18NR5.8NRNRDiemer & Moore 1994 North Florida 6/1-6/298/24-10/25.76NRNRSmith 1995 North Florida 5/27-6/138/18/-10/55.0437.742.2Butler and Sowell 1996 FloridaNR9/4-9/7NR33.5-47.043.5Arata 1958 South Florida NR8/8-9/216.9NRNRMcLaughlin 1990 Central Florida NRNR7.59NRNRGodley 1989 Central Florida NRNR7.838.1NRLinley and Mushinsky 1994 and Linley 1994 Central Florida NRNR7.4638.141.7Demuth 2001 Central Florida 5/27-6/10*8/14-9/137.2940.743.9This Study

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25 Table 1.2. Number of radiographs taken of 47 female G. polyphemus from 2001 and 2002, followed by percentage of total radiographs for the year in parentheses. Multiple radiographs of the same female taken in the same year were included when the status of eggs changed in between radiograph dates (for example, from not visible to visible but not fully shelled). Ten females were x-rayed twice during the same year. Dates (2001)Eggs Not Visible Eggs Visible, Not Fully Shelled Fully Shelled 4/24-4/302 (7.4)0 (0.0)0 (0.0) 5/1-5/149 (33.3)1 (3.7)0 (0.0) 5/15-5/315 (18.5)1 (3.7)1 (3.7) 6/1-6/70 (0.0)1 (3.7)1 (3.7) 6/8-6/140 (0.0)2 (7.4)1 (3.7) 6/15-6/273 (11.1)0 (0.0)0 (0.0) Dates (2002)Eggs Not Visible Eggs Visible, Not Fully Shelled Fully Shelled 4/24-4/301 (3.3)0 (0.0)0 (0.0) 5/1-5/142 (6.6)3 (10.3)0 (0.0) 5/15-5/310 (0.0)8 (26.6)2 (6.6) 6/1-6/74 (13.3)3 (10.3)4 (13.3) 6/8-6/140 (0.0)1 (3.3)2 (6.6) 6/15-6/270 (0.0)0 (0.0)0 (0.0)

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26 Table 1.3. Mean largest egg diameters for individual clutches taken from eggs and x-ray photographs in 2002. T-tests were used to compare the two groups. Asterisk indicates significance of *P 0.05, **P 0.01, ***P 0.001 Clutch ID NEgg Data (Mean Maximum Diameter) X-ray Data (Mean Maximum Diameter) t (d.f., P value) X-ray/Egg Ratio 181842.544.62.714**4.9 5301143.746.94.286**7.3 3781445.046.92.670*4.2 5232040.943.34.676***5.9 4461244.547.76.466***7.2 1531845.248.75.735***7.7 5291842.845.55.524***6.3

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27 Table 1.4. Summary of linear regressions and Pearson Product Moment correlations of adult and clutch characteristics. Independent variables are listed first in regression analysis, followed by the dependent variable. NLinear RegressionPearsons Product Moment R2PRP CL / Clutch Size150.3290.0250.5730.026 CL / Mean Clutch Mass 140.4250.0120.6520.012 CL / Mean Clutch Hatch Mass 60.2740.2860.5240.286 CL / Mean Clutch Max Diameter 80.02950.6840.1720.684 PL / Clutch Size90.4500.0480.6710.048 PL / Mean Clutch Mass 80.0750.5100.2750.510 PL / Mean Clutch Max Diameter 80.0660.5390.2570.539 PL / Mean Clutch Hatch Mass 60.1550.4390.3940.439 Egg Mass / Hatch Mass 330.2400.0040.4900.004 Max Egg Diameter / Egg Mass 660.489<0.0010.699<0.001 Mean Clutch Max Diameter / Clutch Size 80.0330.937 Mean Clutch Hatch Size / Clutch Size 60.3860.449

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28 Chapter Two: Multiple paternity and mating system in a Central Florida Population of Gopher Tortoises, Gopherus polyphemus Introduction The gopher tortoise ( Gopherus polyphemus ) is one of four native North American tortoise species. Gopher tortoise population sizes are in decline, due mostly to extensive habitat loss (Auffenberg and Franz 1982, Diemer 1986). Consequently, the species has gained some form of protection across its range (Ernst et al. 1994), including the listing of species of special concern in Florida (Myers 1990). Because land development in Florida continues at an alarming rate, aggressive conservation efforts are needed to ensure the survival of the species. The formulation of a good conservation plan should include knowledge of the biology and ecology of the species in question. Because of its importance in maintaining genetic variability and for estimating effective population size, a complete understanding of the mating system of a species must be included in the formulation of a conservation plan. Several possible mating systems exist which might be observed in the gopher tortoise, such as monogamy, polygyny, and promiscuity. Monogamy is unlikely, as turtles do not typically display pair-bonds (Pearse and Avise 2001). In the case of polygyny, we expect to see males fertilizing egg clutches of multiple females. In a promiscuous system, we expect to see males fertilizing multiple clutches, as well as multiple paternity among the clutches.

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29 Among reptiles, multiple paternity is known to occur in snakes (McCracken et al. 1999, Prosser et al. 2002), crocodilians (Davis et al. 2001), and lizards (Gullberg et al. 1997). Among turtles, multiple paternity has been reported in most species for which genetic data have been evaluated (summarized by Pearse and Avise 2001). Although, female tortoises probably do not gain any direct benefits such as nuptial gifts or parental care from multiple matings, but there may be indirect benefits to polyandry (Pearse and Avise 2001). Proposed indirect benefits for promiscuous breeders include gaining ‘good genes’ (Kempenaers et al. 1992, Otter and Radcliffe 1996, Watson 1998), avoidance of genetic incompatibility (Zeh and Zeh 1996, Kempenaers et al. 1999, Tegenza and Wedell 2000), increased genetic diversity of offspring (Madsen et al. 1992, Byrne and Roberts 2000). The presence of a dominance hierarchy among male tortoises, with larger males most often proving the victor in aggressive interactions, has been observed among gopher tortoises (Douglass 1976, McRae et al. 1981). While Douglass (1976) hypothesized that dominant males might maintain a loose harem, McRae et al. (1981) suggested that large males would be unable to always defend the females they courted. Instead, they suggested that heightened visitation to females came from increased searching for receptive females, rather than from harem defense. While many instances of male courting behaviors, such as head-bobbing, have been observed and reported in wild populations, few copulations have been observed outside of captive populations (Douglass 1976, Auffenburg 1966, Wright 1982). While field observations may give an indication of which males are dominant in aggressive interactions, it is difficult to determine from observation alone whether dominance interactions lead to a difference in

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30 reproductive success. Using molecular techniques however, it is possible to determine which males actually fertilize a female’s eggs, and to determine which males are most successful in mating. Microsatellites have been used with increasing frequency to investigate the issues of paternity and mating systems. Microsatellites are repeats of short nucleotide sequences, usually 1-6 bp, which are found throughout eukaryote genomes (Chambers and MacAvoy 2000). Because of their extreme variability, microsatellites are especially useful in cases where molecular identification of an individual is necessary (Queller et al. 1993). Microsatellites also are ideal for situations in which non-lethal sampling techniques are preferred. Non-destructive sampling is especially important in species with conservation considerations. Microsatellites have been successfully retrieved from such diverse sources as saliva, hair, feathers, feces, and blood (Queller et al. 1993), many of which are attainable in the field with little negative influence on the individual sampled. Microsatellites provide a great deal of molecular information with minimal sampling impact, making them ideal for paternity study in the gopher tortoise. In my study, I examined the mating system and reproductive behaviors of a population of gopher tortoises in central Florida. In particular, I collected data to address the following questions: Does the gopher tortoise exhibit multiple paternity? If so, is one male responsible for the majority of fertilization in a single clutch? Also, do certain males contribute fertilizations to more female clutches than other males in the population? If so, is it the large males that dominate clutch fertilization?

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31 Methods The study was conducted at the University of South Florida’s Ecological Research Area (ERA), a 200 ha reserve located in Hillsborough County in west-central Florida (28.05oN, 82.20oW). Approximately 20 ha of sandhill habitat within the ECA have been exposed to controlled burning since 1976 (Mushinsky 1992). The controlled burning area is separated into plots which are burned on frequencies of one year, two years, five years, or seven years, or are left as unburned “controls” (Mushinsky 1985). A thriving population of about 280 tortoises occupies the plots (Mushinsky et al. 1994). All plots were trapped for tortoises during the course of the study.From April to August of 2001 and 2002, all active and inactive burrows (classification based on Mushinsky and McCoy 1994) in each plot were located and marked. The width of each burrow was measured at a depth of 500 mm and used as an estimate of the carapace length (CL) of the resident tortoise (Wilson et al. 1991). Burrows greater in width than the minimum CL of sexually mature males in the population, 170mm (Diemer and Moore 1994, Mushinsky et al. 1994) were trapped. Pit traps, consisting of 9.5 L buckets camouflaged with brown fabric and sand, were placed in the ground, level with the burrow entrance. While open, the traps were check every two hours during daytime. Individuals were also gathered by hand when encountered in and around the plots. In trapping all active burrows, I attempted to sample all of the sexually mature males in the population, to insure that all candidate males in the population were sampled. Sexual maturity in gopher tortoises can be readily determined in the field by measurement of the carapace. Mushinsky et al (1994), in a study of the gopher tortoise population at the Ecological Research Area, found that females began reproducing at

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32 around 240 mm carapace length (CL). Data on male tortoises, summarized by, indicate a range from 177-230 mm CL at which sexual maturity is achieved in Florida tortoises. Accordingly, all females greater than 230 mm CL and all males greater than 170 mm CL were considered sexually mature. Mushinsky et al. (1994) found that males and females could be differentiated by measuring plastral concavity (PC) once they had reached a CL of greater than 240 mm. They found that all females had a PC of less than 6 mm, while all males had a PC of greater than 6 mm. Mass and CL were assessed for all sexually mature and sub-adult tortoises captured (Mushinsky et al.1994). A blood sample was obtained from either the brachial sinus or through a subcarapacial approach. After the blood was collected, it was stored in a PVP/BME buffer (10mM Tris-HCl pH8.0, 100mM NaCl, 50mM EDTA, 1% PVP w:v, 0.2% BME) until DNA extraction. All individuals were marked by notching the marginal scutes and released at the burrow where they were captured. Sexually mature female gopher tortoises were radiographed to determine the presence of shelled eggs. The radiographs were made utilizing “The Inspector” X-ray Source, Model 200 (Golden Engineering, Centerville, IN), set to one pulse. The film was processed using the Polaroid 8 x 10 Radiographic Film Processor, Model 85-12 (Polaroid, Waltham, MA). Females with completely shelled eggs were given an injection of 1.5 units per 100g body mass of 3% oxytocin to stimulate oviposition (Ewert and Legler 1978, J. Iverson, personal communication). Egg mass was determined immediately after laying, and diameter (maximum and minimum diameter) was measured with Mitutoyo Absolute Digimatic Calipers (Aurora, IL). Eggs were incubated in nests of vermiculite with a 1:1 w:v ratio of vermiculite to

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33 water (Burke et al. 1996) at temperatures maintained around 30oC (Burke et al 1996, Demuth 2001). Eggs were inspected daily after 75 days of incubation and hatchlings removed when discovered. Eggs that did not hatch after 120 days were removed and opened to extract the embryo. Unhatched embryos were frozen for later DNA extraction. Hatchlings were maintained in aquariums with a 12:12 light cycle and fed a diet of vegetables supplemented with vitamin and mineral powders. Blood samples were obtained from hatchlings by cardiocentesis (E. Jacobson, personal communication). Hatchlings were marked in the same manner as adult individuals and released at the burrow where their mother was captured. DNA extraction followed a standard phenol/chloroform protocol and ethanol precipitation (Schwartz 2003). The 20 l PCR reactions were run with one of two multiplexing primer mixes. Nine microsatellite loci, characterized by Schwartz and Karl (2000), were used for analysis. Multiplexing mix 1 contained the primers GP15F, GP15R-6-FAM, GP30F-TET, GP30R, GP55F-TET, GP55R, GP26F and GP26R-TET. Mix 2 contained the primers GP96F-6-FAM, GP96R, GP61F6-HEX, GP61R, GP19FFAM, GP19R, GP102F-TET, GP102R, GP81F, GP81-R-6FAM. Reaction mixes and cycling parameters on an Omn-e Hybaid thermal cycler followed Schwartz and Karl (2000). PCR samples were run on an ABI Prism 377 automated DNA sequencer (Perkin Elmer, Applied Biosystems, Inc.) at Iowa State University. The program, GENESCAN (Perkin Elmer, Applied Biosystems, Inc.), was used to identify and quantify microsatellite peaks, and all loci were scored three times to insure accuracy in reading and recording results.

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34 Maternal contribution to the clutch was determined by comparing the known maternal genotype to the genotype of her offspring. Paternal genotypes were inferred by removing known maternal contributions from the offspring genotypes where possible. When two or more fathers could be identified for the clutch, the clutch was identified as having multiple sires. Parentage was assigned using exclusion analysis on an individual clutch basis for any clutch with no more than two paternal alleles at all loci. If any locus showed more then two paternal alleles, paternity exclusion was done on an individual hatchling basis. Any candidate male whose genotype mismatched at least one locus was rejected. Individual and combined exclusion probabilities for all loci used in the analysis were calculated with the program CERVUS, version 2.0 (Marshall et al. 1998). Cervus calculates two different exclusion probabilities, exclusion (one) and exclusion (two). Exclusion probability (one) is the power of the locus to exclude a randomly unrelated candidate parent when only the offspring’s genotype is known. Exclusion probability (two) is the calculated power when the genotypes of the offspring and one parent are known. The probability of detecting multiple paternity for individual alleles (d) using the formula of Westneat et al. (1987) equates to the same probability as the individual allele exclusion probability (2) calculated by CERVUS. If more than one male could not be excluded, I performed a likelihood analysis utilizing CERVUS on the remaining candidate males assuming a total of 20 candidate parents (based on field observations), 80% of the candidate parents were sampled (a conservative estimate to account for unsampled males), each individual was genotyped at 98% of the loci, with a 1% genotyping error rate. CERVUS is a likelihood-based program that calculates the log

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35 likelihood of a candidate parent being the true parent compared to an arbitrary individual. The difference ( ) between the two most likely candidates is calculated. Through simulation a critical score is calculated at either relaxed (80%) or strict (95%) confidence. When a male was assigned as the father of a clutch, the probability of another tortoise having the same genotype as that male was calculated as the product across all loci of (p2)2 or (2pq)2. Allele frequencies were determined from the genotypes of the 26 adult females and males captured for the study. The probability of two unrelated individuals sharing the same genotype at all loci (probability of identity) was calculated as in Hanotte et al. (1991). The overall probability of detecting multiple paternity (D) was calculated as in Westneat et al. (1987). This calculation utilizes the frequency of alleles and all possible mating arrangements to compute the probability of the loci used detecting when a female has mating with another male besides the candidate father. Relatedness values were calculated according to the formula of Queller and Goodnight (1989) using the program Relatedness 5.0. Allele frequencies calculated from the adults sampled in the study were used for the calculations. Pairwise R values were calculated between the mother (Px) and the assigned father (Py) and compared to average R values calculated for the mother (Px) and all excluded males combined (Py). The carapace length (CL) and mass of the mothers and assigned fathers for single and multiple-sired clutches and clutch characteristics were compared by t-test. All data were normally distributed and homoscedastic.

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36 Results A total of eight clutches were collected for this study, however, only seven were utilized because of poor embryonic development in most of the eggs in the eighth clutch. The number of offspring genotyped per clutch ranged from four to 11 with a mean of 7.57 2.44. A total of 91 individuals were genotyped, including five sub-adults, 16 adult females, 17 adult males and 53 offspring. The nine microsatellite loci utilized ranged from two to five alleles each, with a mean of 3.44 1.01 alleles per locus. Observed heterozygosity ranged from 0.053 to 0.658 (Table 2.1). The probability of two unrelated tortoises sharing the same genotype for all nine loci (probability of identity) was 3.84 x 10-4 (Table 2.1). The probability of detecting multiple paternity for an individual locus (d) ranged from 0.026 to 0.410 (Table 2.1). Most individual loci (66%) had less than a 20% probability of detecting multiple paternity. For all nine loci combined, however, the chance of detecting multiple paternity was 0.876. Paternal Assignment In most cases, removing the maternal contribution yielded the paternal genotype. In cases where the mother and offspring were both heterozygous for the same alleles and determining which allele was the paternal allele was not possible, males who possessed either allele were not excluded from the analysis. When a father was assigned to a clutch, the probability of another unrelated tortoise having the same genotype as the father was calculated for each assigned male. These probabilities were all extremely low, with a range of 1.55 x 10-6 to 3.83 x 10-13 (Table 2.2). In four of the seven clutches, all candidate males but one were excluded because they mismatched the paternal genotype deduced from the offspring at one or more loci

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37 (Table 2.2). For clutch 378, three candidate males were not excluded in the initial exclusion analysis. These candidate males were compared to the offspring in a likelihood analysis in CERVUS, and male 519 was the most likely candidate male identified for three of the four offspring with 80% confidence for 378-2 and 378-5 ( 2.86 and 2.50 respectively) and with 95% confidence for 378-4 ( 3.13). In addition, Mendelian ratios for clutch 378 differed from expected ( #2 = 4.0, 1d.f., P < 0.05), in at least two different loci for two males not identified as the most likely father by CERVUS. All deviations occurred at loci in which an allele found in the candidate male was not found among the offspring in the clutch. The remaining male (519) was assigned as the father for the clutch. In cases where more than three paternal alleles were detected in the clutch, males were excluded by comparing all male genotypes to each individual offspring. For clutch 446, offspring 446-2, the individual with alleles at three loci which were not found in its clutch mates, had one non-excluded male, male 533 (Table 2.2). Among the remaining individuals in the clutch, male 43 was the only non-excluded male or the only nonexcluded male in common. In clutch 523, male 180 was the common non-excluded male for four of the seven offspring (Table 2.2). In one offspring of the clutch (523-3), male 265 was the only nonexcluded male and in another (523-8) both males 180 and 265 were not excluded. The final individual in the clutch (523-9) did not match any adult male sampled. In fact, this offspring contained two alleles which were unique in the clutch, one of which did not appear in any other tortoise sampled in this study. These alleles could be explained in two ways. Either the maternal or paternal lines mutated at two alleles or an unsampled

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38 male is the father of this individual. If an unsampled male is the father of this individual, the clutch either has three fathers, or the two individuals which are not excluded from male 265, may in fact be the offspring of the unidentified male. Because only half of the father’s alleles are seen in any one offspring, the alleles seen in the offspring assigned to male 265, may be the alleles at which the unknown father is heterozygous. When run through CERVUS, male 265 was assigned as the father of 523-3 with 95% confidence and 523-8 with 80% confidence. Therefore, 265 was assigned paternity of these individuals. Multiple Paternity All mothers were typed and no mismatches between known mother-offspring pairs were observed. The mother’s contribution to each clutch was identified and removed, and all remaining alleles were assigned as paternal alleles. Based on the criteria set for determining multiple paternity, two of the seven clutches (28.6%) appeared to have multiple fathers. In these clutches, the primary male (the male who fertilized the majority of the eggs) fertilized 71.4 % and 80% of the clutch (Table 2.3). Multiple paternity was supported by three paternal alleles at one locus in one clutch and with three or more paternal alleles at two loci in a second clutch. Clutch 446 had three paternal alleles at GP15. Three possible explanations exist for the extra paternal allele. First, it could arise from a mutation in the maternal line. There were no observable mismatches between mother-offspring pairs, which would indicate that a maternal line mutation was not the origination of the allele. The second explanation is that the mutation originated in the paternal line. For mutation to produce the third allele from the other paternal alleles present in either of these clutches, there would have to be

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39 an increase or deletion of five, eight or thirteen repeat units. One of the most commonly accepted mutation model for microsatellites, the stepwise mutation model, predicts an increase or decrease of a single repeat unit (Ohta and Kimura 1973). Multi-step changes have been reported in microsatellites, but they tend to range from two to five repeat units with the lower repeat number being much more common (Ellegren 2000, Hoekert et al. 2002). The repeat unit change necessary to create the mistaken multiple paternity in this case is unlikely. It is also possible for null alleles to lead to the spurious indication of multiple paternity if the mother is heterozygous and the father heterozygous for a different allele and the null allele (Pemberton et al. 1995, Kichler et al. 1999). This situation did not occur in clutch 446. The third possible explanation for the extra allele is multiple paternity. The three paternal alleles are not unusual lengths; all three appear in the adults sampled. Also, the hatchling with the 225 allele had alleles at two other loci which did not appear in any of the other hatchlings in the clutch. When that hatchling was separated from its clutch mates, it was possible through exclusion analysis to assign two fathers, males 43 and 533, for the clutch. In clutch 523, locus GP102 had three paternal alleles and locus GP81 had four paternal alleles. One offspring had alleles at GP102 and GP81 that did not appear in any other offspring in the clutch. The number of eggs in the clutch (t = 0.703, d.f. = 5, P = 0.513) and the total number of eggs genotyped (t = 1.096, d.f. = 5, P = 0.323) did not vary significantly between multiple-sired and single-sired clutches. The number of undeveloped eggs (t = -1.641, d.f. = 5, P = 0.162) also did not vary between single and multi-sired clutches (Table 2.3). It did however appear that there might be a trend towards a negative

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40 relationship between hatching success and multiple paternity (t = 2.254, d.f. = 5, P = 0.074). Mating System and Relatedness Males who were assigned paternity and the other males sampled in the population did not vary significantly in mass (t = 1.335, d.f. = 15, P = 0.202), but differed in carapace lengths (t = 2.400, d.f. = 15, P = 0.030). Only one male (43) appeared to fertilize more than one of the clutches evaluated in this study. Male 43 fertilized 100% of the clutch for one female and 80% of the clutch for another female (Table 2.3). The remaining males fertilized singular clutches or a fraction of a multiple-sired clutch. Male mass (t = -0.967, d.f. = 6, P = 0.371) and CL (t = -0.053, d.f. = 6, P = 0.960) was not significantly different between single-sired clutches and multiple-sired clutches. However, female mass (T = 6.409, d.f. = 5, P = 0.001) and CL (t = 2.682, d.f. = 5, P = 0.044) did vary significantly between females fertilized by a single male and females fertilized by more than one male. Females fertilized by multiple males on average weighed 1320 g less and had carapace lengths 23.1 mm shorter than females fertilized by a single male. In the single-sired clutches, two females were less related to the males who fathered their clutches than to the other males sampled in the population (Table 2.4). Two of the other three females without multiple paternity mated with males more related than the other males, with the third female mating with a male with relatedness similar to the other males sampled. Among the multiple-sired clutches, both females mated with one male with higher than average R values and another male with lower than average R values.

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41 Discussion Reports of aggressive interactions between male gopher tortoises have provoked the question of whether or not these duels give the victor more access to females. Larger males have often been reported as the winners of these conflicts (Douglass 1976). If males winning these contests do receive greater access to females, then larger males might be expected to fertilize a larger portion of females, thus increasing the male’s fitness. If these matches do not lead to a marked increase in access to reproductive females, the pattern of fertilization is expected to be more evenly distributed among males. In this study, fertilization of egg clutches was evenly distributed among the assigned males, with no one male monopolizing fertiliztation. There was a significant relationship between carapace length and fertilization of clutches indicating that larger males have a reproductive advantage over smaller males; however, it is not possible to determine whether or not the males not assigned paternity did not sire clutches among the females not evaluated by this study. Multiple paternity of clutches was observed within a well-defined central Florida population of gopher tortoises. This is perhaps not very surprising considering multiple paternity has been detected in nearly every species of turtle (Testudines) for which it has been assayed (Pearse and Avise 2001). Male size did not affect whether or not a male was the only father of a clutch or one of multiple fathers who sired a clutch. The factor that did relate to whether or not multiple males sired a clutch was the size of the female. Females with multiple-sired clutches weighed at least 1000g less than the females with single-sired clutches. Two possible explanations exist. Perhaps males who win dominance interactions do gain greater access to females, but because the male-male

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42 duels and male-female courtships can take considerable time (McRae et al. 1981, Douglass 1976), the male is constrained to only a few females. If the male is limited to fewer females, there may be preferential guarding of larger females who can produce more eggs. Alternatively, female tortoises also have been observed blocking their burrow entrance and rebuffing males performing courting behaviors (Douglass 1976, personal observation). Large females may be able to turn away attempts by courting males, while small females may have a more limited ability to restrict copulation attempts. The percentage of clutches in which multiple paternity is found varies with species, and even among populations. Among many species included in a number of studies, the percentage of clutches fertilized by multiple males ranged from 4% to 100% (Pearse and Avise 2001, Hoekert et al. 2002, Moore and Ball 2002). While a limited number of clutches were examined in this study, 28.6% showed multiple paternity. This number may be conservative, as the ability of the loci used in this study to detect multiple paternity was only 87.6%. Thus for each clutch in which one father was detected, there was a 12.4% chance that another father went undetected. Multiple paternity may not necessarily be a result of multiple matings in a single season. Many turtle species, including the desert tortoise, store sperm (Palmer et al. 1998, Pearse and Avise 2001). Gopher tortoises exhibit mating behaviors from March to September; however, egg-laying is only known to occur in May and June (Douglass 1976, Iverson 1980, Butler and Hull 1996). Sperm may be stored during fall mating encounters to be used for spring fertilization of eggs. In this study, clutches fertilized by multiple males were skewed towards one male. In both cases of multiple paternity, at least 70% of the eggs were fertilized by one male. One male fertilizing the majority of

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43 the eggs in a clutch may be due to sperm competition (Parker 1970). It is also possible in cases of sperm storage that stored sperm may lose viability or become depleted over time and therefore, the stored sperm fertilizes fewer of the eggs than more recent insemination events (Barnett et al. 1995, Yamagishi et al. 1992, Birkhead 1998). However, in this study it was not possible to determine whether females with multiple sired clutches used stored sperm or mated with multiple males in one season. One offspring, 523-9, could not be assigned to any of the sampled males. It is possible that the unique alleles seen in this offspring were caused by mutation or an unsampled male. Because this population of gopher tortoises has been studied for twenty years, most individuals in the population have been marked (Mushinsky et al. 1994). However, the mother of the unassigned offspring had never been marked. Her lack of marking suggests that she recently immigrated into the population. While it is possible that she was fertilized only by males in the study population, she could also have with a male from outside of the study population prior to immigration. Given the possibility that gopher tortoises may store sperm, the latter scenario is quite likely. Other species of turtles have shown long term sperm storage, including the desert tortoise which can store sperm for more than two years (Palmer et al. 1998, Pearse and Avise 2001). The existence of promiscuous mating is supported by data from central Florida gopher tortoises. While male size did appear to affect opportunity for mating among those males sampled, it was not possible to determine if the unassigned males mated with unsampled females. Among the clutches examined, however, the majority of males fertilized only one clutch, with only one male fertilizing multiple clutches, indicating that at least males of similar size have a more even proportion of the mating, rather than a

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44 harem system. Large males seem to have an advantage in mating. However, the lack of males fertilizing multiple clutches may indicate that it is a costly advantage that allows males only a limited number of copulations events. Relocation is a conservation strategy that has been applied to the gopher tortoise (Diemer 1986). Because no male in this study appeared to monopolize fertilization of clutches, it is possible that movements of males into or out of populations by relocation may not be disruptive to the current mating patterns in the recipient population. However, it appears that small males may not gain as many fertilizations as a large male. Thus the number and size of relocated males may change the impact of relocation on the mating structure of the recipient population. If a relatively large male is moved, there may be a reproductive advantage over the recipient population’s males. When making conservation plans, the possible increase of effective population size caused by multiplesired clutches (Sugg and Chesser 1994) and the possible impacts relocated males on the recipient populations should be considered. It may be more advantageous to move small males who may gradually become a part of the reproductive population, rather than large males who may prove disruptive to the current reproductive population. References Auffenberg, W. 1966. On the courtship of Gopherus polyphemus Herpetologica 22:113-117. Auffenberg, W. and R. Franz. 1982. The status and distribution of the gopher tortoise ( Gopherus polyphemus ). In R.B. Bury (ed.), North American Tortoises: Conservation and Ecology, pp. 95-126. U.S. Department of Interior, Fish and Wildlife Service, Wildlife Research Report 12.

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45 Birkhead, T.R. 1998. Sperm competition in birds. Reviews of Reproduction 3:123-129. Barnett, M., S.R. Telford and B.J. Tibbles. 1995. Female mediation of sperm competition in the millipede Alloporus unicatus (Diplopoda: Spirostreptidae). Behavioural Ecology and Sociobiology 36:413-419. Butler, J.A. and T.W. Hull. 1996. Reproduction of the tortoise, Gopherus polyphemus in northeastern Florida. Journal of Herpetology 30:14-18. Burke, R.L., M.A. Ewert, J.B. McLemore, and D.R. Jackson. 1996. Temperature-dependent sex determination and hatching success in the gopher tortoise ( Gopherus polyphemus ). Chelonian Conservation and Biology 2:86-88. Byrne, P.G. and J.D. Roberts. 2000. Does multiple paternity improve the fitness of the frog Crinia georgiana ? Evolution 54:968-973. Chambers, G.K. and E.S. MacAvoy. 2000. Microsatellites: consensus and controversy. Comparative Biochemistry and Physiology Part B 126:455-476. Davis, L.M., T.C. Glenn, R.M. Elsey, H.C. Dessauers, and R.H. Sawyer. 2001. Multiple paternity and mating patterns in the American alligator, Alligator mississippiensis Molecular Ecology 10:1011-1024. Demuth, J.P. 2001. The effects of constant and fluctuating incubation temperature on sex determination, growth, and performance in the tortoise Gopherus polyphemus Canadian Journal of Zoology 79:1609-1620. Diemer, J.E. 1986. The ecology and management of the gopher tortoise in the southeastern United States. Herpetologica 42:125-133.

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46 Douglass, J.F. 1976. The mating system of the gopher tortoise, Gopherus polyphemus in southern Florida. M.S. thesis, University of South Florida, Tampa, FL. 79 pp. Ellegren, H. 2000. Microsatellite mutations in the germline: implications for evolutionary inference. Trends in Genetics 16:551-558. Ernst, C.H., R.W. Barbour, and J.E. Lovich. 1994. Turtles of the United States and Canada. Smithsonian Institution Press, Washington, USA. Ewert, M.A. and J.M. Legler. 1978. Hormonal induction of oviposition in turtles. Herpetologica 34:314-318. Gullberg, A. M. Olsson, H. Tegelsrom. 1997. Male mating success, reproductive success and multiple paternity in a natural population of sand lizards: behavioral and molecular genetics data. Molecular Ecology 6:105-112. Hanotte, O., T. Burke, J.A.L. Armour, A.J. Jeffreys. 1991. Hypervariable minisatellite DNA sequences in the Indian peafowl Pavo cristatus Genomics 9:587-597. Hoekert, W.E.J., H. Neufglise, A.D. Schouten and S.B.J. Menken. 2002. Multiple paternity and female-biased mutation at a microsatellite locus in the olive ridley sea turtle ( Lepidochelys olivacea ). Heredity 89:107-113. Iverson, J.B. 1980. The reproductive biology of Gopherus polyphemus (Chelonia:Testudinidae). The American Midland Naturalist 103:353-359. Kempenaers, B. G.R. Verheyen, M. Vandenbroeck, T. Burke, C. Vanbroeckhoven, and A.A. Dhondt. 1992. Extra-pair paternity results from female preference for highquality males in the blue tit. Nature 357:494-496.

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47 Kempenaers B., B. Congdon, P. Boag, R.J. Robertson. 1999. Extra-pair paternity and egg hatchability in tree swallows: Evidence for the genetic compatibility hypothesis? Behavioural Ecology 10:304-311. Kichler, K., M.T. Holder, S.K. Davis, R. Mrquez-M, D.W. Owens. 1999. Detection of multiple paternity in the Kemp’s ridley sea turtle with limited sampling. Molecular Ecology 8:819-830. Madsen, T. R. Shine, J. Loman, T. Hakansson. 1992. Why do female adders copulate so frequently? Nature 355:440-441. Marshall, T.C., J. Slate, L.E.B. Kruuk, and J.M. Pemberton. 1998. Statistical confidence for likelihood-based paternity inference in natural populations. Molecular Ecology 7:639-655. McCrae, W.A., J.L. Landers, and J.A. Garner. 1981. Movement patterns and home range of the gopher tortoise. American Midland Naturalist 106:165-179. McCracken, G.F., G.M. Burghardt, S.E. Houts. 1999. Microsatellite markers and multiple paternity in the garter snake, Thamnophis sirtalis Molecular Ecology 8:1475-1479. Moore, M.K. and R.M Ball Jr. 2002. Multiple paternity in loggerhead turtle ( Caretta caretta ) nests on Melbourne Beach, Florida: a microsatellite analysis. Molecular Ecology 11:281-288. Mushinsky, H.R. 1992. Natural history and abundance of southeastern five-lined skinks, Eumeces inexpectatus, on a periodically burned sandhill in Florida. Herpetologica 48:307-312.

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48 Mushinsky, H.R. and E.D. McCoy. 1994. Comparison of gopher tortoise populations on islands and on the mainland in Florida. In R.B. Bury and D.J. Germano (eds.), Biology of North American Tortoises, pp. 39-47. U.S. Fish and Wildlife Service, Fish and Wildlife Research Report 13. Mushinsky, H.R., D.S. Wilson, and E.D. McCoy. 1994. Growth and sexual dimorphism of Gopherus polyphemus in central Florida. Herpetologica 50:119-128. Myers, R.L. 1990. Scrub and high pine. In R.L. Myers and J.J. Ewel (eds.), Ecosystems of Florida, pp. 150-193. Univ. Central Florida Press, Orlando, USA. Ohta, T. and M. Kimura. 1973. A model of mutation appropriate to estimate the number of electrophoretically detectable alleles in a finite population. Genetical Research 22:201-204. Otter, K. and L. Ratcliffe. 1996. Female initiated divorce in a monogamous songbird: Abandoning mates for males of higher quality. Proceedings of the Royal Society London B 263:351-354. Palmer, K.S., D.C. Rostal, J.S. Grumbles, and M. Mulvey. 1998. Long-term sperm storage in the desert tortoise ( Gopherus agassizii ). Copeia 1998:702-705. Parker, G.A. 1970. Sperm competition and its evolutionary consequences in the insects. Biological Review 45:526-567. Pearse, D.E. and J.C. Avise. 2001. Turtle mating systems: Behavior, sperm storage, and genetic paternity. Journal of Heredity 92:206-211.

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49 Pemberton, J.M., J. Slate, D.R. Bancroft and J.A. Barrett. 1995. Nonamplifying alleles at microsatellite loci: a caution for parentage and population studies. Molecular Ecology 4:249-252. Prosser, M.R. 2002. Genetic analysis of the mating system and opportunity for sexual selection in northern water snakes ( Nerodia sipedon ). Behavioral Ecology 13:800-807. Queller, D.C. and K.F. Goodnight. 1989. Estimating relatedness using genetic markers. Evolution 43:258-275. Queller, D.C., J.E. Strassmann, and C.R. Hughes. 1993. Microsatellites and kinship. Trends in Ecology and Evolution 8:285-288. Schwartz, T.S. 2003. Population genetics of the gopher tortoise ( Gopherus polyphemus) in Florida using microsatellites. M.S. thesis, University of South Florida, F.L. 130 pp. Schwartz, T.S. and S.A. Karl. 2000. Genetic structure of Florida gopher tortoise ( Gopherus polyphemus ) populations. American Zoologist 40:1203-1203. Sugg, D.W. and R.K. Chesser. 1994. Effective population sizes with multiple paternity. Genetics 137:1147-1155. Tregenza, T. and N. Wedell. 2000. Genetic compatibility, mate choice and patterns of parentage: Invited review. Molecular Ecology 9:1013-1027. Watson, P.J. 1998. Multi-male mating and female choice increase offspring growth in the spider Neriene litigiosa (Linyphiidae). Animal Behavior 55:387-403.

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50 Westneat, D.F., P.C. Fredrick, and R.H. Wiley. 1987. The use of genetic markers to estimate the frequency of successful alternative reproductive tactics. Behavioral Ecology and Sociobiology 21:35-45. Wilson, D.S., H.R. Mushinsky, and E.D. McCoy. 1991. Relationship between gopher tortoise body size and burrow width. Herp Review 22:122-124. Wright, J.S. 1982. The distribution and population biology of the gopher tortoise ( Gopherus polyphemus ) in South Carolina. M.S. thesis, Clemson University, S.C. 70 pp. Yamagishi,, M., Y. Ito and Y. Tsubaki. 1992. Sperm competition in the melon fly, Bactrocera curcurbitae (Diptera: Tephritidae): effects of sperm ‘longevity’ on sperm precedence. Journal of Insect Behaviour 5:599-609. Zeh, J.A. and D.W. Zeh. 1996. The evolution of polyandry I: Intragenomic conflict and genetic incompatibility. Proceedings of the Royal Society London 263:17111717.

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51 Table 2.1. Allele frequencies, heterozygosities, probability of identity, and exclusion probabilities for nine microsatellites in Gopherus polyphemus LocusAlleleFrequencyExpected Heterozygosity Observed Heterozygosity Probability Of Identity Exclusion (1) Exclusion (2) GP152090.5660.6110.6050.2100.1990.359 2230.079 2250.250 2310.013 2350.092 GP192550.0260.2950.1840.5320.0420.139 2570.829 2590.145 GP263590.0400.3050.2890.5060.0470.163 3650.829 3670.040 3690.092 GP301920.0130.5000.4210.3660.1220.199 1960.395 2120.592 GP552680.0660.1250.1320.7730.0080.058 2740.934 GP611920.0130.1720.1840.7010.0140.082 2010.908 2110.079 GP813970.2240.6620.6580.1680.2390.410 4030.092 4050.158 4070.513 4110.013 GP961500.9740.0520.0530.9010.0010.026 1560.013 1580.013 GP1023090.1840.6130.6320.2270.1830.326 3150.526 3170.290

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52 Table 2.2. Maternal genotypes, inferred paternal genotypes and non-excluded male candidates per clutch. When more than three paternal alleles appeared within a clutch, males were excluded based on individual offspring genotypes. *Males assigned with paternity, followed by the probability of another tortoise sharing the same genotype as that male in parentheses.ClutchMaternal GenotypePaternal GenotypeNon-Excluded Candidate Males LocusAlleleAlleleLocusAlleleAllele 1522322515209235 1925725919257 2636536926367369 3021221230196212 5527427455274 6120120161201 8140340781397407 9615015096150 18 102309317102315 43* (1.14x10-10) 1520922515225235 1925725719257 2636536526365 3019621230196212 5527427455268274 6120120161201 8139739781397407 9615015096150156 201 102317317102309317 343* (4.11x10-13) 1522322515209225 1925725719259 2636536926365 3021221230196212 5527427455274 6120120161201 8140540781397 9615015096150158 253 102315317102315317 441* (5.65x10-13) Continued on the next page.

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53 Table 2.2 (Continued). Maternal genotypes, inferred paternal genotypes and non-excluded male candidates per clutch.ClutchMaternal GenotypePaternal GenotypeNon-Excluded Candidate Males LocusAlleleAlleleLocusAlleleAlleleAlleleAllele 1520920915209 1925725719257 2636536526365 3021221230196 5527427455274 6120120161201 8140540781407 9615015096150 378 102315317102315 P6T1 519* (1.37x10-6) 180 1520923515209225235 1925725719257 2636536526365369 3019621230212 5527427455268274 6120120161201 8140340781397 9615015096150 446 102315315102315 446-1: R1T1, 43* (1.14x10-10), 526 446-2: 533* (7.07x10-12) 446-3: 43* 446-4: 43* 446-6: 43*, GP526, GP462 1520920915209 1925725719257 2636536926365 3021221230196212 6120120161201 5527427455274 8140540781397403405407 9615015096150 523 102315315102303309315 523-3: 265* (1.58x10-8) 523-4: 180* (5.44x10-8) 523-5: P6T1, R1T2, 346, 519, 180* 523-6: 465, 343, 180* 523-7: P6T1, 352, 533, 180* 523-8: P6T1, 265*, 180 523-9: No males matched Continued on the next page.

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54 Table 2.2 (Continued). Maternal genotypes, inferred paternal genotypes and non-excluded male candidates per clutch.ClutchMaternal GenotypePaternal GenotypeNon-Excluded Candidate Males LocusAlleleAlleleLocusAlleleAllele 1520923515209 1925725719257259 2636536526365 3019619630196212 5527427455274 6120120161201 9615015096150 8140740781403405 529 102309315102315102 346* (2.82x10-8)

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55 Table 2.3. Mother, clutch and father characteristics for single-sired and multiple sired clutches. In multiple-sired clutches, the primary male is listed first. Single-SiredMultiple-SiredClutch 18 Clutch 201 Clutch 253 Clutch 387 Clutch 529 Clutch 446 Clutch 523 Mother ID18201253378529446523 Mother CL (mm) 300314306291317276289 Mother Mass (g)4400485041004300445031003100 Mother Location 11W1WRoad2W1E2E5E Total Number of Eggs 912979610 Hatching Success 100%91.6%33.3%57.2%55%33.3%0% Number of Undeveloped Eggs 0102013 Number of Eggs Genotyped 81194957 Father ID4334344151934643 / 533180 / 265 Father CL (mm)290278250264270290 / 263260 / 276 Father Mass (g)380040002800350034003800 / 2800 2850 / 3500 Father Location11E1WUnknownCWRoad1E / 1E1E /2E2 % of Eggs Fertilized by Primary Male 100.0100.0100.0100.0100.080.057.1 1corresponds to those in Figure 2.1

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56 Figure 2.1. Ecological Research Area, Tampa, FL. The plot ID and acres are given for each plot. 660 m

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57 Table 2.4. Relatedness values of mother gopher tortoises and assigned fathers compared to the average relatedness of the mother and other males in the population. The relatedness of the mother to all other individuals sampled in the population (excluding offspring from clutches in this study). MotherAssigned Father R Mother:FatherR Mother:Other Males R Mother:Other Individuals 1843-0.697-0.139-0.037 2013430.238-0.037-0.028 253441-0.459-0.154-0.037 3785190.277-0.250-0.036 44643 / 5330.132 / -0.207-0.030-0.027 523180 / 2650.082 / -0.140-0.129-0.034 5293460.0260.031-0.020

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58 Appendices

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59 Appendix A: Genotyping results for 75 gopher tortoises from the USF Ecological Research AreaIDMotherGP30GP15GP55GP26GP96GP61GP19GP102GP81 18212212223225274274365369150150201201257259309317403407 201196212209225274274365365150150201201257257317317397397 253212212223225274274365369150150201201257257315317405407 378212212209209274274365365150150201201257257315317405407 446196212209235274274365365150150201201257257315315403407 523212212209209274274365369150150201201257257315315405407 529196196209235274274365365150150201201257257309315407407 P1T6196196223225274274365365150150201211257259309315407411 R1T1196212209225274274359369150150201201257259315317407407 R1T2196212209209274274365365150150201211257257315315397405 465212212225225274274365365150150201201257257309317397407 346196212209209274274365365150150201201257259315315403405 519196196209209274274365365150150201201257257315315407407 43196212209235274274367369150150201201257257315315397407 526212212209209274268365369150150201201257259309317397407 265196196209209274274365365150150201201257257309309397407 352212212225225274274365365150150201201257257315315407407 533196212225235274268359365150150201201257259315317407407 JCM196196209213274274365365150150201201257257315317397397 343196212225235274268365365150156201201257257309317397407 441196212209225274274365365150158201201257259315317397405 462196196209209274274365369150150201201257257315315397397 308212212209209274274365365150150201201257257315317405407 P6T1212212209225274274365365150150201201257257315315407407 180196212209225274274365365150150201211257257309315405407 77212212209209274274365365150150200200257257315317405407 453212212209223274274365367150150200200257257315317397407 520196212209209274274365365150150200200257257315317397407 531196212209225274274365365150150200200255255315317405407

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60 Appendix A (Continued)IDMotherGP30GP15GP55GP26GP96GP61GP19GP102GP81 517196212209209274274365365150150200200257257309309403403 536212212209235268274359365150150200200257259315315407407 534196212209225274274365367150150200200259259315317405407 516212212209209274274365365150150200200257257309315405407 160212212209223274274365365150150200200257257315317407407 532196196209223268274365365150150200200257257315317403407 601212212209209274274365365150150200210257257309315397407 133196212225235274274365365150150200200257257315317407407 P6T4212212209225274274365365150150200210257257309315405407 P7192196209225274274365365150150200210257257317317397403 18-218196212223235274274365369150150201201257259309315407407 18-318212212209225274274369369150150201201257257315317397407 18-418196212225235274274365367150150201201257257309315397403 18-518196212209223274274365369150150201201257259309315403407 18-618196212209225274274365369150150201201257259309315403407 18-718196212223235274274369369150150201201257259309315407407 18-818196212223235274274369369150150201201257259315317407407 18-918212212223225274274367369150150201201257259309315397407 2011 201196196209235274274365365150150201201257257309317397407 2012 201212212209225274274365365201201257257317317397407 2013 201212212209225274274365365150156201201257257309317397397 2014 201196212209235274274365365150150201201257257309317397397 2015 201196196209235274268365365150156201201257257309317397407 2016 201196212225235274274365365150156201201257257317317397407 2017 201196196225225274268365365150150201201257257309317397397 2019 201212212209225274268365365150156201201257257317317397397 20110 201196196225225274268365365150150201201257257309317397407 20111 201212212225225274268365365150156201201257257309317397397 20112 201196212209225274268365365150156201201257257317317397407 2531 253196212225225274274365365150158201201257259317317397407 2532 253212212225225274274365369150150201201257259317317405407

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61 Appendix A (Continued)IDMotherGP30GP15GP55GP26GP96GP61GP19GP102GP81 2533 253196212209225274274365365150158201201257259315317405407 2534 253212212223225274274365369150158201201257259317317405407 2535 253212212209223274274365365150158201201257259317317397407 2536 253196212223225274274365365150150201201257259315317405407 2537 253212212209223274274365365150150201201257259315315397407 2538 253196212223225274274365365150158201201257259317317405407 2539 253212212209223274274150150201201 3782 378196212209209274274365365150150201201257257315315405407 3784 378196212209209274274365365150150201201257257315315407407 3785 378196212209209274274365365150150201201257257315317405407 3786 378196212209209274274365365150150201201257257315317407407 4461 446212212209235274274365369150150201201257257 4462 446196212225235274268365365150150201201257257315315407407 4463 446212212209235274274365369150150201201257257315315397407 4464 446196212235235274274365369150150201201257257315315397407 4466 446196212209209274274365369150150201201257257397407 5233 523196212209209274274365365150150201201257257309315397405 5234 523212212209225274274365369150150201201257257309315405405 5235 523196212209209274274365365150150201201257257315315405407 5236 523212212209225274274365369150150201201257257309315407407 5237 523212212209225274274365365150150201201257257315315405407 5238 523196212209209274274365369150150201201257257309315405407 5239 523212212209225274274365365150150201201257257303315403407 5291 529196212209209274274365365150150201201257257315315403407 5292 529196212209209274274365365150150201201257259315315403407 5293 529196196209209274274365365150150201201257257315315405407 5294 529196212209209274274365365150150201201257257315315405405 5295 529196212209235274274365365150150201201257257315315405407 5296 529196212209235274274365365150150201201257259309315405407 5297 529196196209209274274365365150150201201257259309315405407 5298 529196196209209274274365365150150201201257259309315 5299 529196196209235274274365365150150201201257259315315403407