THINKING OUTSIDE THE BOX: EFFECTS OF ENVIRONMENTAL ENRICHMENT AND REARING DURATION ON HEAD-STARTING SUCCESS OF BOX TURTLES

BY

SASHA JAMES TETZLAFF

DISSERTATION

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Natural Resources and Environmental Sciences in the Graduate College of the University of Illinois at Urbana-Champaign, 2019

Urbana, Illinois

Doctoral Committee:

Associate Professor Robert L. Schooley, Chair Adjunct Assistant Professor Jinelle H. Sperry, Director of Research Dr. Brett A. DeGregorio, University of Arkansas Associate Professor Michael P. Ward Professor Bruce A. Kingsbury, Purdue University Fort Wayne

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ABSTRACT

Translocation is the deliberate movement and release of animals and a common management technique in wildlife conservation programs. However, efforts are often unsuccessful because relocated animals have low survival, precluding population establishment.

I reviewed studies using antipredator training, environmental enrichment, and soft release as pre- release behavioral conditioning in translocation programs, and I quantitatively synthesized how these approaches affect post-release success. I then conducted experiments using captive-reared juvenile eastern box turtles (Terrapene carolina) raised with or without naturalistic environmental enrichment to better understand mechanisms influencing habitat preferences in captivity. Next, I investigated if enrichment encouraged natural behaviors before turtles were released into the wild and how being raised in enriched environments affected growth over differing rearing periods (nine vs. 21 months). Finally, I examined how enrichment and captive- rearing duration affected the turtles’ post-release growth, behavior, and survival.

Meta-analysis conducted on 108 effect sizes from 41 studies investigating the effects of pre-release behavioral conditioning on translocation outcomes revealed conditioned animals had higher survival, reduced movement, and greater site fidelity than unconditioned individuals.

Notably, antipredator training, environmental enrichment, and soft release all resulted in improved survival. This suggests pre-conditioning is likely an important tool for improving future wildlife translocations.

When I provided environmental enrichment to captive-born eastern box turtles, they preferred enriched environments, regardless of prior housing experience, suggesting this preference is innate. Thus, enrichment likely enhances welfare of captive box turtles by satisfying an instinctive desire to occupy complex habitat. However, pre-release trials revealed

iii enriched turtles performed no better in ecologically relevant foraging tasks than unenriched turtles. In a predator recognition test, eight-month-old enriched turtles avoided raccoon (Procyon lotor) urine more than unenriched turtles of the same age, but this difference was not apparent one year later. These findings suggest any behavioral benefits conferred by enrichment were modest. Enriched turtles also attained smaller body sizes overall than unenriched turtles pre- release. Enrichment had minimal effects on post-release behavior and survival. However, turtles raised for 21 months moved farther from the release site and had higher post-release survival than those raised for only nine months, regardless of rearing environment. Although raising animals in enriched captivity can increase translocation success for several taxa, my experiments with box turtles and similar previous studies indicate enrichment might have limited utility for enhancing reptile translocations. Instead, implementing a longer rearing period to maximize body size before release appears to most benefit survival by reducing susceptibility to predation, which is commonly cited as a hindrance to post-release success.

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For Kristin

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ACKNOWLEDGEMENTS

Being a graduate student isn’t so much of an island as it is a peninsula; you’re largely out there on your own, but right behind you is support in the form of advisors, lab mates, committee members, professors, family, friends, and many others. I’ve been lucky enough to have support from all such people during my Ph.D. program, and I will be forever grateful for that. There are many individuals and organizations to thank for their contributions that made this dissertation possible.

First, I thank my advisors, Drs. Brett DeGregorio and Jinelle Sperry. It has been an honor to be Brett’s first graduate student, part of Jinelle’s growing list of Ph.D. students, and a

“grandchild” of the Weatherhead Lab. The support from Brett and Jinelle was more than I could have asked for, and their commitment to graduate student training has been inspirational. I am extremely appreciative for the chance they took on me and providing me such amazing opportunities. Brett: thank you for working so closely with me during this time. Your ingenuity, never-ending drive, and patience helped make the most of our work. Jinelle: thanks for being so uplifting and optimistic. The diversity of successful students that have and continue to come through the Sperry Lab clearly demonstrates how great of an environment it is to be in.

I also thank the rest of my dissertation committee members, Drs. Mike Ward, Bob

Schooley, and Bruce Kingsbury. Each brought unique perspective to this work that helped improve it. For their companionship and productive discussions, I thank current and former lab mates Ellery Ruther, Jason Gleditsch, Stephen Tyndel, Sean MacDonald, Dr. Valerie Buxton,

Dr. Jef Vizentin-Bugoni, Dylan O’Hearn, Brittney Graham, Dr. Aron Katz, Libby Sternhagen,

Pat Wolff, and Daniel Kovar. Karen Claus, Courtney Lynch, Lezli Cline, and Kelly Sullan in the

Department of NRES were so patient and helpful with my incessant need for administrative-

vi related assistance.

This work was supported by funds from the United States Department of Defense’s

Environmental Security Technology Certification Program. For assistance in the field and laboratory, I thank Jessie Birckelbaw, Sara Desmond, Odell Encinosa, Alondra Estrada, Richael

Mavesi, David Messmann, Charlotte Robinson, Nicholas Schiltz, Maddie Terlap, David, Kyle, and Jack Tetzlaff, Karen Watson, Pat Wolff, and Wayne, Melinda, and Adrian Yerdon. Everyone who assisted with field and laboratory work deserves my endless gratitude, but Nick’s prodigious ability to locate box turtle nests is the primary reason Chapters 3–5 came to fruition. Charlotte’s hard work in the field made several side projects possible and allowed me to be a less absent husband and father. Dave Messmann also deserves extra recognition; his help and companionship in the field will never be forgotten. Brian Huggett, Jim Langerveld, and Jonathan

Edgerly were instrumental in facilitating fieldwork at Fort Custer Training Center. Dr. T.J.

Benson graciously loaned equipment that facilitated hours of turtle watching during laboratory behavioral trials.

I thank my entire family for their continued support. The Szydlowski family (Mike,

Nicole, Ashleigh, Bradley, and Peyton) and Mitzi Szwajkowski deserve a special shout out for letting Kris and the kids spend so much time with them when I was away in the field. Having the support of family relatively close by while completing a Ph.D. made it more survivable by far.

Lastly and most importantly, thank my wife, Kristin, and our sons, Jack and Luke. Boys:

You have been a source of inspiration throughout this process and a constant reminder of what is truly important. Thanks for being good sports about me having to be away in the field, at a conference, or in the lab writing on weekends, but I’m happy we were able to make some of my dissertation work into family time. Bringing you over to the greenhouse to check on the turtles

vii will be among some of my favorite memories of our young family. Kris: We did it. This dissertation is dedicated to you, as it’s just as much yours as it is mine. Before we met I had considered dropping out of college, but your endless love, support, and selflessness every step of the way is without a doubt the main reason why we got to this point. I can only hope I’ll be able to repay you for the sacrifices you’ve made for our family. I love you and our boys more than I’ll ever be able to express.

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TABLE OF CONTENTS

CHAPTER 1: GENERAL INTRODUCTION ...... 1

CHAPTER 2: EFFECTS OF ANTIPREDATOR TRAINING, ENVIRONMENTAL ENRICHMENT, AND SOFT RELEASE ON WILDLIFE TRANSLOCATIONS: A REVIEW AND META-ANALYSIS ...... 8

CHAPTER 3: CAPTIVE-REARED JUVENILE BOX TURTLES INNATELY PREFER NATURALISTIC HABITAT: IMPLICATIONS FOR TRANSLOCATION ...... 43

CHAPTER 4: TRADEOFFS WITH GROWTH AND BEHAVIOR FOR CAPTIVE BOX TURTLES HEAD-STARTED WITH ENVIRONMENTAL ENRICHMENT ...... 68

CHAPTER 5: CAPTIVE-REARING DURATION MAY BE MORE IMPORTANT THAN ENVIRONMENTAL ENRICHMENT FOR ENHANCING TURTLE HEAD-STARTING SUCCESS ...... 93

CHAPTER 6: SUMMARY ...... 126

APPENDIX: SUPPLEMENTARY DATA FROM CHAPTER 2 ...... 130

1

CHAPTER 1: GENERAL INTRODUCTION

Wildlife conservation translocations entail deliberate movement and release of animals, often for the purposes of augmenting imperiled populations or reintroducing species to areas where they formerly occurred (IUCN/SSC 2013). However, such efforts are often unsuccessful because released animals exhibit behaviors that lead to reduced survival (Seddon et al. 2007).

Conditioning adaptive behavior prior to release using techniques such as actively teaching aversion towards predators (antipredator training), passively promoting natural behaviors in captivity (environmental enrichment), and temporarily holding animals in acclimation pens at release sites (soft release) have been suggested as methods to improve post-release survival

(Reading et al. 2013; Batson et al. 2015). I comprehensively searched, reviewed, and quantitatively synthesized the literature examining how these tactics affected post-release success of wildlife translocations. I then conducted experiments using captive-reared, juvenile eastern box turtles (Terrapene carolina) raised with or without naturalistic environmental enrichment for varying durations to better understand 1) mechanisms influencing habitat preferences in captivity, 2) if enrichment encourages natural behaviors and affects growth before turtles were released into the wild, and 3) whether enrichment or captive-rearing duration affect post-release growth, behavior, and survival.

Several case studies have demonstrated environmental enrichment, antipredator training, and soft release can improve survival, reduce movement, and increase site fidelity of translocated animals (e.g., Biggins et al. 1999; Tuberville et al. 2005; Shier and Owings 2006), but the utility of these tactics has not been broadly quantified. In Chapter 2, I reviewed studies experimentally testing these approaches in translocations and used meta-analysis to examine if they benefit translocations and what factors related to them influence success, such as the

2 translocation type (wild-to-wild or captive releases), age, or taxonomic group. Although understanding how these effects generally influence translocations is useful, the typical high failure rates of translocations requires continued need for experimental data to decipher what aspects of the program contribute to success (Seddon et al. 2007; Moseby et al. 2014).

Releasing animals into nature after extended captive-rearing (head-starting) is generally less successful than moving wild animals directly between natural sites (Griffith et al. 1989;

Fischer and Lindenmayer 2000). Gaining a better understanding of why captive animals prefer certain habitats has implications for improving head-starting because maladaptive habitat selection of released animals or mismatch between cues in captivity and release environments can be reasons for failure (Swaisgood 2010). Yet, mechanisms influencing habitat choice are generally not understood. Early experience of young individuals may affect later preference in that they seek to occupy habitats similar to their rearing environment (Immelmann 1975;

Teuschl et al. 1998). Alternatively, habitat preference may be innate (Partridge 1974). In

Chapter 3, I tested these two competing hypotheses of habitat preference (experiential vs. innate) in the context of informing head-starting efforts using a cohort of captive juvenile eastern box turtles. I hypothesized preference is influenced by rearing environment and predicted turtles would choose a habitat simulating the one they were raised in when provided the choice between different habitats. However, if habitat preference is innate within this species, I expected turtles would prefer to occupy a naturalistic enriched habitat as opposed to a more simplistic, unenriched one regardless of rearing experience.

A common focus in head-starting programs involves rapidly growing individuals to reduce susceptibility to predation or to be closer to reproductive age or size upon release (Daly et al. 2018). However, emphasis on rapid growth may have undesirable consequences, such as

3 the degradation or loss of normal behaviors due to unstimulating captive conditions

(DeGregorio et al. 2013; 2017; Swaisgood et al. 2018). Environmental enrichment can encourage natural behaviors before release (Vargas and Anderson 1999) but potentially comes with a tradeoff of reduced growth in complex enclosures (Krech et al. 1960). Empirical evidence is needed to determine if maximizing growth does indeed come at the cost of beneficial behaviors, which could reduce head-starting success. In Chapter 4, I investigated this potential tradeoff by comparing growth and behavior of environmentally enriched and unenriched captive juvenile eastern box turtles. I predicted enriched turtles reared in structurally complex enclosures, which entailed foraging for spatially variable food in the presence of conspecifics, would grow slower and attain overall smaller body sizes than unenriched turtles.

However, I anticipated this form of enrichment would allow turtles to be more proficient at foraging for novel prey than unenriched turtles, which were housed individually and fed out of simple dishes. I predicted enriched turtles would also show more risk-aversion than unenriched turtles by avoiding a predator cue more often as well as being less likely to emerge from a shelter into a novel environment.

Head-started animals are often thought to lack the necessary skills and experience to successfully transition to post-release environments (Stamps and Swaisgood 2007). For reptiles, the effects of environmental enrichment on translocation success has received minimal attention

(Roe et al. 2015; DeGregorio et al. 2017). In addition to the potential behavioral benefits garnered by enrichment, extended head-starting duration to facilitate growth and maturity could be beneficial (Roe et al. 2010). However, no study has investigated the concomitant influence of enrichment and rearing duration on head-starting outcomes. In Chapter 5, I tested the effects of time in captivity and enrichment on short-term head-starting success for eastern box turtles. I

4 hypothesized enrichment provides opportunities for head-started turtles to develop behaviors that minimize predation risk, as predation is likely the primary cause of mortality for juvenile turtles due to their small body sizes and incomplete hardening of the shell (Dodd 2001). Once turtles were released, I expected enriched turtles would move less, remain less visible, and grow faster due to enhanced foraging skills, leading to higher survival than unenriched turtles. If time in captivity negatively affects behavior, I expected turtles reared for longer would remain exposed and move more than turtles kept in captivity for a shorter duration, leading to lower survival. Alternatively, if larger body size reduces post-release predation (the “bigger is better” hypothesis; Packard and Packard 1988), I expected turtles reared for a longer period would have higher survival.

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LITERATURE CITED

Batson, W.G., I.J. Gordon, and D.B. Fletcher. 2015. Translocation tactics: a framework to support

the IUCN Guidelines for wildlife translocations and improve the quality of applied

methods. Journal of Applied Ecology 52: 1598–1607.

Biggins, D.E., A. Vargas, J.L. Godbey, and S.H. Anderson. 1999. Influence of prerelease

experience on reintroduced black‐footed ferrets (Mustela nigripes). Biological

Conservation 89: 121–129.

Daly, J.A., K.A. Buhlmann, B.D. Todd, C.T. Moore, J.M. Peaden, and T.D. Tuberville. 2018.

Comparing growth and body condition of indoor-reared, outdoor-reared, and direct-

released juvenile Mojave desert tortoises. Herpetological Conservation and Biology 13:

622–633.

DeGregorio, B.A., P.J. Weatherhead, T.D. Tuberville, and J.H. Sperry. 2013. Time in captivity

affects foraging behavior of ratsnakes: implications for translocation. Herpetological

Conservation and Biology 8: 581–590.

DeGregorio, B.A., J.H. Sperry, T.D. Tuberville, and P.J. Weatherhead. 2017. Translocating

ratsnakes: does enrichment offset negative effects of time in captivity? Wildlife Research

44: 438–448.

Dodd, Jr. C.K. 2001. North American Box Turtles: A Natural History. University of Oklahoma

Press, Norman, Oklahoma.

Fischer, J. and D.B. Lindenmayer. 2000. An assessment of the published results of animal

relocations. Biological Conservation 96: 1–11.

Griffith, B., J.M. Scott, J.W. Carpenter, and C. Reed. 1989. Translocation as a species conservation

tool: status and strategy. Science 245: 477–480.

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Immelmann, K. 1975. Ecological significance of imprinting and early learning. Annual Review of

Ecology, Evolution, and Systematics 6: 15–37.

IUCN/SSC. 2013. Guidelines for Reintroductions and Other Conservation Translocations. Version

1.0. Gland, Switzerland: IUCN Species Survival Commission.

Krech, D., M.R. Rosenzweig, and E.L. Bennett. 1960. Effects of environmental complexity and

training on brain chemistry. Journal of Comparative and Physiological Psychology 53:

509–519.

Moseby, K.E., B.M. Hill, and T.H. Lavery. 2014. Tailoring release protocols to individual species

and sites: one size does not fit all. PLoS ONE 9: e99753.

Packard, G.C. and M.J. Packard. 1988. Physiological ecology of reptilian eggs and embryos. Pages

523–605 In: Gans, C. and R.B. Huey (eds) Biology of the Reptilia, vol 16. Liss, New York.

Partridge, L. 1974. Habitat selection in titmice. Nature 247: 573–574.

Reading, R.P., B. Miller, and D. Shepherdson. 2013. The value of enrichment to reintroduction

success. Zoo Biology 32: 332–341.

Roe, J.H., M.R. Frank, S.E. Gibson, O. Attum, and B.A. Kingsbury. 2010. No place like home:

An experimental comparison of reintroduction strategies using snakes. Journal of Applied

Ecology 47: 1253–1261.

Roe, J. H., M.R. Frank, and B.A. Kingsbury. 2015. Experimental evaluation of captive-rearing

practices to improve success of snake reintroductions. Herpetological Conservation and

Biology 10: 711–722.

Seddon, P.J., D.P. Armstrong, and R.F. Maloney. 2007. Developing the science of reintroduction

biology. Conservation Biology 21: 303–312.

Stamps, J.A and R.R. Swaisgood. 2007. Someplace like home: Experience, habitat selection and

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conservation biology. Applied Animal Behaviour Science 102: 392–409.

Shier, D.M. and D.H. Owings. 2006. Effects of predator training on behavior and post-release

survival of captive prairie dogs (Cynomys ludovicianus). Biological Conservation 132:

126–135.

Swaisgood, R.R. 2010. The conservation-welfare nexus in reintroduction programmes: a role for

sensory ecology. Animal Welfare 19: 125–137.

Swaisgood, R.R., M.S. Martin‐Wintle, M.A. Owen, X. Zhou, and H. Zhang. 2018. Developmental

stability of foraging behavior: evaluating suitability of captive giant pandas for

translocation. Animal Conservation 21: 474–482.

Teuschl, Y., B. Taborsky, and M. Taborsky. 1998. How do cuckoos find their hosts? The role of

habitat imprinting. Animal Behaviour 56: 1425–1433.

Tuberville, T.D., E.E. Clark, K.A. Buhlmann, and J.W. Gibbons. 2005. Translocation as a

conservation tool: site fidelity and movement of repatriated gopher tortoises (Gopherus

polyphemus). Animal Conservation 8: 349–358.

Vargas, A. and S.H. Anderson 1999. Effects of experience and cage enrichment on predatory skills

of black-footed ferrets (Mustela nigripes). Journal of Mammalogy 80: 263–269.

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CHAPTER 2: EFFECTS OF ANTIPREDATOR TRAINING, ENVIRONMENTAL ENRICHMENT, AND SOFT RELEASE ON WILDLIFE TRANSLOCATIONS: A REVIEW AND META-ANALYSIS1

ABSTRACT Wildlife translocations can have conservation value but results have been mixed regarding animal behavior and survival post-release. Practitioners have adopted antipredator training, environmental enrichment, and soft release as pre-release conditioning tactics to encourage adaptive behavior and improve post-release survival, but their utility has not been broadly quantified. We performed a formal literature review and conducted meta-analysis on 108 effects from 41 studies experimentally testing how these tactics affected survival, movement, or site fidelity compared to unconditioned animals. We further investigated how each conditioning tactic, animal source (wild-to-wild translocated or captive-released), age, and taxonomic group

(birds, fish, mammals, and reptiles) influenced outcomes. Relative to unconditioned animals, conditioned individuals were 1.5 times more likely to survive, had reduced movement, and were three times more likely to show site fidelity. Each of the three conditioning tactics resulted in improved survival. Juveniles released from captivity derived the greatest survival benefit from conditioning. Across taxa, conditioning most benefitted survival of fish. Conditioning also had positive effects on survival of mammals and reptiles, albeit with less certainty than for fish.

Estimates comparing survival of conditioned to unconditioned birds were much more variable, suggesting avian translocation programs using conditioning generally need improvement. Soft release consistently reduced movement and increased site fidelity; this was an especially viable

1This chapter has been published in Biological Conservation. Full citation: Tetzlaff, S.J., J.H. Sperry, and B.A. DeGregorio. 2019. Effects of antipredator training, environmental enrichment, and soft release on wildlife translocations: a review and meta-analysis. Biological Conservation 236: 324–331.

9 technique for adult wild-to-wild translocated animals. We provide quantitative evidence that behavioral conditioning can aid wildlife translocations, and we encourage continued experiments to further elucidate how refined tactics could advance conservation efforts using translocation as a management tool.

INTRODUCTION

Conservation translocations entail deliberate movement of organisms from one site for release in another, often with the aim of augmenting imperiled populations or reintroducing species to areas where they have been extirpated (IUCN/SSC, 2013). Conservation translocations

(hereafter “translocations”) have been used for decades as a wildlife management tactic, but success of such efforts has generally been low or uncertain (Griffith et al., 1989; Fischer and

Lindenmayer, 2000; Seddon et al., 2007). Failure of released populations to establish and persist could result from animals exhibiting behaviors associated with low post-release survival such as maladaptive habitat selection and atypical movement (Einum and Fleming, 2001; Jule et al.,

2008; Swaisgood, 2010). The effectiveness of translocation is still critically questioned (Pérez et al., 2012; Taylor et al., 2017), and the utility of long-standing tactics thought to contribute to success needs to be quantified to advance the field of reintroduction biology (Armstrong and

Seddon, 2008). Tactics aimed at conditioning the behavior of translocated animals, such as environmental enrichment, antipredator training, and soft release are rapidly gaining popularity as means to improve translocation success (Reading et al., 2013; Batson et al., 2015; Shier,

2016), but their utility has not been broadly quantified. We evaluated the effectiveness of behavioral conditioning on translocations releasing captive and wild animals to examine which factors related to these approaches influence success.

Captive animals are an important source for translocations (Kleiman, 1989; Brichieri-

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Colombi et al., 2018), but captivity can have detrimental effects on behavior (DeGregorio et al.,

2017; Swaisgood et al., 2018). Offsetting these effects is often accomplished using environmental enrichment. This can take on numerous forms but generally entails providing environmental stimuli necessary for optimal psychological and physiological wellbeing

(Shepherdson, 1998). Enrichment can enhance translocations by providing essential learning opportunities pre-release (Reading et al., 2013). For example, rearing black-footed ferrets

(Mustela nigripes) in outdoor pens and giving them experience killing live prey was shown to reduce dispersal and improve survival compared to ferrets reared in simplistic cages (Biggins et al., 1999). Enrichment also might help captive animals avoid developing maladaptive behaviors.

For instance, practitioners captive-rearing birds for translocation frequently use puppets, costumes, or parent-rearing to reduce the likelihood of juveniles imprinting on human caretakers

(Valutis and Marzluff, 1999; Utt et al., 2008). Likewise, stranded southern sea otter (Enhydra lutris nereis) pups had better foraging skills and increased survival when reared with a surrogate adult female than if rehabilitated using traditional methods relying heavily on human care

(Nicholson et al., 2007). Yet, it is unclear how broadly effective environmental enrichment is for enhancing translocation success, as it had minimal to no effects on post-release behavior and survival for other mammals (Rogers et al., 2016) as well as for some fish (Carrera-García et al.,

2017) and reptiles (Roe et al., 2015; DeGregorio et al., 2017).

Although environmental enrichment provides opportunities for captive animals to express several natural behaviors, it might be ineffective at directly teaching individuals to be wary of predators (Griffin et al., 2000). Naïve animals often do not behaviorally demonstrate recognition of relevant predators (Blumstein et al., 2002), which is concerning because predation is a prevailing cause of translocation failure (Fischer and Lindenmayer, 2000). Antipredator training

11 using live or model predators as well as predator cues (e.g., scent) can be an effective approach for conditioning aversive behavior towards predators. For example, juvenile black-tailed prairie dogs (Cynomys ludovicianus) exposed to live predators before translocation had higher post- release survival than untrained conspecifics (Shier and Owings, 2006), and Chinook salmon

(Oncorhynchus tshawytscha) had greater post-release recovery rates if fish were exposed to live predators prior to release (Maynard et al., 1998). However, antipredator training does not always improve post-release survival. For instance, Lopes et al. (2017) used model predators and human contact paired with aversive stimuli to condition blue-fronted Amazon parrots (Amazona aestiva), and Moseby et al. (2012) used model, tactile, and olfactory predator stimuli to train greater bilbies (Macrotis lagotis); neither study found apparent benefits of conditioning.

Whether animals are released from captivity or free-ranging individuals are translocated between natural sites, challenges incurred with moving them to novel environments such as stress (Teixeira et al., 2007) and lack of familiar sensory cues (Swaisgood, 2010) could be associated with failure. As such, animals often rapidly disperse from release areas, which could limit reproductive potential and reduce survival by unnecessarily expending energy and increasing exposure to predators or vehicles (Le Gouar et al., 2012). To combat such effects, practitioners have employed “delayed” or “soft” release measures to aid animals’ transition to release sites. This typically involves temporarily confining animals in pens, providing supplementary resources (e.g., food, water, access to shelter), or implementing a combination of penning and resource provisioning at the release site (Cid et al., 2014). The rationale for soft release is that animals have greater chance to acclimate and become anchored to their new surroundings and could be less apt to immediately disperse once released. However, results from studies using soft release have also been mixed. Compared to animals that are immediately

12 released unrestrained into the recipient site and not provisioned with supplementary resources

(i.e., hard released), some authors reported soft release reduced dispersal, increased site fidelity, and improved survival (Tuberville et al., 2005; Mitchell et al., 2011; Knox and Monks, 2014).

Yet, others reported minimal to no effects (Hardman and Moro, 2006; de Milliano et al., 2016;

Bannister et al., 2018) or even suggest it can be a detriment to success (Thompson et al., 2001;

Richardson et al., 2013; Batson et al., 2017).

Clearly, there is a considerable discrepancy regarding the efficacy of conditioning on behavior and survival of translocated animals, perhaps because the basis for using these tactics often comes from intuition rather than evidence (Seddon et al., 2007). Quantitatively evaluating the effectiveness of pre-release conditioning will help identify successful tactics from those needing refinement and provide valuable insight to inform future translocations. To this end, we reviewed published translocation literature, and used meta-analysis of data from studies experimentally testing the effects of environmental enrichment, anti-predator training, and soft release to ask: Does conditioning broadly benefit translocations, and if so, is success affected by factors such as conditioning tactic, translocation type (wild-to-wild or captive releases), age, or taxonomic group? Given the variable nature of translocations and conditioning tactics used

(Batson et al., 2015), we expected effect sizes generated from meta-analysis would be generally small. Based on benchmarks commonly used to evaluate short-term success, we predicted conditioned animals would have higher post-release survival, move less, and show greater fidelity to release areas than unconditioned animals.

METHODS

Data sources, inclusion criteria, and extraction

We searched for literature current to May 2018 in Google Scholar using the following

13 terms singly and in combination: “antipredator training”, “captiv-”, “condition”, “conservation”,

“delayed release”, “enrich-”, “enrichment”, “environmental”, “head-start”, “headstart”,

“reinforc-”, “reintroduc-”, “soft release”, “tactic”, “translocat-”, “translocation”, and “wildlife”.

This search returned peer-reviewed journal articles, technical reports, book chapters, and graduate theses and dissertations. We scanned titles and abstracts to determine if articles might be eligible for inclusion after further reading and applying our criteria (see below). To gather other potential sources for our dataset, we reviewed reference lists in (backward search) and citations of (forward search) articles meeting our criteria as well as relevant reviews (Reading et al., 2013; Batson et al., 2015). Although our search likely missed some applicable studies, we are confident the resulting dataset from our systematic search is a robust representation of the available literature (Nakagawa et al., 2017).

Many translocations are not conducted as experiments, precluding the ability to challenge historic assumptions about what interventions are beneficial and limiting mechanistic understanding of outcomes (Seddon et al., 2007; Swaisgood, 2010; Bannister et al., 2018). Thus, to be included in meta-analysis, a study had to experimentally compare survival, movement, or site fidelity between conditioned animals and unconditioned conspecifics. We categorized conditioning tactics based on distinctions described in previous reviews (Reading et al., 2013;

Batson et al., 2015). We included studies that used only environmental enrichment, antipredator training, or soft release as these are the most regularly tested tactics. However, because each of these tactics can entail a wide range of methods we restricted the studies used in our meta- analysis by narrowly defining the tactics as follows: We defined environmental enrichment as providing animals stimulation in captivity that gave them opportunities to express species-typical behaviors that are expected to confer post-release benefits. For example, housing animals in

14 naturalistic enclosures, giving opportunities to forage like they would in nature, and interaction with conspecifics all constituted enrichment. We considered antipredator training separate from enrichment since this usually entails brief, infrequent sessions (van Heezik et al., 1999; Alonso et al., 2011; Cortez et al., 2015) whereas enrichment is often passively provided. We thus defined antipredator training as situations where researchers actively attempted to condition this specific behavior by simulating predators or providing exposure to live predators/predator sensory cues.

We defined soft release by when animals were temporarily confined at the release site in some form of enclosure rather than being immediately released. We did not include studies that used other soft release measures such as resource supplementation because this is rarely experimentally implemented (e.g., a treatment group with provisioning compared to an unprovisioned group). We were necessarily restrictive in our data inclusion to protect the integrity of the analyses, and as a result, we excluded several studies that nearly, but not entirely, met our inclusion criteria. Forty articles were retained for data extraction after applying our inclusion criteria (Appendix A). We also included data from an unpublished study on translocation of eastern box turtles (Terrapene carolina) conducted by the authors in Michigan,

USA.

We created a separate dataset for each of our main effects of interest (survival, movement, and site fidelity) and analyzed these datasets independently. In several cases, a study investigated more than one of these effects and was thus used in multiple databases. Sample sizes of monitored released animals in each group (conditioned and unconditioned) had to be reported for inclusion so we could calculate effect sizes for analyses. Data were generally provided from animals monitored with telemetry, but if effects were estimated from other methods (e.g., recapture rates as a survival proxy); we converted proportions to whole numbers based on release

15 sample sizes (Barron et al., 2010). To be included in the survival dataset, studies had to report the number or proportion of animals known or estimated to have survived and died during the post-release monitoring period. To be included in the movement dataset, studies had to report mean and standard deviation (SD) of metrics used to evaluate movement (e.g., distance dispersed from release sites, total area used, or daily movement rate). If SD was not reported but standard errors or symmetric confidence intervals were, we converted them to SD. To be included in the site fidelity database, authors had to report the number or proportion of animals that did or did not exhibit site fidelity according to their definition of what constituted fidelity. For instance, authors have defined site faithfulness as staying within 1 km of the release area (Tuberville et al.,

2005; Hardman and Moro, 2006). For 10 studies where data were not readily extractable but met our inclusion criteria, we contacted authors up to two times for clarification; authors of four articles responded to our requests.

We made judgments of whether to retain effects from a given study on a case-by-case basis. If a study simultaneously conditioned a group with multiple tactics, we did not include effects from this treatment. For example, D'Anna et al. (2012) had three treatment groups for conditioning white seabream (Diplodus sargus): environmental enrichment, antipredator training, or both enrichment and predator training. Disentangling the effects of simultaneous conditioning from enrichment and antipredator training would not be possible, so we did not include effects from this group. If a study had replication such as multiple release cohorts or sites, we treated them as separate effects if authors reported this level of information. For example, Jonssonn et al. (1999) soft released brown trout (Salmo trutta) in multiple areas, so we included effects from each site. Similarly, Maynard et al. (1996) reared Chinook salmon with either sand or gravel substrate. We considered both of these groups environmentally enriched but

16 treated them as independent effects since the authors reported results for each group.

In addition to the abovementioned data, we assigned each study a unique ID and noted citation information such as authors, journal title (if a peer-reviewed article), and publication year; continent where the research was conducted; how long animals were monitored post- release; Latin and common name of the focal species; the main conditioning tactic as environmental enrichment, antipredator training, or soft release; whether translocations were wild-to-wild (free-ranging animals moved directly between natural sites) or captive releases. In nearly all cases, animals released from captivity were captive-born, but some wild-born animals were held and conditioned in captivity prior to translocation (e.g., Nicholson et al., 2007;

DeGregorio et al., 2017). Only four vertebrate classes were present in our datasets, so we noted

Class as Actinopterygii, Aves, Mammalia, or Reptilia. We recorded age as adult, juvenile, both if multiple age classes were translocated, or unknown/not stated. We also noted sex as male, female, both if multiple sexes were translocated, or unknown/not stated.

Effect size calculation and meta-analyses

We calculated effect sizes and conducted all analyses in R version 3.4 (R Core Team,

2017). We used the metafor package (Viechtbauer, 2010) to calculate effect sizes and associated variances from extracted data using the escalc function and to construct meta-analytical models.

We generated effect sizes for survival and site fidelity as odds ratios (OR) using the numbers of conditioned and unconditioned animals known or estimated to have survived and died and that did or did not exhibit site fidelity, respectively. Since we combined multiple metrics for our movement analysis, we calculated standardized mean difference of movement between conditioned and unconditioned animals as Hedge’s g (also commonly referred to as Hedge’s d) from means and SD. We considered effect sizes of 0.2, 0.5, and 0.8 as small, medium, and large,

17 respectively, for Hedge’s g (Nakagawa and Cuthill, 2007).

Controlling for phylogenetic non-independence is important when conducting meta- analyses with multiple species (Nakagawa et al., 2017). We created a phylogenetic tree of all species used in analyses with the rotl package (Michonneau et al., 2016). This package does not calculate tree branch lengths, so we estimated them using the compute.brlen function in the ape package (Paradis and Schliep, 2018). We then created phylogenetic correlation matrices from the resulting trees (Figs. A1–A3) if needed for use in models.

To determine if the random factors “phylogeny” and “study ID” were needed to control for phylogenetic non-independence or multiple effects analyzed from the same study, we used likelihood ratio tests (LRT) comparing models including these random factors to a null model with no random factors. Accepting statistical significance at α ≤ 0.05, the LRTs suggested both factors were needed for survival (P ≤ 0.04) and movement (P ≤ 0.002), so we used multilevel models to conduct meta-analyses of these effects. The LRTs suggested neither random factors were needed for site fidelity (P ≥ 0.60), so we used a random effects meta-analytical model for this analysis.

Beyond estimating the overall magnitude of an effect, meta-analysis can be used to quantify the level of consistency among effect sizes used in analysis (Senior et al., 2016). We determined how much heterogeneity (inconsistency) existed among effects in each dataset using the I2 statistic, which ranges from 0 to 100% (Nakagawa et al., 2017). Values of 25%, 50%, and

75% broadly suggest low, moderate, and high amounts of heterogeneity, respectively (Higgins et al., 2003). I2 values >80% are common in ecological and evolutionary meta-analyses since they often deal with multiple species and systems (Senior et al., 2016). We thus a priori anticipated there would be moderate to high levels of heterogeneity among effect sizes in our coarse meta-

18 analyses, and this often suggests more detailed subsequent analyses will be useful for deciphering potential causes of variation among effect sizes (Senior et al., 2016; Nakagawa et al.,

2017).

In addition to the overall effects of behavioral conditioning on survival, movement, and site fidelity, we were also interested in determining what factors might influence these metrics.

We thus conducted several independent meta-analyses on subsets of effects in each of our datasets. Using the same model structures as in our main meta-analyses, we conducted subgroup analyses to investigate if survival, movement, or site fidelity were affected by conditioning tactic, translocation type, age, or Class. We conducted subgroup analyses only if there were at least five effects comprised of effects from three independent studies per subgroup within each dataset. Thus, we did not conduct subgroup analyses for certain categorical variables due to it not being tested, lack of reported information, or low numbers of effects within each category. Our subgroups for survival were conditioning tactic (environmental enrichment, antipredator training, or soft release), translocation type (wild-to-wild or released from captivity), age (adult or juvenile), and Class (Actinopterygii, Aves, Mammalia, or Reptilia). Subgroups for movement were conditioning tactic (environmental enrichment and soft release only), translocation type, age, and Class (Mammalia and Reptilia only). Wild-to-wild translocations within translocation type were the only factor where enough effects existed in the site fidelity dataset to conduct subgroup analysis.

We report effect sizes with 95% confidence intervals (CI) for all meta-analytical models.

We considered effects to be statistically significant if CIs for odds ratios of survival and site fidelity did not overlap one. For movement, we considered effects to be statistically significant if

CIs did not overlap zero for Hedge’s g estimates. However, we also interpret results in terms of

19 biological importance even if some estimates had CIs overlapping traditional significance threshold criteria (Nakagawa et al., 2017).

Publication bias can arise when studies with non-significant results are less likely to be published. We investigated for the potential of publication bias in each dataset by inspecting funnel plots for asymmetry, running Egger’s regression tests (Egger et al., 1997), and conducting trim-and-fill analyses to determine the number effects that might have been “missing” from our datasets due to publication bias (Duval and Tweedie, 2000). We included effects only from peer- reviewed studies in these analyses.

RESULTS

Summary of all effects

In total, we analyzed 108 effects from 41 studies meeting our inclusion criteria: 59 effects from 36 studies of survival, 41 effects from 18 studies of movement, and eight effects from six studies of site fidelity. There was a range of 1–5 effects per study for survival, 1–6 effects per study for movement, and 1–2 effects per study for site fidelity. The most commonly used conditioning tactic was soft release. We analyzed 73 effects for soft release, 28 effects for environmental enrichment, and seven effects for antipredator training. There were 70 effects for captive releases and 38 for wild-to-wild translocations. Fifty effects were for adults, 49 effects were for juveniles, and nine effects were from multiple age groups. There were 36 effects for reptiles, 33 for mammals, 22 for fish, and 17 for birds. Most studies released both males and females (72 effects), only one study released solely males (one effect) or females (three effects), but many studies did not report or were uncertain of sexes (32 effects). Forty-six effects were from studies conducted in North America, 29 from Australia/New Zealand, 25 from Europe, seven from Asia, and only one from South America. Eighty-seven effects were from peer-

20 reviewed articles in 23 different journals, 16 were from gray literature (three from books, two from technical reports, and 11 from graduate theses), and five were from an unpublished dataset.

Publication dates spanned 37 years (1981–2018).

Survival

Soft release had the most effects for survival, followed by environmental enrichment, and antipredator training had the least (Table 2.1). There were more effects on captive releases than wild-to-wild translocations. There were slightly more effects for juveniles than adults, and only a few effects were from multiple age classes. The largest number of effects was for mammals, followed by fish, birds, and reptiles.

Overall, translocated animals were 1.55 (95% CI: 1.23 to 1.95) times more likely to survive post-release if they had been conditioned (Fig. 2.1). As expected, we detected moderate heterogeneity between effects (I2 = 42.16%). The application of any of the three conditioning tactics resulted in higher survival relative to unconditioned animals: Animals receiving antipredator training were 2.14 (95% CI: 1.34 to 3.40) times more likely to survive than untrained ones, environmentally enriched animals were 1.55 (95% CI: 1.01 to 2.39) times more likely to survive than unenriched individuals, and soft released animals were 1.47 (95% CI: 1.07 to 2.02) times more likely to survive than hard released animals. Animals released from captivity were 1.73 (95% CI: 1.25 to 2.40) times more likely to survive if they had been conditioned, but we found minimal evidence suggesting conditioning benefited survival for wild-to-wild translocations (OR = 1.09, 95% CI: 0.64 to 1.87). Juveniles were 1.94 (95% CI: 1.17 to 3.21) times more likely to survive if they had been conditioned, but conditioning had more variable effects for adults (OR = 1.31, 95% CI: 0.82 to 2.09). Conditioning led to improved survival for fish (OR = 1.51, 95% CI: 1.13 to 2.03). Conditioning also had generally positive benefits for

21 mammals (OR = 1.38, 95% CI: 0.85 to 2.24) and reptiles (OR = 1.95, 95% CI: 0.78 to 4.88), but not with the precision it did for fish. Although the mean effect size was similar to other taxa, there was much more uncertainty in the effectiveness of conditioning for birds (OR = 1.59, 95%

CI: 0.25 to 9.95). We found no evidence of publication bias from the funnel plot or the Egger’s regression test (z = 0.33, P = 0.74). The trim-and-fill analysis estimated no effects were missing

(Fig. A4).

Movement

Distributions of effects for movement based on conditioning tactic, translocation type, age class, and taxonomic group are shown in Table 2.2. Soft release had the most effects, followed by environmental enrichment, and there was only one effect for antipredator training.

There were more effects for captive releases than wild-to-wild translocations. There were slightly more effects for adults than juveniles, and only a few effects were from multiple age classes. The largest number of effects was for reptiles, followed by mammals, then fish, and there was only one effect for birds.

There was a small-to-moderate overall negative effect of conditioning on movement

(Hedge’s g = ‐0.34, 95% CI: ‐0.63, ‐0.06), indicating conditioned animals moved less than unconditioned ones on average post-release (Fig. 2.2). We detected moderate heterogeneity between effects (I2 = 51.56%). Soft released animals had reduced movement compared to hard released animals (Hedge’s g = ‐0.51, 95% CI: ‐0.84 to ‐0.19). Compared to unenriched animals, environmental enrichment appeared to have minimal effects on reducing movement (Hedge’s g =

0.12, 95% CI: ‐0.37 to 0.61). Conditioning reduced movement for wild-to-wild translocated animals compared to unconditioned ones (Hedge’s g = ‐0.60, 95% CI: ‐1.17 to ‐0.04), but conditioning had minimal overall effects for animals released from captivity (Hedge’s g = ‐0.10,

22

95% CI: ‐0.44 to 0.24). Conditioning reduced movement for adults (Hedge’s g = ‐0.48, 95% CI:

‐0.87 to ‐0.08) but not juveniles (Hedge’s g = ‐0.01, 95% CI: ‐0.27 to 0.24). Movement of mammals was reduced if they were conditioned (Hedge’s g = ‐0.35, 95% CI: ‐0.69 to 0.00).

There was more uncertainty regarding the effects of conditioning on movement for reptiles

(Hedge’s g = ‐0.32, 95% CI: ‐0.82 to 0.17). We found minor evidence of publication bias from funnel plots, but the Egger’s regression test was nonsignificant (z = ‐0.37, P = 0.71). We found some potential for publication bias from the trim-and-fill analysis, which estimated four effects were missing (Fig. A5).

Site fidelity

Soft release was the only conditioning tactic used to increase site fidelity of translocated animals. There were slightly more effects for wild-to-wild translocations than captive releases

(Table 3). Most effects were from adults, two were for multiple age classes, and there was only one effect for juveniles. The largest number of effects was for reptiles, followed by mammals.

There was only one effect for birds and none for fish.

Soft released animals were 3.20 (95% CI: 1.23 to 8.34) times more likely to show site fidelity than hard released animals (Fig. 2.3). We detected no evidence of heterogeneity in the data (I2 = 0.00%). Wild-to-wild translocated animals were 3.50 (95% CI: 1.48 to 8.30) times more likely to show site fidelity when soft released than if hard released, and this was the only subgroup comparison we were able to make for the site fidelity variable. We found minor evidence of publication bias from the funnel plot, likely due to the low number of effects analyzed, but the Egger’s regression test was not significant (z = 1.37, P = 0.17). The trim-and- fill analysis estimated one effect was missing (Fig. A6).

23

DISCUSSION

Outcomes of wildlife translocations are variable but often result in failure (Fischer and

Lindenmayer, 2000; Germano and Bishop, 2009). To date, several reviews have espoused the benefits of conditioning to improve conservation translocations (Fischer and Lindenmayer, 2000;

Swaisgood, 2010; Reading et al., 2013), and some programs have seen high success, such as for recovery of black-footed ferrets (Dobson and Lyles, 2000) and whooping cranes (Grus americana) (Urbanek et al., 2010). However, a meta-analytical assessment to evaluate the efficacy of various conditioning tactics had not yet been conducted, likely due to the complicated nature of quantitatively synthesizing such a diverse literature. Put simply, the manner in which translocation programs implement conditioning varies widely between studies, making comparisons difficult. Despite these inherent difficulties, we believe there is value in making broad comparisons between translocations using conditioning tactics and those that do not. Our results provide evidence that behavioral conditioning is on average beneficial for multiple benchmarks used to gauge translocation success. Although more demonstration and validation is necessary before wide-spread adoption can be advocated, our results indicate many translocation programs would benefit from the use of one or more conditioning tactics and could help guide practitioners in choosing the most appropriate tactic for their situation.

It is encouraging our analyses revealed both “animal-focused” (antipredator training and enrichment) and “environment-focused” (soft release) conditioning tactics (Batson et al., 2015) broadly led to higher survival for translocated animals. These effects were most pronounced when practitioners released juveniles from captivity. Naïve captive-reared animals might fail to develop essential learned behaviors that could enhance survival probability once released (Einum and Fleming, 2001; Mathews et al., 2005; Reading et al., 2013). Efforts expended in promoting

24 natural behaviors appears to have positive effects on survival and is an area deserving of further attention. In contrast, conditioning had minimal effects on survival for wild-to-wild translocated animals, which commonly involve adults. Wild-to-wild translocations are often more successful than those releasing captive animals (Griffith et al., 1989; Fischer and Lindenmayer, 2000).

Captive animals thus likely receive the greatest benefits from conditioning whereas practitioners translocating wild animals might experience less payoff. Additionally, young animals might exhibit greater behavioral plasticity than adults and thus be more likely to respond and change their behaviors in response to conditioning stimuli, whereas adults (particularly wild adults) might have more fixed behaviors. In short, practitioners could have the ability to condition beneficial behaviors for captive-reared juveniles whereas translocation of adults should focus on preventing degradation of behavior if animals are held in captivity for long periods (DeGregorio et al., 2017).

Translocated animals often make large, erratic movements as they orient to their new surroundings or attempt to home to their capture locations (Swaisgood, 2010). Our results support the idea that soft release via temporary penning is effective for confining wild adults until they develop some affinity for the release site and thus are less likely to make large movements upon release. We did not find consistent effects of soft release on the movements of captive or juvenile animals. This approach might not have as much impact on these individuals because they generally do not have established home ranges like many wild adults do and are thus likely not attempting to home. Soft release also had the desired effect of increasing site fidelity. The influence of conditioning on site fidelity had the lowest number of effects across our datasets, so this is a focal area that deserves further investigation.

Our results also suggest the effectiveness of conditioning varies by taxon. We found the

25 most pronounced effects of conditioning enhancing survival in fish, with somewhat less overall survival benefits observed for reptiles and mammals. We suggest the narrower confidence intervals around observed effect sizes for fish is a result of greater sample sizes. In our datasets, studies translocating birds, mammals, or reptiles generally released tens of animals, whereas most fish studies released hundreds or thousands of individuals. Translocating large numbers of animals has been associated with success (Wolf et al., 1998; Fischer and Lindenmayer, 2000), so releasing large groups of conditioned fish could have greater relative advantages compared to other vertebrate taxa. Conditioning had the most variable effects on survival for birds, demonstrating that avian translocation programs broadly need refinements to conditioning protocols. There have been several successes involving conditioning of birds, as has been demonstrated for Andean condors (Vultur gryphus) (Wallace and Temple, 1987), California condors (Gymnogyps californianus) (Meretsky et al., 2000), whooping and sandhill cranes

(Antigone canadensis) (Duff et al., 2001; Urbanek et al., 2010), and Puerto Rican parrots

(Amazona vittata) (White et al., 2005). These programs frequently rely upon time- and labor- intensive protocols, but more passive conditioning is unlikely to be successful for many bird translocations. Conditioning was effective for reducing movement of mammals but less so for reptiles. Limited evidence suggests lengthier conditioning programs might be most beneficial for reptiles. For example, soft release can be effective at reducing movement if reptiles are held in pens for several months to upwards of a year or more (Tuberville et al., 2005; Knox and Monks,

2014; Knox et al., 2017).

The limited number of studies we were able to include in our analyses compared to myriad conducted translocations raises two important points: First, our findings demonstrate the broad benefits of behavioral conditioning, so more research using these and other tactics (sensu

26

Batson et al., 2015) is needed. Translocations are most frequently conducted using imperiled or game species, but future studies using common species would allow practitioners to experimentally assess the effects of these tactics in a scientifically rigorous manner, which could aid in refining our ability to successfully implement them. Second, several studies did experimentally implement the conditioning tactics we evaluated but reported insufficient information for inclusion in meta-analysis. We urge authors to report necessary information so analyses such as ours can be updated as more studies are conducted (Moseby et al., 2014;

Nakagawa et al., 2017). Additionally, although translocations are diverse in their design and execution, adhering to existing frameworks such as the Translocation Tactics Classification

System could facilitate standardization of tactics and thus data collation in future meta-analyses

(Batson et al., 2015).

We identified several gaps in the literature that limit our ability to broadly evaluate the effects of behavioral conditioning on translocation success. We are unaware of or were unable to include experimental studies that used antipredator training for reptiles (antipredator training experiments were generally scant), translocations of conditioned adult fish, wild-to-wild translocations of conditioned fish, or wild-to-wild translocations comparing solely conditioned and unconditioned juveniles. Conditioning experiments with amphibians and invertebrates are also deficient despite potential for propagation of large release cohorts, similar to fish. We also found geographic gaps in the literature, with very few studies reported from Asia or South

America. We do not suspect this biased the generality of our findings, but practitioners conducting translocations in these areas might consider increased adoption of conditioning experiments. Finally, based on available literature we were limited to using short-term success measures in our analyses. Post-release monitoring lasted 157 days on average and ranged from

27

10 to 825 days across studies. Establishment and persistence of a translocated population without the need for continued human support is the ultimate goal of translocations (Seddon, 1999), so more long-term studies on the effects of conditioning on translocation success are needed.

The potential conservation value of wildlife translocations is great, but they can be risky and high-cost endeavors (Kleiman, 1989; Fischer and Lindenmayer, 2000). Lengthy and labor- intensive conditioning programs can also be very costly yet provide minimal benefits, as was found for initial pre-release conditioning efforts for reintroduction of golden lion tamarins

(Leontopithecus rosalia) (Kleiman et al., 1991). Thus, deciding whether to incorporate a conditioning component could be an important consideration when designing translocation programs. Our meta-analyses suggest conditioning animals with any of the tactics we investigated has overall positive effects on survival, particularly for captive juveniles. Soft release might be the most effective tactic for reducing movement for wild-to-wild translocated adults. Soft release also appears useful for increasing site fidelity, but more experiments are needed to corroborate the limited number of studies we analyzed. Although environmental enrichment had minimal effects on movement, coupling enrichment with soft release could be an effective tactic for reducing movement and improving survival of animals released from captivity. Conditioning fish, reptiles, and mammals is generally effective, but behavioral conditioning associated with avian translocations needs refinement. Although our results show broad positive effects of behavioral conditioning on translocations, we emphasize previous authors in that a “one size fits all” approach does not apply for translocations given their system- specific attributes (Moseby et al., 2014). Nevertheless, we encourage continued conditioning experiments and adaptive management (Armstrong et al., 2007) to further illuminate how improved tactics could lead to even greater success in future efforts.

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TABLES AND FIGURES

Table 2.1. Contingency tables showing the distribution of effects for survival of conditioned and unconditioned translocated animals based on age class, taxonomic group, conditioning tactic, and translocation type. NA refers to nonsensical effects (e.g., wild-to-wild translocations would not use environmental enrichment since this conditioning occurs in captivity).

Taxa Age class Actinopterygii Aves Mammalia Reptilia adult 0 6 15 2 juvenile 18 7 3 4 multiple 0 2 2 0

Age class Conditioning tactic adult juvenile multiple antipredator training 2 4 0 soft release 19 15 4 environmental enrichment 2 13 0

Taxa Conditioning tactic Actinopterygii Aves Mammalia Reptilia antipredator training 1 4 1 0 soft release 8 10 17 3 environmental enrichment 9 1 2 3

Translocation type Conditioning tactic captive release wild-to-wild antipredator training 5 1 soft release 23 15 environmental enrichment 15 NA

Age class Translocation type adult juvenile multiple captive release 10 32 1 wild-to-wild 13 0 3

Taxa Translocation type Actinopterygii Aves Mammalia Reptilia captive release 18 10 10 5 wild-to-wild 0 5 10 1

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Table 2.2. Contingency tables showing the distribution of effects for movement of conditioned and unconditioned translocated animals based on age class, taxonomic group, conditioning tactic, and translocation type. NA refers to nonsensical effects (e.g., wild-to-wild translocations would not use environmental enrichment since this conditioning type occurs in captivity).

Taxa Age class Actinopterygii Aves Mammalia Reptilia adult 0 1 10 11 juvenile 4 0 0 12 multiple 0 0 1 2

Age class Conditioning tactic adult juvenile multiple soft release 18 6 3 environmental enrichment 3 10 0

Taxa Conditioning tactic Actinopterygii Aves Mammalia Reptilia soft release 0 1 10 16 environmental enrichment 4 0 0 9

Translocation type Conditioning tactic captive release wild-to-wild soft release 11 16 environmental enrichment 13 NA

Age class Translocation type adult juvenile multiple captive release 8 16 0 wild-to-wild 14 0 3

Taxa Translocation type Actinopterygii Aves Mammalia Reptilia captive release 4 1 3 16 wild-to-wild 0 0 8 9

30

Table 2.3. Contingency tables showing the distribution of effects for site fidelity of conditioned and unconditioned translocated animals based on age class, taxonomic group, and translocation type. Only soft release was used as a conditioning tactic.

Taxa Age class Aves Mammalia Reptilia adult 1 2 2 juvenile 0 0 1 multiple 0 0 2

Age class Translocation type adult juvenile multiple captive release 2 1 0 wild-to-wild 3 0 2

Taxa Translocation type Aves Mammalia Reptilia captive release 1 1 1 wild-to-wild 0 1 4

31

Figure 2.1. Forest plot of all effects (n = 59) from 36 studies included in meta-analysis of survival for conditioned and unconditioned translocated animals. Points represent odds ratios and lines are 95% confidence intervals (CI) for study-level effects. The diamond represents the overall estimated odds ratio and 95% CI showing the odds of survival was higher for conditioned animals on average.

32

Figure 2.2. Forest plot of all effects (n = 41) from 18 studies included in meta-analysis of movement for conditioned and unconditioned translocated animals. Points represent standardized mean difference (Hedge’s g) and lines are 95% confidence intervals (CI) for study-level effects. The diamond represents the overall estimated standardized mean difference and 95% CI showing conditioned animals moved less than unconditioned ones on average.

33

Figure 2.3. Forest plot of all effects (n = 8) from six studies included in meta-analysis of site fidelity for soft released relative to hard released translocated animals. Points represent odds ratios (OR) and lines are 95% confidence intervals (CI) for study-level effects. The diamond represents the overall estimated OR and 95% CI showing the odds of exhibiting site fidelity was higher for soft released animals on average.

34

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CHAPTER 3: CAPTIVE-REARED JUVENILE BOX TURTLES INNATELY PREFER

NATURALISTIC HABITAT: IMPLICATIONS FOR TRANSLOCATION2

ABSTRACT

Habitat choice has broad repercussions for animals, but mechanisms influencing such choices are generally not understood. When conducting conservation translocations using captive-reared animals, elucidating mechanisms influencing habitat preference pre-release could inform rearing methods and post-release behavior. We raised 32 captive-born eastern box turtles

(Terrapene carolina) for eight months—16 in naturalistic enriched enclosures and 16 in unenriched enclosures. We then let each turtle choose between environments simulating both rearing conditions in a novel enclosure to evaluate the hypothesis that rearing environment influences habitat selection. Preference for the enriched environment was exhibited by enriched

(selection probability = 0.81, 95% CI: 0.54–0.96) and unenriched (selection probability = 0.88,

95% CI: 0.62–0.98) turtles. Those that chose the enriched habitat (n = 27) did so in less than half the time ( = 504.8 sec, 95% CI: 266.49–743.04) than those that selected the unenriched one (n =

5, = 1339.0 sec, 95% CI: 783.84–1894.07, P = 0.009). Selection latency did not differ between rearing treatments (P = 0.871). We conducted a second experiment to clarify if preferences were based on familiarity or novelty by using the same enclosure, but we gave individuals a choice between the habitat representing their rearing condition and a novel object (empty tissue box).

Preference for respective rearing environment was exhibited by enriched (selection probability =

0.87, 95% CI: 0.59–0.98) and unenriched (selection probability = 0.81, 95% CI: 0.54–0.96) individuals. Selection latency did not differ based on side chosen (P = 0.439)

2This chapter has been published in Applied Animal Behaviour Science. Full citation: Tetzlaff, S.J., J.H. Sperry, and B.A. DeGregorio. 2018. Captive-reared juvenile box turtles innately prefer naturalistic habitat: implications for translocation. Applied Animal Behaviour Science 204: 128–133.

44 or rearing treatment (P = 0.764). Selection latency was repeatable by individuals (R = 0.47, 90%

CI = 0.24– 0.71, P = 0.003), and this personality trait may be heritable since it was also repeatable by clutch mates (R = 0.36, 90% CI = 0.03–0.62, P = 0.001). Preference for a naturalistic environment by captive-born eastern box turtles appears to be driven by an innate mechanism rather than based on prior experience. Housing this species in enriched enclosures should increase welfare. Moreover, our collective findings highlight the importance of evaluating how intrinsic effects could affect translocation outcomes.

INTRODUCTION

An animal’s choice of habitat has broad repercussions for survival and fitness

(Rosenzweig, 1981). Despite this importance, the mechanisms influencing habitat choice are generally not understood. Multiple hypotheses may explain why animals prefer certain habitats.

For example, early experience of young individuals may affect later preference in that they seek to occupy habitats similar to their rearing environment (Immelmann, 1975; Teuschl et al., 1998).

Furthermore, selection of familiar habitat may confer a performance advantage relative to novel ones (Aubret and Shine, 2008). Alternatively, habitat preference may be innate (Partridge, 1974).

In such instances, individuals may choose habitats based on the presence of particular sensory cues despite never having been exposed to them (Dixson et al., 2008). We tested these two competing hypotheses of habitat preference (experiential vs. innate) in the context of informing wildlife translocations using a cohort of captive-reared juvenile eastern box turtles (Terrapene carolina).

Understanding why animals prefer certain habitats could be useful for designing more biologically realistic studies on laboratory animals (Arnold and Estep, 1994) or for informing conservation programs where captive-reared individuals are released into nature. These “head-

45 starting” efforts often have low success rates, and an inability of translocated animals to discover, recognize, and settle in suitable habitat post-release is one prevailing reason for failure

(Fischer and Lindenmayer, 2000). If individuals search for and occupy habitats most similar to those they were reared in (Stamps and Swaisgood, 2007), substantial effort should be made to ensure rearing environments adequately mirror suitable habitat at the release site (Reading et al.,

2013). This approach could condition animals to occupy appropriate habitat upon release and increase survival and reproductive rates of released individuals.

Providing naturalistic enclosures to promote welfare has been utilized by animal caretakers for decades (Sheperdson et al., 1998). This practice is a form of environmental enrichment and is becoming progressively adopted by conservation biologists as a means of increasing translocation success (Reading et al., 2013). However, post-release habitat selection of conspecifics exposed to enriched captive environments may not differ from those subjected to unenriched environments (e.g., Roe et al., 2015). One underlying reason for this is that innate habitat preference may supersede preference for familiar habitat. Currently, it is unknown if most captive animals actively prefer familiar habitats (i.e., those resembling their captive environments) or have innate preference for particular aspects of a habitat. Experimental manipulation of captive-reared animals should help elucidate such trends and inform management. Previous work demonstrated adult eastern box turtles prefer an enriched environment over an unenriched one (Case et al., 2005), but how prior captive conditions affect preference has not been considered.

Additionally, although preference for and performance within particular environments can be tightly linked with rearing environment (Aubret and Shine, 2008), individual variation in behavior (i.e., personality) can also be influential. Measuring individual consistency (i.e.,

46 repeatability) of a given behavior is a popular method for quantifying animal personality (Bell et al., 2009). Recent work demonstrated free-ranging adult eastern box turtles exhibit personality via individual consistency in boldness (Kashon and Carlson, 2018), but it is unknown how rearing condition or genetic mechanisms might affect its expression for captive individuals.

By raising naïve animals with known histories in controlled settings, one can remove the confounding effects of prior experience and explore how factors such as environment, personality, and heritability affect habitat choice (Mason et al., 2013). We tested how these effects impact habitat preference of eastern box turtles raised in either an enriched or unenriched environment. We hypothesized preference is influenced by rearing environment and predicted turtles would choose a habitat simulating the one they were raised in when provided an alternate choice. However, if habitat preference is innate within this species, we expect that turtles would prefer to occupy a naturalistic habitat as opposed to a more simplistic one regardless of rearing experience. Given the mental and physical benefits of enrichment (Burghardt, 2013), we also anticipated turtles reared in enriched environments would take less time to choose a habitat than those reared in unenriched environments. Finally, we used repeated measurements of habitat selection latency to test the hypotheses that captive-reared juveniles exhibit personalities that are influenced by heritability.

METHODS

This research was conducted under an approved protocol by the University of Illinois

Institutional Animal Care and Use Committee (#16017) and Scientific Collector’s Permits granted by the States of Michigan and Illinois (#NH17.5980).

Study species

Eastern box turtles are long-lived (regularly >50 years) reptiles that inhabit temperate and

47 subtropical regions of the eastern United States. Much less is known about juveniles compared to adults, but eastern box turtles are primarily terrestrial and occupy forests with shrubby and herbaceous understories (Dodd, 2001). Due to population declines resulting from habitat loss, road mortality, intense predation (particularly of nests and juveniles), and collection for the pet trade, the species is listed as Vulnerable by the IUCN and is included in CITES Appendix II (van

Dijk, 2011).

Animal acquisition and rearing conditions

We collected eastern box turtle eggs in-situ by excavating recently-laid nests from 4–17

June 2016 at Fort Custer Training Center (FCTC), an Army National Guard training facility located near Battle Creek, Michigan, USA. Although this species can lay more than one clutch per year in some populations (Dodd, 2001), our short duration of egg collection excluded the possibility of collecting more than one clutch from a given female. After collection, we used graphite pencils to assign a clutch and within-clutch ID to each egg (e.g., 1.1). We incubated eggs indoors at FCTC at a constant temperature of 26.7 oC in egg incubators (Hova-Bator, Model

1602N; GQF Manufacturing Company Inc., Savannah, Georgia, USA). We incubated eggs grouped by clutch ID on incubation medium (HatchRite incubation bedding) in plastic deli cups.

Larger clutches were split between deli cups so that no more than four eggs occupied a cup. We hatched 32 turtles from 11 clutches of eggs. Once eggs began hatching, we allowed neonates to remain in the incubator until they fully emerged from their egg (~48 hrs).

We transported neonates to a greenhouse on the University of Illinois at Urbana-

Champaign campus for rearing. We initially housed hatchlings individually in 60.3 cm long x

42.2 cm wide x 27.9 cm tall transparent plastic tubs with an 11.5 cm long x 8.5 cm wide x 8 cm tall plastic hide box, reptile cage carpet (Zoo Med Eco Carpet; Zoo Med Laboratories, Inc., San

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Luis Obispo, California, USA), and a shallow food bowl. We kept these tubs on a slight angle to hold fresh standing water (ca. 4 cm deep) in the lower end for drinking and soaking. We offered each turtle commercially purchased live blackworms (Lumbriculus variegatus) and canned cat food at least two times daily until they began reliably eating. Once we confirmed that each turtle was healthy and regularly eating (less than two weeks after hatching), we randomly assigned each turtle to an enriched or unenriched environment.

We raised 16 enriched turtles in naturalistic enclosures and 16 unenriched turtles in comparably simplistic enclosures (Fig. 3.1). Unenriched turtles were kept individually in enclosures consisting of the housing methods described above, but once these individuals were two months old we added a ca. 42 x 42 cm piece of plastic shelf liner to rest on the cage carpet at the high end of each tub to provide a dry area. Enriched turtles were housed in groups of four in

132 cm long x 79 cm wide x 30 cm deep Rubbermaid® stock tanks. We designed these enclosures to reasonably mimic natural habitat, consisting of structural features functionally similar to those commonly used by wild box turtles. Each enclosure had substrate of ca. 6 cm deep of coconut fiber (Zoo Med Eco Earth®) to promote digging and burrowing, shrubby and herbaceous artificial plants, sphagnum moss, two half logs (Zoo Med Habba Hut) for additional hiding places, and two naturalistic shallow rock water dishes. We should note that although enriched turtles had opportunities for social interactions that unenriched turtles did not, we considered this as a general component of environmental enrichment, which likely did not affect our results given the typical asocial behavior of box turtles (Dodd, 2001).

We offered the same amount of food per turtle at each feeding. All individuals were fed the same diet, which initially consisted of live blackworms and mealworms (Tenebrio molitor).

We transitioned turtles to live superworms (Zophobas morio) and then solely to live redworms

49

(Eisenia foetida) after several months. We also offered fresh mixed greens (excluding spinach) and Zoo Med Gourmet Box Turtle Food—a commercial diet consisting of pellets and dehydrated mealworms, strawberries, and mushrooms. Unenriched turtles were provided food on 10 cm diameter petri dishes that were placed in the same spot within each tub at each feeding. Enriched turtles were offered fresh greens and the commercial diet on naturalistic shallow food dishes, but we also scattered those items and live food throughout their enclosures to promote active foraging. Turtles were offered fresh food five days per week, and we dusted food with calcium powder three days per week. We also provided enriched turtles with cuttlebones to chew on.

Fresh water was provided ad libitum. We provided all turtles with UVB light daily from 0630–

1630 hours using Zoo Med ReptiSun® 5.0 bulbs. We affixed a 25 cm diameter light hood with a

13 W compact fluorescent bulb to each enriched tank. We hung a continuous row of 117 cm fluorescent linear tubes housed in fixtures (Zoo Med T5 High Output Terrarium Hood) spanning the lower (wet) ends of the rows of unenriched tubs. Being raised in a greenhouse, all turtles were exposed to the same natural photoperiod that varied seasonally in addition to receiving artificial light. Similarly, temperature inevitably fluctuated on a daily and seasonal basis, but we attempted to regulate ambient temperature in the greenhouse between 21–29 oC.

General testing procedures

We tested the behavior of each turtle in the greenhouse where they were reared when eight months old in April 2017. We tested turtles individually in a 60 cm long x 42 cm wide x

16.5 cm tall transparent plastic bin (hereafter “enclosure”). We placed solid white plastic covers around the outside and under the enclosure to reduce external stimuli. We recorded all trials using a video camera (Lorex LNB3163B bullet surveillance camera; Lorex Technology, Inc.,

Markham, ON, Canada) mounted above the enclosure. An observer, situated out of view of the

50 turtles, recorded data while watching trials on a television monitor (Eyoyo 20.32 cm portable

HDMI LCD monitor; Shenzhenshi cai hui Technology Co., Ltd, Shenzhen, China). Only one observer (SJT) conducted trials to eliminate interobserver variability. We ensured intraobserver reliability by comparing response variables measured during experiments to recorded videos after trials concluded. We placed each individual back in its primary enclosure immediately after a trial was completed. Between trials, we rinsed the enclosure with hot water and switched which side of the enclosure cues were presented. We conducted tests only during 1000–1500 hours since box turtles are predominantly diurnal (Dodd, 2001).

Experiment 1: Choice between enriched and unenriched habitat

This experiment tested which habitat turtles would select when given a choice between an enriched or unenriched habitat. A 12 x 42 cm rectangular area at each end of the enclosure constituted the space considered selected. The remaining 36 x 42 cm space in the mid-portion was barren (Fig. 3.2). The unenriched side of the enclosure simulated the environment unenriched turtles were reared in and consisted of the same carpet and a plastic hide box. The habitat on the opposite end simulated the enriched rearing condition consisting of coconut fiber substrate, artificial herbaceous plants, and sphagnum moss. A turtle was placed in the center of the enclosure perpendicular to the habitats so it had equal opportunity to see each side before making a selection. We recorded the choice each turtle made (a move into the enriched side, the unenriched side, or no choice) and the latency (seconds) to select a side using a stopwatch. An individual was considered to have selected a side of the enclosure once its entire body crossed the threshold of barren territory to one of the two habitats. Individuals, which did not move into either habitat within one hour were considered to not have made a selection and excluded from analyses.

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Experiment 2: Choice between rearing environment and novel object

We conducted a second experiment to explore if preferences were based on familiarity or novelty. Using the same enclosure, we gave individuals a choice between the habitat representing their rearing condition (hereafter “home” when referring to this experiment) or an object novel to all turtles: a 10 cm long x 22.5 cm wide x 12 cm tall empty cardboard tissue box

(hereafter “box”). The box had a 12 cm wide x 10 cm tall opening, which the turtles could easily enter. Similar objects are commonly used as refuges for captive reptiles. We considered a choice made when the turtle’s entire body crossed the threshold into one of the two sides.

Data analyses

We conducted statistical analyses using R version 3.3 (R Core Team, 2016). For both

Experiments 1 and 2, we used exact binomial tests to calculate within-treatment probability and

95% confidence interval (CI) of turtles selecting a given habitat. To determine if habitat selection within-treatments was non-random (i.e., preferences existed); we set hypothesized probability of choice as 0.5. We used linear models to test if latency (sec) to make a choice was dependent on rearing treatment or the side of the enclosure selected. We initially tested these as an interactive effect but found no meaningful interaction (the 95% CI of the parameter estimate [β] considerably overlapped zero), so we tested them as main effects. We examined Q-Q plots to ensure residuals approximately conformed to a normal distribution and assessed equality of variances by examining boxplots based on Brown-Forsyth tests using the HH package

(Heiberger, 2017); we detected no violations of assumptions. If we found evidence suggesting selection latency differed between treatments or based on the side selected, we compared estimated marginal (least squares) means and 95% confidence limits using the lsmeans package

(Lenth, 2016).

52

A behavior is considered repeatable if within-individual variance is low compared to high among-individual variance (Bell et al., 2009). We calculated a repeatability estimate (R) for selection latency of individual turtles with a linear mixed-effects model assuming a Gaussian distributed error using the rptR package (Stoffel et al., 2017). Values of R range from 0–1, where increasing values suggest stronger repeatability. We controlled for rearing treatment and trial order (Experiment 1 or 2) by using these variables as fixed effects with individual turtle identity as a random effect. We quantified uncertainty (90% confidence limits) in R based on 1,500 iterations of parametric bootstrapping. Because this modeling framework in general describes the proportion of among-group variance compared to the total sample variance, we also tested if selection latency was repeatable by turtles from the same clutch using the same fixed effects and bootstrapping procedure described above, but we used clutch identity as a random effect.

RESULTS

Choice between enriched and unenriched habitat

All 32 turtles made a choice within one hour. Thirteen enriched turtles chose the enriched side of the enclosure and three chose the unenriched side. The probability of enriched turtles choosing enriched habitat differed from 0.5 (P = 0.021; Fig. 3.3). Two unenriched turtles chose the unenriched side and 14 chose the enriched side. The probability of unenriched turtles choosing unenriched habitat differed from 0.5 (P = 0.004; Fig. 3.3). Latency to select a side of the enclosure did not differ between rearing treatments (Table 3.1). Turtles that chose the enriched side did so in less time than those that selected the unenriched side (Table 3.1; Fig. 3.4).

Choice between rearing environment and novel object

One enriched turtle did not select a habitat within the one-hour time limit, but all unenriched individuals made a choice. Thirteen enriched turtles chose home and two chose the

53 box. The probability of enriched turtles choosing home differed from 0.5 (P = 0.007; Fig. 3.5).

Thirteen unenriched turtles chose home and three chose the box. The probability of unenriched turtles choosing home also differed from 0.5 (P = 0.021; Fig. 3.5). Turtles did not differ in selection latency based on rearing treatment or the habitat selected (Table 3.1).

Repeatability of behavior

Selection latency was repeatable by the 31 individual turtles that made a choice in both trials (R = 0.47, 90% CI: 0.23–0.70, P = 0.003). This behavior was also repeatable by turtles from the same clutch (R = 0.36, 90% CI: 0.03–0.61, P = 0.001), but we restricted this analysis to

26 individuals from six clutches because five turtles came from single-egg clutches and had no siblings for comparison. We conducted these analyses without including rearing treatment and trial order as fixed effects (i.e., unadjusted repeatability estimates) and observed no qualitative differences to these results.

DISCUSSION

Our findings suggest captive-born juvenile eastern box turtles prefer to occupy naturalistic, complex environments relative to simplistic ones, and even turtles, which had experienced only an unenriched enclosure predominantly chose the enriched environment. These results lend support to the hypothesis that this species has an innate habitat selection mechanism rather than based on prior experience. Similar innate behavior has been observed for other animals. For instance, captive-raised juvenile coal tits (Periparus ater) and blue tits (Cyanistes caeruleus) with no experience of vegetation demonstrated innate preference for the vegetation type each respective species commonly selects in nature (Partridge, 1974). In an experiment similar to ours, adult eastern box turtles spent over 90% of their time in an enriched environment when given the choice between enriched and unenriched sides of an enclosure, regardless of

54 previous short-term housing condition (Case et al., 2005). However, the prior experiences of turtles used in that study were not entirely known, like they were for our captive-born turtles.

Most turtles selected their rearing habitat over a novel object, supporting our conclusion that preference for the enriched habitat (by unenriched turtles in particular) was not based on neophilia. This somewhat contrasts with results from a similar study on birds. Raach and Leisler

(1989) reared moustached warblers (Acrocephalus rnelanopogon) in either an enriched or unenriched condition and found enriched birds preferred their rearing environment when both were simultaneously presented, but birds reared in unenriched conditions were indifferent in their preference. The authors assumed this was due to some unenriched individuals exhibiting neophobia towards the enriched habitat. Thus, display of innate habitat preference as opposed to that based on previous experience likely varies taxonomically, and may have implications for wildlife conservation translocations.

Understanding how captive-rearing conditions affects both intra (Aubret and Shine,

2008) and interspecific variation in developmental and behavioral plasticity could be informative for head-starting efforts. For some species, environmental enrichment may be a valuable tool for positively influencing post-release behavior (Reading et al., 2013). However, the returns for others may be rather limited if animals have innate preference for particular habitats that cannot be shaped by captive-rearing conditions. Regardless, addressing basic animal welfare concerns during captive-rearing can be easily accomplished by conducting such experimental assessments of captive habitat preference (Case et al., 2005).

Although there is ample evidence demonstrating the positive effects of environmental enrichment for captive animals (Sheperdson et al., 1998), many zoo, research, and pet animals are housed in simplistic conditions for ease of maintenance, cost-effectiveness, or the perceived

55 health benefits of a sterile environment. “Paper and plastic husbandry” is a common practice when keeping captive reptiles, whether permanently housed in captivity (Burghardt, 2013) or utilized in head-starting programs (e.g., King and Standford, 2006). It is clear that enriched housing generally benefits reptiles (Burghardt, 2013), and unenriched conditions may promote behavioral deprivation (Dawkins, 1988). Translocation candidates raised in these environments may not be able to express desired behaviors (Hughes and Duncan, 1988) that may be critical to survival or reproduction upon release. Our findings thus represent an opportunity for caregivers to promote welfare by allowing animals to express preferences (Arnold and Estep, 1994).

The fact our captive-reared turtles, particularly those never exposed to vegetation or natural substrate, predominantly chose to occupy complex habitat has promise for these individuals making appropriate habitat use decisions post-release, which could benefit survival.

Having no known parental care, juvenile box turtles likely best avoid predation from a staggering number of predators by spending much of their time in microhabitats favoring crypsis, such as dense vegetation (Dodd, 2001). Furthermore, turtles that chose the enriched habitat did so in less time than those that chose the unenriched habitat, suggesting the presentation of this environment triggered a prompt selection response. Because turtles were fully exposed when placed in the center of the enclosure at the start of trials, rapidly retreating to complex cover could further benefit predator avoidance (Dodd, 2001). Vegetative and substrate structure appeared to serve as the predominant visual cues for turtles to select the enriched habitat since visual and olfactory cues from other potential attractants such as food or water were absent.

We cannot rule out social enrichment as a factor contributing to our results since there was potential for enriched turtles to interact with conspecifics during captive-rearing whereas unenriched turtles were housed individually. The impacts of social interaction on vertebrate

56 development and behavior has been well-studied (e.g., Dreosti et al., 2015). However, because we did not detect an effect of treatment on behavior and conspecific cues were not present in our experimental trials, it is unlikely that social stimulation of enriched turtles substantially influenced our results. Furthermore, box turtles are generally regarded as asocial (Dodd, 2001) and, anecdotally, enriched individuals were rarely documented interacting in their rearing tubs.

The repeatability of selection latency by turtles demonstrates personality can be apparent early in life, and this trait may be heritable since it was repeatable by turtles from the same clutch. These individuals will eventually be released into the wild and monitored to gauge translocation success. Previous work on translocated snakes subjected to similar captive treatments as our turtles showed no effect of enrichment (DeGregorio et al., 2017; Roe et al.,

2015). If we do not observe discernable differences in post-release behavior and survival between enriched and unenriched turtles, this might suggest innate preferences and variability in temperament could broadly be important for translocation outcomes (Merrick and Koprowski,

2017) and more influential than extrinsic factors such as holding environment. Despite accumulating evidence showing reptiles exhibit personalities (Waters et al., 2017), relating this to translocation outcomes has only been recently assessed for turtles and for a single species

(Germano et al., 2017; Steele, 2017). Further investigation regarding how rearing and release techniques influence behavioral types (Watters and Meehan, 2007) and if this affects translocation success is warranted.

CONCLUSION

Juvenile eastern box turtles preferred a naturalistic enriched habitat over a simplistic, unenriched one in experimental trials regardless if they had been raised in an enriched or unenriched environment. This suggests these turtles conform to an innate habitat selection

57 mechanism as opposed to one driven by previous experience. Therefore, rearing this species in enriched enclosures consisting of naturalistic features should benefit welfare by satisfying this instinctive desire to occupy complex habitat. Providing such enclosures to head-starting candidates may better prepare them for exhibiting behaviors critical to survival once released into natural habitat, but the inherent behavior we observed should also be considered when conducting wildlife conservation translocations.

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TABLE AND FIGURES

Table 3.1. Estimated mean difference (β) with 95% lower (LCL) and upper (UCL) confidence limits of habitat selection latency for juvenile eastern box turtles (Terrapene carolina) based on rearing treatment or habitat selected in an experimental enclosure. The unenriched group was the reference variable when testing for rearing treatment differences. When testing for differences based on the habitat selected, the unenriched side was the reference variable in Experiment 1 and the home side was the reference variable in Experiment 2. Test statistics (t) and P-values are also shown.

Experiment Parameter β LCL UCL t P

1 rearing treatment 35.29 -403.83 474.40 0.16 0.871

1 habitat selected 834.19 229.50 1438.87 2.82 0.009

2 rearing treatment 94.83 -546.29 735.95 0.30 0.764

2 habitat selected 334.20 -536.91 1205.31 0.79 0.439

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Figure 3.1. Rearing conditions for enriched (left) and unenriched (right) juvenile eastern box turtles (Terrapene carolina).

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Figure 3.2. Schematic of experimental apparatus (not drawn to scale) used to test habitat selection by juvenile eastern box turtles (Terrapene carolina). The 12 x 42 cm area on each end of the enclosure contained a habitat representing each rearing condition (enriched or unenriched) in Experiment 1. In Experiment 2, one end contained a novel object and the opposite end contained a habitat representing a given turtle’s rearing condition. There were no materials present in the 36 x 42 cm mid-portion. A turtle had to completely cross the threshold simulated by the dotted vertical lines to have been considered selecting a habitat.

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Figure 3.3. Probability with 95% confidence interval of enriched (n = 16) and unenriched (n = 16) juvenile eastern box turtles (Terrapene carolina) selecting an enriched or unenriched side of an experimental enclosure when simultaneously presented.

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Figure 3.4. Mean latency (sec) with 95% confidence limits of juvenile eastern box turtles (Terrapene carolina) to select either an enriched (filled circle, n = 27) or unenriched (filled triangle, n = 5) habitat simulating each rearing condition in an experimental enclosure.

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Figure 3.5. Probability with 95% confidence interval of enriched (n = 15) and unenriched (n = 16) juvenile eastern box turtles (Terrapene carolina) selecting either a novel object (empty tissue box) or an environment representing their respective rearing condition (home) when simultaneously presented in an experimental enclosure.

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CHAPTER 4: TRADEOFFS WITH GROWTH AND BEHAVIOR FOR CAPTIVE BOX

TURTLES HEAD-STARTED WITH ENVIRONMENTAL ENRICHMENT3

ABSTRACT

Head-starting is a conservation strategy that entails releasing captive-reared animals into nature at sizes large enough to better resist post-release predation. However, efforts to maximize growth in captivity may jeopardize development of beneficial behaviors. Environmental enrichment can encourage natural behaviors before release but potentially comes with a tradeoff of reduced growth in complex enclosures. We compared growth and behavior of enriched and unenriched captive-born juvenile box turtles (Terrapene carolina). Enriched turtles grew slower than unenriched turtles during the first eight months in captivity, although growth rates did not differ between treatments from 9–20 months old. After five months, post-hatching, unenriched turtles became and remained larger overall than enriched turtles. During two foraging tasks, unenriched turtles consumed more novel prey than enriched turtles. In a predator recognition test, eight-month-old enriched turtles avoided raccoon (Procyon lotor) urine more than unenriched turtles of the same age, but this difference was not apparent one year later. The odds of turtles emerging from a shelter did not differ between treatments regardless of age. Although our results suggest turtles raised in unenriched environments initially grew faster and obtained larger overall sizes than those in enriched conditions, tradeoffs with ecologically-relevant behaviors were either absent or conditional.

3This chapter has been published in the Special Issue “Advances in the Biology and Conservation of Turtles” in Diversity. Full citation: Tetzlaff, S.J., J.H. Sperry, and B.A. DeGregorio. 2019. Tradeoffs with growth and behavior for captive box turtles head-started with environmental enrichment. Diversity 11: 40.

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INTRODUCTION

Head-starting, a practice that involves captive-rearing animals for release into nature, is a popular approach for augmenting or reintroducing imperiled wildlife populations. A common focus in these programs involves rapidly growing individuals to reduce susceptibility to predation or be closer to reproductive age or size upon release [1,2,3]. However, emphasis on rapid growth may have undesirable consequences, such as the degradation or loss of normal behaviors. Behavior may be especially compromised the longer individuals are kept in captivity

[4,5]. Empirical evidence is needed to explore if maximizing growth does indeed come at the cost of beneficial behaviors, which could reduce head-starting success. We investigated this potential tradeoff by comparing growth and behavior of environmentally enriched and unenriched captive-reared juvenile eastern box turtles (Terrapene carolina).

Head-started animals are often raised in simplistic enclosures, which can be cost- efficient, are relatively easy to maintain, and allow caretakers to closely monitor food intake.

With easy access to food, individuals housed in such conditions often experience rapid growth

[6]. However, because captivity can have detrimental effects on development, behavior of captive animals often differs from wild-bred conspecifics [5]. Such findings have implications for outcomes of wildlife translocations using captive animals. For instance, carnivores translocated from captivity may have reduced ability to capture prey compared to those directly translocated from the wild [7]; suggesting captive animals are often unprepared for life in nature.

Practitioners attempting to promote ecologically relevant behaviors and improve welfare for head-started animals have incorporated environmental enrichment into rearing protocols, such as raising animals in enclosures with naturalistic features simulating release sites, providing foraging opportunities like those experienced in nature, and communally housing conspecifics to

70 promote social skills [8,9,10,11]. Assessing behavior of head-started animals prior to release often suggests enrichment can prepare animals to succeed in post-release environments [12,13].

A potential conflict with using environmental enrichment to condition adaptive behavior is that it may come at the cost of accelerated growth rates in captivity. If the enrichment regime requires animals to search for spatially variable food in structurally complex enclosures and/or compete for food with conspecifics, growth may be reduced compared to unenriched individuals

[14]. Animals in complex enclosures may simply also choose to spend more time hidden or exploring their surroundings and less time eating than conspecifics raised in simplistic enclosures. Thus, maximizing growth and promoting ecologically relevant behaviors could be mutually exclusive goals, presenting potentially conflicting rationale for deciding how individuals should be reared.

Tradeoffs between growth and behavior may be particularly relevant to turtle head- starting programs due to the life-history attributes of most Chelonians [15]. Juvenile turtles

(order Testudines) often have lower estimated survival rates than adults, with predation assumed to be the primary cause of mortality [16,17]. Older, larger animals are less vulnerable to predation, in part due to a larger and thicker shell that can be a successful deterrent for many predators [3,18]. Additionally, wild turtles often do not reach reproductive sizes for upwards of

1–2 decades, so increasing juvenile growth rates should allow individuals to be closer to reproductive size upon release [19]. Success of turtle head-starting efforts has been mixed.

Although this approach involves considerable time and expense and survival of released juveniles can be low [20], in some cases it may be one of the most effective methods for re- establishing viable populations [21]. Studies explicitly evaluating a tradeoff between a naturalistic rearing environment and one that maximizes growth rate on behaviors that are

71 important for survival in the wild are lacking.

We hypothesized a tradeoff exists between growth and behavior for turtles raised with and without environmental enrichment. We predicted enriched turtles reared in structurally complex enclosures, which entailed foraging for spatially variable food in the presence of conspecifics, would grow slower and attain overall smaller body sizes than unenriched turtles.

However, we anticipated this form of enrichment would allow turtles to be more proficient at foraging for novel prey than unenriched turtles, which were housed individually and fed out of simple dishes. We predicted enriched turtles would also show more risk-aversion than unenriched turtles by avoiding a predator cue more as well as being less likely to emerge from a shelter into a novel environment.

METHODS

Study Species and Animal Husbandry

Eastern box turtles inhabit temperate and subtropical regions over much of the eastern

United States [16]. The species is of conservation concern because populations have declined from habitat loss, road mortality, intense predation (particularly of nests and juveniles), and collection for the pet trade [22]. As such, the species is listed as Vulnerable by the IUCN and is included in CITES Appendix II [23].

This research was conducted under an approved protocol (#16017) by the University of

Illinois Institutional Animal Care and Use Committee and Scientific Collector’s Permits granted by the States of Michigan and Illinois (#NH17.5980). Subjects for this study were acquired as eggs from nests laid by free-ranging female eastern box turtles at Fort Custer Training Center, an

Army National Guard training facility located near Battle Creek, Michigan, USA. We artificially incubated eggs indoors and raised hatchlings (n = 32) in a greenhouse on the campus of

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University of Illinois at Urbana-Champaign. The transparent greenhouse ceiling allowed exposure to natural photoperiods. Similarly, temperature inevitably fluctuated on a daily and seasonal basis, but we attempted to regulate ambient temperature in the greenhouse between 21–

29 °C. We raised neonates in either an enriched or unenriched environment beginning in mid-

August 2016 (within two weeks of hatching). Enriched turtles (n = 16) were communally housed in 132 cm long × 79 cm wide × 30 cm deep Rubbermaid® stock tanks (Rubbermaid Commercial

Products, Huntersville, NC, USA) (n = 4–5 individuals per replicate) with naturalistic features designed to mimic vegetation and substrate commonly utilized by wild eastern box turtles [16]

(Figure 4.1). Unenriched turtles (n = 16) were housed individually in comparably simplistic enclosures consisting of a 60 cm long × 42 cm wide × 28 cm tall transparent plastic tub with reptile cage carpet (Zoo Med Eco Carpet; Zoo Med Laboratories, Inc., San Luis Obispo, CA,

USA) and a 42 × 42 cm piece of plastic shelf liner resting on the carpet. We gave these turtles a small plastic hide box and kept tubs on a slight angle to hold fresh standing water (ca. 4 cm deep) in the lower end for drinking and soaking (Figure 4.1). We provided all turtles with UVB light daily from 0630–1630 h using Zoo Med ReptiSun® 5.0 bulbs (Zoo Med Laboratories, Inc.). We affixed a 25 cm diameter light hood with a 13 W compact fluorescent bulb to each enriched tank.

We hung a continuous row of 117 cm fluorescent linear tubes housed in fixtures (Zoo Med T5

High Output Terrarium Hood) spanning the lower (wet) ends of the rows of unenriched tubs. We used Zoo Med Reptitherm® (Zoo Med Laboratories, Inc.) under tank heat pads from October–

March to maintain temperatures and facilitate activity and foraging throughout the winter months.

The type and amount of food provided to individuals at each feeding was similar between rearing treatments. However, we fed enriched turtles in a manner that we anticipated would

73 entail challenges leading to improved foraging, as this is a skill frequently cited as being deficient for translocated animals released from captivity [11]. Because wild box turtles actively forage for items such as invertebrates, vegetation, and fungi in complex terrestrial microhabitats

[16], we predominantly fed enriched turtles by scattering food throughout their enclosures.

Unenriched turtles were provided food on 10 cm diameter petri dishes placed in the same spot in enclosures at each feeding. We initially fed live blackworms (Lumbriculus variegatus) and mealworms (Tenebrio molitor). We then transitioned turtles to live superworms (Zophobas morio) and then solely to live redworms (Eisenia foetida) after several months. We also offered fresh mixed greens (excluding spinach) and Zoo Med Gourmet Box Turtle Food—a commercial diet consisting of pellets and dehydrated mealworms, strawberries, and mushrooms. Turtles were offered fresh food five days per week, and we dusted food with calcium powder three days per week. Because wild box turtles often scavenge animal carcasses and consume bones to obtain minerals [24,25], we provided enriched turtles with commercially available cuttlebones to chew on as an additional component to their enrichment. Fresh water was provided ad libitum. Further details of study animal acquisition and husbandry methods are described elsewhere [26].

Growth

We recorded individuals’ mass (g) using a digital scale (Sartorius M-PROVE Portable

Scale; Sartorius AG, Göttingen, Germany) approximately once per week. To compare growth between rearing treatments at the time behavior was assessed in captivity (see below), we calculated daily growth rate for all 32 turtles when eight months old using each turtles’ mass after hatching in August 2016 and mass in April 2017, divided by the number of days between measurements. Twelve turtles (six enriched and six unenriched) were randomly selected for release into the wild in May 2017. We recalculated daily growth rates for the 20 turtles (10 in

74 each treatment) retained in captivity for an additional year, when 20 months old. We compared growth rates between treatments for each age range (0–8 and 9–20 months) with linear models in

R version 3.4 [27], which we used for all analyses. We analyzed the interactive effect of treatment and time (number of months post-hatching) on mass using a linear model.

Behavioral Assays

General Information

We conducted several behavioral assays in the greenhouse where turtles were reared during late March–early April of 2017 and 2018 when turtles were eight and 20 months old, respectively. All 32 turtles (16 in each treatment) were tested in 2017. The twenty remaining individuals (10 in each treatment) were re-tested in the same assays one year later. We randomly selected the testing order of individuals. Turtles were tested individually, placed back in primary enclosures once a trial was completed, and were tested in only one assay per day. We affixed covers around the sides and bottom of testing arenas to reduce the influence of external stimuli.

Between trials, we cleaned arenas with ethanol and rinsed them with hot water. We conducted assays during daylight hours (0800–1600) because this species is predominantly diurnal [16].

Relevant substrates and food items were novel to all turtles at the first time of testing. For trials involving food, each turtle was fasted for 48 h prior to testing to encourage an appetitive response, but all individuals were offered their normal diet in primary enclosures for five consecutive days before the fasting period began.

For trials requiring direct observation to record response variables, we placed video cameras (Lorex LNB3163B bullet surveillance camera; Lorex Technology, Inc., Markham, ON,

Canada) above testing arenas and filmed trials using a digital video recorder (Yoko Technology

RYK-9122 DVR; Yoko Technology Corp., New Taipei City, Taiwan). Observers recorded data

75 while situated out of view from turtles by watching trials on a television monitor (Eyoyo 20.32 cm portable HDMI LCD monitor; Shenzhenshi cai hui Technology Co., Ltd, Shenzhen, China).

All timed response variables were recorded in seconds using stopwatches. Observers were at times aware of a turtle’s identity. To ensure this did not bias our results, an observer unaware of turtles’ identities scored a randomly selected sample of the same response variables from recorded trials. We found strong agreement between response variables recorded at the time of testing and those scored from video footage (Pearson’s r > 0.9 in all cases).

Foraging Behavior

We designed two assays to test turtles’ foraging efficiency in contrasting environments so that each generally differed from a respective treatment’s rearing conditions. The first tested their ability to detect and capture relatively immobile prey but in a more challenging environment

(hereafter “simple prey, complex environment”). We placed turtles in a 35 cm long × 21 cm wide

× 12 cm tall, open-topped plastic arena with 2 cm of cypress mulch uniformly spread over the floor. We put a turtle in the center of the arena under a dark 10 cm3 cup, where it acclimated for 5 min. We then placed one commercially-raised live black soldier fly (Hermetia illucens) larva

(0.5–2 cm in length) slightly hidden in each of three corners of the arena just before removing the cup covering the turtle. We recorded the number of larvae consumed by each turtle within 4 h once unconstrained to move about the arena. Because our only response variable for this assay were count data, we did not collect video footage for this trial. For each age group (8 and 20 months), we used generalized linear models assuming a Poisson distribution to examine if the number of larvae consumed differed between treatments.

To complement the simple prey, complex environment test, we conducted a second assay to test turtles’ ability to procure more mobile prey but in a relatively simplistic environment

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(hereafter “complex prey, simple environment”). We used a slightly larger plastic arena (60 cm long × 42 cm wide × 17 cm) than that used in the other foraging task because we used more agile prey for this assay. We placed turtles in the center of the arena and allowed them to explore their surroundings for 5 min. We then placed an individual under a dark 10 cm3 cup at one end of the arena, where it was held for an additional 5 min. We placed two commercially-raised live dubia cockroaches (Blaptica dubia; ca. 0.5 cm in length) at the opposite end of the arena (one in each corner) after the acclimation period and immediately removed the cup covering the turtle. We chose dubia cockroaches because they were fast, active prey that would represent a challenge for turtles to capture and were morphologically and behaviorally different from any live prey they had previously encountered. For this test, we provided no substrate in the arena for prey to hide in, but we roughed the floor with sandpaper to better enable turtles and prey to locomote. Within a 30 min time-limit, we recorded latency to strike at a cockroach as well as the number consumed. Any individual that did not strike at prey within the time-limit during this assay was given the full trial length as its strike latency. We used generalized linear models assuming a

Poisson distribution to test if the number of cockroaches consumed differed between treatments in each age group. We used linear models to examine if strike latency differed between treatments at each age.

Antipredator Behavior

We designed this assay to test turtles’ recognition and avoidance of an olfactory predator cue. Although neither group of turtles had ever been exposed to predators, we predicted enriched turtles may be more likely to recognize and respond to this cue than unenriched turtles because they were continually stimulated by their surroundings. We placed two evenly-sized paper towels covering all but approximately 4 cm of the center of the floor in a 35 cm long × 21 cm

77 wide × 12 cm tall, plastic arena. One paper towel was sprayed with diluted raccoon (Procyon lotor) urine (hereafter “scented”). Raccoons are a well-known predator of eastern box turtles [16] and are abundant at the site where subjects in this study were collected (unpublished data). The other paper towel was sprayed with distilled water as an unscented liquid control (hereafter

“unscented”). Similar approaches have been used to test antipredator behavior in other taxa

[28,29]. This 20 min trial began immediately after placing a turtle in the center of the arena, where their plastron was not in contact with either paper towel. Turtles thus had opportunity to inspect either side before making a choice but had to fully move onto one of the paper towels to have been considered choosing. Any individual that did not respond within the time-limit for this assay (i.e., remained motionless) was excluded from analysis. We used generalized linear models assuming a binomial distribution to examine if the odds of choosing the unscented side of the arena differed between treatments at each age. We also recorded how much time individuals spent away from the scented side of the arena as a measure of avoidance of the cue. We tested if this response differed between treatments at each age using linear models.

Shelter Emergence

This assay was designed to evaluate propensity to emerge from shelter into a novel environment. We placed turtles in a clear 60 cm long × 42 cm wide × 17 cm plastic arena with a

10 cm3 PVC shelter placed at one end. There was no substrate in the arenas. We put turtles inside shelters facing the entrance and allowed them to acclimate for 5 min by covering the entrance with cardboard. We then removed the cover, allowing the turtles the option of exiting the shelter.

Our response variable was a binary effect of “emerged” or “did not emerge”. We used generalized linear models assuming a binomial distribution to examine if the odds of emerging differed between treatments at each age.

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For general linear models, we ensured data residuals approximated a Gaussian distribution by inspecting quantile-quantile plots, and we assessed homogeneity of variances using Brown-Forsyth tests. We assessed overdispersion for generalized linear models by dividing the residual deviance by the degrees of freedom. We considered effects to be statistically significant when P ≤ 0.05.

RESULTS

Growth

We recorded mass an average of 28.72 ± 1.37 standard deviation (SD) times for 32 turtles from August 2016 to April 2017. We recorded mass an average of 44.15 ± 0.99 SD times for 20 individuals kept in captivity for an additional year. Enriched turtles grew significantly slower than unenriched turtles during the first eight months in captivity (P < 0.001), but we found no difference in growth rates between treatments for turtles kept in captivity an additional year (P =

0.27; Figure 4.2a). We found a significant interaction between treatment and time for predicting body mass (P < 0.001). Unenriched turtles became larger than enriched turtles after the first five months in captivity and remained overall larger for the duration of rearing (Figure 4.2b).

Foraging Behavior

In the simple prey, complex environment task, where turtles were placed in enclosures with mulch substrate and given 4 h to find and consume black soldier fly larvae, they consumed an average of 1.13 ± 1.33 SD larvae (range 0–3). Eight-month-old enriched turtles consumed significantly fewer larvae on average compared to unenriched turtles of the same age (P < 0.001;

Figure 4.3). We found a similar pattern between treatments when turtles were 20 months old (P <

0.01; Figure 4.3). The average number of larvae eaten during a trial by enriched turtles was low due to only three enriched turtles consuming any prey during trials, whereas only five unenriched

79 turtles did not consume larvae.

In the complex prey, simple environment test, where turtles were placed in simplistic enclosures and given 30 min to strike at and consume dubia cockroaches, they consumed an average of 1.06 ± 0.96 SD larvae (range 0–2). Like the previous foraging assay, many enriched turtles (n = 12) did not respond to the prey and remained motionless during trials, whereas only three unenriched turtles did not consume a cockroach. Eight-month-old enriched turtles consumed significantly fewer cockroaches on average compared to unenriched turtles of the same age (P < 0.001; Figure 4.4a). Again, we found a similar pattern between treatments when turtles were 20 months old, where enriched turtles consumed few cockroaches and all ten unenriched turtles consumed both cockroaches (P < 0.01; Figure 4.4a). Unenriched turtles were significantly quicker to strike at prey than enriched turtles when eight (P < 0.001) and 20 months old (P < 0.001; Figure 4.4b).

Antipredator Behavior

When given 20 min to choose between initially occupying a side of an arena scented with raccoon urine or sprayed with a water control, turtles appeared to choose randomly with no apparent initial distinction between sides. When eight months old, eight of fifteen (53%) enriched turtles (one enriched turtle did not make a choice) chose the unscented side and six of sixteen (37.5%) unenriched turtles chose the unscented side. When tested at 20 months in age, five of 10 (50%) enriched turtles chose the unscented side, and four of 10 (40%) unenriched chose the unscented side. Thus, the odds of selecting the unscented side did not differ significantly between treatments at eight (P = 0.38) or 20 months old (P = 0.65). Eight-month- old enriched turtles spent significantly more time away from the scented area than unenriched turtles of the same age (P = 0.05), but we found no difference in time spent away from the

80 scented area between treatments when turtles were 20 months old (P = 0.35; Figure 4.5).

Shelter Emergence

When given 20 min to emerge from shelter into a novel environment, two of 16 (12.5%) enriched turtles emerged, and six of sixteen (37.5%) unenriched turtles emerged when eight months old. When 20 months old, five of 10 (50%) enriched and seven of 10 (70%) unenriched emerged. Thus, the odds of emerging did not differ significantly between treatments when eight

(P = 0.12) or 20 months old (P = 0.37).

DISCUSSION

Although experiments investigating the effects of environmental enrichment on behavior and morphology for captive reptiles are increasing, this taxon has been historically neglected compared to others such as birds or mammals [10]. Furthermore, studies investigating such effects in the context of informing wildlife reintroductions in general are lacking [11]. We explored a potential tradeoff between growth and ecologically relevant behaviors in captivity for head-started box turtles and found this tradeoff is most apparent for younger turtles. Our prediction that enriched turtles would grow slower than unenriched turtles was supported only during the first eight months of rearing. Growth rates did not differ between rearing treatments from 9–20 months in age, suggesting growth lags from experiencing more natural foraging conditions began to dissipate for enriched turtles towards the end of the study. In a similar study, head-started smooth green snakes (Opheodrys vernalis) experiencing restricted foraging during simulated winter in captivity to prepare them for natural conditions exhibited compensatory growth and were of similar size compared to snakes kept active year-round by the time of release

[30]. However, it is important to note that our enriched turtles had consistently smaller body sizes than unenriched turtles from a few months after hatching to the end of the study and were

81 ultimately a third smaller, on average. If larger body size allows head-started turtles to be less vulnerable to certain predators once released, as has been suggested, raising them in conditions to facilitate growth may be a preferred rearing method [3].

We predicted enriched turtles would excel over unenriched turtles in foraging assays because they had been raised to find spatially variable food in complex enclosures. However, unenriched turtles consumed more novel prey than enriched turtles in both tests and were quicker to strike at cockroaches in the complex prey, simple environment task. We suggest the novelty of active foraging during assays may have promoted unenriched turtles to have such a strong response. Although subadult enriched ratsnakes (Elaphe obsoleta) did not differ from unenriched conspecifics in a laboratory foraging task [31], findings similar to ours indicative of behavioral deprivation were reported for domestic animals raised in unstimulating environments. Rats reared in unenriched conditions pressed a bar to turn on a light in an operant conditioning chamber more often than enriched ones, even if the light did not come on [32]. Pigs (Sus scrofa domesticcus) raised in unenriched conditions had abnormally greater dendritic growth in the somatosensory cortex than enriched individuals, which was thought to be a function of environmental deprivation leading pigs to exhibit hyper stimulus-seeking behavior [33]. It is unclear why there was a general lack of response by enriched turtles, but their behavior could indicate they were more willing than unenriched turtles to forgo foraging in unfamiliar environments.

Although all our captive turtles had never been exposed to predators, we presumed enriched turtles would show greater response to a predator cue given the cognitive benefits enrichment can provide [8,9,10,11]. Contrary to our prediction, turtles did not show aversion to the area scented with raccoon urine when making a primary choice, which could suggest a lack

82 of ability in naïve individuals to recognize predator urine as a potential risk. We acknowledge this result could be related to limitations of our methodology in that we used small testing apparatuses, so odor from the urine may have been broadly distributed and detectable throughout the arena. Additionally, we did not provide food or refuge in this assay that may motivate turtles to assess tradeoffs between predator cue avoidance and other survival attributes. Enrichment may provide earlier developmental advantages for avoiding predators because eight-month-old enriched turtles avoided the urine more than unenriched turtles of the same age, providing some evidence of aversion to this scent. Treatments did not differ in how long they avoided raccoon urine when 20 months old; enriched turtles’ behavior did not change from the previous year, whereas older unenriched individuals exhibited avoidance behavior like enriched turtles. The mechanisms of predator recognition for captive-born turtles are not well understood, but general stimulation from enrichment may be beneficial in this regard [34], at least in the short-term.

Because enriched turtles had greater hiding opportunities in their enclosures, we expected this group would be less likely to emerge from shelter. However, we did not find any treatment difference in turtles’ propensity to emerge, perhaps because there was no potential cost associated with not emerging. Additionally, hiding in cover is likely innate for this species, as this is probably the most efficient antipredator behavior for juveniles which are smaller and have less rigid shells than adults [3,18,26]. As with our predator recognition assay, future experiments might show greater treatment differences if shelter emergence tests incorporate aspects of our other assays, such as determining if turtles will differ in emergence propensity based on whether food or predator cues are present.

Despite mixed success and considerable expense [35], head-starting is likely to remain popular given its intuitive nature and public support [21]. Given the increasing global reduction

83 of turtle populations [36], experimentally evaluating captive-rearing practices as we did here is vital for advancing head-starting efforts [3,9]. Our results suggest environmental enrichment may come at the cost of growth and total size attainment for turtles, and the expected behavioral benefits were modest or absent. However, our investigation looked at only a small number of behaviors in a laboratory setting, and it remains to be seen if enriched and unenriched turtles exhibit differing behaviors or survival post-release.

84

FIGURES

Figure 4.1. Rearing conditions for enriched (left) and unenriched (right) head-started eastern box turtles (Terrapene carolina). Enriched turtles were housed in groups of 4–5, provided with coconut fiber substrate to bury in, artificial plants and half logs for additional hiding areas, and naturalistic water dishes. Unenriched turtles were housed individually and provided with carpet for substrate and a small plastic hiding structure.

85

Figure 4.2. (a) Growth rates (g/day) of enriched and unenriched eastern box turtles (Terrapene carolina) during captive-rearing. Thirty-two turtles (16 in each treatment) were raised for eight months. Twenty turtles (10 in each treatment) were kept in captivity for an additional year. Symbols represent means and bars are 95% confidence intervals. (b) Mass (g) of enriched and unenriched turtles during captive-rearing. Lines are means fit by loess smoothing, and ribbons represent 95% confidence intervals.

86

Figure 4.3. Number of black soldier fly larvae (Hermetia illucens) consumed by enriched and unenriched captive-reared eastern box turtles (Terrapene carolina) when eight and 20 months old. Symbols represent means and bars are 95% confidence intervals.

87

Figure 4.4. Number of dubia cockroaches (Blaptica dubia) consumed (a) and latency to strike at a cockroach (b) by enriched and unenriched eastern box turtles (Terrapene carolina) when eight or 20 months old. Symbols represent means and bars are 95% confidence intervals. There is no confidence interval for twenty-month-old unenriched turtles in panel (a) because all these turtles consumed both cockroaches.

88

Figure 4.5. Time (seconds) enriched and unenriched captive-reared eastern box turtles (Terrapene carolina) spent away from a side of an experimental arena scented with northern raccoon (Procyon lotor) urine when eight and 20 months old. Symbols represent means and bars are 95% confidence intervals.

89

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93

CHAPTER 5: CAPTIVE-REARING DURATION MAY BE MORE IMPORTANT THAN ENVIRONMENTAL ENRICHMENT FOR ENHANCING TURTLE HEAD-STARTING

SUCCESS4

ABSTRACT

Raising captive animals past critical mortality stages for eventual release (head-starting) is a common conservation tactic. Counterintuitively, post-release survival can be low. Post- release behavior affecting survival could be influenced by captive-rearing duration and housing conditions. Practitioners have adopted environmental enrichment to promote natural behaviors during head-starting such as raising animals in naturalistic enclosures. Enrichment might be especially beneficial for animals held in captivity long-term to prevent degradation of adaptive behaviors. Using 32 captive-born turtles (Terrapene carolina), half of which were raised in enriched enclosures, we employed a factorial design to explore how enrichment and rearing duration affected post-release growth, behavior, and survival. Six turtles in each treatment

(enriched or unenriched) were head-started for nine months (cohort one). Ten turtles in each treatment were head-started for 21 months (cohort two). At the conclusion of captive-rearing, turtles in cohort two were overall larger than cohort one, but unenriched turtles were generally larger than enriched turtles within each cohort. Once released, enriched turtles grew faster than unenriched turtles in cohort two, but we otherwise found minimal evidence suggesting enrichment affected post-release survival or behavior. Cohort two dispersed farther and had generally higher active season survival than cohort one (0.50 vs. 0.33). Body mass was positively associated with daily survival probability. Our findings suggest attaining larger body sizes from

4This chapter has been published in Global Ecology and Conservation. Full citation: Tetzlaff, S.J., J.H. Sperry, B.A. Kingsbury, and B.A. DeGregorio. Captive-rearing duration may be more important than environmental enrichment for enhancing turtle head-starting success. Global Ecology and Conservation: e00797.

94 longer captive-rearing periods to enable greater movement and alleviate susceptibility to predation (the primary cause of death) could be more effective than environmental enrichment alone in chelonian head-starting programs where substantial predation could hinder success.

INTRODUCTION

Wildlife translocation, the deliberate human-facilitated movement and release of animals, is a common management technique aimed at augmenting imperiled populations or reintroducing species to areas where they have been extirpated (Seddon et al., 2007). Despite the potential conservation value of translocations, many fail because animals have low survival when released in novel environments, precluding successful establishment of released populations (Fischer and

Lindenmayer, 2000; Germano and Bishop, 2009). In particular, releasing animals from captivity is typically less successful than translocating wild animals directly between natural sites (Griffith et al., 1989; Fischer and Lindenmayer, 2000). This is in part thought to be a function of captive animals lacking the necessary experience to successfully transition to post-release environments

(Stamps and Swaisgood, 2007). Head-starting entails rearing captive animals for an extended period of time to allow them to grow until they reach a size threshold in which predation vulnerability is greatly reduced. However, these efforts often fail because individuals disperse from release sites and struggle with avoiding predators, acquiring food, or selecting suitable habitats (Einum and Fleming, 2001; Jule et al., 2008; Le Gouar et al., 2012). Two fundamental factors that might impact head-starting success are how long juveniles are raised before release and the captive-rearing conditions.

The behavior of captive animals often differs from wild-bred conspecifics because captivity can have detrimental effects on development (Mathews et al., 2005; Swaisgood et al.,

2018). Potential conflicts could thus exist when deciding how long animals should be held prior

95 to translocation. Longer head-starting periods generally result in larger or more mature animals being released, which could provide a survival advantage if such individuals are less susceptible to predation (Nagy et al., 2015; Daly et al., 2018). However, behaviors critical to survival such as foraging and predator avoidance might degrade with longer captivity duration (DeGregorio et al.,

2013, 2017; Swaisgood et al., 2018).

Attempts to promote ecologically relevant behaviors and improve welfare for head- started animals have been accomplished by incorporating environmental enrichment into rearing protocols (Swaisgood, 2010). This could include raising animals in enclosures with naturalistic features simulating release sites, providing foraging opportunities like those experienced in nature, and communally housing conspecifics to promote social skills (Reading et al., 2013).

Indeed, enrichment appears to broadly have positive effects on translocations, as post-release survival is generally higher for animals that are enriched compared to unenriched conspecifics

(Tetzlaff et al., 2019a).

The effects of environmental enrichment and captivity duration on reptile head-starting success has received minimal attention. Roe et al. (2015) found no differences in post-release growth, behavior, or survival between captive-born common watersnakes (Nerodia sipedon), which were provided with enrichment for several months prior to release relative to unenriched conspecifics. However, the same study reported that larger and older snakes had higher survival compared to younger and smaller individuals (Roe et al., 2010), indicating extended head- starting duration to facilitate growth and maturity could be beneficial. Enriching captive-born reptiles from birth might show more pronounced differences. A factorial experiment investigating the impacts of time in captivity and environmental enrichment would better enable mechanistic understanding of post-release behavior (e.g., foraging, movement, exposure) and

96 survival for head-started animals.

Head-starting is an intuitive and attractive option for conserving chelonians (turtles, tortoises, and terrapins; order Testudines) given this is one of the most threatened vertebrate groups globally, with approximately 60% of species threatened with extinction or having gone extinct in recent times (Lovich et al., 2018). However, head-starting efforts have met with mixed success and considerable debate exists regarding the efficacy of this practice (Burke, 2015).

Before adopting head-starting practices, rearing facilities should evaluate both the positive and negative effects of various rearing durations and the use of environmental enrichment for promoting behaviors that lead to improved survival once released.

We experimentally tested the effects of time in captivity and enrichment on short-term head-starting success for eastern box turtles (Terrapene carolina). We raised one cohort of turtles, half in enriched conditions and half in unenriched conditions, for nine months before release. We chose this duration because it is representative of several published turtle head- starting efforts (e.g., Buhlmann et al., 2015; Daly et al., 2018; Quinn et al., 2018). We raised another cohort of turtles, half in enriched conditions and half in unenriched conditions, for an additional year before release (21 months total). We hypothesized enrichment provides opportunities for head-started turtles to develop behaviors that minimize predation risk, as predation is likely the primary cause of mortality for juvenile turtles due to their small body sizes and incomplete hardening of the shell (Dodd, 2001). Once released we expected enriched turtles would move less, remain less visible, and grow faster due to enhanced foraging skills, leading to higher survival than unenriched turtles. If time in captivity has negative effects on behavior, we expected turtles reared for longer would remain exposed and move more than turtles kept in captivity for a shorter duration, leading to lower survival. Alternatively, if larger body size

97 reduces post-release predation (sensu the “bigger is better” hypothesis; Packard and Packard,

1988; Janzen, 1993), we expected turtles reared for a longer period would have higher survival.

METHODS

Study species and site

Eastern box turtles inhabit temperate and subtropical regions over much of the eastern

United States (Dodd, 2001). This species is typically associated with forested habitats but also occupies forest edges, shallow wetlands, and grasslands such as old field and prairie (Dodd,

2001; Gibson, 2009). The species is of conservation concern because populations have declined from habitat loss, road mortality, intense predation (particularly of nests and juveniles), and collection for the pet trade (Kiester and Willey, 2015). As such, the eastern box turtle is listed as

Vulnerable by the IUCN and is included in CITES Appendix II (van Dijk, 2011).

We conducted fieldwork at Fort Custer Training Center, an Army National Guard training facility located in southwest Michigan, USA, near the northern range limit for eastern box turtles. The approximately 3,000 ha installation is comprised primarily of woodlands

(2,023 ha), wetlands (485 ha), and old field/prairie (485 ha). Fort Custer is enclosed by chain-link fence, and unpaved dirt roads intersect the site at approximately 1 km intervals. Most of the site has minimal human disturbance and vehicle traffic is limited.

Husbandry practices

Subjects for this study were acquired as eggs from nests laid by free-ranging females at

Fort Custer. We artificially incubated eggs indoors and raised hatchlings (n = 32) in a greenhouse on the campus of University of Illinois at Urbana-Champaign. The transparent greenhouse ceiling allowed exposure to natural photoperiods. Similarly, temperature inevitably fluctuated on a daily and seasonal basis, but we attempted to regulate ambient temperature in the greenhouse

98 between 21 and 29 °C. We raised hatchlings in either an enriched or unenriched environment beginning in mid-August 2016 (within two weeks of hatching). Enriched turtles (n = 16) were communally housed in 132 cm long x 79 cm wide x 30 cm deep Rubbermaid® stock tanks

(n = 4–5 individuals per replicate) with naturalistic features designed to mimic vegetation and substrate commonly utilized by wild eastern box turtles (Dodd, 2001, Fig. 5.1). Unenriched turtles (n = 16) were housed individually in comparably simplistic enclosures consisting of a

60 cm long x 42 cm wide x 28 cm tall transparent plastic tub with reptile cage carpet (Zoo Med

Eco Carpet; Zoo Med Laboratories, Inc., San Luis Obispo, CA, USA) and a 42 × 42 cm piece of plastic shelf liner resting on the carpet. We provided these turtles a small plastic hide box and kept tubs on a slight angle to hold fresh standing water (ca. 4 cm deep) in the lower end for drinking and soaking (Fig. 5.1). We note that although enriched turtles had opportunities for social interactions that unenriched turtles did not, we considered this as a general component of environmental enrichment. We observed no agonistic interactions between enriched turtles, and regardless of treatment, all turtles survived during captive-rearing.

The type and amount of food provided to individuals at each feeding was similar between rearing treatments, but we predominantly fed enriched turtles by scattering food throughout their enclosures to promote active foraging. Unenriched turtles were provided food on 10 cm diameter petri dishes placed in the same spot in enclosures at each feeding. We fed turtles live invertebrate prey such as blackworms (Lumbriculus variegatus), mealworms (Tenebrio molitor), superworms

(Zophobas morio), and redworms (Eisenia foetida). We also offered thawed frozen berries, fresh mixed greens (excluding spinach), and Zoo Med Gourmet Box Turtle Food—a commercial diet consisting of pellets and dehydrated mealworms, strawberries, and mushrooms. Turtles were offered fresh food daily, five days per week. Fresh water was provided ad libitum. We generally

99 weighed (g) and measured carapace length (mm) of each turtle weekly and conducted brief behavioral trials prior to releasing turtles (Tetzlaff et al., 2019b), but we otherwise limited handling. Additional details of study animal acquisition and husbandry methods have been previously described (Tetzlaff et al., 2018, 2019b).

Release procedures

We released 12 turtles (six enriched and six unenriched) at Fort Custer in May 2017, after approximately nine months of head-starting. We hereafter refer to this release group as cohort one. We retained the remaining 20 turtles (cohort two: 10 enriched and 10 unenriched) in captivity for an additional year and released these individuals in the same area as cohort one in

May of 2018 after approximately 21 months of head-starting. We released turtles into a roughly

450 m2 area in hardwood forest dominated by an overstory of maples (Acer spp.) and oaks

(Quercus spp.) and an understory dominated by a diverse community of herbs and shrubs.

Several wetlands were adjacent to the forest patch. We chose the release site based on previous work suggesting resident box turtles at Fort Custer prefer these habitat types (Gibson, 2009). It is also adjacent to a heavily-used box turtle nesting site, so the general area is likely occupied by resident juveniles (Laarman et al., 2018).

We anticipated initial post-release mortality might be high, so we placed all turtles in acclimation pens in the forest patch to ease their transition to the wild (Tuberville et al., 2005).

This also provided us daily opportunity to observe well-being, behavior, and growth of turtles without the risk of rapid mortality. Pens were 1.8 m long x 1 m tall x 1 m wide and constructed using approximately 4 cm diameter PVC pipe. We enclosed the top and sides of each pen with plastic poultry netting. We buried the legs and netting of each pen approximately 10 cm into the ground to keep predators from entering and prevent turtles escaping. We installed three pens

100 approximately 45 m apart in the release area in April 2017 and placed four turtles in each pen on

19 May. We repeated a similar release procedure for cohort two in 2018 and we built two additional pens for release of this cohort to accommodate the larger number of individuals being released. To track growth rates during the acclimation period, we measured straight carapace length to the nearest 0.01 mm of each turtle once per week using digital calipers (Fisherbrand™

Traceable™ Digital Calipers; Fisher Scientific, Hampton, New Hampshire, USA).

We opened pens at the conclusion of the acclimation period by cutting away an approximately 10 cm tall section of the netting from ground level on each of the pens, which allowed turtles to exit pens at-will. In 2017, we opened the pens after 38 days on 26 June. All turtles in cohort one aside from one individual exited pens within three days after pens were opened; the last turtle left its pen on 5 July. In 2018, we opened the pens after 34 days on 8 June.

All, except one turtle, in cohort two left their release pens within one day; the last turtle left its pen on 14 June. Although we released each cohort in different years and at slightly different times within each year, the general climatic conditions were similar between release years. For example, we used loggers (Thermochron iButton model DS1921G, Fondriest Environmental,

Inc., Fairborn, OH, USA; ± 0.5 °C error) to monitor hourly temperature in the forest patch where turtles were released, and the mean temperature difference between years when turtles were undergoing acclimation was 1.22 °C. We thus do not expect minimal climatic differences influenced behavior or survival of turtles in pens or once released.

Post-release monitoring

We used radio-telemetry to monitor turtles once they exited pens until they either died during the active season (generally April–October at Fort Custer; Gibson, 2009) or initiated overwintering. Depending on a turtle's size, we affixed a 0.9 or 1.2 g transmitter (Advanced

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Telemetry Systems, Inc., Isanti, MN, USA) to the carapace of each turtle using epoxy.

Transmitters were no more than 7% of a turtle's mass. We generally located individuals five days per week during daylight hours (0700–1800) from June to August and once every 1–2 weeks from September to November each year using a handheld receiver (R-1000, Communications

Specialists, Inc., Orange, CA, USA) and 3-element mini Yagi antenna. Each time we located a turtle, we recorded its position in Universal Transverse Mercator units (UTM, North American

Datum of 1983) with a handheld GPS (Garmin eTrex 30; 3 m accuracy). We visually confirmed if turtles were alive at least two times per week on non-successive tracking days. During these occasions, we estimated the proportion of a turtle's body that was exposed to the nearest 25%

(Harvey and Weatherhead, 2010). To minimize disturbing turtles on days we did not estimate exposure, we radio-tracked individuals to within approximately 1 m of their location but did not visually confirm survival status. We measured each turtle's straight carapace length to the nearest

0.01 mm using digital calipers and mass to the nearest 0.01 g using a digital scale (Sartorius M-

PROVE Portable Scale; Sartorius AG, Göttingen, Germany) every two weeks.

We inferred turtles had initiated overwintering by their consistent lack of aboveground activity for more than two weeks each fall. Transmitter batteries would not last through the winter, so we placed approximately 15 cm tall x 100 cm long x 50 cm wide wire cages over each turtle once we were confident they were overwintering, which aided in determining overwinter survival the following spring. We placed cages on turtles in cohort one on 10 November 2017 and turtles in cohort two on 2 November 2018. We replaced transmitters on any turtles from cohort one that survived into the second monitoring season in 2018 and radio-tracked these turtles along with cohort two that year. We ceased monitoring in April 2019 after we confirmed which turtles survived the 2018–19 winter.

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Data analyses

We conducted all statistical analyses in R version 3.5.1 (R Core Team, 2018). Where appropriate, we confirmed data residuals approximated a Gaussian distribution by inspecting quantile-quantile plots and ensured variances were homogeneous using Brown-Forsythe tests.

We calculated daily growth rate for each turtle while in acclimation pens using the difference of the first and last carapace measurement divided by the number of days between measurements.

Using similar methods, we calculated daily growth rate for each turtle post- release—from when pens were opened until death or overwintering initiated. We used linear models to analyze the effects of treatment and cohort on individual daily growth rates while in pens and post-release.

We calculated daily movement rate for each turtle, defined as the summed distance between subsequent tracking events divided by the number of days monitored. We also calculated how far each turtle dispersed during radio-tracking by measuring the straight-line distance between a turtle's release pen and its furthest location from the pen. We used linear models to analyze the effects of treatment and cohort on these movement metrics. To control for the varying survival rates between turtles, we included individual probability of surviving the active season monitoring period as a covariate in our dispersal model. We also used a linear model to determine if dispersal distance was associated with individual survival probability within each cohort.

To analyze exposure of turtles, we used a linear mixed model with the package nlme

(Pinheiro et al., 2018). We included treatment and cohort as fixed effects and turtle identity as a random effect to account for the repeated measurements per individual. For this and the abovementioned analyses, we initially modeled treatment and cohort as an interactive effect.

However, we found no significant interactions (see Results) and thus tested treatment and cohort

103 as main effects for each response (growth, movement rate, dispersal, and exposure).

To assess the influence of turtle- and study-specific effects on daily survival probability during the active season, we used generalized linear mixed models implemented in the package lme4 (Bates et al., 2015). Our response variable was the binary response of “survived” or “died” for each individual radio-telemetry location. We implemented a modified version of the traditional logit link function used in binary logistic regression, where models were weighted for the time interval between radio-telemetry locations (Shaffer, 2004). This approach also allowed us to utilize a turtle's most recent mass measurement for a given telemetry location, an important consideration because turtles were presumably experiencing weight changes once released that could be related to survival. We evaluated a candidate model set for the fixed effects of treatment, cohort, time (days since release), and body mass—both independently and as varying additive combinations. Mass and time since release were not highly correlated (|r| ≤ 0.70). We also included an intercept-only model (i.e., a null model with no predictors) in our candidate set, for a total of 11 models. To control for the non-independent multiple observations per turtle, we used turtle identity as a random effect in all survival models. We used Akaike's Information

Criterion (Akaike, 1973) corrected for small sample sizes (AICc) to rank candidate models and evaluated their support based on model weight (i.e., the relative probability a given model is the

“best”; Burnham and Anderson, 2002). If a single model was not highly ranked (e.g., received

∼90% of the AICc weight), we used multimodel inference and generated model-averaged estimates for parameters of interest. We did not evaluate models for overwinter survival probability because all but one turtle survived winter (see Results).

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RESULTS

Growth rates

All turtles survived the on-site acclimation period. Average daily growth rate in pens was modest (0.04 mm/day ± 0.02 SD). Growth did not differ between treatments while in acclimation pens but differed between cohorts (Table 5.1). Turtles in cohort two grew faster while in acclimation pens than those in cohort one (Fig. 5.2).

We radio-tracked 12 turtles, 276 times in 2017 and 24 turtles, 1,459 times in 2018

(including three from the previous year). Post-release growth was on average 0.10 mm/day ±0.05

SD. We found evidence that daily growth was influenced by the additive effects of treatment and cohort (Table 5.1). This relationship appeared to be driven by enriched turtles in cohort two, which grew faster than unenriched turtles in the same cohort (Fig. 5.3). Growth rates of unenriched turtles in both cohorts and enriched turtles in cohort one were similar (Fig. 5.3). We were unable to calculate post-release growth rate for one unenriched turtle in cohort two because this individual died shortly after release.

Movement

Turtles moved 9.78 m/day ± 6.19 SD on average, but movement rates were quite variable between individuals (range: 4.25–27.28 m). Movement rates did not differ between treatments or cohorts (Table 5.1). Turtles dispersed an average of 205.98 m ± 191.79 SD from release pens, but dispersal distances were also highly variable between individuals (range: 31.78–828.90 m). We found no differences in dispersal distance between treatments, but dispersal differed between cohorts (Table 5.1). Turtles in cohort two dispersed more than twice the distance as those in cohort one on average (Fig. 5.4). Dispersal distance was not associated with survival probability in cohort one (R2 = −0.001, P = 0.36) or cohort two (R2 = 0.02, P = 0.27).

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Exposure

We recorded exposure 146 times for turtles in cohort one and 420 times for turtles in cohort two. Exposure levels did not differ between treatments or cohorts (Table 5.1). In cohort one, enriched turtles were fully exposed on 45 of 84 (54%) locations, and unenriched turtles were fully exposed on 31 of 62 (50%) locations. In cohort two, enriched turtles were fully exposed on 73 of 185 (39%) locations, and unenriched turtles were fully exposed on 93 of 235

(40%) of locations.

Survival

In total, 14 of the 32 (0.44, 95% confidence interval [CI]: 0.26–0.62) released turtles survived their first active season post-release. Two of six turtles in each treatment (0.33, 95% CI:

0.04–0.78) of cohort one survived the 2017 active season. These four turtles all survived winter and emerged in spring 2018. Three of these individuals (one enriched and two unenriched) also survived the 2018 active season and were alive at the end of this study in April 2019. The proportion of surviving enriched turtles was higher than unenriched turtles for several weeks post-release in 2017, but their survival eventually fell to levels similar to unenriched towards the end of the active season (Fig. 5.5). In cohort two, four of 10 (0.40, 95% CI: 0.12–0.74) enriched turtles and six of 10 (0.60, 95% CI: 0.26–0.88) unenriched turtles survived the 2018 active season. The proportion of enriched turtles surviving post-release was always lower than unenriched turtles in this cohort as the season progressed (Fig. 5.5). All turtles in cohort two that survived the 2018 active season were alive in April 2019 except for one enriched individual; only its intact shell with the transmitter attached was found in its post-overwintering cage on the surface after the 2018–19 winter, so we could not determine when it died.

The main cause of active season mortality in both cohorts was presumed to be predation,

106 accounting for all mortality in cohort one and most in cohort two. Two individuals (one from each treatment) in cohort two were run over by vehicles on dirt road edges. One enriched turtle in this cohort died of unknown causes but was not depredated as its intact, undamaged carcass was recovered. We rarely recovered intact carcasses of depredated turtles but frequently found either shell fragments or only a carapace as well as teeth impressions in epoxy coating transmitters suggestive of mesopredators and rodents as likely dominant predators.

Survival analyses were based on 2,740 turtle “exposure” or “tracking” days. Model selection using AICc suggested body mass was more important for predicting daily survival than treatment, cohort, or time since release (Table 5.2). There was a modest positive association between survival probability and body mass (model-averaged regression coefficient and 95% unconditional CI: 0.05, 0.01–0.1; Fig. 5.6). The model with mass as a sole predictor received

33% of the weight of evidence and was 3.5 delta AIC units above the first model that did not include body mass (intercept-only model). All models that included the effects of mass and other additive variables cumulatively received 71% of the weight of evidence and were all ranked above the null model. Models for the independent or additive effects of treatment, cohort, or time since release received little support (≤6%) and were all ranked below the null model.

DISCUSSION

We explored if head-starting juvenile eastern box turtles with environmental enrichment could lead to behaviors that would enhance survival post-release compared to turtles raised in more traditional, simplistic (unenriched) conditions. We expected the potential deleterious effects of captivity would be offset with enrichment the longer turtles were held in captivity.

However, overall we found limited evidence of enrichment improving post-release behavior and survival. Instead, turtles head-started for 21 months grew faster in acclimation pens on the

107 release site, dispersed farther, and had generally higher active season survival probability (0.50,

95% CI: 0.28–0.72) than turtles head-started for nine months (0.33, 95% CI: 0.07–0.60), which we attribute to body size.

Our findings are largely in line with the “bigger is better” hypothesis, which suggests larger juvenile body size increases survival and performance (Packard and Packard, 1988;

Janzen, 1993; Kissner and Weatherhead, 2005). Given nest predation can be a serious threat to chelonian population viability (Dodd, 2001; Spencer et al., 2017), head-starting might be an effective strategy for enhancing recruitment (Carstairs et al., 2019). Estimated annual adult survival for T. carolina can be very high (e.g., >0.95, Currylow et al., 2011), whereas at another study site in Michigan, Altobelli (2017) found estimated survival probability of radio-tracked hatchling T. carolina was zero at upwards of one-year in age. Similar to our results, larger hatchlings were more likely to survive over a longer time period (Altobelli, 2017). At the time of each cohort's release, turtles in cohort two were generally larger than cohort one, but unenriched turtles were overall larger than enriched turtles at each release point (Tetzlaff et al., 2019b).

However, there was still variation in body sizes among turtles in each treatment within cohorts.

This variation could partially explain why the effects of treatment and cohort were not ranked as highly in model selection predicting daily survival probability as models containing the effect of body mass. Although confidence intervals for survival estimates overlapped considerably, we suggest there were likely still biologically relevant effects of treatment and cohort on survival; in cohort two, the proportion of surviving enriched turtles was nearly ten percent higher than cohort one (0.40, 95% CI: 0.12–0.74 vs. 0.33, 95% CI: 0.04–0.78), and the proportion of surviving unenriched turtles (0.60, 95% CI: 0.26–0.88) was nearly twice that of cohort one. Additionally, turtles with the highest daily survival probability were the heaviest (approximately 70 g) and

108 therefore were from cohort two because no turtle in cohort one exceeded 50 g in the first season post-release.

Accumulating evidence from this and other studies suggests attaining larger body sizes over a longer rearing period could be an effective method for increasing survival of head-started reptiles. For instance, head-started common watersnakes had greater survival if reared for a longer period (Roe et al., 2010, 2015). Size at release might be especially important for turtles because juveniles are vulnerable to numerous predator species (Dodd, 2001; Nagy et al., 2015).

We randomly selected turtles for release in each cohort to reduce potential biases related to behavior and survival. However, our results collectively suggest intentionally selecting the largest individuals for release at a given point may be most beneficial for increasing survival, and this may require rearing smaller individuals for longer periods before release. Additionally, juvenile turtles have softer shells than adults and full hardening of the carapace does not naturally occur until several years after birth (Arsovski et al., 2018). By growing turtles at an advanced rate in captivity, their carapace hardens earlier and might provide resistance to predation leading directly to higher survival (Daly et al., 2018).

We predicted enriched turtles would be more efficient foragers and thus grow faster than unenriched turtles once released because of their experience searching for spatially variable prey in their more complex rearing environments (Reading et al., 2013). Although growth rates did not differ between treatments in cohort one, enriched turtles in cohort two grew faster than unenriched turtles despite being overall smaller at release (Tetzlaff et al., 2019b). In addition to the potential behavioral benefits conferred by enrichment, being able to eat more diverse prey from having a larger gape could also explain why turtles in cohort two generally grew faster in acclimation pens than those in cohort one (Tucker et al., 1995).

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Although unenriched turtles were able to seek cover in an artificial shelter, we expected more naturalistic hiding opportunities during captive-rearing would better condition enriched turtles to remain hidden more often once released. In turn, we predicted this could be a mechanism for enhancing survival because juvenile terrestrial turtles have limited defenses from predators aside from exhibiting cryptic behavior (Dodd, 2001). However, exposure levels did not differ between treatments or cohorts, and turtles were frequently observed fully exposed. Wild- caught adult captive ratsnakes provided enrichment prior to translocation were also no less visible overall than unenriched conspecifics (DeGregorio et al., 2017). Snakes released from captivity were found exposed more often than wild conspecifics, which seemingly increased vulnerability to predators (DeGregorio et al., 2017). If an extended rearing duration to maximize body size is not logistically or financially feasible when head-starting reptiles, practitioners might consider implementing training programs such as those that have been successful for conditioning antipredator behavior in other taxa (Reading et al., 2013; Tetzlaff et al., 2019a).

Daily movement rates did not differ between rearing treatments or release cohorts, but turtles in cohort two dispersed farther on average than those in cohort one, regardless of rearing treatment. This suggests all turtles had similar activity patterns, but those in cohort two moved farther across the landscape. Larger overall body sizes from a longer rearing period likely facilitated greater movement (Janzen et al., 2000), as we found no evidence suggesting captivity duration affected boldness or exploratory behavior of turtles prior to release (Tetzlaff et al.,

2019b).

Dispersal-related mortality is commonly linked with translocation failure because animals could be more susceptible to predators or vehicle mortality, expend energy reserves while forgoing feeding, or leave high-quality release sites and move to lower quality areas (Le

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Gouar et al., 2012; Attum and Cushall, 2015). Although two (10%) turtles in cohort two were killed by vehicles when dispersing, an unexpected finding in our study was that dispersal largely did not come at a short-term cost to survival. Similar results were also found for translocated voles (Microtus rossiaemeridionalis), which were thought to benefit from dispersal by reducing odor concentrations near the release site that attracted predators hunting via olfaction (Banks et al., 2002). Olfactory-hunting mammals are also major predators of turtles (Dodd, 2001).

Dispersal of translocated juvenile turtles could thus not only confer survival benefits but also enhance gene flow in populations with low genetic diversity (Kimble et al., 2014). Further, terrestrial chelonians have important movement-dependent ecological functions such as seed dispersal (Lovich et al., 2018). For example, eastern box turtles are the only known effective dispersal agent of mayapple (Podophyllum peltatum) (Braun and Brooks, 1987).

Head-starting is a common management practice in turtle conservation programs, but traditional captive-rearing practices thought to increase success are often based on intuition rather than quantitative evidence (Seddon et al., 2007). Our experimental approach allowed for stronger inference regarding mechanisms influencing success and provides insight for future efforts. Because longer rearing duration came at no apparent cost to adaptive behavior or survival, our results indicate raising turtles for several years in captivity to maximize size at release could be valuable for practitioners attempting to restore imperiled populations. Future studies might investigate if post-release survival rates asymptote or perhaps even decline again after a given length of time of captive-rearing. This could be important for determining if survival rates are not improved after turtles exceed a certain size threshold before release or whether captivity duration eventually has negative effects on behavior and post-release survival

(DeGregorio et al., 2013, 2017). Such investigations could aid in striking a balance between

111 maximizing success with cost-effectiveness for head-starting efforts (Fischer and Lindenmayer,

2000; Canessa et al., 2016). Finally, we suggest longer term (i.e., >1 year) post-release monitoring should be conducted when possible. This is necessary for measuring more ultimate outcomes of conservation translocations, particularly for long-lived species such as chelonians and because post-release effects may last for several years (Bertolero et al., 2018).

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TABLES AND FIGURES

Table 5.1. Parameter estimates with 95% lower (LCL) and upper (UCL) confidence limits and associated P-values for growth and behavior metrics for head-started eastern box turtles (Terrapene carolina). Estimates are mean differences between rearing treatments (enriched or unenriched) or cohorts (released after nine or 21 months of head-starting). Unenriched turtles are the reference group for treatment, and turtles released when 21 months old (cohort two) are the reference group for cohort. Turtles were held in acclimation pens for approximately one month before being fully released and radio-tracked. An asterisk indicates an interactive effect.

Metric Predictor Estimate LCL UCL P Growth rate (mm/day) in Treatment -0.01 -0.02 0.01 0.41 acclimation pens Cohort 0.02 0.01 0.04 0.01 Treatment*Cohort 0.01 -0.03 0.04 0.71 Post-release growth rate Treatment -0.03 -0.07 0.00 0.04 (mm/day) Cohort 0.04 0.00 0.07 0.03 Treatment*Cohort -0.04 -0.10 0.02 0.21 Movement rate (m/day) Treatment -0.47 -5.04 4.10 0.84 Cohort 3.03 -1.66 7.72 0.20 Treatment*Cohort -3.82 -13.27 5.63 0.41 Dispersal distance (m) Treatment -30.03 -157.53 97.47 0.63 Cohort 132.91 -8.41 274.23 0.06 Treatment*Cohort -167.42 -432.57 97.72 0.21 Exposure Treatment 0.82 -5.93 7.56 0.81 Cohort -6.17 -13.74 1.39 0.11 Treatment*Cohort 7.22 -7.70 22.14 0.33

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Table 5.2. Model selection results for predicting daily survival of head-started eastern box turtles (Terrapene carolina) based on body mass, time (days since release), rearing treatment (enriched or unenriched), cohort (released in 2017 or 2018), and an intercept-only (i.e., null) model. K is the number of parameters in each model. ΔAICc is the difference in AICc values from a given model to the top-ranked model. AICc weight shows the relative likelihood a given model is the most supported. + indicates an additive effect.

Model K ΔAICc AICc weight Log likelihood mass 3 0.00 0.33 -85.66 mass + time 4 1.72 0.14 -85.51 mass + cohort 4 1.99 0.12 -85.65 mass + treatment 4 2.01 0.12 -85.66 intercept-only 2 3.49 0.06 -88.41 cohort 3 3.64 0.05 -87.48 time 3 3.77 0.05 -87.55 cohort + time 4 4.09 0.04 -86.70 treatment 3 5.19 0.02 -88.26 treatment + cohort 4 5.39 0.02 -87.35 treatment + time 4 5.41 0.02 -87.36

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Figure 5.1. Rearing conditions for enriched (left) and unenriched (right) head-started eastern box turtles (Terrapene carolina). Enriched turtles were housed in groups of 4–5, provided with coconut fiber substrate to bury in, artificial plants and half logs for additional hiding areas, and naturalistic water dishes. Unenriched turtles were housed individually and provided with carpet for substrate, a small plastic hiding structure, and standing water to soak in.

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Figure 5.2. Average daily growth rates of head-started eastern box turtles (Terrapene carolina) while being held in acclimation pens for approximately one month before release. Turtles in cohort one and two had been head-started for nine and 21 months, respectively, prior to being placed in pens. Points are means and bars are 95% confidence intervals.

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Figure 5.3. Daily post-release growth rates of head-started eastern box turtles (Terrapene carolina). Turtles were captive-reared in either an enriched or unenriched condition for nine months (cohort one) or 21 months (cohort two). Points are means and bars are 95% confidence intervals.

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Figure 5.4. Mean dispersal distances of head-started eastern box turtles (Terrapene carolina). Turtles in cohort one and two had been head-started for nine and 21 months, respectively, prior to release. Points are means and bars are 95% confidence intervals.

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Figure 5.5. Proportion of head-started eastern box turtles (Terrapene carolina) surviving over time during the active season based on being raised in enriched or unenriched conditions for either nine (left panel) or 21 months (right panel) before release.

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Figure 5.6. Estimated daily survival probability (solid line) fit by loess smoothing with 95% confidence interval (ribbon) of head-started eastern box turtles (Terrapene carolina) as a function of body mass.

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CHAPTER 6: SUMMARY

The general goal of my dissertation was to further discern factors contributing to success of wildlife translocations, particularly how efforts aimed at conditioning adaptive behavior prior to translocation influences post-release outcomes. By conducting a systematic literature search for and meta-analysis of effects from such studies, I found antipredator training, environmental enrichment, and soft release can enhance survival, reduce movement, and increase site fidelity of translocated animals (Chapter 2). Juveniles released from captivity derived the greatest survival benefit from these conditioning tactics. Across taxa, conditioning most benefitted survival of fish, followed by mammals and reptiles, and least for birds. Soft release consistently reduced movement and increased site fidelity; this was an especially viable technique for adult wild-to- wild translocated animals.

My second specific goal was to determine whether raising turtles in contrasting conditions affected their habitat preferences in captivity, with the intention of informing efforts that rely on captive-rearing and release animals into nature for conservation purposes (head- starting). In Chapter 3, I examined if captive-born juvenile eastern box turtles (Terrapene carolina) exhibited preference for their respective rearing environment or an alternative habitat when provided a choice between the two. I found turtles predominantly preferred to occupy a naturalistic enriched habitat over comparably simplistic, unenriched environment, regardless if they were raised in enriched or unenriched captivity. I found no evidence suggesting this preference was due to novelty for unenriched turtles because nearly all turtles exhibited preference for their respective rearing environment when given a choice between this and a habitat novel to all turtles (an empty tissue box). Together, these results suggest preference for enriched habitats is innate for this species and could thus enhance welfare of turtles during head-

127 starting.

A common focus in head-starting programs involves rapidly growing individuals to reduce susceptibility to predation or be closer to reproductive age or size upon release. However, emphasis on rapid growth may have undesirable consequences such as the degradation or loss of normal behaviors, particularly as animals spend longer time in captivity. In Chapter 4, I evaluated if efforts to maximize growth might come at the expense of developing natural behaviors in captivity for head-started eastern box turtles. I found enriched turtles grew slower than unenriched turtles during the first eight months in captivity, although growth rates did not differ between treatments from 9–20 months old. After five months post-hatching, unenriched turtles became and remained larger overall than enriched turtles. During two foraging tasks, unenriched turtles consumed more novel prey than enriched turtles. In a predator recognition test, eight-month-old enriched turtles avoided raccoon (Procyon lotor) urine more than unenriched turtles of the same age, but this difference was not apparent one year later. The odds of turtles emerging from a shelter did not differ between treatments regardless of age. Although turtles raised in unenriched environments initially grew faster and obtained larger overall sizes than those in enriched conditions, results from the suite of behavior trials suggest tradeoffs with ecologically-relevant behaviors were either absent or conditional. However, this investigation looked at only a small number of behaviors in a laboratory setting.

Adequate post-release monitoring is necessary to understand ultimate outcomes of translocations. In Chapter 5, I tested whether head-starting eastern box turtles in an enriched or unenriched environment for nine months (cohort one) or 21 months (cohort two) affected post- release growth, behavior, and survival. By radio-tracking released turtles, I found enriched turtles grew faster than unenriched turtles in cohort two, but I otherwise detected no evidence

128 suggesting enrichment affected post-release survival or behavior. Cohort two dispersed farther and had generally higher active season survival than cohort one (50% vs. 33%). Body mass was positively associated with daily survival probability. These findings are generally in line with the

“bigger is better” hypothesis, which suggests larger juvenile body size increases survival and performance.

Head-starting is a common management tactic in conservation programs for numerous species, but traditional captive-rearing practices thought to increase success are often based on intuition rather than quantitative evidence. My meta-analysis demonstrates raising animals in enriched captive environments can increase translocation success for several taxa. I found captive eastern box turtles show strong instinctive preference for enriched habitat, suggesting raising turtles in such conditions could benefit welfare while being head-started. However, my experiments with box turtles and similar previous studies indicate enrichment might have limited utility for enhancing reptile translocations. I found no evidence suggesting being raised in an enriched environment promoted adaptive behavior or improved survival post-release compared to turtles raised in less stimulating conditions. Thus, attaining larger body sizes from a longer captive-rearing period to enable greater movement and alleviate susceptibility to predation (the primary cause of death in my study) could be more effective than environmental enrichment in head-starting programs for turtles and similar taxa where substantial predation could hinder success. I suggest raising turtles for several years in captivity to maximize size at release could be valuable for practitioners attempting to restore imperiled populations. Future studies might investigate if post-release survival rates asymptote after a given length of captive-rearing. This could be important for determining if captivity duration eventually has negative effects on behavior and post-release survival or if survival rates are not improved after turtles exceed a

129 certain size threshold before release. Furthermore, this could aid in striking a balance between maximizing success with cost-effectiveness for head-starting efforts.

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APPENDIX: SUPPLEMENTARY DATA FROM CHAPTER 25

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Supplementary figures

Figure A1. Phylogenetic tree of species used in meta-analysis of survival.

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Figure A2. Phylogenetic tree of species used in meta-analysis of movement.

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Figure A3. Phylogenetic tree of species used in meta-analysis of site fidelity. Note phylogeny was not included as a random factor in this analysis (see Methods).

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Figure A4. Funnel plot of effects from studies investigating the influence of behavioral conditioning on survival. Filled circles are actual effect sizes estimated from a trim-and-fill analysis (see Results).

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Figure A5. Funnel plot of effects from studies investigating the influence of behavioral conditioning on movement. Filled circles are actual effect sizes and hollow circles are four missing effect sizes estimated from a trim-and-fill analysis (see Results).

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Figure A6. Funnel plot of effects from studies investigating the influence of behavioral conditioning on site fidelity. Filled circles are actual effect sizes and the hollow circle is one missing effect size estimated from a trim-and-fill analysis (see Results).