GOPHERUS AGASSIZII

CHANGING THE SURVIVAL FORMULA FOR THE MOJAVE DESERT TORTOISE

(GOPHERUS AGASSIZII) THROUGH HEAD-STARTING

by

PEARSON A. MCGOVERN

(Under the Direction of Tracey D. Tuberville and Clinton T. Moore)

ABSTRACT

Mojave desert tortoise populations are in decline and improving juvenile survival pre-

release (head-starting) is being evaluated to augment populations. We released three

treatment groups to evaluate the potential of combination head-starting. Treatment groups

consisted of tortoises reared outdoors for 6-7 years (n = 30), outdoors for two years (n =

24), or indoors for one year followed by outdoors for one year (‘combination head-

started’; n = 24). Combination head-starts were smaller than 6-7-year-old outdoor reared

animals at release, and both groups were significantly larger than animals reared solely

outdoors for two years. All treatment groups had nearly identical body conditions, while

two-year-old outdoor animals had significantly softer shells than either of the other

treatments pre-release. Combo head-starts exhibited strong post-release site-fidelity in

comparison to the solely outdoor reared treatments. Size was a significant predictor of

survival, with combo head-starts and 6-7-year-old outdoor head-starts exhibiting

particularly high survival rates 10-months post-release.

INDEX WORDS: Reptile, turtle, desert tortoise, Gopherus agassizii, population

augmentation, head-starting, applied conservation, survivorship,

recruitment



by

PEARSON A. MCGOVERN

B.S., Texas A&M University, 2017

A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial

Fulfillment of the Requirements for the Degree

MASTER OF SCIENCE

ATHENS, GEORGIA

2019

© 2019

PEARSON A. MCGOVERN

All Rights Reserved



by

PEARSON A. MCGOVERN

Major Professors: Tracey D. Tuberville

Clinton T. Moore

Committee: Kurt A. Buhlmann

Jeff Hepinstall-Cymerman

Electronic Version Approved:

Ron Walcott

Interim Dean of the Graduate School

The University of Georgia

December 2019

iv

ACKNOWLEDGEMENTS

Any use of trade, firm, or product names is for descriptive purposes only and does not

imply endorsement by the United States Government. Funding for this research was

provided by the National Park Service (PSAC‐CESU Cooperative Agreement numbers

P08AC00193 and P17AC01606), California Energy Commission (Agreement numbers

500‐10‐20 and EPC-16-038), and by the U.S. Department of Energy under award DE-

FC09-07SR22506 and DE-EM0004391 to the University of Georgia Research

Foundation. All procedures were conducted in accordance with permits issued by the

U.S. Fish and Wildlife Service (TE-17838A-3), U.S. National Park Service (MOJA-

2018-SCI-0016 [under study MOJA-00258]), and California Department of Fish and

Wildlife (SC-0011221). Animal protocols were approved by the University of Georgia

under Animal Use Permits A2013 02-017-Y1-A0 and A2017 01-021-Y3-A3. I thank the

Warnell School of Forestry and Natural Resources at the University of Georgia for

funding through the Warnell Assistantship program. Roy C. Averill-Murray (USFWS -

Desert Tortoise Recovery Office) and Rebecca Jones (California Department of Fish and

Wildlife) provided assistance with permitting. Drs. Brian Todd and Mark Peaden

provided immeasurable support and commitment to the completion of this project. Jacob

Daly was instrumental in providing both field and analytical support. I also want to thank

Melia Nafus, Max Steele, Carmen Candal, and Collin Richter for their valuable

contributions in both husbandry and field work.

v

It was my choice of committee that made this project and my entire master’s

experience such an unforgettable time in my life. To Dr. Tracey Tuberville, a huge thank

you for the time and effort you put into making this thesis a valuable addition to the

conservation of the desert tortoise. The thoughtfulness and care that you place into each

detail of the scientific process is something I will draw from for the rest of my career. To

Dr. Kurt Buhlmann, I will always remember the many talks we shared on the excitement

and joy that all things nature bring to us both. There is still so much to be seen and

discovered, and I look forward to pursuing these countless opportunities with the

unceasing excitement with which you live life. You and Tracey have solidified my love

for turtles while expanding my skills and knowledge to really make a change! To Dr.

Clint Moore, I have been impressed with you since the first day I walked into your office,

and I will be forever thankful that you were willing to serve as my co-advisor. This

project benefitted greatly from your perspective and unrivaled skill. To Dr. Jeff

Hepinstall-Cymerman, incorporating your expertise was a crucial component of not only

this study but my growth as a scientist.

Dr. Mark Peaden (‘Marky mark’), you already know how much you’ve done for

me and this project, so instead my thanks is for the friend you have been since day one.

Having you and Hankster around made the field feel like home. A huge thanks to my lab

mates, all of whom embraced me into their lab and always had my back. I also want to

thank Adam Clause for showing me how to be a true professional in this field.

When contemplating the pursuit of this degree, it was the support of loved ones

that made the decision a no-brainer. My parents have supported me from day one and for

vi

that I will be forever grateful. Cam, your love and endless patience deserve so much

credit in the completion of this project.

vii

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ............................................................................................... iv

LIST OF TABLES ............................................................................................................. ix

LIST OF FIGURES .............................................................................................................x

CHAPTER

1 INTRODUCTION AND LITERATURE REVIEW .........................................1

Literature Cited ..........................................................................................10

2 COMPARING OUTDOOR ONLY REARING VERSUS A COMBINATION

OF INDOOR-OUTDOOR REARING FOR HEAD-STARTING THE

MOJAVE DESERT TORTOISE (GOPHERUS AGASSIZII) .........................19

Introduction ................................................................................................19

Methods......................................................................................................23

Results ........................................................................................................34

Discussion ..................................................................................................38


3 SIZE AS A PREDICTOR OF POST-RELEASE SURVIVAL FOR HEAD-

STARTED MOJAVE DESERT TORTOISES (GOPHERUS AGASSIZII) ....65

Introduction ................................................................................................65

Methods......................................................................................................69

Results ........................................................................................................78

viii

Discussion ..................................................................................................80


4 SUMMARY AND CONCLUSIONS ..............................................................99

Literature Cited ........................................................................................102

ix

LIST OF TABLES

Page

Table 2.1: Summary statistics of growth metrics for head-started Mojave desert tortoises

(Gopherus agassizii) reared under two different husbandry

treatments………………………………………………………………………...56

Table 2.2: Post-release movement metrics for head-started Mojave desert tortoises

(Gopherus agassizii) from two treatment groups (Combination head-started and

Outdoor head-started) released in September 2018 in the Mojave National

Preserve, CA, USA ................................................................................................57

Table 3.1: Candidate models used to evaluate which predictors (Age, MCL, Total

Displacement, Surface activity, Burrow switching) have the greatest effect on the

post-release fate of head-started Mojave desert tortoises released into the Mojave

National Preserve, CA, USA. ................................................................................94

Table 3.2: Predicted 10-month survival rates and 95% confidence intervals for juvenile

Mojave desert tortoises derived using a generalized linear model with fate as a

binary response variable and both MCL at release (mm) and proportion of surface

activity as predictor variables ................................................................................95

x

LIST OF FIGURES

Page

Figure 2.1: Midline carapace length (MCL) of 2016 cohort juvenile Mojave desert

tortoises (Gopherus agassizii) in two treatment groups ........................................58

Figure 2.2: Mass of 2016 cohort juvenile Mojave desert tortoises (Gopherus agassizii) in

two treatment groups..............................................................................................59

Figure 2.3: Body condition of 2016 cohort juvenile Mojave desert tortoises (Gopherus

agassizii) in two treatment groups .........................................................................60

Figure 2.4: Shell hardness index (SHI) versus midline carapace length (MCL) for two

treatment groups of juvenile Mojave desert tortoises (Gopherus agassizii)

compared after both one year and two years of pre-release head-starting. ............61

Figure 2.5: Shell hardness index (SHI) versus midline carapace length (MCL) for 2011,

2012, and 2016 cohort juvenile Mojave Desert Tortoises (Gopherus agassizii) in

September 2018 .....................................................................................................62

Figure 2.6: Mean distance moved from release site during pre-dormancy dispersal (left)

and mean distance moved from dormancy burrow after 10 months post-release

(right) by juvenile Mojave desert tortoises (Gopherus agassizii) from two

treatment groups.....................................................................................................63

Figure 2.7: Kaplan-Meier survival curves for head-started Mojave desert tortoises

(Gopherus agassizii) in two treatment groups released in the Mojave National

Preserve, CA, USA. ...............................................................................................64

xi

Figure 3.1: Map of release site with 78 release points for head-started Mojave desert

tortoises released in the Mojave National Preserve, CA ........................................96

Figure 3.2: Kaplan-Meier survival curve for head-started Mojave desert tortoises

(Gopherus agassizii) released into the Mojave National Preserve, CA, USA. ......97

Figure 3.2: Observed survival fates and predicted survival probabilities based on size

(MCL) at release for juvenile Mojave desert tortoises (Gopherus agassizii)

released into the Mojave National Preserve, CA, USA. ........................................98

1

CHAPTER 1

INTRODUCTION AND LITERATURE REVIEW

Wildlife species worldwide are experiencing severe population declines (Gibbons et al.

2000, Vie et al. 2009). Particularly at risk are turtles and tortoises, with nearly half of all

species threatened with extinction (Turtle Taxonomy Working Group 2017). Threats

include habitat loss and degradation (Dodd 1990, Gibbons et al. 2000, Gibbs and Shriver

2002), poaching for consumption and the illegal pet trade (Shiping et al. 2006), invasive

species (Buhlmann and Coffman 2001, Cadi and Joly 2004, Bolton and Brooks 2010),

subsidized predators (Browne and Hecnar 2007, Esque et al. 2010), and a changing

climate (Ihlow et al. 2012). The effects of these widespread threats are often exacerbated

by the suite of life history traits that characterize most chelonians (Congdon et al.

1993,1994, Tucker et al. 2001). Chelonians typically follow a pattern of slow growth,

delayed sexual maturity, high adult survivorship, and low rates of hatchling recruitment

(Iverson 1991, Turner et al. 1987, Congdon et al. 1993), traits which greatly increase

vulnerability to continued losses and contribute to markedly slow population recovery

(Congdon et al. 1994, Hall et al. 1999, Dorcas et al. 2007). Studies have shown that turtle

populations are predominantly reliant on and sustained by high rates of adult survival

(Doak et al. 1994, Heppell 1998). However, if strategies for mitigating losses to the adult

population are combined with efforts to bolster multiple life-stages, population recovery

may be expedited (Crawford et al. 2014, Spencer et al. 2017). In response, turtle

conservation programs have begun promoting and implementing management techniques

2

aimed at improving neonate and juvenile survival alongside efforts that reduce adult

mortality (Burke 2015, USFWS 2011, Crawford et al. 2014, Buhlmann et al. 2015).

Head-starting - the process of rearing offspring in captivity through their most

vulnerable life-stages to increase survivorship (Burke 2015) - is experiencing a recent

resurgence in turtle conservation after a period of discordant skepticism (Frazer 1992,

Heppell et al. 1996, Seigel and Dodd 2000). Head-starting programs have been

implemented for a variety of turtle species, including western pond turtles (Actinemys

marmorata; Haegen et al. 2009), redbelly turtles (Pseudemys rubriventris; Haskell et al.

1996), Blanding’s turtles (Emydoidea blandingii; Buhlmann et al. 2015), European pond

turtles (Emys orbicularis; Mitrus 2005), gopher tortoises (Gopherus polyphemus;

Tuberville et al. 2015), and Galapagos giant tortoises (Chelonoidis spp.; Milinkovitch et

al. 2013, Jensen et al. 2015). The controversial history of head-starting stems from its

expensive implementation, its concentrated focus in which only one element (juvenile

survival) of the larger conservation problem is addressed, and the limited post-release

monitoring data available to formally evaluate its effectiveness (Frazer 1992, Morafka et

al. 1997, Seigel and Dodd 2000). However, studies have shown that using head-starting

concurrently with other management initiatives can expedite population stability as well

as improve recovery (Crawford et al. 2014, Spencer et al. 2017). Solely using head-

starting to combat population declines without first ameliorating the causes of the

declines is ineffective; however, if used as part of a toolbox for managing population

viability it may play a vital role in conserving imperiled populations.

As part of the process of developing and refining species-specific head-starting

programs, evaluations should consider the effects of both captive and release conditions

3

on head-started animals. When augmenting wildlife populations through the release of

head-started individuals, habitat quality at the release site must be a priority (Griffith et

al. 1989). Habitat quality considers not only habitat requirements such as availability of

refugia, nest sites, and forage, but also more broadly whether predator abundances and

levels of intra- and interspecific competition approximate natural conditions. Through

ensuring that animals are released into optimal habitat, practitioners encourage fidelity to

the release site (Griffith et al. 1989, Larkin et al. 2004). In turn, increasing site fidelity

diminishes energetically costly movements away from the intended site, which is the

single greatest reason for failure in reptile release projects (Germano and Bishop 2009).

Long bouts of searching or dispersal from the release site, most often observed

immediately post-release, can also increase risk of environmental exposure and predation

(Hazard and Morafka 2002, Attum et al. 2010). Thus, post-release monitoring should

address both movement and survival following release.

Another important metric to consider when evaluating head-starting is individual

health. Individual health can be used to assess the quality of husbandry treatments but

may also be an important predictor of post-release performance and success (Booth and

Hixon 1999, Schmutz and Ely 1999, Garner et al. 2011). Evaluating pre-release health is

vital in avoiding disease transfer to previously unaffected populations (Woodford and

Rossiter 1994), as well as improving post-release survival by releasing individuals with

high body condition (Booth and Hixon 1999). Body condition, calculated as a ratio of

mass to some measure of length or volume, has been used extensively as a metric of

individual health (Stevenson et al. 2006). In tortoises specifically, a high body condition

translates to greater water balance and storage capacity, which in turn increases an

4

individual’s ability to withstand periods of drought (Nagy et al. 2002, Loehr et al. 2007).

For species inhabitating resource limited environments such as deserts, conservation of

limited water resources is integral for continued survival (Averill-Murray 2002).

The Mojave desert tortoise (Gopherus agassizii, hereafter ‘desert tortoise’ or

‘tortoise’) is a fossorial species endemic to the Mojave and Colorado deserts in the

southwest U.S.A. (Murphy et al. 2011). Desert tortoises confront a myriad of threats,

including habitat loss (Berry 1984), road mortality (Boarman and Sazaki 1996, Peaden et

al. 2015), anthropogenically subsidized predators (Esque et al. 2010), disease (USFWS

2011), and a changing climate (Lovich et al. 2014), all of which may act independently or

in concert (Foreman 1986, USFWS 2011). These threats have caused severe and

continued population declines (Allison and McLuckie 2018), leading to the development

and implementation of strategies aimed at recovering populations. Specific strategies

include fencing of roadways, habitat restoration, translocation of displaced wild

individuals, and head-starting (USFWS 2011). Head-starting, as previously mentioned,

focuses on improving survival of the vulnerable hatchling age-class (Burke 2015).

Annual survivorship and recruitment of hatchlings in the wild is naturally low, while

likely highly episodic (Gibbons 1987, Morafka 1994). Thus, management techniques that

focus on improving survival of this naturally at-risk age-class may help contribute to

population recovery (Heppell 1998, Tuberville et al. 2009).

Most formal head-starting programs for the desert tortoise have focused on

outdoor rearing in predator-proof enclosures in natural habitat (Morafka et al. 1997).

These initial projects sought to determine whether outdoor predator-proof enclosures

could be used to successfully rear juvenile desert tortoises (Morafka et al. 1997) and if

5

supplemental rain had an effect on growth or survivorship of tortoises while in captivity

(Nagy et al. 2015a). Annual survivorship of captive-reared juveniles has been shown to

be high (>90%) in outdoor predator-proof pens (Nagy et al. 2015a, Spangenberg 1996,

Tuberville et al. 2019). Supplemental rain increased growth during captivity and was

especially important in improving survival of older juveniles during drought conditions

(Nagy et al. 2015a). However, because desert tortoises living outdoors undergo both

winter dormancy and summer estivation, growth was still relatively slow, requiring 5-9

years of outdoor rearing for juveniles to reach a size considered to be predator-resistant

(Hazard et al. 2015, Nagy et al. 2015b). Post-release monitoring of juveniles found that a

minimum release size of 84-110 mm midline carapace length (MCL) conferred an

advantage in avoiding predation. These initial studies recommended use of supplemental

rain during captive rearing to increase growth and selecting release sites at least 546 m

from the head-start facility to preclude homing (Hazard et al. 2015, Nagy et al. 2015a,

Nagy et al 2015b).

In 2011, University of Georgia and University of California, Davis initiated a

joint long-term project in the Mojave National Preserve, CA to experimentally evaluate

head-starting as a recovery tool for the desert tortoise. Initial experiments at our site

focused on quantifying survival and growth differences between directly released

hatchling tortoises and tortoises head-started for 6-18 months under two different levels

of supplemental rain (Tuberville et al. 2019). Doubling the level of supplemented rain

significantly increased growth, although the modest improvement underscored the

lengthy head-starting period required for juveniles reared solely outdoors to attain

recommended release sizes (Tuberville et al. 2019). However, annual survival during

6

captivity averaged 94% across rain treatments (Tuberville et al. 2019), corroborating

findings from previous studies that annual survival of juvenile desert tortoises in outdoor

predator-proof pens is high (Nagy et al. 2015a, Spangenberg 1996).

Tuberville et al. (2019) monitored post-release survival of animals released at age

0-18 months, providing baseline estimates for future comparisons at our site. Although

the release sizes (40.8-61.5 mm MCL) were smaller than the release sizes recommended

in previous studies (Nagy et al. 2015b, Hazard et al. 2015), the goal was to incorporate an

experimental control (by including directly released hatchlings) and provide pilot data for

refining head-starting protocols. None of the factors examined - including size at release,

time of year released, maternal effects, or season - were found to have a significant effect

on post-release survival, with annual survival estimates averaging 48-49% across all

animals (Tuberville et al. 2019). Collectively, these results suggest that unmeasured

factors play an important role in survival of head-starts reared outdoors for only short

periods in captivity (6-18 mo). Alternatively, the effects of these factors may not have

been evident at the range of sizes compared.

Due to the considerable investment necessary to rear animals outdoors to

recommended release sizes, Tuberville et al. (2019) suggested that indoor rearing should

be investigated as a potential head-starting technique for desert tortoises. Indoor head-

starting has been used successfully with both aquatic and terrestrial turtle species

(Buhlmann et al. 2015, Green 2015, Quinn et al. 2018), and has the potential to greatly

increase the efficiency of head-starting programs. Animals reared indoors remain active

and feeding year-round and thus exhibit accelerated growth, leading to a decrease in the

7

duration of the head-start period required for animals to attain the recommended release

size.

A recent study by Daly et al. (2018) implemented this recommendation by

evaluating the effects of indoor head-starting on growth, body condition and shell

hardness in desert tortoises. The study compared direct released hatchlings to both

outdoor-reared and indoor-reared tortoises head-started for seven months, all from the

same year cohort, making it the first study to include an indoor husbandry component for

head-starting the Mojave desert tortoise. Indoor-reared tortoises grew over 16 times faster

than direct-released animals and over eight times faster than those reared in outdoor

predator-proof enclosures, clearly showing the growth advantage of foregoing

hibernation in a stable indoor environment (Daly et al. 2018). The study also evaluated

body condition and shell hardness for all released individuals. Body condition values for

solely indoor head-started tortoises were lower than those of their solely outdoor-reared

siblings, although still within the range of values calculated for wild desert tortoises

(Nagy et al. 2002). Shell hardness was compared between seven-month-old indoor-reared

tortoises and similarly sized but older (3-4yr old) outdoor-reared tortoises. Tortoises

reared indoors had softer shells than older tortoises raised outdoors, although this may be

the result of the shell ossification process needing to develop over a period greater than

that of the study (Daly et al. 2018, Nagy et al. 2011). In fact, shell hardness values for all

indoor animals were within the normal range for this species when compared to estimates

for wild tortoises of this age class (Daly et al. 2018, Nagy et al. 2011). Shell hardness,

which increases with both size and age, is a key metric in head-starting due to its

presumed correspondence with vulnerability to predation (Nagy et al. 2011), particularly

8

ravens (Kristan & Boarman 2003). Daly et al. (2019) evaluated the post-release survival

of these indoor head-starts, comparing them to both directly released hatchlings and

animals head-started solely outdoors for seven months. Treatment groups exhibited no

difference in post-release survival (Daly et al. 2019). In contrast to a previous release at

the same site, in which only one mortality (2.2% of animals released) was attributed to

avian predation (Tuberville et al. 2019), common ravens (Corvus corax) were found to be

the main cause of death (26.5% of animals released) for head-starts released by Daly et

al. (2019). To reduce the risk of raven predation, Daly et al. (2019) recommended

releasing head-starts in the fall to avoid brooding ravens and at least 1.6 km from possible

raven nesting structures (powerlines, billboards, etc.).

To follow up on the findings of these prior studies and to continue evaluating

head-starting as a viable management technique for the Mojave desert tortoise, this study

incorporated the use of combination head-starting. Combination head-starting (hereafter

“combo” head-starting), as defined in this study, is the combination of initial rearing

indoors followed by outdoor rearing prior to release. This afforded “combo” head-started

tortoises the advantage of increased growth during the indoor phase while also allowing

them time outdoors for increased shell ossification, as well as physical and behavioral

acclimatization to their natural environment. The objectives of my thesis research are to

evaluate this novel head-starting method by comparing pre- and post-release metrics of

success between two year old “combo” head-started tortoises, tortoises of their same age

but smaller size reared solely outdoors, and older tortoises of the same size reared solely

outdoors. In Chapter 2, I assigned animals from the same cohort to one of two treatments:

1) “combo” head-started animals reared indoors for one year followed by outdoors for

9

one year, and 2) animals reared solely outdoors for two years. I compare their pre-release

morphometrics, body condition, and shell hardness as well as their post-release

movement and survival for 10 months following release. In Chapter 3, I compare survival

and release size between animals reared solely outdoors for different head-starting

durations (two years or 6-7 years) to refine the recommended size at release for this

threatened species. In the concluding chapter (Chapter 4), I synthesize the results of my

research, place them in the context of previous findings, and make recommendations for

their implementation in future head-starting programs for desert tortoises. Given the dire

condition of turtle populations worldwide (Turtle Taxonomy Working Group 2017),

contributing to the knowledge of turtle population augmentations is critical for future

conservation efforts.

10

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CHAPTER 2

COMPARING OUTDOOR ONLY REARING VERSUS A COMBINATION OF

INDOOR-OUTDOOR REARING FOR HEAD-STARTING THE MOJAVE DESERT

TORTOISE (GOPHERUS AGASSIZII)

INTRODUCTION

Many wildlife populations are in severe decline globally due to anthropogenic

threats (Gibbons et al. 2000, Loh et al. 2008, Vie et al. 2009). In response,

conservationists and management agencies have implemented diverse management

strategies to recover populations (Myers et al. 2000, Lindenmayer et al. 2006, USFWS

2011, Haag et al. 2014). While strategies aimed at acquiring and protecting natural

habitat remain paramount in conserving wildlife, species with depleted populations often

require additional interventions (Ellis et al. 2000, Jachowski et al. 2009, Milinkovitch et

al. 2013). Head-starting is an approach in which offspring are protected through their

early life stages before release at a size presumed to be less prone to mortality (USFWS

2011, Burke 2015). When used alongside management techniques that mitigate the

original causes of decline, head-starting has the potential to expedite population recovery

(Tomillo et al. 2008, Crawford et al. 2014, Spencer et al. 2017). Head-starting has been

used in the recovery of a variety of taxa (e.g., Mona Island iguanas [Cyclura cornuta

stejnegeri], Pérez-Buitrago et al. 2008; Philippine crocodiles [Crocodylus mindorensis],

van de Ven et al. 2009; mangrove finches [Camarhynchus heliobates], Cunninghame et

al. 2015; and hellbenders [Cryptobrancus alleganiensis], Crane and Mathis 2011), but to

increase success and efficiency, it is important to continue evaluating, improving, and

customizing this management technique for each at-risk species.

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The Mojave desert tortoise (Gopherus agassizii) is a keystone species of the

Mojave and Colorado deserts of the southwest U.S.A. that has experienced severe

population declines (Luckenbach 1982, Murphy et al. 2011, USFWS 2011, Allison and

McLuckie 2018) and has been classified as threatened under the U.S. Endangered Species

Act. Populations of this desert endemic are affected by habitat loss, road mortality,

subsidized predators, and the direct and indirect effects of changing climate (Berry 1986,

Barrows 2011, USFWS 2011, Peaden et al. 2015). To combat these threats and recover

depleted populations, a wide range of management strategies has been suggested and

employed (USFWS 2011), including head-starting.

The Mojave desert tortoise has been the subject of head-starting efforts for nearly

30 years (Morafka et al. 1997). Initial projects involved the rearing of hatchlings in

outdoor predator-proof enclosures situated in natural habitat to evaluate the effects of

captive rearing enclosures on nest success and hatchling survival and to gather insight

into unknown aspects of juvenile tortoise ecology (Morafka et al. 1997). Juvenile survival

in predator-proof enclosures was high (>90%; Morafka et al. 1997, Spangenberg 1996),

leading to additional head-starting projects (Nagy et al. 2015a, Tuberville et al. 2019).

These subsequent projects incorporated the use of supplemental rain in outdoor

enclosures to increase growth and improve survival during periods of natural drought.

Recent studies continue to build on these earlier findings to identify possible avenues for

further improvement of these metrics.

Slow growth rates have burdened conventional outdoor captive-rearing efforts for

the desert tortoise (Hazard et al. 2015, Nagy et al. 2015a). Desert tortoises spend roughly

95% of their lives below ground during the thermal extremes of winter and summer

21

(Woodbury and Hardy 1948, Ernst and Lovich 2009). These prolonged bouts of inactivity

during inhospitable ambient conditions result in restricted opportunities for foraging and

growth (Medica et al. 2012). Previous studies have found that 5–9 years of outdoor

captive-rearing are required to raise juveniles to a size that confers a survival advantage

upon release into the wild (84-110 mm midline carapace length (MCL); Nagy et al. 2011,

Hazard et al. 2015, Nagy et al. 2015a). To reduce this extended captivity, indoor captive-

rearing has recently been used to enhance growth by allowing head-starts to remain

active and foraging year-round (Daly et al. 2018). The expedited growth exhibited by

tortoises reared under this technique was significant, producing 7-month-old captive-

reared tortoises equivalent in size to 5-6-year-old wild animals (Daly et al. 2018).

However, indoor captive-reared tortoises had softer shells and slightly lower body

condition compared to older animals (3-4 yrs old) of similar size reared solely outdoors

(Daly et al. 2018). Rearing hatchlings solely indoors also holds the potential of raising

tortoises naïve to their surroundings upon release into the wild (Grueber et al. 2017).

Incorporating an outdoor rearing component may serve as pre-release enrichment by

providing head-starts with native forage, opportunities for burrowing, and exposure to

natural environmental cues. Environmental enrichment has been shown to improve the

survival of captive reared animals released to the wild (Tetzlaff et al. 2019). In this study,

we introduce the use of combination head-starting, defined as initial captive-rearing

indoors followed by conventional outdoor captive-rearing prior to release. This novel

approach maximizes tortoise growth during indoor rearing and exposes tortoises to

environmental cues during outdoor rearing. The outdoor rearing period should also

promote shell ossification through exposure to natural UV light (Acierno et al. 2006).

22

We use shell hardness and body condition as metrics for monitoring the possible

effects of accelerated growth during the captive period in desert tortoises. Shell hardness

has been found to increase with both body size and age in juvenile desert tortoises (Nagy

et al. 2011, Daly et al. 2018). Although we did not find studies showing a direct

correlation between shell hardness and increased survival, an increase in shell hardness

presumably reduces the vulnerability to certain predators, such as ravens (Corvus corax;

Kristan and Boarman 2003, Nagy et al. 2015b). Body condition, an estimate of body

density, has been used as a physiological metric of health in previous tortoise studies

(Nagy et al. 2002, Loehr et al. 2007, McCoy et al. 2011). Nagy et al. (2015a) found that

increased body condition from supplemental watering resulted in higher survival in

outdoor predator proof enclosures during a natural drought. Body condition values may

also increase concurrently with the shell ossification process (as harder shells weigh

more), highlighting the value of monitoring both metrics.

Long-distance dispersal away from the release site is the primary reason for

failure in herpetofaunal release programs (Germano and Bishop 2009). Thus, as a

possible indicator of short-term success, we assessed post-release movement. For head-

starting to ultimately succeed, released animals must survive to maturity and reproduce

successfully (Pritchard 1981). However, monitoring a released juvenile to reproductive

age may take over 10 years post-release. As a more immediate metric of this ultimate

goal, we monitored survival during the first 10 months post-release.

In this study, we compared two head-starting treatments for the Mojave desert

tortoise: 1) solely outdoor captive-rearing for two years; and 2) combination head-

starting, consisting of one year of indoor captive-rearing followed by one year of outdoor

23

captive-rearing. We compared size, body condition and shell hardness between the two

treatment groups using animals from the same year cohort. We also compared movement

and survival from release to dormancy and during the first 10 months post-release.

Ultimately, by reducing the duration of the captive rearing period and decreasing the cost

of implementing this technique, combination head-starting may increase the efficiency

and success of population augmentation efforts.

METHODS

Study Site

This study was conducted in the Ivanpah Valley in the Mojave National Preserve

(MNP), San Bernardino County, California, which is within the Eastern Mojave recovery

unit of the Mojave desert tortoise (Nussear et al. 2009, USFWS 2011). The husbandry

portions of this study were conducted at the Ivanpah Desert Tortoise Research Facility

(IDTRF) at 820 m elevation. The release site, located approximately 15 km away,

occurred at 1000-1100 m elevation. The release site was dominated by Yucca woodland

habitat composed primarily of creosote (Larrea tridentata) and white bursage (Ambrosia

dumosa), with Yucca (Y. brevifolia, Y. shidigera), pencil cholla (Cylindropuntia

ramosissima), and littleleaf ratany (Krameria erecta) heavily intermixed (Todd et al.

2016). Limited anthropogenic disturbances at the release site included abandoned cattle

corrals, a powerline, and seldom-used dirt roads.

Obtaining hatchlings

In April 2011, we initiated the IDTRF head-starting program by capturing,

attaching transmitters (RI-2B, Holohil Systems Ltd., Ontario, Canada), and radio-tracking

30 adult female desert tortoises in the Ivanpah Valley of the MNP. We tracked females at

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least monthly using a 3-element Yagi antenna (AF Antronics, Inc., Urbana, IL) and a

R1000 receiver (Communications Specialists, Inc., Orange, CA). In April 2016, we

brought all radio-tracked females to the IDTRF and radiographed them to check for the

presence of calcified eggs (Diagnostic Imaging Systems, Poskam, Colorado, USA; 60

kvp, 0.8 mAS, 74 cm focal length; Gibbons and Greene 1979). We immediately released

all non-gravid females at their point of capture. We placed gravid females in individual

nesting enclosures and allowed them to nest naturally. These predator-proof nesting

enclosures measured 5 x 9 m and included pre-made burrows, natural cover and forage,

as well as sprinkler systems for supplemental watering (Tuberville et al. 2019). After

initial placement in the enclosures, we radiographed females weekly to monitor for egg

deposition. If nesting was confirmed, we immediately released females at their point of

capture. In accordance with our permits, we released all females that did not nest within

30 days at their last known burrow location.

We allowed eggs to incubate naturally in the predator-proof enclosures.

Approximately 80 days after the estimated nesting date, we began searching pens daily

for emerged hatchlings. We removed hatchings as they emerged from nests and marked

them by notching unique combinations of marginal scutes using numerical codes

assigned by USFWS (modified from Cagle 1939). We also searched nests for any

unhatched eggs or unemerged hatchlings. After all hatchlings from the 2016 cohort had

emerged and been uniquely marked, we used a stratified random design to assign all

healthy neonates (evenly separating siblings between treatments; Daly et al. 2018,

Tuberville et al. 2019) from our 2016 cohort to one of two treatment groups: solely

outdoor head-started (hereafter Outdoor HS) or combination (indoor + outdoor) head-

25

started (hereafter Combo HS). Both treatment groups were reared in captivity for two

years prior to release.

Outdoor HS

On 07 September 2016, we moved the 38 hatchlings assigned to the Outdoor HS

treatment to outdoor predator-proof enclosures at the IDTRF. Captive rearing enclosures

measured 9 m x 9 m and included pre-made starter burrows (53-cm long, 10-cm diameter

perforated plastic pipes buried at 20o angles), natural cover and forage, as well as rotating

sprinkler systems (Nafus et al. 2015b, Daly et al. 2018, Tuberville et al. 2019). We placed

a maximum of 10 animals into each pen (0.125 tortoises/m2), and siblings from the same

clutch were separated into different pens. We provided supplemental food as well as

artificial rain once every 1-2 weeks for 30-min during the active season (March-October).

Rain supplementation was provided using rotating garden sprinklers to sustain native

plant growth (Beatley 1974), allow hatchlings to drink, and to encourage tortoises to feed

on supplemental food.

Tortoises were fed a mixture of leafy greens and water-soaked Mazuri® Tortoise

Diet (Mazuri Exotic Animal Nutrition, St. Louis, MO). The greens included dandelion

(Taraxacum officinale), mustard greens (Brassica juncea), turnip greens (Brassica rapa),

collards (a cultivar of Brassica oleracea), endive (Cichorium endivia), and escarole (C.

endivia latifolia). The weekly combination of greens varied by availability, though

always consisted of at least three different types in an attempt to approximate the

nutritional properties of the desert tortoise’s natural diet (Jarchow et al. 2002). We

monitored the amount provided to each pen to avoid attracting ants and rodents with

excess food.

26

Combo HS

We reared 30 Combo HS tortoises in mesocosms in the indoor climate-controlled

IDTRF for the first year (September 2016-September 2017) before moving them to an

outdoor predator-proof enclosure for the second year (September 2017-September 2018).

We set ambient temperature inside the IDTRF to a constant 24.4 °C. Indoor mesocosms

were constructed from 50-gallon (189 L) Rubbermaid (Atlanta, Georgia, USA) stock

tanks (132 x 79 x 30.5 cm) and lined with natural substrate (Daly et al. 2018). We

prepared six identical tanks, each of which held five tortoises (n = 30). Each tank

contained three hides made from longitudinally halved plastic pipe (11.5 cm in diameter

and 12-cm in length), a paper feeding plate, and a humid hide box. Humid hide boxes

have been shown to promote smooth shell growth in tortoises (Wiesner and Iben 2003).

Hide boxes were made from Rubbermaid Roughneck (Atlanta, Georgia, USA) tote boxes

(40 x 26 x 18 cm) and filled with approximately 4 cm of peat moss, which we moistened

every 3-4 days and replaced every two weeks. Tortoises were able to enter the hide box

through a burrow-shaped entrance hole cut into each tote. We suspended Mini Combo

Deep Dome Dual Lamp Fixtures (ZooMed Laboratories Inc., San Luis Obispo,

California) 30 cm over each tank with a 50 W ZooMed Repti Basking Spot Lamp bulb

for daytime basking (37 °C) and a ZooMed 50 W Infrared Basking Spot bulb for night-

time heat (32 °C). In addition to the dual basking lights, we provided each tub with a 26

W Exo-Terra Reptile UVB150 bulb (45 cm above substrate; Rolf C. Hagen Corp.,

Mansfield, MA., U.S.A) for optimal calcium metabolism and D3 conversion. We

connected all lights to automatic timers, regulated monthly to reflect the natural

27

photoperiod. Indoor conditions were held constant to allow juveniles to remain active and

forage year-round (Diez et al. 2009).

We fed the Combo HS group ad libitum 4-5 times per week during the indoor

rearing period (Diez et al. 2009), using the same food mixture as described above for the

Outdoor HS treatment. Twice per week, we supplemented their food with a light dusting

of Rep-Cal Calcium with Vitamin D3 (Rep-Cal Research Labs, Los Gatos, California).

We soaked Combo HS tortoises weekly for 15-30 minutes in 1-2 cm of water. In

September 2017, we moved all Combo HS tortoises outdoors to a single 10 m x 30 m

predator-proof enclosure at the IDTRF for their second year of rearing. This larger

enclosure contained all of the previously mentioned enclosure components (refugia,

natural vegetation, sprinklers) while maintaining a similar tortoise density (0.11

tortoises/m2) to the Outdoor HS treatment. Supplemental rain was provided weekly

during the activity season (March – October) in this pen.

Morphometrics

We weighed and measured (hereafter ‘measured’) all hatchlings immediately after

emergence from the nest. We then measured Outdoor HS tortoises once each spring and

fall during the captive rearing period. We measured Combo HS tortoises monthly while

indoors (September 2016-September 2017) and then on the same cycle as the Outdoor

HS treatment thereafter. We recorded mass to the nearest 0.1 g (Model HH320, OHAUS

Corporation, Parsippany, NJ, USA); when tortoise mass exceeded 300 g, we recorded

mass to the nearest 1 g (My Weigh 6001, HBI Technologies, Phoenix, AZ, USA). We

recorded midline carapace length (MCL), maximum shell height, and maximum shell

28

width on the bridge to the nearest 0.1 mm using dial calipers (Series 505, Mitutoyo,

Aurora, IL, USA).

Body Condition

We calculated body condition (BC) for all animals from both treatment groups

using measurements taken at hatching (‘initial’), one-year post-hatching (‘Year 1’; when

the Combo HS group was transitioned outdoors), and immediately prior to release

(‘release’; 2 years post-hatching) based on the formula described by Nagy et al. (2002):

𝐵𝑜𝑑𝑦 𝑐𝑜𝑛𝑑𝑖𝑡𝑖𝑜𝑛 (𝑔

𝑐𝑚3) =

𝑤𝑒𝑖𝑔ℎ𝑡 (𝑔)

𝑠ℎ𝑒𝑙𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 (𝑐𝑚3)

𝑆ℎ𝑒𝑙𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 (𝑐𝑚3) = 𝑀𝐶𝐿(𝑐𝑚) × 𝑤𝑖𝑑𝑡ℎ(𝑐𝑚) × ℎ𝑒𝑖𝑔ℎ𝑡(𝑐𝑚)

Shell Hardness

We measured shell hardness of all juveniles (n = 48) after one year of captive

rearing (September 2017) and again just prior to release (September 2018). To calculate

shell hardness index (SHI) we first used a 4-inch (10.2 cm) tension-calibrated micrometer

(L.S. Starrett Company, Athol, Massachusetts, USA; model: 3732XFL-4) to measure

each tortoise’s uncompressed shell height (UCSH) at the center of the third vertebral

scute (Nagy et al. 2011). We then measured compressed shell height (CSH) by turning

the micrometer spindle and compressing each tortoise’s shell between the two measuring

faces until the spindle ratchet slipped continually for approximately 270 degrees (Daly et

al. 2018). We used these measurements to calculate a shell hardness index as described

by Nagy et al. (2011), in which a SHI value of 100 corresponds to maximum hardness

(i.e., an incompressible shell):

𝑆ℎ𝑒𝑙𝑙 𝐻𝑎𝑟𝑑𝑛𝑒𝑠𝑠 𝐼𝑛𝑑𝑒𝑥 (𝑆𝐻𝐼) =𝐶𝑆𝐻

𝑈𝐶𝑆𝐻 × 100

29

Experimental Releases

We released the 2016 cohort Outdoor HS (n = 24) and Combo HS animals (n = 24)

concurrently and in the same area as another release group of older solely outdoor-reared

juveniles (n = 30; 2011-2012 cohorts), discussed separately in Chapter 3. Our release

location was chosen based on the 1.6 km minimum recommended distance from raised

structures such as powerlines that could serve as perching sites for ravens (Daly et al.

2019), accessibility for frequent tracking, and habitat quality (Baxter 1988, Nafus et al.

2015a, Todd et al. 2016). At our study site, juvenile desert tortoises select for

microhabitats with sandy soils, high density perennial shrubs, abundant rodent burrows,

and washes (Todd et al. 2016). Washes have been shown to influence movement (Nafus

et al. 2017, Peaden et al. 2017) and provide increased forage (Jennings and Berry 2015).

Using ArcGIS (version 10.5; ESRI, Redlands, CA), we first created a 0.6-km2 rectangular

release plot (300 m X 2000 m) parallel to an access road. We then used the ‘Fishnet’

function to generate release points every 50 m throughout the plot. From there, we set an

interior buffer (buffer in from plot edge) of 50 m and removed all points within the

buffered area. We then selectively removed interior points to create three equally spaced

blocks 300 m apart and each containing 26 release points for a total of 78 release points.

We buffered 10 m around each release point to maximize our ability to select a release

refugium consisting of a large perennial shrub and an intact kangaroo rat (Dipodomys

spp.) burrow (Nafus et al. 2015a, Todd et al. 2016, Daly et al. 2019). Prior to release, we

enlarged the burrow to provide a refuge large enough to fully conceal the released

animal. Under this release protocol the minimum possible distance between release

burrows was 30 m. Final release points ranged between 150-350 m from the access road

30

and between 1.9-2.5 km to the closest powerline. We chose to separate release blocks to

allow us to track blocks individually, facilitating the tracking of this many animals, and to

minimize our activity in each block.

Post-release monitoring

We attached Advanced Telemetry Systems (ATS; Advanced Telemetry Systems, MN,

U.S.A) radio-transmitters to the fifth vertebral scute with gel epoxy (Devcon 5-minute

epoxy gel, ITW Engineered Polymers, County Clare, Ireland). We fitted Outdoor HS

with R1670 transmitters (3.1 g) and Combo HS with the slightly larger R1680 model (3.6

g). Before the epoxy hardened, we applied a thin coat of sand to camouflage the

transmitter package (Kazmaier et al. 2002). We checked each animal within 24 hours of

release to make sure their transmitters were working. After the initial 24-hour post-

release check, we tracked animals within each release block twice per week for the first

three weeks (25 September 2018-15 October 2018) before reducing our tracking

frequency to once per week until dormancy. During the dormancy period (November

2018-February 2019) we tracked juveniles every 10-14 days before returning to a weekly

tracking schedule in March for the remainder of the study (March 2019-July 2019). We

continued tracking during the winter dormancy period, though at a reduced rate, because

juvenile desert tortoises may be surface active when weather is favorable in winter

months and because we were interested in monitoring winter mortalities (Morafka 1994,

Wilson et al. 1999). At each tracking occasion, we recorded the tortoise’s location to the

nearest ±3 m using a handheld GPS (Garmin model GPSmap 76CSx, Olathe, Kansas).

31

Statistical methods

All statistical tests were performed in Program R (R Core Team 2017), and all inferences

were supported at a threshold type I error rate (alpha) of 0.05. Data are presented as

group means ± 1 standard error (SE). Graphical visualization of model residuals and

Shapiro-Wilk tests were used to test model assumptions of normally-distributed residuals.

When necessary, we transformed the data to meet model assumptions.

Morphometrics

We used linear mixed effects models (LME; ‘nlme’ package, ‘lme’ function) to

test for differences between treatment groups in MCL, mass, BC, and SHI. We compared

these response variables at hatching (‘initial’; except SHI), after 1 year of captive rearing

(‘Year 1’), and at the end of the two-year captive-rearing period (‘release’). We set the

metric of interest as the response variable and included mother identification number (ID)

as a random effect in all models to account for maternal effects (Steyermark and Spotila

2001, Nafus et al. 2015). We included MCL as a continuous covariate in models testing

the effect of treatment on SHI, allowing us to evaluate whether treatments varied at

similar sizes. We also tested for interaction between MCL and SHI in both (‘Year 1’ and

‘release’) SHI models. In any model in which the interaction was not significant, we

retained MCL as a covariate but removed the interactive term from the final model.

Movement

We used the Spherical Law of Cosines (Movable Type Ltd. 2015) to calculate

straight-line distances between successive tracking locations. For each individual, we

calculated mean distance moved per tracking event (excluding dormancy tracking events

from 01 November 2018 – 05 March 2019), final displacement at dormancy (31 October

32

2018) from the original release burrow, final distance from the dormancy burrow at the

end of the study (23 July 2019; for all animals surviving through dormancy), and final

displacement from release burrow until the animal died, went missing or until the end of

the study (23 July 2019; for all animals surviving >25 days post-release). We chose to

include all animals surviving at least 25 days post-release in the latter analyses because

animals in both treatment groups reached a clear asymptote for displacement from the

release site within 25 days. We did not include the dormancy period (01 November 2018

– 05 March 2019) in our movement analyses due to the fact that no movement was

recorded during this time (also seen in Rautenstrauch et al. 1998), though we did

document scat on burrow aprons throughout the winter which can be presumed to be

evidence of winter basking activity during sunny winter days.

We used a gamma hurdle model (Mullahy 1986) to compare distance moved per

tracking event (log link for nonzero distance component, logit link for binary outcome of

moved/not moved) between treatment groups (for all animals that emerged post-

dormancy; Daly et al. 2019). We fit this model under a Bayesian estimation framework

using the ‘brms’ package in R (R version 3.5, www.r-project.org, accessed 05 Sep 2019)

which implements Markov chain Monte Carlo (MCMC) sampling of the posterior

distributions of model parameters. To account for the non-independence of repeated

measures on the same individuals, we included individual identification number (ID) as a

random effect in our model. We used noninformative improper flat priors (uniform

distribution on the real line) to sample treatment group effects. We ran 4 chains of the

MCMC sampler, each with 10,000 iterations and a thinning rate of 1, while discarding

the first 1,000 iterations as burn-in (Daly et al. 2019). We considered treatment group

33

differences to be significant if 95% Bayesian credible intervals (BCIs) of model

predictions did not overlap.

To compare pre-dormancy movement distance, post-dormancy movement

distance, and final displacement between treatment groups, we used a generalized linear

model (Gamma family, log link) with the movement metric as the response variable and

treatment group as a categorical predictor variable. For both pre-dormancy movement

distance and final displacement, we included only those animals surviving >25 days post-

release (21 October 2019; n = 46). To be included in the post-dormancy movement

comparison, animals had to survive the dormancy period and emerge in Spring 2019 (n =

45). We added 0.1 m to all pre-dormancy and post-dormancy movement data to eliminate

zeros contained in the data. We report data as raw means ± 1 SE in text and tables and

figures for ease of interpretation, unless otherwise noted.

Survivorship

Upon finding a deceased tortoise, we thoroughly examined the surrounding area

for clues of cause of death. We photographed each carcass, including any signs of

predation, and removed carcass remnants for further analysis. We attributed deaths to one

of five sources of mortality: mammal depredated, bird depredated, unidentified predator,

exposure, or unknown fate. Signs of predation included digging, tracks, scat, and

condition of the carcass or transmitter. We classified carcasses that were found near clear

digging activity or that had obvious tooth marks as “mammal predated.” Remains near

perch sites or with clear signs of being punctured by a beak were classified as “bird

depredated.” Remains found without tooth marks or lacking evidence of avian predation

were considered depredated by an “unidentified predator.” Animals found intact on the

34

surface or dead in their burrow were presumed to have died due to “exposure.” An

“unknown fate” classification was given to animals lost due to radio failure (faulty

batteries or the antenna became damaged), and these animals were right censored (no

assumption of fate) in our survival analysis. We estimated survival rates in the field from

release until dormancy (25 September 2018 – 31 October 2018) and from release until the

end of the tracking period (September 2018 – July 2019) using the Kaplan-Meier

estimator in the R package ‘survival’ for each treatment group.

RESULTS

Sixteen females collectively laid 92 eggs, resulting in 68 successfully emerged hatchlings

(73.9% emergence) after an average incubation period of 87 days (range 74-106 days).

We randomly selected 48 hatchlings (which represented 15 of the 16 females that

successfully nested) for inclusion in this study.

Morphometrics

Neither mean MCL (45.2 ± 0.3 mm) nor mean mass (20.8 ± 0.3 g) at hatching

(‘initial’) differed between treatment groups (MCL: F1,32 = 0.442, p = 0.51; mass: F1,32 =

0.235, p = 0.63; Table 2.1). After one year of captive rearing (‘Year 1’; September 2017),

when the Combo HS treatment was transitioned to outdoor rearing pens, Combo HS

tortoises (MCL: 102.5 ± 1.5 mm; mass: 205.8 ± 7.2 g) were significantly longer and

heavier than Outdoor HS tortoises (MCL: 66.5 ± 1.5 mm, F1,32 = 291.9, p < 0.001; mass:

62.6 ± 7.4 g, F1,32 = 194.0, p < 0.001; Table 2.1). Combo HS tortoises (MCL: 117.2 ± 1.7

mm, mass: 300.6 ± 10.7 g) retained their size advantage through release (September

2018; Outdoor HS MCL: 83.7 ± 1.7 mm, F1,32 = 193.9, p < 0.001; Outdoor HS mass:

115.9 ± 10.7 g, F1,32 = 148.6, p < 0.001; Table 2.1; Figures 2.1, 2.2), with Combo HS

35

tortoises weighing on average nearly three times that of Outdoor HS tortoises and

achieving the shell length of an 8-year-old wild tortoise (Medica et al. 2012).

Body condition

Using the formula from Nagy et al. (2002), average initial BC was 0.53 ± 0.004

g/cm3 (n = 48) and did not differ between treatment groups (F1,32 = 2.965, p = 0.10; Table

2.1). Body condition values at both Year 1 (September 2017; Combo HS: 0.59 ± 0.007;

Outdoor HS: 0.57 ± 0.007; F1,32 = 2.267, p = 0.14; Table 2.1) and at release (September

2018; Combo HS: 0.57 ± 0.007; Outdoor HS: 0.57 ± 0.007; F1,32 = 0.008, p = 0.93; Table

2.1; Figure 2.3) remained similar between treatment groups. We also found no difference

when comparing mean BC at release for animals found dead prior to spring emergence (n

= 3; 0.58 ± 0.009) to those known to survive dormancy (n = 45; 0.57 ± 0.007; F1,32 =

0.523, p = 0.40).

Shell hardness

Year 1 SHI did not vary between Combo HS (x̄ = 95.9 ± 0.6%) and Outdoor HS

tortoises (x̄ = 94.8 ± 1.31%; t = -0.81, df = 30, p = 0.43; Table 2.1; Figure 2.4). Two

Outdoor HS animals were unavailable for measurements in Year 1 due to early

hibernation. After two years of captive rearing, Combo HS tortoises (x̄ = 97.9 ± 0.4%)

had significantly harder shells than Outdoor HS tortoises (x̄ = 95.7 ± 0.6%; t = -3.86, df =

31, p = 0.0005; Table 2.1, Figure 2.4) at release. Size (MCL) was not a significant

predictor of SHI in the models (p > 0.77 for both ‘Year 1’ and ‘release’), nor was the

interaction between treatment and MCL (p > 0.34 for both ‘Year 1’ and ‘release’). After

two years, Combo HS animals had the shell hardness values of 6-7-year-old outdoor-

reared tortoises at our site (Figure 2.5).

36

Movement

Combo HS and Outdoor HS tortoises did not differ in probability of moving or in

distance moved per movement event during the 10-month study period based on

overlapping Bayesian Credible Intervals [95% BCI]. Probability of moving between

tracking events was 0.46 (95% BCI = 0.42 - 0.50) for the Combo HS group, and 0.42

(95% BCI = 0.38 - 0.47; Table 2.2) for the Outdoor HS group. During movement events,

Combo HS tortoises moved an average of 36.2 m (95% BCI = 29.7 - 44.7 m) per event,

compared to 43.4 m (95% BCI = 34.1 - 54.6 m; Table 2.2) by Outdoor HS tortoises.

Between release and dormancy (‘pre-dormancy’; 25 September 2018 – 31

October 2018), Outdoor HS tortoises dispersed farther from their release burrows (n=23;

x̄ = 255 ± 89 m; t = 1.63, df = 45, p = 0.11), moving nearly twice the distance moved by

Combo HS tortoises (n=23; x̄ = 114 ± 40 m; Table 2.2; Figure 2.6). However, between

emerging from dormancy and the end of the study (‘post-dormancy’; 05 March 2019 – 25

July 2019), Outdoor HS tortoises reduced their movement away from their release

location (n=22; x̄ = 92 + 34 m) and exhibited similar displacement distances between

their dormancy location and final location when compared to Combo HS tortoises (n=23;

x̄ = 54 + 19 m; t = 1.05, df = 44, p = 0.30; Table 2.2; Figure 2.6). However, when the

two largest post-dormancy movers from the Outdoor HS group (896 m and 534 m) were

removed from the data, mean post-dormancy movement distance was reduced to 30 m for

the Outdoor HS treatment group. Total displacement from release burrow until the end of

the study (25 September 2018 – 23 July 2019) was heavily influenced by pre-dormancy

dispersal away from the release burrow, leading to a significant difference in final

displacement between treatment groups (t = 2.05, df = 45, p = 0.047; Table 2.2). Outdoor

37

HS tortoises (n=22; x̄ = 326 ± 93 m) dispersed over two times the distance of Combo HS

tortoises (n=23; x̄ = 143 ± 41 m) during the 10-month post-release monitoring period.

Notably, within treatment groups, there was large variation in final displacement, ranging

from 9–1178 m (Combo HS) and 11–1041 m (Outdoor HS) based on all animals that

survived dormancy.

Post-release survivorship

Survival from release to dormancy (5 weeks post-release; 25 September 2018 - 31

October 2019) was equally high for both treatment groups at 96% (n = 23/24 Combo HS

and n = 23/24 Outdoor HS; Figure 2.7). During dormancy, one Outdoor HS tortoise was

found dead after having been removed from its winter burrow by a mammalian predator,

as evidenced by signs of digging at the burrow. At the end of the 10-month post-release

monitoring period (25 September 2018 - 25 July 2019), 38 (79.2%) of the 48 released

head-starts were confirmed alive. All ten mortalities were attributed to predation; no

animals died due to exposure. Six (12.5% of released tortoises) head-starts were mammal

predated, three (6.2% of released tortoises; all from Outdoor HS group) were killed by

avian predators, and one (2.1% of released tortoises) was killed by an unidentified

predator. The head-start killed by an unidentified predator was found dead and buried in

its burrow. The burrow entrance showed signs of mammalian digging, while both the

burrow and tortoise carcass were covered in harvester ants (Veromessor spp.). Overall

estimated survival for the 48 head-starts during the 10-month post-release monitoring

period (September 2018 - July 2019) was 0.79 (95% CI, 0.69-0.92; Figure 2.7). Ten-

month survival was 0.88 (95% CI, 0.75-1.0) for the Combo HS treatment and 0.71 (95%

38

CI, 0.55-0.92; Figure 2.7) for the Outdoor HS treatment, with overlapping 95%

confidence intervals indicating no difference between treatment groups.

DISCUSSION

After two years of captive rearing, Combo HS tortoises were both larger and had

harder shells than Outdoor HS tortoises from the same cohort. Combo HS tortoises were

the size of an 8-year-old wild juvenile with the shell hardness of an 11-year-old wild

tortoise (Nagy et al. 2011, Medica et al. 2012), and weighed nearly three times more than

same-aged Outdoor HS tortoises. However, Outdoor HS tortoises were also large for their

age when compared to wild counterparts, attaining the size of 4-5-year-old wild tortoises

with just two years of captive rearing (Medica et al. 2012). Outdoor HS animals in this

study grew 19.3 mm MCL/yr, an enhanced rate that can be attributed to the supplemental

food and water they were provided weekly during the activity season (March-October).

Tuberville et al. (2019) reported growth rates of 12.7 mm MCL/yr for juvenile tortoises

reared outdoors without supplemental food at the IDTRF. Collectively, these results lend

support for providing supplemental food to desert tortoises during outdoor captive

rearing, particularly as vegetation in the pens becomes depleted over time.

The growth rate of Combo HS tortoises during their year indoors (4.7 mm/month)

was similar to that found by Daly et al. (2018) for tortoises reared indoors under nearly

the same protocols (4.3 mm/month). Collectively, the findings of Daly et al. (2018) and

those of this study highlight the significant growth advantage of indoor captive rearing

for head-starting efforts for desert tortoises. Combo HS tortoises surpassed the Nagy et

al. (2015) recommended release size of 100 mm MCL after just one year of indoor

rearing (x̄ = 102.5 mm MCL), while Outdoor HS tortoises reached a mean size equivalent

39

to the Hazard et al. (2015) recommendation of 84 mm MCL after two years outdoors (x̄ =

83.7 mm MCL; Figure 2.1).

Throughout the two-year captive rearing period, both treatment groups exhibited

similar body conditions. In contrast, Daly et al. (2018) found that tortoises reared indoors

for seven months had lower body condition than same-aged outdoor-reared individuals

(although this relationship diminished when comparing larger individuals). Daly et al.

(2018), the only other study to incorporate an indoor captive-rearing component for the

desert tortoise, posited that the decreased body condition exhibited by indoor head-starts

may have been a result of decreased bone density in relation to body size. By

incorporating UV lighting and a calcium supplement for the duration of the indoor

captive-rearing period, our husbandry protocols may have enhanced calcium assimilation

during the indoor rearing phase of the Combo HS treatment in comparison to tortoises

reared indoors by Daly et al. (2018), a possibility corroborated by our shell hardness

measurements (see below).

Body condition of head-started tortoises ranged from 0.47-0.65 g/cm3 across

treatments and during the captive rearing period. Nagy et al. (2002) determined prime

body condition values for wild desert tortoises to be between 0.6-0.7 g/cm3, which 21-

27% of individuals in our study attained. However, prime body condition values

represent peak nutritional status and thus do not correspond with the post-summer

(September) body condition values obtained in this study. In fact, it has been shown that

desert tortoises often emerge from summer aestivation weighing less than they did the

previous spring (Nagy et al. 1986). Nonetheless, after initial measurements, all

individuals in our study had body condition values above the Nagy et al. (2002) threshold

40

value for a dehydrated desert tortoise (0.45 g/cm3). Likewise, using the body condition

formula from Loehr et al. (2004), the tortoises in our study were well within the range of

body condition values for both adult speckled tortoises (Chersobius signatus; Loehr et al.

2004) and western pond turtles (Actinemys marmorata; Ashton et al. 2015).

The slow growth and delayed maturity exhibited by chelonians stem from their

investment in maintenance and growth of a protective shell (Klemens 2000). The level of

protection, and presumably the ability to withstand a predatory attack, can best be

measured by quantifying shell hardness, which increases with both size and age in

healthy turtles (Hill 1999, Nagy et al. 2011). Though significantly larger, Combo HS

tortoises at Year 1 had shell hardness values similar to Outdoor HS tortoises at Year 1. It

seems that one year of indoor conditions (i.e. UV lighting, calcium supplementation, and

accelerated growth) or one year of outdoor conditions (i.e. supplemental food and water

as well as natural sunlight) at our site both result in nearly equal shell hardness values.

However, Combo HS tortoises had significantly harder shells at release than 2-year old

Outdoor HS tortoises. Two-year old Combo HS tortoises had shell hardness values equal

to those calculated for an 11-year-old wild desert tortoise (Nagy et al. 2011). Through

combining the accelerated growth of indoor captive rearing and the natural shell

ossification of outdoor captive rearing, we were able to significantly increase the shell

hardness of two-year-old captive reared desert tortoises relative to both wild counterparts

as well as tortoises captive reared solely outdoors.

Extreme post-release dispersal can remove individuals from optimal habitat, result

in higher incidence of exposure and predation, and ultimately impede translocation

efforts (Stamps and Swaisgood 2007, Germano and Bishop 2009, Roe et al. 2010). In our

41

study, with the exception of 2 individual outliers (> treatment mean + 2 SD), released

head-starts did not move far from their release burrows. Both treatment group means

were within the range (~100-400 m) of mean initial (< 2mo) dispersal distances reported

for head-started or translocated juvenile desert tortoises (Nafus et al. 2017, Germano et

al. 2017). However, in our study, pre-dormancy dispersal distance of Outdoor HS

tortoises was twice that of Combo HS tortoises. This increased movement by Outdoor HS

tortoises may be related to their smaller release size, allowing them to heat up faster each

morning (Berry and Turner 1986, Geffen and Mendellssohn 1988, Harris et al. 2015) and

possibly take advantage of a longer period of favorable surface temperatures each day to

disperse farther than the larger Combo HS tortoises.

Roughly 80% of the total dispersal by both treatment groups occurred in the first

three weeks post-release, supporting previous studies of post-release movement of head-

started desert tortoises (Hazard et al. 2015, Nagy et al. 2015, Germano et al. 2017, Daly

2017) and gopher tortoises (Quinn et al. 2018). Germano et al. (2017) also reported a

secondary dispersal event after the first dormancy period, during which 13% of their

juveniles engaged in movements away from the release site. Similarly, four tortoises

(9%) in our study dispersed >100 m (range 104-896 m) away from their dormancy

burrows during the spring activity period. Post-dormancy dispersal was slightly less (~20

m) for Outdoor HS tortoises, when two extreme movers were removed from the data,

possibly due to the larger size of Combo HS tortoises necessitating a slightly larger home

range to acquire all necessary resources (Lindstedt et al. 1986, though see Harless et al.

2009). However, total displacement during the 10-month study period was predominantly

driven by pre-dormancy dispersal from the release burrow. Combination head-starting

42

significantly decreased this initial dispersal, resulting in Combo HS tortoises settling

nearly 200 m closer to their release burrows than Outdoor HS tortoises after ten months.

Future studies should investigate the replicability of this result, alongside the potential

physiological explanations for its manifestation. Reducing initial dispersal allows refined

management within conservation areas as well as continued monitoring of population

recovery.

When compared to previous releases of juvenile desert tortoises (Morafka et al.

1997, Hazard and Morafka 2002, Nagy et al. 2015, Germano et al. 2017, Nafus et al.

2017), including those from our release area (Tuberville et al. 2019, Daly et al. 2019),

tortoises in our study exhibited particularly high survival rates. This finding was

especially pronounced when considering the survival of our Combo HS group, whose

88% 10-month survival marks the highest survivorship ever reported for any size-class of

juvenile desert tortoises. Mortality risks for juvenile tortoises released at our sites in the

Ivanpah valley have changed over time. Original releases (Tuberville et al. 2019) focused

on locating appropriate microhabitats for release as well as the effects of short-term

outdoor captive rearing. Short-term (outdoor) captive rearing resulted in the release of

smaller head-starts (40.8–61.5 mm MCL), a possible explanation for the significant

losses as a result of exposure in that study. Smaller juveniles experience increased rates

of water loss and are thus at a greater risk for desiccation during post-release activities

(Wilson et al. 2000). Over 15% of head-starts released by Tuberville et al. (2019) died

due to suspected exposure, greatly outnumbering the losses attributed to avian predators

(n = 1, 2.2%). Ravens became a significant source of mortality in the subsequent release

at our site by Daly et al. (2019), with nearly 27% of all released tortoises lost to these

43

opportunistic predators in the first year post-release (Sherman 1993, Boarman 2003). In

turn, Daly et al. (2019) recommended releasing tortoises over 1.6 km away from

powerlines, a known nesting structure for ravens. Incorporating this recommendation as

part of our release protocols coupled with the larger size of our combo HS tortoises may

account for the higher survival rates documented in our study. Building on previous

efforts has increased the success and efficiency of our head-starting program.

Management implications

In just two years of captive rearing, combination head-starting produced tortoises

larger than all previously published recommendations for minimum size at release for the

desert tortoise (Nagy et al. 2015, Hazard et al. 2015). Combo HS tortoises, equivalent in

size to 8-year-old wild tortoises (Medica et al. 2012), had nearly prime body condition

values (Nagy et al. 2002) and a shell hardness index similar to an 11-year-old wild

tortoise (Nagy et al. 2011). In addition, Combo HS tortoises dispersed shorter distances

than Outdoor HS tortoises.

Both post-release site fidelity and survival were high for both treatment groups.

By releasing tortoises in late September, we were able to minimize the amount of time

available for dispersal prior to dormancy, increasing the probability that animals would

settle near the release area. An early fall release also allowed tortoises sufficient time to

establish burrows and become familiar with areas for resource acquisition before the

especially vulnerable spring activity period documented in this and in other desert

tortoise post-release studies (Nagy et al. 2015a, Daly et al. 2019). The combination of a

fall release when raven activity is lower and selecting release sites >1.6 km from

potential perching sites (Daly et al. 2019), likely contributed to the high post-release

44

survival exhibited by both treatment groups. In agreement with previous studies (Hazard

et al. 2015, Daly et al. 2019), the initial results of our study support releasing head-starts

during the fall activity period. To determine the effect of annual climatic variation on our

findings, replication as well as continued monitoring should be prioritized.

45

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56

TABLES

Table 2.1. Summary statistics of growth metrics for juvenile Mojave desert tortoises

(Gopherus agassizii) reared under two different husbandry treatments (Outdoor head-

started, Combination head-started) including measurements taken shortly after hatching

in Fall 2016 (‘initial’), measurements taken after one year of captive rearing in Fall 2017

(‘Year 1’; when Combination head-starts were transitioned outdoors), and measurements

taken before release in Fall 2018 (‘Release’). Intervals are reported at 95% confidence. P-

values (significant values in bold) correspond to linear mixed effects models (with

mother ID as random effect). MCL= midline carapace length.

Metric Treatment n Mean SE Lower CI Upper CI Min Max p-value

Initial MCL (mm) Outdoor HS 24 45.4 0.4 44.6 46.1 41.4 47.6 0.51

Combo HS

24 45.0 0.4 44.2 45.8 41.0 47.7

Initial mass (g) Outdoor HS 24 21.1 0.5 20.1 22.1 16.5 23.6 0.63

Combo HS

24 20.9 0.5 19.8 21.9 16.8 25.0

Initial BC (g/cm3) Outdoor HS 24 0.54 0.01 0.53 0.55 0.49 0.60 0.10

Combo HS

24 0.53 0.01 0.51 0.54 0.45 0.58

Year 1 MCL (mm) Outdoor HS 24 66.5 1.5 63.4 69.5 52.2 76.9 <0.0001

Combo HS

24 102.5 1.5 99.6 105.5 84.9 116.6

Year 1 mass (g) Outdoor HS 24 62.6 7.4 47.9 77.5 34.3 85.5 <0.0001

Combo HS

24 205.8 7.2 191.4 220.3 124 302.0

Year 1 BC (g/cm3) Outdoor HS 24 0.57 0.01 0.56 0.58 0.51 0.63 0.14

Combo HS

24 0.59 0.01 0.57 0.60 0.50 0.65

Year 1 SHI Outdoor HS 24 94.8 1.31 92.1 97.4 90.2 97.2 0.39

Combo HS

24 95.9 0.6 94.8 97.1 92.9 98.2

Release MCL (mm) Outdoor HS 24 83.7 1.7 80.2 87.1 68.6 97.8 <0.0001

Combo HS

24 117.2 1.7 113.7 120.6 102.3 136.7

Release mass (g) Outdoor HS 24 115.9 10.7 94.3 137.4 67.3 175.9 <0.0001

Combo HS

24 300.6 10.7 279.0 322.2 200.0 421.0

Release BC (g/cm3) Outdoor HS 24 0.57 0.01 0.56 0.58 0.46 0.61 0.93

Combo HS

24 0.57 0.01 0.56 0.58 0.52 0.63

Release SHI Outdoor HS 24 95.7 0.6 94.5 96.8 92.5 97.2 0.0005

Combo HS 24 97.9 0.4 97.0 98.7 94.9 99.1

57

Table 2.2. Post-release movement metrics for 48 juvenile Mojave desert tortoises

(Gopherus agassizii) from two treatment groups (Combination head-started and Outdoor

head-started) released in September 2018 in the Mojave National Preserve, CA.

Displacement pre-dormancy represents dispersal from release burrow until dormancy for

all animals surviving to dormancy (n = 46; 25 September 2018 – 31 October 2019).

Displacement post-dormancy represents movement from dormancy burrow until the end

of the study for all animals that emerged post-dormancy (n = 45; 05 March 2018 – 23

July 2019). Total displacement represents the distance from release burrow until the end

of the study for all animals that emerged post-dormancy (n = 45; 25 September 2018 – 23

July 2019). Movement probability is defined as the probability of moving during any

weekly tracking event. BCI = Bayesian credible interval. We accepted significance at

alpha = 0.05 or if BCIs did not overlap. Significant p values are in bold.

Metric Treatment n Mean SE Min Max p-value

Displacement pre-

dormancy

Outdoor HS

Combo HS

23

23

255

114

89

40

26

0

1084

1183

0.11

Displacement post-

dormancy

Outdoor HS

Combo HS

22

23

92

53

34

19

0

0

896

231

0.299

Displacement 10-

months

Outdoor HS

Combo HS

22

23

326

143

93

41

11

9

1041

1178

0.047

n Estimate Lower

BCI

Upper Result

BCI

Movement probability

Outdoor HS

Combo HS

22

23

.42

.46

.38

.42

.47

.50

Not

different

Distance moved

during movement

events

Outdoor HS

Combo HS

22

23

43.4

36.2

34.1

29.7

54.6

44.7

Not

different

58

FIGURES

Figure 2.1. Midline carapace length (mm) at release for 2016 cohort juvenile Mojave

desert tortoises (Gopherus agassizii) from two treatment groups: Combination head-

started (“Combo HS”) and Outdoor head-started (“Outdoor HS”) in September 2018.

Dashed horizontal lines represent previously published release size recommendations

(Nagy et al. 2015, Hazard et al. 2015). The “whiskers” correspond to + 1.5 times the

interquartile range (IQR).

59

Figure 2.2. Mass (g) at release for 2016 cohort juvenile Mojave desert tortoises

(Gopherus agassizii) from two treatment groups: Combination head-started (“Combo

HS”) and Outdoor head-started (“Outdoor HS”) in September 2018. The “whiskers”

correspond to + 1.5 times the interquartile range (IQR).

60

Figure 2.3. Body condition (g/cm3) at release for 2016 cohort juvenile Mojave desert

tortoises (Gopherus agassizii) from two treatment groups: Combination head-started

(“Combo HS’) and Outdoor head-started (“Outdoor HS”) in September 2018. Body

condition calculated using Nagy et al. (2002) formula. The “whiskers” correspond to +

1.5 times the interquartile range (IQR).

61

Figure 2.4. Shell hardness index (SHI) versus midline carapace length (MCL) at Year 1

(left) and Year 2 (‘Release’; right) for 2016 cohort juvenile Mojave desert tortoises

(Gopherus agassizii) from two treatment groups: Combination head-started (“Combo

HS”) and Outdoor head-started (“Outdoor HS”) in September 2017 and September 2018.

Shell hardness index calculated using Nagy et al. (2011) protocols.

62

Figure 2.5. Shell hardness index (SHI) versus midline carapace length (MCL) for 2011,

2012, and 2016 cohort juvenile Mojave desert tortoises (Gopherus agassizii) from three

treatment groups: Combination head-started (“Combo HS”), 2016 Outdoor head-started

(“2016 Outdoor HS”), and 2011-2012 Outdoor head-started (“2011-2012 Outdoor HS”)

in September 2018. All three treatment groups were released concurrently in this study

and “2011-2012 Outdoor HS” animals are compared in Chapter 3. Shell hardness index

calculated using Nagy et al. (2011) protocols.

63

Figure 2.6. Mean distance moved from release site during pre-dormancy dispersal (left;

25 September 2018 – 31 October 2019) and mean distance moved from dormancy

burrow after 10 months post-release (right; 05 March 2019 – 25 July 2019) by juvenile

Mojave desert tortoises (Gopherus agassizii) from two treatment groups: Combination

head-started (“Combo HS”) and Outdoor head-started (“Outdoor HS”). The “whiskers”

correspond to + 1.5 times the interquartile range (IQR).

64

Figure 2.7. Kaplan-Meier survival curves for 48 Mojave desert tortoises (Gopherus

agassizii) in two treatment groups: Combination head-started (“Combo HS”; n = 24) and

Outdoor head-started (“Outdoor HS”; n = 24). Estimates are shown from 25 September

2018 until 23 July 2019 (43 weeks). Data were right-censored, meaning no assumptions

were made about the fate of lost or missing animals. Dashed vertical lines represent the

beginning and end of the dormancy period (31 October 2018 – 05 March 2019). Shaded

bands are 95% confidence intervals.

65

CHAPTER 3

SIZE AS A PREDICTOR OF POST-RELEASE SURVIVAL FOR MOJAVE DESERT

TORTOISES (GOPHERUS AGASSIZII)

INTRODUCTION

Positive relationships between size and survival have been demonstrated in a variety of

taxa (Wilbur and Collins 1973, Swingland et al. 1979, Murie and Boag 1984, Williams et

al. 1993). This relationship is especially evident in long-lived organisms exhibiting type

III survivorship, where mortality is inversely related with age and thus often, size

(Iverson 1991). Chelonians, generally characterized by low hatchling survival, slow

growth, delayed maturity, and high adult survival, are a prime example of an organism

with type III survivorship (Iverson 1991, Congdon et al. 1993, Heppell 1998). The

elevated survivorship of adult chelonians is a result of ontogenetic advances in size,

thermal and hydric homeostasis, mobility, and shell hardness (Wilson 1991, Haskell et al.

1996, Wilson et al. 2001, Nagy et al. 2011). Though the highest rates of survival in

chelonians are typically exhibited by adults, survival increases with each successive size-

class (Iverson 1991). This relationship presumably leads to differential rates of survival

across the size range of the juvenile life-stage (Haegen et al. 2009, Nagy et al. 2015a).

Growth rates, which affect the age at which juveniles reach a given size, can vary

widely as a result of environmental, climatic, demographic, and genetic factors (Ernst

1975, Rhen and Lang 1995, Steyermark and Spotila 2001, Mack et al. 2018). Ernst

(1975) measured the growth rates of a northern population of spotted turtles (Clemmys

guttata), determining that growth was limited by the length of the active season and the

66

corresponding water temperatures. Koper and Brooks (2000) measured growth rates of

juvenile painted turtles (Chrysemys picta) and corroborated previous findings that

warmer, longer active seasons increase growth (Parmenter 1980, Frazer et al. 1993).

Studies have also found that snapping turtles (Chelydra serpentina) grow faster in

habitats with high productivity (Brown et al. 1994), and that rapid growth in gopher

tortoises (Gopherus polyphemus) may be attributed to high quality habitat achieved

through active management (Mushinsky et al. 1994). These studies suggest that certain

factors, such as habitat quality and food availability, could potentially be managed to

improve growth in wild populations. In turn, captive reptile populations were studied to

determine whether husbandry conditions could be manipulated to accelerate growth rates

(Reiber et al. 1999, Jarvie et al. 2015, Daly et al. 2018).

Head-starting, the rearing of offspring in captivity until they are presumably more

likely to survive in the wild (Burke 2015), is gaining prominence among chelonian

recovery efforts and is predicated on the assumption that juvenile size at release is

positively correlated with post-release survival (Haegen et al. 2009, Buhlmann et al.

2015, Quinn et al. 2018). In addition to attempting to increase post-release survival by

releasing larger individuals, head-starting can increase the number of juveniles recruited

into the population at the release site. This is achieved by removing the threat of

predation on the vulnerable hatchling size class while providing optimal conditions for

growth during captivity. Although prioritizing habitat protection and high adult

survivorship remains paramount in conserving populations, head-starting may help

stabilize declining populations if implemented alongside other conservation measures

(Tomillo et al. 2008, Crawford et al. 2014, Spencer et al. 2017). This multi-faceted

67

approach, where head-starting is used as a short-term augmentation tool together with

more broadly focused conservation initiatives, is being applied in the recovery of the

Mojave desert tortoise (Gopherus agassizii; USFWS 2011).

The Mojave desert tortoise (hereafter ‘desert tortoise’), federally listed as

threatened in 1990, has experienced concerning population declines for the past half-

century (USFWS 2011). Contributing to these declines are multiple factors, including

habitat destruction, increased incidence of drought, and growing populations of

subsidized predators (Berry 1986, Foreman 1986, USFWS 2011). In 1989, researchers

began evaluating head-starting as a potential addition to the wide range of management

strategies used to mitigate desert tortoise population declines (Morafka et al. 1997).

Early head-starting projects focused on evaluating the health and survival of head-

starts during captivity (Morafka et al. 1997, Nagy et al. 2015a), but researchers soon

transitioned to assessing which factors most affected post-release success (Nagy et al.

2015b, Hazard et al. 2015). Based on three months of monitoring post-release

survivorship, Hazard et al. (2015) recommended the release of head-starts measuring at

least 84 mm midline carapace length (MCL). Subsequently, Nagy and colleagues (2015b)

found that annual survivorship reached 100% during non-drought years at their study site

for animals >100 mm MCL at time of release. Drought years induced prey-switching

behavior in coyotes (Canis latrans), resulting in losses of the larger individuals (>100

mm MCL) as well (Nagy et al. 2015b). Though these recommended release sizes may

seem similar, a 16-mm difference in MCL represents a minimum of one additional year

of growth in captivity under conventional outdoor rearing conditions (for growth rates see

Tuberville et al. 2019, Daly et al. 2018, Nagy et al. 2015b). As a result, 5-9 years of

68

outdoor rearing are required to reach previously recommended release sizes (Hazard et al.

2015, Nagy et al. 2015b). Therefore, refining the recommended release size has the

potential to both increase post-release success and maximize the efficiency of head-

starting desert tortoises.

In addition to the direct effects of size on survival, certain post-release behaviors

may affect survival of released head-starts, and these behaviors might be expected to vary

depending on an individual’s size or age. Long distance dispersal from the release site is

the primary elevator of mortality risk in herpetofaunal translocations (Germano and

Bishop 2009). Dispersal increases risk of exposure to both thermal extremes and

predation (Hazard and Morafka 2002, Stamps and Swaisgood 2007), thus distance moved

from release site may negatively correlate with survival probability. Increased exposure

and predation may also result from longer or more frequent bouts of surface activity.

Daly et al. (2019) found that all surviving tortoises released in their study were found out

of their burrows on less than 30% of tracking events, and decreased surface activity had a

positive effect on survival probability. Desert tortoises spend a large proportion of their

time inactive in their burrows (Zimmerman et al. 1994) but individuals vary in the

number of burrows used in an activity season (Harless et al. 2009). Because moving

between or relocating to a different burrow requires tortoises to be surface active, the

number of burrows used by an individual may be negatively associated with survival.

Alternatively, burrow switching could also be regarded as a form of predator avoidance

(Henen et al. 2017), with greater survival probability associated with individuals that

employ this strategy. Thus, both the tendency to be surface active and variation in burrow

switching may be important predictors of post-release survival.

69

Since 2011, our team has been evaluating the potential role of head-starting in

contributing to the recovery of the desert tortoise. During that time, we have conducted a

series of experimental studies (Nafus et al. 2015, Todd et al. 2016, Daly et al. 2018,

Tuberville et al. 2019, Daly et al. 2019) focused on identifying combinations of both pre-

and post-release conditions that maximize tortoise survival and the efficiency of head-

starting. The goal of our study is to evaluate the effect of release size on survival of head-

started desert tortoises during their first year following release. We examined this effect

by releasing captive-reared tortoises spanning a wide range of body sizes (68-145 mm

MCL). Along with size at release, we also assessed the effect of time spent in captivity,

post-release dispersal distance, surface activity, and burrow switching behavior on post-

release survival.

METHODS

Study site

Both the captive husbandry and field components took place within the Mojave

National Preserve (MNP), a 650,000 ha preserve in San Bernardino County, California.

Specifically, our captive-rearing facility, the Ivanpah Desert Tortoise Research Facility

(IDTRF), and our release site were in the Ivanpah Valley in the northeastern most corner

of the MNP. The primary habitat at our release site was “Yucca Woodland,” dominated

by creosote bush (Larrea tridentata), white bursage (Ambrosia dumosa), littleleaf ratany

(Krameria erecta), and heavily intermixed Yucca (Y. schidigera, and Y. brevifolia;

Turner et al. 1984; Todd et al. 2016). Although habitat suitability remains relatively high

in the Ivanpah Valley (Nussear et al., 2009), current tortoise densities (2.8 per km2 in

2012; Allison and McLuckie 2018) are much lower than they were historically (77 – 85

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per km2 in 1977 to 1980; Turner et al. 1984). The captive-rearing portion of the study,

carried out at the IDTRF, made use of indoor rearing facilities and outdoor predator-proof

enclosures (further details below) located 15 km from our release site.

Obtaining hatchlings

In April 2011, we began tracking 30 adult female desert tortoises from our field

site in the Ivanpah Valley. We tracked females monthly using a 3-element Yagi antenna

(AF Antronics, Inc., Urbana, IL) and a R1000 receiver (Communications Specialists,

Inc., Orange, CA). Each April (except 2014) we brought all females to the IDTRF and

radiographed them to check for the presence of calcified eggs (Diagnostic Imaging

Systems, Poskam, Colorado, USA; 60 kvp, 0.8 mAS, 74 cm focal length; Gibbons and

Greene 1979). We placed gravid females in individual nesting enclosures (5 x 9 m) and

allowed them to nest naturally. Nesting enclosures were constructed to deter both avian

and mammalian predators and contained natural vegetation for shade and forage. We

provided females with artificial burrows and supplemental water. Weekly, we monitored

for egg deposition by radiographing females in the pens. If nesting was confirmed, we

immediately released the female at her point of capture. Accordant with our permits, we

released, at their last known burrow location, any females that had not nested within 30

days.

Eggs incubated naturally in the enclosures. After approximately 80 days, we

began searching the pens daily for emerged hatchlings. We marked all hatchlings by

notching unique combinations of marginal scutes using codes assigned by USFWS

(Cagle 1939). We carefully excavated all nests with un-emerged hatchlings to recover

any unhatched eggs or entombed hatchlings. We randomly assigned all healthy neonates

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to rearing enclosures at the conclusion of the hatching season. This study includes

juveniles from the 2011, 2012 and 2016 cohorts.

Pre-release husbandry

Each year we placed all (prior to 2015) or a subset (2015 and later) of the available

hatchlings in predator-proof enclosures at the IDTRF to be reared solely outdoors. The

enclosures contained natural substrate and vegetation (Alberts 2007) which we

supplemented with rocks, downed Yucca logs, and “starter burrows” as shelter sites. We

also outfitted each enclosure with rotating garden sprinklers to provide artificial rain

every 1-2 weeks during the tortoise active season (March – October; for more

information see Tuberville et al. 2019, Daly et al. 2018). Husbandry protocols within the

enclosures have been modified over the course of the head-starting program, as noted

below, to respond to changing habitat conditions in enclosures and different research

objectives.

Initially, tortoises from the 2011-2015 cohorts received only supplemental rain

and relied solely on natural vegetative growth in the pens for forage (Beatley 1974).

Starting in March 2016, due to the depletion of native forage within the pens, we began

providing supplemental food during each artificial watering event. Supplemental food, a

measured mix of leafy greens and Mazuri® Tortoise Diet (Mazuri Exotic Animal

Nutrition, St. Louis, MO; see Chapter 2 and Daly et al. 2018), approximated the

nutritional properties of the desert tortoise’s natural diet (Jarchow et al. 2002). Thus, the

2011-2015 cohorts initially received only supplemental water but starting in Spring 2016

also received supplemental food, whereas the 2016 cohort received both supplemental

water and food throughout their outdoor rearing.

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Beginning in 2015, we began rearing a subset of each cohort indoors in an attempt

to accelerate their growth while in captivity (Daly et al. 2018). Daly et al. (2018)

compared indoor captive-rearing to conventional outdoor captive-rearing, finding that

indoor-reared tortoises (7 mo) grew significantly faster (8x) than their outdoor-reared

siblings but lacked the shell hardness of similar sized, yet older (3-4 yrs old) outdoor-

reared individuals. To combine the increased growth of indoor rearing and the increased

shell hardness of outdoor rearing, in fall 2016 we initiated a novel method of combination

rearing. Combination rearing consisted of one year of indoor rearing followed by one

year of outdoor rearing in predator-proof enclosures (Chapter 2), with the outdoor rearing

portion including both supplemental water and food as previously described.

As part of the current study, we released 78 tortoises from three different cohorts,

representing a wide range of release sizes. We released 30 tortoises from our 2011-2012

cohorts. We released 48 tortoises from our 2016 cohort – half of which were reared solely

outdoors while the other half was combination reared. By releasing animals from these

three cohorts (2011, 2012, 2016) reared under the protocols described above, we were

able to release tortoises at sizes that bracketed the previously recommended release sizes

(84 mm MCL, Hazard et al. 2015; 100 mm MCL, Nagy et al. 2015b). We did not

consider husbandry treatment in analyzing individual fate because previous releases at

our site have failed to detect differences in post-release survival and behavior based on

husbandry treatment groups (Tuberville et al. 2019, Daly et al. 2019, Chapter 2).

Morphometrics and growth

We measured all hatchlings immediately after they emerged from nests. After

these initial measurements, we measured animals each fall (September), including

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immediately prior to release (25 September 2018). At each measuring period, we

recorded midline carapace length (MCL, straight-line distance from the anterior edge of

the nuchal scute to the inside of the natural notch in the supracaudal scute) to the nearest

0.1 mm using vernier calipers. We compared mean MCL at hatching among cohorts

using linear mixed effects models (‘nlme’ package, ‘lme’ function in R) with mother

identification number as a random effect. Similarly, we compared mean MCL at release

among cohorts. When a significant difference was detected (α = 0.05 rate of type I error),

we used Tukey’s post-hoc comparisons (‘glht’ function in the ‘multcomp’ package) to

investigate pairwise differences.

To calculate mean annual growth rates for each individual, we separated the

captive-rearing period into three husbandry stages; 1) outdoor rearing with no

supplemental food, 2) outdoor rearing with supplemental food, and 3) indoor rearing.

Thus, for tortoises from our 2011-2012 cohorts, we calculated separate mean annual

growth rates for their time spent in pens with no supplemental food and for their time

spent in pens with supplemental food. Additionally, we calculated mean annual growth

rates for their first two years of life, allowing us to compare growth of same-aged

tortoises reared outdoors with (2016 cohort) and without (2011-2012 cohorts)

supplemental food. For the combination-reared tortoises, we calculated separate annual

growth rates for the indoor and outdoor rearing stages. To calculate annual growth rates,

we divided the change in MCL between initial and last fall measurements by the number

of years reared under that husbandry stage. For the 2011-2012 cohorts, for which we

modified outdoor husbandry protocols to include supplemental feeding during spring

2016, we used fall 2015 measurements as final measurements for calculating growth for

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outdoor rearing with no supplemental feeding and used those same measurements as

initial sizes to calculate growth resulting from outdoor rearing with supplemental food.

We felt comfortable using fall 2015 measurements despite the March 2016 change in

husbandry, as previous studies have found that desert tortoises exhibit almost no growth

before April and after July each year (Medica et al. 1975). Data are presented as means ±

1 SE.

Experimental releases

In fall 2018, we released all juveniles into Yucca woodland habitat at our site. The

specific release site was chosen based on habitat quality, its accessible yet remote

location, and previously published recommendations on minimum distance from potential

perch sites for avian predators, namely ravens (Nafus et al. 2015, Todd et al. 2016, Daly

et al. 2019). Using ArcGIS (version 10.5; ESRI, Redlands, CA), we first delineated a 0.6

km2 rectangular release plot (300 m X 2000 m) parallel to our lone sandy access road,

with the nearest corner >1.9 km from a powerline near our site. We then used the

‘Fishnet’ function to generate release points throughout the plot at 50-m intervals. Next,

we removed points to create three equally spaced blocks of release points within the

rectangular plot. Points were then labeled 1–78 to correspond with a release location for

each juvenile tortoise. Each block, separated from each other by approximately 350 m,

comprised 26 points. Release points were buffered 10 m to maximize our ability to select

a release refugium for each juvenile that contained a large perennial shrub and an intact

kangaroo rat burrow (Dipodomys spp.; Nafus et al. 2015, Todd et al. 2016) at each

generated release location. Each juvenile tortoise was released head-first into a kangaroo

rat burrow that we had previously enlarged as needed to safely conceal the released

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animal. Under this release protocol, taking into account the buffered limit of each release

point, the minimum possible distance between release burrows was 30 m. We conducted

bouts of tracking effort by replicate release block to most efficiently track the large

number of released animals.

Prior to release, we held all juveniles indoors for 3–5 days at the IDTRF, allowing

us to soak and feed tortoises, visually assess their health, and attach transmitters. We

randomly assigned each juvenile, irrespective of treatment, to one of the 78 release

centers (Figure 3.1). We released all juveniles on 25 September 2018. We released

animals in their pre-selected refugium between 0600 and 1000 h to avoid extreme

temperatures.

Post-release monitoring

We attached radio-transmitters to juveniles prior to their release. We affixed

transmitters (3.1 g R1670 on 2016 animals reared solely outdoors; 3.6 g R1680

transmitters on all others; Advanced Telemetry Systems, MN, USA) to the fifth vertebral

scute with 5-minute epoxy (Devcon 5-minute epoxy gel, ITW Engineered Polymers,

County Clare, Ireland). Transmitters weighed no more than 5% of the body mass at time

of release of the recipient tortoise (apart from the three smallest 2016 outdoor animals for

which the transmitter weighed 6% of the pre-attachment body mass). To help camouflage

the transmitter bundle, we coated all transmitters with a thin layer of sand before the

epoxy hardened (Kazmaier et al. 2002).

We tracked each animal 24 hours after initial release and then twice per week for

the next three weeks. We then tracked juveniles weekly until dormancy (31 October

2018), then every 10-14 days during dormancy (November 2018 – February 2019). As

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tortoises became active in the spring (March 2019), we resumed weekly tracking for the

remainder of the study (March 2019 – July 2019). At each tracking occasion, we recorded

the tortoise’s location to the nearest ±3 m using a handheld GPS (Garmin model

GPSMAP 76, Olathe, Kansas). We used these tracking occasions to monitor post-release

movement and behavior (surface activity, burrow switching), which were subsequently

used as predictors in our survival models (details below).

We used the Spherical Law of Cosines (Movable Type Ltd. 2015) to calculate, for

each individual tortoise, the straight-line distance between its release burrow and its final

location (total displacement). We set its final location as where it was found on the last

day of the study (23 July 2019) for all individuals surviving the study or, for those that

died, their last known live location. We calculated surface activity as the number of times

a tortoise was found outside a burrow or pallet (burrow with width longer than length)

divided by the total number of tracking occasions. We defined burrow switching as the

number of unique burrows used by an individual divided by the total number of tracking

occasions that individual was found in a burrow; the proportion thus accounted for

differences in number of tracking occasions among individuals due to mortalities during

the study.

Survivorship

We estimated post-release survival rates through the end of the monitoring period

(September 2018–July 2019) using the Kaplan-Meier estimator in the R package

‘survival’ (R Core Team 2017). When a deceased tortoise was found, we carefully

inspected the surrounding area for any signs of what may have caused the death of the

individual. Signs of predation included digging, tracks, scat, and teeth marks or beak

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punctures on the carcass or transmitter. We photographed each carcass in situ and

collected them for further analysis. We attributed mortalities to one of five

classifications: mammal depredated, bird depredated, unidentified predator, exposure, or

unknown fate. Clear digging activity or obvious tooth marks suggested mammal

predation. We classified remains near perch sites with clear signs of being struck by a

beak as bird depredated. If the carcass was damaged but remains showed no evidence of

tooth marks or avian predation, we considered it to be depredated by an unidentified

predator. We presumed dead tortoises found intact either on the surface or in their

burrows to have been killed by exposure. We classified animals lost due to radio failure

(faulty batteries or the unit fell off) as unknown fate, and we right censored (no

assumption of fate) these animals in our survival analysis.

We evaluated the effect of several factors on individual fate, using only the

individuals with a known fate at the end of the study. We included time spent in captivity,

MCL at release, total displacement, surface activity, and burrow switching as potential

predictors in a combination of generalized linear models (GLMs) with a binary response

(alive = 1 and dead = 0). Coefficients of correlation (r) among predictor variables were

all <0.65, with time in captivity and MCL at release being the most highly correlated (r =

0.63). We then used Akaike information criterion (AIC) values to compare among the

competing models and evaluate model fit. To account for uncertainty in model selection,

we used multi-model averaging (R package ‘AICcmodavg’). We generated model-

averaged predictions and 95% confidence intervals (CIs) of survival across a range (60-

150 mm) of MCL values, holding all other predictor variables to their average values. We

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kept all predictor variables other than MCL constant because MCL at release is the sole

predictor under a managers control.

RESULTS

Morphometrics and growth

Mean MCL at hatching was 45.1 ± 0.2 mm (n = 78), though it differed among

cohorts (F2,48 = 9.022, p < 0.0001). Mean hatching MCL for the 2011 cohort (45.9 ± 0.3

mm) and 2016 cohort (45.2 ± 0.2 mm) was significantly larger than for the 2012 cohort

(43.9 ± 0.2 mm; p < 0.032 in both cases). For the 2011-2012 cohorts, growth during

outdoor rearing averaged 11.4 ± 0.4 mm MCL/yr with no supplemental food (2011–

2015) and 13.5 ± 0.6 mm MCL/yr with the addition of supplemental food (2016–2018).

During their first two years of life (2011-2013 and 2012-2014 for our 2011 and 2012

cohorts, respectively), 2011-2012 animals grew 11.0 ± 0.4 mm MCL/yr. Juveniles from

our 2016 cohort reared solely outdoors with supplemental food grew 19.2 ± 0.7 mm

MCL/yr. Tortoises reared using combination rearing grew 57.5 ± 2.0 mm MCL/yr while

indoors (September 2016 – September 2017) and 14.7 ± 1.3 mm MCL/yr while outdoors

with supplemental food (September 2017 – September 2018). The overall combination of

rearing conditions and cohorts used in this study resulted in release sizes ranging from

68–145 mm MCL. Mean time spent in captivity among released animals was 3.64 years

(range = 2 – 7).

Surface activity and burrow switching

Mean total displacement over the course of the 10-month post-release monitoring

period was 295 ± 55 m (n = 76; range = 9 – 3062 m). Proportion of surface-active

tracking events averaged 0.43 ± 0.03 (n = 76; range = 0.13 – 1.0) among all released

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head-starts with a known fate at the end of the study. The mean number of unique

burrows used by each individual over the monitoring period (25 September 2018 – 23

July 2019), or until the animal died, was 5.3 ± 0.21 (range = 0 – 9). Mean burrow

switching by tortoises in our study was 0.35 ± 0.03 (range 0.0 – 1.0).

Survivorship

Of the 78 released tortoises, 63 (80.8%) were confirmed alive at the end of the

study (23 July 2019). Based on analysis of each deceased animal in the field as well as

the surroundings of each carcass, all mortalities were attributed to predation. Mammal

predators were responsible for the largest percentage of mortality (12.8% of released

tortoises). Three (3.8%) head-starts were bird predated and two (2.6%) were lost due to

radio failure. Notably, all tortoises depredated by avian predators were 2016 solely

outdoor reared animals (68-98 mm MCL at release). Based on the 78 tortoises released in

this study, total 10-month post-release survival was estimated at 0.83 (95% CI, 0.75-0.92;

Figure 3.2).

No model for survival in the candidate set gathered >0.25 of AIC model weight,

indicating high uncertainty in model selection (Table 3.1). In each of the 4 most

competitive models in the set (△AIC < 2.0), only MCL was a significant predictor of fate

(p 0.009). Model-averaged predictions over the range of MCL (with all other measured

covariates held constant at their respective means) showed that survival probability

increased with size at release (Table 3.2; Figure 3.3). Ten-month predicted survival

probability ranged from 0.65 (0.42 – 0.83) to 0.93 (0.78 – 0.98) at the MCL limits used in

this study (60 – 150 mm MCL). Head-starts released at our study site had predicted

survival probabilities of 0.76 (0.64 – 0.86) and 0.82 (0.71 – 0.89) at the previously

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recommended release sizes of 84 mm MCL and 100 mm MCL (Hazard et al. 2015, Nagy

et al. 2015b, respectively).

DISCUSSION

Among chelonians, smaller individuals often have higher rates of mortality than larger

conspecifics (Haskell et al. 1996, O’Brien et al. 2005). The mortalities documented in our

study followed this trend. Over half (7/13; 54%) of the mortalities we documented were

of animals smaller than 91 mm MCL, a disproportionate amount given that only 27%

(21/78) of the animals released were below this size threshold. Furthermore, the three

smallest individuals released in this study were all found depredated. Though we

evaluated a suite of possible predictors on individual fate, size was the sole significant

predictor among those we examined of whether an animal lived or died in our study.

A positive relationship between size and survival has been previously reported for

desert tortoises (Hazard et al. 2015, Nagy et al. 2015b). Nagy et al. (2015b) found that

survivorship reached 100% for tortoises > 100 mm MCL in their first-year post-release,

adding that mortality was highest amongst the smallest individuals (<80 mm MCL), none

of which survived longer than four years post-release. Another study at that same site in

the western Mojave Desert found that no animal above 84 mm MCL died during the short

three-month monitoring period following release (Hazard et al. 2015). Notably, both

studies were predominantly affected by raven predation. In fact, Hazard et al. (2015)

reported ravens as the sole source of mortality, whereas only three (23% of mortalities)

individuals in our study were concluded to have been killed by avian predators

(presumably ravens). The size of our largest raven-depredated individual (90 mm MCL)

was similar to that reported by both Nagy et al. (2015b; 80 mm MCL) and Hazard et al.

81

(2015; 84 mm MCL) for raven predation at their site. Notably, Nagy et al. (2015b) found

that raven predation was likely the cause for the near complete eradication of tortoises

below 80 mm MCL from their study over a three-year period. The three raven-depredated

juveniles in our study ranged between 68-90 mm MCL, the two smallest of which

showed clear signs of being punctured by a beak while the largest individual lacked

puncture wounds. Our results, coupled with previous findings of size thresholds for avian

predation (Hazard et al. 2015, Nagy et al. 2015b), suggest that risk of raven-related

mortality is reduced as tortoises grow and their shells harden (Nagy et al. 2011).

Age and size are often too correlated to reliably differentiate their relative effects

on response variables. However, as a result of overlapping sizes among cohorts due to

differential husbandry practices used in this study, we were able to separately evaluate

size and age (time spent in captivity) as predictors of post-release fate. We found that

size, rather than age, was the sole significant predictor of fate at our site, with larger

juveniles exhibiting greater survival than same-aged, smaller individuals. This finding

suggests that accelerating growth of hatchling head-starts holds the potential to maximize

the success and efficiency of head-starting programs. For example, simply by

incorporating both an indoor and outdoor component in the pre-release husbandry of

head-started desert tortoises, our study shows that head-starting programs can rear

tortoises equivalent in size to six-year-old outdoor reared animals in just two years. This

reduction in the captive period equates to four years of savings on husbandry- and

personnel-related expenditures, greatly increasing the potential for programs to expand

the number of head-starts that can be reared each year or result in cost-savings that can be

invested in additional management practices for the benefit of the species (i.e. restoration,

82

habitat protection). Importantly, based on our releases to date, we have found no evidence

that the accelerated growth rates associated with indoor rearing, at least under the

husbandry practices we used, result in physiological impairment or differences in post-

release behavior or survival (Chapter 2, Tuberville et al. 2019, Daly et al. 2018, 2019).

Reducing time spent in captivity may also hold physiological benefits, in addition

to saving limited conservation dollars (Warkentin et al. 1990, Hermes et al. 2004, de

Assis et al. 2015). A study on the effects of long-term captivity on the stress response of

the Cururu toad (Rhinella icterica), found that prolonged captivity led to elevated stress

hormone levels over the duration of the captive period as well as decreased immune

response (de Assis et al. 2015). Long-term captivity has also been implicated in increased

basal metabolic rate and decreased antimicrobial fighting ability in birds (Warkentin et al.

1990, Buehler et al. 2008). In reptiles specifically, time spent in captivity has been found

to alter feeding behaviors and negatively affect post-release survival in rat snakes

(Pantherophis obsoletus; DeGregorio et al. 2013, 2017). Our finding – that time in

captivity can be reduced by incorporating an indoor rearing component – limits the

potential for manifestation of any negative consequences that might be associated with

prolonged captivity.

Tortoise growth responds to husbandry manipulations, as seen by the variable

growth rates of tortoises reared under the differing husbandry protocols implemented in

this study. Maximum growth was observed when using indoor rearing, during which

head-starts grew nearly three times faster (57.5 mm MCL/yr) than the fastest growing

cohort of solely outdoor reared juveniles (19.2 mm MCL/yr) at our site. Alternatively, for

programs focused on or logistically constrained to solely using outdoor rearing, we found

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that growth was vastly improved in same-aged juvenile tortoises by the addition of

supplemental food, coupled with supplemental watering. While using indoor rearing is

clearly the most efficient way to accelerate growth, outdoor rearing allows head-starts to

acclimate to outdoor conditions and develop natural thermoregulatory behaviors. Given

that we did not release solely indoor-reared tortoises as part of this study, we cannot be

certain how lack of outdoor conditioning would have affected post-release survival

relative to the head-starts released in our study. Solely indoor-reared tortoises released by

Daly et al. (2019) at our study site exhibited survival rates similar to both outdoor reared

and direct released siblings, although any potential differences may have been masked by

high predation rates associated with a single brooding pair of ravens that became

established shortly after the tortoises were released. We were able to minimize this source

of mortality in our study by releasing animals in the fall after raven nesting was

completed and by releasing animals > 1.9 km away from powerline pylons that serve as

raven perch structures (Daly et al. 2019).

In summary, our study lends further support for the positive relationship between

size and post-release survival in head-started juvenile desert tortoises, while highlighting

how the higher growth rates associated with the indoor phase of combination rearing can

improve the efficiency of head-starting programs. We found that size, not age, best

predicts post-release survival and corroborated that indoor rearing allows for significantly

increased growth in comparison to conventional outdoor rearing (Daly et al. 2018). We

include estimated survival probabilities over a range of juvenile desert tortoise sizes,

providing managers with an increased ability to predict outcomes of subsequent head-

start releases based on release size. At our study site, raven predation only affected

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animals < 90 mm MCL, corroborating previous size thresholds for raven predation at a

study site in the western Mojave Desert (Hazard et al. 2015, Nagy et al. 2015b). Though

raven predation ceased above 90 mm MCL, predicted survival continued to increase

incrementally over the entire MCL range used in this study. We provide survival

estimates over a wide range of potential release sizes because recommending a single

specific size at release for future desert tortoise head-starting programs fails to recognize

the complexity of variable release site conditions and logistical constraints faced by

individual projects. In producing our survival estimates, we acknowledge that survival

will vary depending on habitat quality, predator abundance, climatic conditions, and other

sources of variability. Practitioners must factor in their own site-specific conditions and

program-specific objectives when determining the ideal size at release and the most

efficient, logistically feasible method of attaining that size.

85

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94

TABLES

Table 3.1. Twenty-one candidate models to evaluate which predictors (Time in captivity,

MCL, Total displacement, Surface activity, Burrow switching) have the greatest effect on

the post-release fate (1=alive, 0=dead) of head-started Mojave desert tortoises released

into the Mojave National Preserve, San Bernandino, CA, USA. The most parsimonious

model is indicated in bold. Model terms included: TIC (Time in captivity in years; 2,6, or

7), MCL (Midline carapace length in mm at release, September 2018), TDisp (Total

displacement from release burrow until death or the end of the study on 23 July 2019),

SA (surface activity, or the proportion of tracking events that an individual was on the

surface), and Burr (Burrow switching, calculated as the number of unique burrows used

divided by the total number of tracking events found in a burrow). Values presented

include log likelihood (LL), model degrees of freedom (K), Akaike Information Criteria

(AIC), delta AIC (△AIC), and Akaike weights (AICWt), which display the weight of

each model in the candidate set.

Model LL K AIC △AIC AICWt

MCL + SA

MCL

TIC + MCL + SA

MCL + Burr + SA

MCL + TDisp

MCL + Burr

TIC + MCL

TIC + MCL + Burr + SA

MCL + TDisp + Burr + SA

MCL + TDisp + Burr

TIC + MCL + TDisp

TIC + MCL + Burr

TIC + MCL + TDisp + Burr + SA

TIC + MCL + TDisp + Burr

TIC

TIC + TDisp

TIC + Burr

TIC + SA

TIC + TDisp + Burr

TIC + Burr + SA

TIC + TDisp + BuSw + SA

-31.9

-33.6

-31.7

-31.9

-33.2

-33.3

-33.6

-31.7

-31.7

-33.0

-33.0

-33.3

-31.4

-32.8

-35.9

-35.4

-35.5

-35.8

-35.0

-35.4

-34.9

2

1

3

3

2

2

2

4

4

3

3

3

5

4

1

2

2

2

3

3

4

67.9

69.2

69.5

69.9

70.3

70.7

71.1

71.4

71.4

71.9

72.0

72.6

72.8

73.6

73.7

74.9

75.0

75.7

75.9

76.7

77.6

0.0

1.3

1.6

2.0

2.4

2.8

3.2

3.5

3.5

4.0

4.1

4.7

4.9

5.7

5.8

7.0

7.1

7.8

8.0

8.8

9.7

0.25

0.13

0.11

0.09

0.07

0.06

0.05

0.04

0.04

0.03

0.03

0.02

0.02

0.01

0.01

0.008

0.007

0.005

0.004

0.003

0.002

95

Table 3.2. Predicted 10-month survival rates and 95% confidence intervals for head-

started juvenile Mojave desert tortoises (Gopherus agassizii) derived using a generalized

linear model with fate as a binary response variable (0 = “alive”, 1 = “dead”) and MCL,

time in captivity, total displacement, surface activity, and burrow switching as predictor

variables. Time in captivity, total displacement, surface activity, and burrow switching

were kept constant at their overall means when generating survival estimates over the

range of MCL values (60–150 mm). MCL = midline carapace length in mm.

MCL Survival Probability (%) Lower 95% CI Upper 95% CI

60

65

70

75

80

85

90

95

100

105

110

115

120

125

130

135

140

145

150

64.6

67.2

69.6

72.0

74.2

76.3

78.3

80.2

81.9

83.5

85.0

86.3

87.5

88.6

89.7

90.6

91.4

92.1

92.8

41.9

46.7

51.4

55.8

59.9

63.5

66.6

69.2

71.2

72.8

74.0

75.0

75.7

76.3

76.8

77.2

77.5

77.8

78.0

82.7

83.1

83.6

84.2

84.9

85.8

86.8

88.0

89.3

90.6

91.8

93.0

94.1

95.1

95.9

96.7

97.3

97.8

98.2

96

FIGURES

Figure 3.1. Map of release site with 78 release centers for juvenile Mojave desert

tortoises (Gopherus agassizii) in the Mojave National Preserve, San Bernardino, CA. All

78 tortoises were released on 25 September 2018. Animals from three different cohorts

(2011, 2012, 2016) were released.

97

Figure 3.2. Kaplan-Meier survival curve for 78 head-started Mojave desert tortoises

(Gopherus agassizii) released into the Mojave National Preserve, San Bernardino, CA,

USA. Estimates are based on monitoring from 25 September 2018 until 23 July 2019 (43

weeks). Data were right-censored, in that no assumptions were made about the fate of lost

or missing animals (n = 2). Shaded bands are 95% confidence intervals.

98

Figure 3.3. Observed survival fates (open circles) and predicted survival probabilities

based on size (midline carapace length, MCL) at release for juvenile Mojave desert

tortoises (Gopherus agassizii) released into the Mojave National Preserve, San

Bernardino, CA, USA. We predicted survival probability by setting time in captivity

(3.64 yrs), total displacement (295 m), surface activity (0.43), and burrow switching

(0.35) to the overall mean for all animals released. We then generated predictions of

survival probability over a range (50-160 mm) of MCL values. Solid lines are predicted

model estimates and dashed lines are upper and lower limits of 95% confidence intervals.

The vertical dashed line (MCL = 90) represents the threshold for raven predation in our

study.

99

CHAPTER 4

SUMMARY AND CONCLUSIONS

Head-starting has been evaluated by desert tortoise researchers for nearly three decades,

resulting in continued and substantial increases in knowledge. Outdoor predator-proof

enclosures were the first test of the feasibility of rearing the desert tortoise (Morafka et al.

1997), followed by the addition of supplemental water and food to encourage growth and

boost survival in the enclosures (Nagy et al. 2015, Germano et al. 2017, Tuberville et al.

2019). Growth was still slow however, which led Daly et al. (2018) to incorporate the use

of indoor rearing to bypass winter dormancy and greatly enhance the growth advantage

of being reared under captive conditions. These previous studies formed the foundation

for the continued improvement of this technique.

This thesis evaluates the potential role of combination rearing, a novel method of

indoor rearing coupled with outdoor rearing, in the short-term recovery of the Mojave

desert tortoise. To maximize the ability of head-starting to make an impact on reversing

population declines, programs must evaluate all aspects of their protocols, leading to

continued improvement and increased efficiency. Head-starting alone will not be enough

to undo the range-wide declines sustained by this species, however, as part of a holistic

conservation program (Klemens 2000), head-starting can play a vital role in expediting

population stability (Crawford et al. 2014, Spencer et al. 2017).

In Chapter 2, we experimentally evaluated the pre- and post-release effects of

combination (indoor/outdoor) rearing as compared to the conventional method of strictly

outdoor rearing. We found that two years of combination rearing resulted in juveniles

100

equivalent in size to an 8-year-old wild tortoise with the shell-hardness of an 11-year-old

wild individual (Nagy et al. 2011). For rearing conducted strictly outdoors, we confirmed

that the continued use of supplemental feeding to expedite outdoor head-start growth is

warranted and recommended during this phase. Beyond increased size and shell-

hardness, combination reared juveniles exhibited minimal dispersal tendencies and,

following release, had one of the highest annual survival rates documented in head-

started desert tortoises. All in all, combination rearing was found to be an efficient and

successful technique for both maximizing growth during captivity and subsequent

survival during the first-year post-release. However, only continued monitoring of

released head-starts, including to maturity, will provide the ultimate answers on the long-

term viability of this technique (Lindenmayer et al. 2012, Bertolero et al. 2018). In

addition to long-term monitoring, we also recommend the implementation of

combination rearing and post-release studies in other parts of the desert tortoise’s range

to test the replicability of our findings throughout the diverse habitats inhabited by this

species (USFWS 2011).

In Chapter 3, we sought to test the effect of several predictor variables on post-

release fate of head-started Mojave desert tortoises. We released juveniles ranging in size

from 68-145 mm midline carapace length (MCL) and reared under variable husbandry

protocols. Among our potential predictors (size, time in captivity, total displacement,

surface activity, and burrow switching), only size at release had a significant effect on the

survivorship of released head-starts. This finding, which suggests that two-year-old

combination reared individuals have the same survival probability as 6-7-year-old

outdoor reared tortoises, has the potential to markedly increase the utility of head-

101

starting. Even without considering the financial savings associated with expediting the

head-starting process by 4-5 years, this reduction in the time that it takes to rear robust

head-starts can increase the capacity of current head-starting programs, thereby greatly

accelerating the contribution of head-starting to the recovery of the desert tortoise. Most

importantly, by increasing the efficiency with which funds are used in head-starting,

programs will be able to devote resources to ensuring that the original causes of decline

are also ameliorated (Frazer et al. 1992).

102

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