1 AUTHORS’ ACCEPTED VERSION 2 Version of record: Grueber CE, Peel E, Wright B, Hogg CJ, Belov K (2019) A breeding 3 program to support wild recovery. Reproduction, Fertility and Development, 31, 1296-1304. DOI: 4 https://doi.org/10.1071/RD18152

5 A Tasmanian devil breeding program to * 6 support wild recovery 7 Grueber CE1,2, Peel E1, Wright B1, Hogg CJ1, Belov K1,3

8 1 The University of Sydney, School of Life and Environmental Sciences, Faculty of Science, Sydney, 9 NSW 2006, 10 2 San Diego Zoo Global, PO Box 120551, San Diego, CA 92112, USA 11 3 Corresponding author. Email: [email protected]

12 Word count: 5,242 (main text) + 82 references and 3 figures

13 Keywords: adaptation to captivity, conservation, insurance population, microsatellites, pedigree, 14 reproductive success, translocation,

15 * This article was written as an outcome of the Reproduction Down Under meeting held in 2017 to 16 celebrate the career of our friend and mentor Professor Marilyn Renfree. Marilyn shattered the glass 17 ceiling and paved the way for female marsupiologists in Australia. We are pleased to share the 18 Tasmanian devil story in this special issue as our tribute to the effect Marilyn has had on our careers. 19 Her impact on the field (and us) is immense.

20 Abstract 21 Tasmanian devils are threatened in the wild by devil facial tumour disease: a transmissible cancer 22 with high fatality rate. In response, the Save the Tasmanian Devil Program (STDP) established an 23 “insurance population” breeding program across Australia. The program includes a range of 24 institutions, from zoo‐like intensive enclosures to larger, more natural environments. This structure 25 is designed to enable preservation of genetic diversity as well as natural behaviours of devils. In our 26 genetic research, we have provided data to help the STDP reach its goals for the breeding program. 27 These include studies of the determinants of breeding success in captivity and the wild, as well as 28 genetic analyses to provide resources for management of the species and support devil 29 conservation. In this review, we highlight the variety of valuable questions that have been addressed 30 by this work and the conservation outcomes that have resulted. Overall, the devil breeding program 31 provides a valuable example of how genetic research can be used to understand and improve 32 reproductive success of threatened species.

33 Introduction 34 The Tasmanian devil Sarcophilus harrisii (Figure 1) is an iconic Australian native marsupial, and the 35 world’s largest extant marsupial carnivore. Restricted in the wild to the island state of , 36 devils are now threatened with extinction by an unusual transmissible cancer, devil facial tumour 37 disease (DFTD). Most devils that contract DFTD die within 6 – 9 months of lesions appearing (Pearse 38 & Swift 2006). DFT1 was first recorded in the 1990s in the north‐east of Tasmania (Hawkins et al. 39 2006), with a second strain (DFT2) appearing in the 2010s (Pye et al. 2015). These two strains are 40 now known collectively as DFTD. DFT1 has spread throughout the devil population, causing dramatic 41 population declines and fragmentation across most of Tasmania, with confirmed disease‐free 42 pockets remaining only in the north west (Lazenby et al. 2018) and in the remote south‐west 43 (DPIPWE, unpublished data). Due to its recent emergence, DFT2 is still contained to the Channel 44 Peninsula Area south of , and is currently under investigation by the Save the Tasmanian Devil 45 Program in collaboration with the University of Tasmania (STDP, pers. comm.).

46 Devils breed annually between March and June each year (Guiler 1970) although captive devils 47 appear to have a more staggered breeding season compared with wild devils (Keeley et al. 2012). 48 Devils are seasonally polyoestrous and typically breed between 2 and 5 years of age (Guiler 1970; 49 Keeley et al. 2012), although “precocial breeding” (at age 1) can occur in low‐density populations 50 (Jones et al. 2008; Lachish et al. 2009). Devils have a short life‐span, up to 7 years in the wild; age 51 distributions in DFTD‐affected sites are skewed towards younger animals (Grueber et al. 2018; 52 Lazenby et al. 2018). As marsupials, devils give birth after a short gestation time of approximately 12 53 days, and the young continue development in their mother’s pouch (Keeley et al. 2012). Devils give 54 birth to around 20 joeys, however only four survive due to the number of teats; subsequent losses at 55 the pouch‐young stage are thought to be very low (Guiler 1970). Mean litter sizes in the wild vary 56 around 2.9 – 3.0 (Guiler 1970; Pemberton 1990), with significant year‐to‐year variation in the 57 proportion of females breeding and litter sizes, even in DFTD‐free populations (Farquharson et al. 58 2018a).

59 Knowledge of reproductive success in natural wild devil populations is limited to a handful of 60 studies. In the 1960’s, female breeding rates averaged 49% in the north‐west of Tasmania (Guiler 61 1970). Similarly, just prior to the emergence of DFTD in the 1980’s, on average 75% of females were 62 breeding in the north‐east (Pemberton 1990). Wild devil populations continue to face population 63 decline, including DFTD‐free populations such as Woolnorth in the north‐west (Farquharson et al. 64 2018a). Comparing data from 2004‐2009 versus 2014‐2016, there was a statistically significant 65 decline in female reproductive rate from 75% to 19.4% at Woolnorth, as well as a statistically 66 significant decrease in litter size (Farquharson et al. 2018a). This change could not be attributed to 67 changes in population size, sex ratio or body condition, but may be associated with environmental 68 variation, as measured using the Southern Oscillation Index (Farquharson et al. 2018a). In 69 populations affected by DFTD, females are more likely to show evidence of breeding compared to 70 females at DFTD‐free sites, although the reasons for this pattern are unclear (Grueber et al. 2018; 71 Lazenby et al. 2018). One hypothesis is that difference in breeding rates in response to DFTD 72 presence may emerge due to changes in aggressive interactions and reduced competition for dens 73 (Grueber et al. 2018). Additional research is required to understand the role of other biological and 74 environmental factors that impact reproductive success of healthy and DFTD‐affected wild 75 populations.

76 The Save the Tasmanian Devil Program (STDP) was established in 2003 as the Tasmanian and 77 Australian Governments’ response to DFTD. The STDP are tasked with the ongoing management of 78 Tasmanian devils in the wild to ensure they maintain ecological function in the landscape (Save the 79 Tasmanian Devil Program 2014). The overall strategy consists of the following areas: 80  Breed and maintain devils in the insurance population to provide offspring with new genetic 81 diversity for release to the wild (detailed below), 82  Monitor devil roadkill and hotspots and mitigate against roadkill when releasing devils, 83  Utilise both and Forestier Peninsula as translocation source populations, while 84 ensuring that these activities do not compromise the genetic integrity of the sites, 85  Identify populations harbouring unsampled genetic diversity across the state and acquire 86 new founders to improve genetic diversity within the insurance metapopulation, 87  Continue annual monitoring at sites across the state to determine when and where wild 88 populations are persisting, recovering or going locally extinct, and 89  Undertake advocacy to inform local, national and international stakeholders on the plight of 90 Tasmanian devils and their survival.

91 As part of their ongoing management of the species, both in captivity and the wild, the STDP 92 undertake frequent field trips to monitor population and individual health (Lazenby et al. 2018). 93 Along with physical observations of body condition, age estimation from tooth eruption and wear, 94 and breeding estimates based on female pouch condition, tissue samples are collected from 95 individually‐identified devils to enable genetic analysis (Figure 2). Working in collaboration with the 96 STDP, we have undertaken a suite of molecular genetic studies to help inform management and 97 conservation of the species. Our work has targeted immunogenetic and genomic research into the 98 causes and consequences of DFTD (e.g. Siddle et al. 2007b; Woods et al. 2007; Ujvari et al. 2012; 99 Ujvari et al. 2013; Howson et al. 2014), as well as the drivers of reproductive success in the insurance 100 population and the wild. The aim of this collaborative partnership has been to bridge the “research‐ 101 implementation gap” by combining research and conservation in real time to improve population 102 recovery (Hogg et al. 2017a). In this review, we describe the Tasmanian devil insurance population, 103 and the ways in which genetic and genomic data have been developed and used to improve 104 reproductive outcomes for this iconic threatened species.

105 The Tasmanian devil insurance population

106 Origins and structure 107 The Tasmanian devil insurance population commenced in 2006 with four zoos on the Australian 108 mainland, and now encompasses nearly 40 Australian institutions (Hogg et al. 2017b). The Zoo and 109 Aquarium Association (ZAA; previously ARAZPA) is tasked with managing the insurance population 110 on behalf of the Save the Tasmanian Devil Program. In 2008, a population habitat and viability 111 analysis (PHVA) workshop was held in Hobart to discuss alternate conservation scenarios and 112 develop a metapopulation management framework (CBSG 2008). Since that time, the 113 metapopulation has grown in size and now consists of over 700 devils in zoo‐based housing 114 (intensive and group enclosures) in addition to free‐range enclosures (22 ha in size), Maria Island 115 and the fenced Forestier Peninsula (Hogg et al. 2017b). The Tasmanian devil insurance 116 metapopulation framework has moved from a traditional model of ex situ versus in situ 117 management, to one that is a continuum between the traditional zoo enclosure (housing 1 or 2 118 devils) to large free‐ranging semi‐wild populations on Maria Island and the fenced Forestier 119 Peninsula. Different enclosure types offer different advantages and disadvantages to the program, 120 and so the use of a range of facilities enables flexibility to the STDP (Figure 3). 121 The insurance metapopulation has been a successful breeding program since its establishment. 122 Between 2006 and 2017, 1,060 weaned offspring have been produced (Srb 2017) representing 160 123 founders with more than 98% of the wild‐sourced gene diversity maintained (Biggs et al. 2017). The 124 key objectives (gene diversity retained, number of founders, island site, free‐range enclosures) 125 outlined in the 2009 Insurance Metapopulation Strategy (CBSG/DPIPWE/ARAZPA 2009) have all been 126 met and in many instances exceeded (Hogg et al. in press).

127 The overarching goal of the insurance metapopulation is to maintain genetic diversity and provide 128 individuals for release to support dwindling wild populations. Ensuring that the population remains a 129 valuable source for these wild releases involves detailed information on reproduction and breeding. 130 Close management of breeding in captivity is undertaken using a pedigree‐based “mean kinship” 131 strategy, based on records held in the Tasmanian devil studbook (Srb 2017). In this approach, 132 population managers select breeding pairs based on their level of relatedness to the population as a 133 whole, so that underrepresented lineages are targeted first, while also ensuring minimal inbreeding 134 (Montgomery et al. 1997). In facilities where multiple males and females share a pen, the devils are 135 housed such that breeding amongst any pairs of males and females within the group would be 136 acceptable from a genetic standpoint. These approaches are guided by population management 137 software (Lacy et al. 2011), and are widely used for the intensive management of genetic diversity in 138 zoo‐based threatened species breeding programs around the world. For Tasmanian devils, the scale 139 of the insurance population has enabled (and arguably, requires), a nuanced approach to managing 140 the reproductive objectives of the program. Through a combination of data types, particularly 141 genetic and pedigree analysis, conservation scientists are examining the determinants of variation in 142 reproductive outcomes across the program. Together these results provide information that helps to 143 maintain a genetically healthy and robust insurance population that can be used to support devil 144 conservation.

145 Factors influencing captive breeding rates 146 Many factors influence devil reproductive success in captivity, including captive housing, genetic 147 provenance, birth origin, changes to the microbiome and adaptation to captivity. This section details 148 the data that have been collected to examine these processes, and recommendations that have 149 been put forward to improve genetic management of the insurance population.

150 Previous genetic analysis of wild devils has shown subtle population structure on an east‐west 151 gradient (Jones et al. 2004; Miller et al. 2011), so one early question was whether devils from 152 different parts of the range would successfully breed together in the insurance population. The 153 insurance population was established using devils largely from the west of Tasmania, as these sites 154 were ahead of the disease at the time and it was of paramount importance to ensure that only 155 healthy animals were brought into captivity (Hogg et al. 2017b). In subsequent years, devils from 156 eastern parts of the range were brought into the population via the “orphan program”, wherein the 157 offspring of injured or sick mothers were housed in strict quarantine procedures before eventually 158 contributing to the larger program. Fortunately, genetic provenance of devils has little effect on their 159 breeding success in captivity (Hogg et al. 2015). There was no significant difference in the number of 160 joeys produced from the mixed pairing of individuals with east and west provenance, compared to 161 pairing individuals with like provenance, although, west‐west pairings had a higher success rate 162 (Hogg et al. 2015). It is unclear why these differences occur, although it is possible that, because 163 eastern populations had undergone declines due to DFTD prior to their inclusion in the insurance 164 population, inbreeding may play a role.

165 Research has shown that the types of enclosures used can cause reproductive skew (variation 166 among individuals in breeding success). As described above, captive devils are housed in a variety of 167 pens, ranging from intensive housing with forced monogamy (i.e. single male with a single female 168 with no offer of choice), to group housing where multiple males and females are housed in large 169 fenced enclosures (Figure 3). Group housing enables mate choice, and more male devils fail to 170 reproduce in this scenario compared to intensive enclosures (Gooley et al. 2018). It is probable that 171 this male reproductive skew is a result of competition. Body weight is a positive predictor of 172 reproductive success amongst group housed devils, and so large dominant males likely prevent 173 smaller males from breeding (Gooley et al. 2018). Group housing provides additional benefits over 174 intensive breeding, such as opportunities for devils to maintain their natural behaviours (Figure 3). 175 Modelling shows that maintaining group and intensively managed devils separately would result in 176 loss of genetic diversity and increase in inbreeding across the program as a whole (Gooley et al. 177 2018). Because all sites are managed collectively as a metapopulation, individuals are frequently 178 moved between intensive and group housed facilities, which can maximise reproductive success and 179 genetic diversity.

180 Because captive environments differ from the wild in many ways, especially the more intensive 181 enclosures, it is possible that evolutionary processes may drive changes in the genetic variation that 182 underpins traits associated with reproductive outcomes. Captive environments can have significant 183 impacts on productivity across many species and contexts (Farquharson et al. 2018b). Adaptation to 184 captivity involves selection of genetic variants suited to the captive environment, which may be 185 disadvantageous in the wild (Frankham 2008). Experimental studies have shown that changes can 186 occur rapidly (Horreo et al. 2017; Le Luyer et al. 2017; Willoughby et al. 2017), and in some species 187 have negative impacts on the success of releases (e.g. Evans et al. 2014). As such, a main goal of 188 conservation breeding programs is to minimise adaptation to captive environments. This is 189 particularly relevant to devils, given that reintroduction to the wild is a crucial component of the 190 program. In devils, pairing success is higher for wild‐born/wild‐born pairings, compared to captive‐ 191 born pairings (Hogg et al. 2015), and wild‐born females produce significantly more joeys, regardless 192 of the birth‐origin of the sire (Hogg et al. 2015; Farquharson et al. 2017). Over multiple generations, 193 linear modelling predicted that, for a first‐time breeder, wild‐born female devils had 56.5% chance 194 of producing a litter, which declined to 2.8% for fifth generation captive‐born females (Farquharson 195 et al. 2017). Genetic change in a population occurs over generations, and so conservation breeding 196 programs will sometimes attempt to slow change by breeding animals when they are older, and thus 197 extending the generation time. For devils, females that are given their first breeding attempt when 198 they are older are less likely to breed (Farquharson et al. 2017). Taken together, these results 199 indicate that extending generation time to minimise adaptation to captivity would require a greater 200 number of breeding pairs than would be needed if animals are bred earlier, in order to account for 201 the reduced productivity of older females.

202 One possible mechanism of adaptation to captivity is early viability selection, the differential survival 203 of offspring due to genotype that occurs prior to management observations (Grueber et al. 2015a). 204 For example, a reduction in brood or litter sizes due to genotype may arise in utero due to genetic 205 disorders or genetic incompatibility of mother and foetus, as well as post‐partum early sibling 206 competition. In devils, more than 20 joeys are born, but only a maximum of four are raised in the 207 pouch (Guiler 1970). The survivors may not be a random sample of the genotypes present within the 208 litter at birth. This may impact estimates of inbreeding depression and maintenance of genetic 209 diversity that are both crucial to genetic management of captive breeding programs (Grueber et al. 210 2015a).

211 Health and fitness of wild and captive animals has been linked to fluctuations in the gut microbiome 212 (Bahrndorff et al. 2016; Stumpf et al. 2016). The microbiome constitutes the genomes of all the 213 microorganisms which live in, or on the host (Grice & Segre 2012). The Tasmanian devil gut, skin, 214 pouch and oral microbiomes were sequenced in 2015, and showed significant compositional 215 differences between wild and captive devils (Cheng et al. 2015). The diversity and abundance of 216 bacteria in the gut, skin and pouch of captive devils was significantly lower than wild individuals, 217 particularly for devils housed in intensive facilities (Cheng et al. 2015). Devils in captivity are exposed 218 to multiple factors which may influence their microbiome: veterinary treatments, changes to diet 219 and restricted access to other animals and environments. The impact of these changes is unknown, 220 but may cause obesity and reduce breeding success in captive individuals within the insurance 221 population (Cheng et al. 2015).

222 Factors influencing breeding rates at semi‐wild sites 223 Important components of the insurance metapopulation are devil releases to Maria Island and the 224 fenced Forestier Peninsula. Once animals are released from the more intensive captive 225 environments, their ability to integrate into their target populations is of key importance. 226 Specifically, devils must survive, and breed. Release of Tasmanian devils onto Maria Island (where 227 there were no devils previously), in 2012 and 2013, was very successful (Rogers et al. 2016; 228 Thalmann et al. 2016; Wise et al. 2016). Maria Island devils are managed at the population level: 229 individuals may be translocated to or from the population, as required by the needs of the program 230 as a whole. Unlike more intensive management, there is no day‐to‐day management of individuals. 231 For example, Maria Island devils make their own choices about where to range, what to eat, and 232 who to interact or breed with. From the devils’ perspective, this is a wild population. The survival 233 rate of devils released to Maria Island was high (Rogers et al. 2016), and the original founding cohort 234 bred well (McLennan et al. 2018). Using genetic data to reconstruct the pedigree of devils breeding 235 on Maria Island provided valuable insights into the reproductive behaviour of devils after release. 236 The analysis found that supplemented males (i.e. those introduced in the year following population 237 establishment) had a reduced probability of breeding relative to males that were in the initial 238 colonising cohort (McLennan et al. 2018). Further analysis of the Maria Island pedigree indicated a 239 decline in genetic diversity across the four years since the population was established, that was not 240 detectable using molecular methods (McLennan et al. 2018). Thus, genetic supplementation of the 241 population was recommended, to maintain diversity at the site, but local males are likely to 242 outcompete the new males for mating opportunities. The recommended solution was to 243 supplement using females. Ongoing monitoring of the population will determine the efficacy of this 244 approach.

245 Genetic and genomic data to improve breeding 246 Maintaining reliable reproduction in captive and wild populations of threatened species is crucial to 247 maintaining genetic diversity. The influence of stochastic processes on genetic diversity is related to 248 the effective population size. In captivity, effective population size is maximised by minimising the 249 variation in family sizes and reproductive output of breeders. In the wild, where there is no such 250 control, the only option is to attempt to improve population growth. In the following section, we 251 examine how molecular genetic data are being used to monitor breeding outcomes, and their 252 impact on diversity, in the devil insurance metapopulation.

253 Species of conservation concern, such as the Tasmania devil, are often suffering from a litany of 254 small population pressures by the time a captive breeding program is initiated. Low success rates in 255 captive breeding programs, especially when using captive‐bred animals for wild reintroductions, 256 have been attributed to the captive population being founded by a small number of animals and/or 257 the source population suffering from low genetic diversity (Hogg et al. under review). Understanding 258 the genetic composition of the founding population is paramount to a breeding program’s success 259 (Grueber et al. 2015b). Accurately reconstructing pedigrees to prevent inbreeding and maximise 260 genetic diversity in the captive population should increase productivity and adaptive potential of 261 animals once released.

262 Pedigree reconstruction with neutral markers reveals breeding patterns 263 At the time the devil breeding program was established only a handful of microsatellite loci were 264 available for genotyping the devil (Jones et al. 2003). With genetic diversity in the devil being low 265 (Jones et al. 2004; Siddle et al. 2007b; Miller et al. 2011; Cheng et al. 2012; Cui et al. 2015; Morris et 266 al. 2015; Hendricks et al. 2017) more markers were required to assess the captive population of 267 devils. Development of a further 33 microsatellite loci was instrumental in pedigree reconstruction 268 within a “managed environmental enclosure” (see Figure 3) (Gooley et al. 2017). The group breeding 269 enclosures house multiple males and females, which means that both paternity and maternity 270 testing is required. Without the increased allelic diversity provided by the new microsatellite 271 markers, pedigree reconstruction would not be possible as many devils share common alleles. The 272 insights described above, namely that male devils experience higher reproductive skew in group 273 breeding conditions as compared to intensive breeding, would not have been possible without the 274 pedigree reconstruction that these new markers enabled.

275 Pedigree reconstruction also enables researchers to evaluate the role of inbreeding in captive 276 programs. Microsatellite data have shown that devils in captivity appear to have variation in 277 inbreeding that was not detectable using the pedigree (Gooley et al. 2017). It is possible that this 278 variation in inbreeding arises from unidentified relationships among the population founders. This 279 latter hypothesis is currently being tested using additional molecular analysis (Hogg et al. under 280 review). The results will determine whether the variation in breeding success seen amongst pairs, 281 within the insurance population, is attributable to previously unidentified close relationships among 282 the founding animals, and thus inbreeding depression.

283 Targeting immune genes to learn about mate choice and changes in diversity 284 Further research has used microsatellites that are linked to important immunogenetic regions, the 285 major histocompatibility complex (MHC) to investigate functional diversity in the devil (Cheng & 286 Belov 2012). The major histocompatibility complex genes encode proteins that are involved with 287 self/non‐self recognition and therefore play an important role in pathogen recognition and also 288 mate choice (Jordan & Bruford 1998; Kamiya et al. 2014; Brandies et al. 2018). These markers have 289 been used to assess mate choice in captive devils (Russell et al. 2018), and more work is currently 290 underway in our research group. Understanding the role that MHC diversity plays in devil mate 291 choice is expected to improve breeding success in the program. Furthermore, because MHC diversity 292 in the devil is low (Siddle et al. 2007a; Cheng & Belov 2012), maximising diversity at the MHC may be 293 possible through appropriate pairings and/or enabling natural mate choice to occur. Determining 294 the consequences of such management, for genetic diversity overall, is the subject of substantial 295 ongoing research.

296 MHC is not the only immunogenetic region that is important for resilience to pathogens, and 297 therefore of interest in conservation biology (Acevedo‐Whitehouse & Cunningham 2006). Low 298 genetic diversity in the devil extends to other key immune regions (Cui et al. 2015; Morris et al. 299 2015). Diversity at a range of immune genes is vital for adaptive capacity of devil populations once 300 they are reintroduced to the wild and begin to face new disease challenges.

301 In general, mate choice typically plays a role in maintaining diversity at immune regions (Westneat & 302 Birkhead 1998), but is restricted within the captive environment. The differing management 303 practices across the types of housing facilities are expected to have varying impacts on mate choice 304 and subsequent offspring viability and genetic diversity (Figure 3). There is ongoing debate amongst 305 conservation scientists in regards to whether mate choice would permit greater or lesser diversity 306 retention in captive breeding programs, relative to controlled breeding (Asa et al. 2011). On one 307 hand, mate choice in captivity increases overall breeding success in several species (e.g. Ihle et al. 308 2015; Martin‐Wintle et al. 2015; Hartnett et al. 2018), and could plausible help maintain genetic 309 diversity if breeders mate disassortatively (choose partners that are different from themselves, e.g. 310 Isles et al. 2001). On the other hand, reproductive skew may be increased if some “disfavoured” 311 individuals fail to breed (e.g. Gooley et al. 2018) or if certain “attractive” genotypes are favoured 312 (e.g. Cutrera et al. 2012), lowering diversity as a whole. The complexity of genetically driven mate 313 choice means that intermediate outcomes are plausible (e.g. Eizaguirre et al. 2009). Concerns about 314 adaptation to captivity also apply in a mate choice context: if breeding behaviour is altered relative 315 to the wild, changes in diversity that result may negatively impact the success of animals that are 316 released to the wild. Empirical data from devils suggests that minimising inbreeding and maximising 317 genetic diversity overall remain the best options for a healthy and viable population that will have 318 the best outcomes upon reintroduction to the wild. Ongoing studies examining mate choice across 319 more of the program, and more of the genome, will better inform this conclusion.

320 The sequencing of the Tasmanian devil genome (Murchison et al. 2012) provided a vital resource for 321 much research investigating both neutral and functional diversity in the devil (Grueber et al. 2015b; 322 Morris et al. 2015; Wright et al. 2015). By resequencing a number of devil genomes and aligning 323 them to the devil reference genome (Miller et al. 2011; Murchison et al. 2012), we were able to 324 investigate diversity at specific loci in silico (Morris et al. 2015), before designing targeted markers to 325 assess these regions in a wider sample of devils from the captive population (Wright et al. 2015). We 326 have used this information to investigate relatedness amongst the founders of the captive devil 327 population (Hogg et al. under review), vital knowledge for the management of subsequent 328 generations. For species suffering from low genetic diversity, such as the devil, genetic data are vital 329 to ensuring that we preserve as much genetic variation as possible through captive breeding. 330 Release to the wild 331 The purpose of the insurance population when it was established was to repopulate mainland 332 Tasmania with devils (CBSG 2008) once DFTD had caused the species go extinct in 25‐30 years 333 (McCallum et al. 2007). In order to understand the changes occurring in the landscape as a result of 334 DFTD, the STDP has undertaken monitoring of wild devils since 2003 (Grueber et al. 2018; Lazenby et 335 al. 2018) and maintain a comprehensive database of DFTD spread across the state. As populations 336 continue to persist in some areas (Lazenby et al. 2018), there is increasing concern about the ability 337 of devil populations to persist in the face of ongoing small‐population pressures. Small populations 338 are vulnerable to stochastic (chance) events and genetic diversity loss. For example, preliminary 339 modelling has shown that in the presence or absence of DFTD, the largest factor affecting the 340 retention of allelic diversity in the wild is population size (Grueber et al. under review). In 2015 the 341 STDP commenced the trial Wild Devil Recovery Project to answer a series of questions regarding the 342 release of devils from the insurance population into the wild. These include:

343  Do feed stations and scat manipulation dampen the dispersal of released devils, thus 344 allowing released animals to make a greater reproductive contribution to the recipient 345 populations (STDP unpubl data)? 346  Do dispersal patterns differ following a hard release (animals are released immediately at 347 the new site) versus a soft release (animals are held in pens for a period of a period, prior to 348 release at the new site)? 349  Will released devils successfully breed with incumbent animals, enabling genetic rescue of 350 the target population (e.g. Grueber et al. under review)? 351  What processes impact the survival of captive versus wild born devils immediately post‐ 352 release (e.g. Grueber et al. 2017)? 353  Does immunotherapy provide released devils with the ability to mount an immune response 354 to DFTD (e.g. Pye et al. 2018)?

355 To address these questions, the STDP undertook four release events between 2015 and 2017 at 356 Narawntapu National Park (Aug 2015), Forestier Peninsula (Nov 2015), Stony Head (Sept 2016) and 357 wukalina (May 2017). To ensure that each release event would be of maximal genetic benefit to the 358 incumbent population, devils were selected from the zoo‐based insurance population or Maria 359 Island using all available information, including pedigree and molecular data (Hogg, unpubl data). 360 Particular care was taken to select a balanced sex ratio for release, a staggered age distribution, a 361 combination of known and unknown breeders, and individuals that represented the distribution of 362 observed heterozygosity across the genetic marker set used. The outcomes of these release events 363 are still being monitored and investigated, particularly in relation to any breeding success 364 differences between incumbent and released devils.

365 Devils released to wild sites on the Tasmanian mainland, particularly those with DFTD present, 366 encounter more challenges than they experience in captivity, or even on Maria Island. Wild devils 367 encounter many environmental and host factors that are controlled in captivity: disease, weather, 368 predation and anthropogenic risks such as vehicles. The separation of captive devils from these risks 369 through long‐term captive breeding may produce naïve individuals not suited to survival in the wild 370 (reviewed in Grueber et al. 2017). Roads are a major threatening process for devils, as they travel 371 long distances at night, scavenge on roadkill and use roads for dispersal (Owen & Pemberton 2005). 372 Of 69 captive devils reintroduced to Narawntapu National Park and Forestier Peninsula, the fate of 373 50 was known, 19 of which were killed by vehicle strikes within 6 weeks post‐release (Grueber et al. 374 2017). The authors of that study acknowledge that, because the Forestier Peninsula was a 375 reintroduction, and NNP had very low incumbent density, it was not possible to compare the road 376 deaths of released devils to “normal” roadkill rates. However, anecdotal evidence from the STDP 377 indicated the roadkill rate post release were high (Grueber et al. 2017). Multiple generations in 378 captivity increased the probability of fatal vehicle strike post‐release (Grueber et al. 2017). Sex, age 379 and release location had no impact on survival. Given strikes occurred in the period immediately 380 following release, captive devils may lack behaviours such as vehicle avoidance used to explore novel 381 environments (Grueber et al. 2017). A variety of conservation tools have now been deployed to 382 prevent these losses. For example, animals used for subsequent releases have been selected 383 primarily from Maria Island, where there are no vehicles and so devils do not become habituated.

384 Conclusions 385 The Tasmanian devil insurance population is a large breeding program established to help prevent 386 extinction of this important and iconic species. Through a diversity of enclosure types, species 387 managers achieve a variety of reproductive benefits, from preserving genetic diversity to allowing 388 natural mate choice behaviours to persist. The consequences of these management decisions are 389 revealed through detailed genetic studies and inform policy. For example, the observation that 390 increasing generations in captivity increased the probability of fatal vehicle strike upon release led to 391 immediate changes in the way animals were sourced for releases. Similarly, observations of 392 reproductive skew in male breeding enclosures have led population managers to consider how the 393 devil groups housed in these pens are structured in future. Detailed pedigree and molecular genetic 394 studies of reproduction in captivity and the wild are helping to inform the devil breeding program, to 395 the benefit of the species. The long‐term benefits to wild populations of releasing devils from the 396 insurance population to genetically augment sites is yet to be realised and monitoring these release 397 events continues.

398 More generally, our genetic studies of the determinants of reproduction in the devil insurance 399 metapopulation informs the conservation of other important threatened species. Captive breeding 400 for reintroduction to the wild is a valuable conservation strategy (Frankham 2008; Conde et al. 2011; 401 Conway 2011); the International Union for Conservation of Nature (IUCN) recommends captive 402 breeding as component of the conservation of over 2,000 species around the world (CBSG 2017). In 403 Australia, fifty‐five mammal species are currently listed as vulnerable, endangered or critically 404 endangered (IUCN 2017), and captive management is intended to reduce the extinction risk of many 405 of this country’s charismatic and highly endangered native species (Harley et al. 2018). Restoration 406 in the wild is the goal for these programs. We know that, in general, captive‐to‐wild releases fail 407 more often than wild‐to‐wild translocations (Williams & Hoffman 2009); this is where lessons from 408 the devil program can help. As one of the largest conservation breeding programs in the region, the 409 wide diversity of genetic, ecological, disease, and husbandry research that has been undertaken 410 (much of which is described above) informs the way we approach reproductive management of 411 threatened species. The results are therefore beneficial not just to devil conservation, but help guide 412 the effective use of managed breeding programs to preserve global biodiversity. 413 Acknowledgements 414 Our research into the genetic processes affecting Tasmanian devil productivity captivity and the wild 415 has been supported by many wonderful people over the years. In particular, we thank the members 416 of the Australasian Wildlife Genomics Group past and present, and our partners in the Save the 417 Tasmanian Devil Program, the Zoo and Aquarium Association, San Diego Zoo Global, Toledo Zoo, and 418 the University of Sydney. We thank our collaborators at universities and zoos and wildlife parks in 419 Australia and worldwide. Our research program has been supported by the Australian Research 420 Council (LP0989727, LP0989613, FT0992212, DP110102656, DP110102731, LP140100508, 421 DP140103260, DP170101253, DP180102465), the Morris Animal Foundation, the Holsworth Wildlife 422 Research Endowment, San Diego Zoo Global, and the University of Sydney.

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650 651 652 Figures 653

654

655 Figure 1 656 Tasmanian devil released after a health check on Maria Island (photo credit: CJH)

657

658

659 Figure 2 660 Genetic sampling of devils is undertaken using a small ear‐punch biopsy. The animal is held securely 661 in a hessian sack in a calm, dark environment to minimise stress (photo credit: CJH) 662

663 Figure 3 664 The Save the Tasmanian Devil Program Insurance Metapopulation comprises institutions of several 665 types, which collectively offer a range of benefits to the program. One the left of the diagram, 666 smaller enclosures provide the opportunity for more intensive management control, which improves 667 retention of genetic diversity. On the right of the diagram are larger, more “hands‐off” populations, 668 where natural behaviours are more readily expressed.