1 AUTHORS’ ACCEPTED VERSION 2 Version of record: Grueber CE, Peel E, Wright B, Hogg CJ, Belov K (2019) A Tasmanian devil 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, Australia 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 Tasmania, 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 Hobart, 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 Maria Island 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.