CSIRO PUBLISHING
Reproduction, Fertility and Development, 2021, 33, 573–587 https://doi.org/10.1071/RD21058
Integrating biobanking could produce significant cost benefits and minimise inbreeding for Australian amphibian captive breeding programs
Lachlan G. Howell A,B,I, Peter R. MawsonC, Richard FrankhamD,E, John C. RodgerA,B, Rose M. O. Upton A,B, Ryan R. WittA,B, Natalie E. CalatayudF,G, Simon Clulow H and John ClulowA,B
ASchool of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia. BFAUNA Research Alliance, Kahibah, NSW 2290, Australia. CPerth Zoo, Department of Biodiversity, Conservation and Attractions, PO Box 489, South Perth, WA 6951, Australia. DDepartment of Biological Sciences, Macquarie University, Sydney, NSW 2019, Australia. EAustralian Museum, Sydney, NSW 2010, Australia. FSan Diego Zoo Institute for Conservation Research, San Pasqual Valley Road, Escondido, CA 92027, USA. GConservation Science Network, 24 Thomas Street, Mayfield, NSW 2304, Australia. HCentre for Conservation Ecology and Genomics, Institute for Applied Ecology, University of Canberra, Bruce, ACT 2617, Australia. ICorresponding author. Email: [email protected]
Abstract. Captive breeding is an important tool for amphibian conservation despite high economic costs and deleterious genetic effects of sustained captivity and unavoidably small colony sizes. Integration of biobanking and assisted reproductive technologies (ARTs) could provide solutions to these challenges, but is rarely used due to lack of recognition of the potential benefits and clear policy direction. Here we present compelling genetic and economic arguments to integrate biobanking and ARTs into captive breeding programs using modelled captive populations of two Australian threatened frogs, namely the orange-bellied frog Geocrinia vitellina and the white bellied frog Geocrinia alba. Back-crossing with frozen founder spermatozoa using ARTs every generation minimises rates of inbreeding and provides considerable reductions in colony size and program costs compared with conventional captive management. Biobanking could allow captive institutions to meet or exceed longstanding genetic retention targets (90% of source population heterozygosity over 100 years). We provide a broad policy direction that could make biobanking technology a practical reality across Australia’s ex situ management of amphibians in current and future holdings. Incorporating biobanking technology widely across this network could deliver outcomes by maintaining high levels of source population genetic diversity and freeing economic resources to develop ex situ programs for a greater number of threatened amphibian species.
Keywords: anuran, artificial reproductive technologies, captive survival-assurance colonies, cryopreservation, frog, genome resource banking, IVF.
Received 12 February 2021, accepted 23 March 2021, published online 18 May 2021
Introduction significant in situ declines across multiple taxa due to expanding Ex situ conservation tools, particularly captive breeding and and persistent anthropogenic threats, policy failings and funding assisted reproductive technologies (ARTs) are becoming neglect (Woinarski et al. 2017; Howell and Rodger 2018; Wintle increasingly important for amphibian species recovery (Bishop et al. 2019). Threatening processes have driven some Australian et al. 2012; Kouba et al. 2012; Clulow and Clulow 2016; species to an increasing reliance on ex situ management across Browne et al. 2019; Clulow et al. 2019). Australian animals face the country’s network of institutions holding captive collections
Journal compilation Ó CSIRO 2021 Open Access CC BY-NC www.publish.csiro.au/journals/rfd 574 Reproduction, Fertility and Development L. G. Howell et al.
(Harley et al. 2018). This is especially true for amphibians, A suggested global target for captive breeding programs is to which face considerable in situ declines globally (Scheele et al. retain 90% of source population heterozygosity (Ht/Ho) for 2019) and domestically (Scheele et al. 2017) due to infectious 200 years (Soule´ et al. 1986), later adjusted to 90% for 100 years fungal diseases (Bower et al. 2017). For example, the amphibian (Frankham et al. 2010). However, it is unlikely this target has chytrid fungus Batrachochytrium dendrobatidis alone has been met within any captive breeding program due to the large resulted in the decline or extinction of more than 500 species in colony sizes and resources that would be required to avoid just a few decades (Scheele et al. 2019), with little that can be heterozygosity loss through inbreeding and genetic drift done to combat ongoing declines in the wild (although see (Howell et al. 2020). Howell et al. (2020) provided a strong Clulow et al. 2018a). Several Australian amphibian species are argument based on modelling that biobanking technology could at immediate risk of extinction (Skerratt et al. 2016; Gillespie address the challenges of high costs and genetics common to et al. 2020), some of which rely on ex situ conservation tools captive breeding programs and allow captive programs to meet (particularly captive breeding) as a last line of defence (Skerratt (and surpass) the suggested genetic retention benchmark et al. 2016; Harley et al. 2018). (,10% Ht/Ho loss after 100 years). Amphibians account for .27% of all native species held in Biobanking technology (frozen living cell repositories com- historic and on-going captive breeding programs in Australia, bined with ARTs) has long been proposed as a complementary representing 11 of the 40 species held in significant captive tool that may reduce space and holding requirements, as well as programs between 1980 and 2018, including six on-going labour and other resources, required to run captive breeding amphibian programs (a modest number compared with the programs (Holt et al. 1996; Clulow and Clulow 2016; Ananjeva quantum of species facing in situ declines; Harley et al. 2018; et al. 2017; Silla and Byrne 2019). Howell et al. (2020) Gillespie et al. 2020). The modest number of programs quantified the potential cost and genetic benefits of biobanking (particularly on-going programs) can be attributed to the technology using genetic and economic modelling and demon- logistical challenges and financial costs of captive breeding strated that supplementing captive-bred populations of Oregon (Mawson and Lambert 2017; Harley et al. 2018), particularly spotted frogs Rana pretiosa with frozen founder spermatozoa the high average annual costs (.A$200 000) that are often through IVF every generation could result in massive short- and required for on-going multiyear (or multidecade) programs long-term cost reductions, minimise inbreeding and greatly (Harley et al. 2018). External multisectoral funding support is reduce required live colony sizes. That modelling suggested oftenrequiredtomeetthesehighcosts(Mawson and Lambert that the reductions in inbreeding were potentially significant 2017; Harley et al. 2018) and, in the absence of external enough to exceed the genetic retention benchmark of 90% of funding, captive breeding is often self-funded by the institution source population heterozygosity within a captive amphibian responsible for the program (Harley et al. 2018). As a result of population, and even allow 95% or 99% heterozygosity targets limited funding, it has been estimated that there are only to be realistically achieved. Howell et al. (2020) proposed that resources to hold approximately 50 amphibian species globally amphibians are an ideal taxon to demonstrate the synergies and in captive programs, which is in stark contrast to the at least benefits of biobanking in captive breeding due to a combination 3000 amphibian species currently threatened with extinction of ideal life history traits (e.g. minimal post-fertilisation parental (Gagliardo et al. 2008; Zippel et al. 2011; Bishop et al. 2012; support, short generation length and high fecundity; Bloxam and Clulow et al. 2014; Murphy and Gratwicke 2017; Gonza´lez- Tonge 1995), comparatively low ex situ holding costs for del-Pliego et al. 2019). individual animals (Conde et al. 2015) and recent advances in Aside from economic challenges, captive breeding programs the underlying reproductive sciences in amphibians (Kouba and face significant challenges managing genetic diversity within Vance 2009; Kouba et al. 2013; Clulow et al. 2014, 2018b, captive collections. Captive breeding programs are criticised for 2019; Clulow and Clulow 2016). the theorised or realised deleterious genetic effects associated A consensus on the potential merits of biobanking tech- with sustained captivity (Snyder et al. 1996), including domes- nology and ARTs is not universal among the Australian tication and adaptation to captivity (Frankham 2008), inbreed- conservation community (Skerratt et al. 2016), and uptake ing depression (Ralls et al. 1988), reduced reproductive fitness of the technology has been largely absent in Australia and and success (Farquharson et al. 2018) and loss of fitness when elsewhere (Monfort 2014; Clulow et al. 2019). No examples reintroduced or translocated (Robert 2009). There are various yet exist of biobanking technology leading to measurable examples of these genetic problems occurring within Australian outcomes in an Australian amphibian captive breeding pro- captive breeding programs across multiple taxa. Even some of gram, and there are few, if any, examples globally (Howell Australia’s most well-resourced captive programs (e.g. Tasma- et al. 2020). Without a paradigm shift from practitioners, the nian devils Sarcophilus harrisii and orange-bellied parrots benefits of biobanking will not be realised in Australian Neophema chrysogaster) have theorised or realised genetic amphibian conservation despite clear economic and genetic issues (Farquharson et al. 2017; Grueber et al. 2017; Morrison fitness benefits. Nevertheless, stretched budgets for conser- et al. 2020). There is a clear focus within many programs on vation, the challenges of captive breeding with standard understanding and maximising genetic diversity (Hogg 2013), husbandry and the persistent in situ decline of amphibians including for captive amphibians (Lees et al. 2013). Genetic provide Australian ex situ conservation practitioners and diversity is consistently a key consideration and desired perfor- institutions with a strong impetus to develop and adopt cost- mance outcome when planning and running captive breeding effective biobanking and ART tools for the ex situ manage- programs (Harley et al. 2018). ment of Australian amphibians. Biobanking reduces captive costs and inbreeding Reproduction, Fertility and Development 575
This study provides support for integrating biobanking access to captive breeding costs and colony sizes from Perth technology into Australian amphibian captive breeding pro- Zoo’s Native Species Breeding Program (Supplementary grams by testing economic and genetic models targeted towards Tables S1–S3). G. vitellina and G. alba are currently main- the network of Australian captive breeding institutions. Here, tained as part of a successful population supplementation we model the end-stage cost benefits, reductions in required program in which spawn are collected from the wild, raised to colony sizes and genetic diversity outcomes achieved with metamorphosis with a high survival rate and returned to sup- biobanking technology in modelled populations of orange- plement (head-start) the wild source population (with only a bellied frogs Geocrinia vitellina and white-bellied frogs Geo- small component of ex situ breeding). In the present study we crinia alba using cost data from the Perth Zoo Native Species use data for the cost of husbandry derived from these captive Breeding Program. As in Howell et al. (2020), we modelled the holdings, but model a more conventional captive breeding economic cost and required colony sizes to meet different colony in which there is a single founder event from a source heterozygosity (Ht/Ho) targets (90%, 95% and 99% of source population and subsequent generations are derived from the F0 population Ht/Ho) under scenarios of no biobanking and bio- founders. banked colonies of G. vitellina and G. alba where the captive populations are supplemented with frozen founder spermatozoa Models using existing or developing ARTs at every generation. We have adopted the methodologies presented in Howell et al. We also provide a roadmap to incorporate biobanking (2020) to model the potential cost and genetic benefits of technology into existing and novel ex situ amphibian captive incorporating biobanking technology into the captive breeding breeding programs in Australian institutions. We propose of G. vitellina and G. alba. We incorporate actual captive required inputs (through economic resources, strategic partner- breeding program costs (2017–18 financial year breeding sea- ship generation, infrastructure usage and/or leverage from son of G. vitellina and G. alba within Perth Zoo’s Native Species existing programs) across a set of actions for G. vitellina and Breeding Program) and genetic modelling (Howell et al. 2020) G. alba that would also be applicable to other Australian to model the long-term cost benefits and genetic outcomes amphibian species. We provide budgets and timelines for achieved if biobanking were successfully implemented and significant actions along the pathway, including closing could be incorporated with minimal additional infrastructure species-specific knowledge gaps and biobanking protocol investment and labour costs into existing funded programs. development for candidate amphibian species. Finally, we We modelled various 100-year captive breeding cost scenar- outline a new paradigm for the ex situ management of Australian ios in which the size of live G. vitellina and G. alba captive amphibians under which biobanking technology could free colonies reflects the colony sizes required to maintain different economic resources to generate an ex situ network in Australia proportions of the source population heterozygosity (90%, 95% with the flexibility and resource capacity to allow the inclusion and 99% Ht/Ho retention), with or without the use of biobanking of a greater number of species in captive programs in response to technology, as proposed in other studies (Soule´ et al. 1986; the continued in situ declines of Australian amphibians. Frankham et al. 2010; Howell et al. 2020). For these hypotheti- cal populations, we model biobanking strategies in which cryopreserved founder spermatozoa of G. vitellina and G. alba Materials and methods are used to reduce the size of live colonies while meeting genetic Study species retention targets by the reintroduction of founder genes at each Australia’s orange-bellied frog G. vitellina and white-bellied generation via IVF up to 100 years after the colony is estab- frog G. alba are small, terrestrial breeding frogs endemic to a lished. We assume 4-year generational intervals for G. vitellina small area of south-west Western Australia (Clulow and Swan and G. alba (Table 2). 2018). The two species are listed as vulnerable and critically We adjusted the genetic back-crossing model presented in endangered respectively under the International Union for the Howell et al. (2020) to account for the different life history Conservation of Nature (IUCN) Red List (Hero and Roberts traits, required back-cross frequency and Ne of G. vitellina and 2004; Roberts and Hero 2004), and Australia’s Environment G. alba. Our model determines the census size (N)ofthe Protection and Biodiversity Conservation (EPBC) Act 1999 captive colony required to maintain various levels of genetic Threatened Species List (https://www.environment.gov.au/ diversity (with heterozygosity values derived from inbreeding cgi-bin/sprat/public/publicthreatenedlist.pl). Both species coefficients) under two scenarios: (1) non-back-crossed popu- are facing combined persisting threats of disease lations representing the minimum number of individuals (chytridiomycosis), land use and habitat alterations, climate required to meet heterozygosity targets in a conventional change (Hoffmann et al. 2021) and invasive species (Wardell- captive breeding program without biobanking; and (2) popula- Johnson et al. 1995; Roberts et al. 1999). Each species is tions with back-crossing to founder genotypes every genera- reduced to small fragmented metapopulations within narrow tion at 4-year intervals sourced from biobanked founder home ranges, and both have documented in situ genetic issues spermatozoa. For back-crossed populations, Ne was generated (Driscoll 1997, 1998, 1999; Roberts et al. 1999; Conroy 2001). by random substitution into iterative genetic models until the We selected G. vitellina and G. alba as case study species due 100-year Ht/Ho met desired genetic retention targets. We to the availability of the primary data required for the model- derived a range of colony size numbers (N) using various ling presented in Howell et al. (2020), G. vitellina and G. alba published and assumed Ne/N ratio estimates for G. vitellina effective population sizes (Ne; Table 1; Driscoll 1997)and and G. alba (Table 1). Modelled Ne/N ratios included: (1) a 576 Reproduction, Fertility and Development L. G. Howell et al.
Table 1. Effective population size (Ne) to census population size (N) ratios used for different modelling scenarios for modelled captive biobanked and non-banked populations of orange-bellied frogs Geocrinia vitellina and white-bellied frogs Geocrinia alba
The Ne/N ratios presented here were used to perform Ne to N conversions for modelled non-banked populations designed to meet different genetic retention targets (90%, 95% and 99% Ht/Ho retention)
Modelling scenario Description and rationale Ne/N ratios sampled Ne/N value used in Source modelling
1. Mean ratio for captive The mean Ne/N ratio for captive vertebrates Red-crowned crane Grus japonensis 0.3 Mace (1986) vertebrates across various taxa, used in the absence of (Ne/N ¼ 0.45) Ne/N ratios for captive amphibians. We Grevy’s zebra Equus grevyi consider this modelling scenario to be the (Ne/N ¼ 0.28) most realistic for biobanked populations Scimitar-horned oryx Oryx dammah
among the published captive Ne/N values (Ne/N ¼ 0.20) 2. Median temporal and The median Ne/N ratio from a range of pub- Cane toad Rhinella marina 0.141 Frankham (1995), demographic Ne/N lished estimates of amphibian Ne/N values; (Ne/N ¼ 0.052) Frankham et al. values for amphibians these values are derived from wild popula- Crested newt Triturus cristatus (2019)
tions and provide lower estimates than (Ne/N ¼ 0.185) captive-derived estimates from other taxa European common frog Rana tempor-
aria (Ne/N ¼ 0.705) Marbeled newt Triturus marmoratus
(Ne/N ¼ 0.185) Natterjack toad Bufo calamita
(Ne/N ¼ 0.097) Red-spotted newt Notophalmus
viridescens (Ne/N ¼ 0.073) 3. Species-specific Ne/N Only available species-specific Ne/N ratios Orange-bellied frog G. vitellina 1.17 and 1.21 Driscoll (1999) values for G. vitellina and G. alba. Estimates are (Ne/N ¼ 1.17) demographic and wild derived, and although White-bellied frog G. alba
these estimates do account for variables (Ne/N ¼ 1.21) typically affecting Ne/N ratios discussed in Frankham (1995), the approach taken may
overestimate Ne/N. In fact, these values are considerably higher than other estimates for amphibian species that account for the same variables (see Scenario 2). We present these estimates as published values for G. vitellina and G. alba while recognising they represent
extremes of Ne/N estimates that generate much lower estimates of required census captive population sizes
mean Ne/N ratio for captive vertebrate populations of 0.3 Cost modelling (presented in Mace 1986); (2) a median Ne/N value of All cost modelling was based on all fixed and variable program 0.141 calculated using range of wild-derived temporal Ne/N costs from Perth Zoo’s Native Species Breeding Program for the estimates for amphibian species (Table 1; Frankham 1995; 2017–18 breeding seasons of G. vitellina and G. alba. We used a Frankham et al. 2019); and (3) species-specific wild-derived microcosting approach whereby all fixed and variable costs demographic Ne/N estimates for G. vitellina and G. alba of 1.17 (expressed in nominal Australian dollars) associated with the and 1.21 respectively (Table 1; Driscoll 1999). The range of Ne/ captive breeding of G. vitellina and G. alba were calculated or N values modelled here includes extremes of published values estimated using face-to-face interviews with personnel as well and assumptions that generate a wide range of census popula- as access to financial, logistical and operations reporting. tion sizes for captive G. vitellina and G. alba; we assume that Year 1 economic ex situ holding cost per individual (C)is true captive Ne/N values for G. vitellina and G. alba values given by Eqn 1: would lie somewhere between the extremes modelled here. Derived N values were substituted into an economic costing C ¼ ðÞþ½ I þ G þ E þ L þ U þ F =N M ð1Þ model based on costs for the 2017–18 breeding season of G. vitellina and G. alba at Perth Zoo. This was used to estimate where all costs are in nominal 2018 Australian dollars, and an economic ex situ holding cost per individual animal for each where I represents facilities costs (set-up and recurring species and 100-year captive colony costs for each derived maintenance), G represents fixed founder collection costs colony size (N) with or without biobanking technology in (field collection costs including vehicles, accommodation and various modelling scenarios. food for staff), E represents minor equipment and consumables Biobanking reduces captive costs and inbreeding Reproduction, Fertility and Development 577
Table 2. General genetic modelling assumptions and species-specific parameters used to adapt modelling approaches for captive colonies of orange- bellied frogs Geocrinia vitellina and white-bellied frogs Geocrinia alba
General genetic modelling assumptions and parameters for captive colonies