Molecular Ecology (2008) 17, 3448–3463 doi: 10.1111/j.1365-294X.2008.03841.x AssessmentBlackwell Publishing Ltd of genetic diversity in the critically endangered Australian corroboree frogs, Pseudophryne corroboree and Pseudophryne pengilleyi, identifies four evolutionarily significant units for conservation MATTHEW J. MORGAN,*§ DAVID HUNTER,† ROD PIETSCH,† WILLIAM OSBORNE‡ and J. SCOTT KEOGH* *School of Botany and Zoology, Australian National University, Canberra, ACT 0200, Australia, †New South Wales Department of Environment and Climate Change, PO Box 2115, Queanbeyan, NSW 2620, Australia, ‡Institute for Applied Ecology, University of Canberra, Canberra, ACT 2106, Australia Abstract The iconic and brightly coloured Australian northern corroboree frog, Pseudophryne pengilleyi, and the southern corroboree frog, Pseudophryne corroboree are critically endan- gered and may be extinct in the wild within 3 years. We have assembled samples that cover the current range of both species and applied hypervariable microsatellite markers and mitochondrial DNA sequences to assess the levels and patterns of genetic variation. The four loci used in the study were highly variable, the total number of alleles observed ranged from 13 to 30 and the average number of alleles per locus was 19. Expected heterozygosity of the four microsatellite loci across all populations was high and varied between 0.830 and 0.935. Bayesian clustering analyses in STRUCTURE strongly supported four genetically distinct populations, which correspond exactly to the four main allopatric geographical regions in which the frogs are currently found. Individual analyses performed on the separate regions showed that breeding sites within these four regions could not be separated into distinct populations. Twelve mtND2 haplotypes were identified from 66 individuals from throughout the four geographical regions. A statistical parsimony network of mtDNA haplotypes shows two distinct groups, which correspond to the two species of corroboree frog, but with most of the haplotype diversity distributed in P. pengilleyi. These results demonstrate an unexpectedly high level of genetic diversity in both species. Our data have important implications for how the genetic diversity is managed in the future. The four evolutionarily significant units must be protected and maintained in captive breeding programmes for as long as it is possible to do. Keywords: Anuran, Australia, conservation genetics, frog, microsatellite, mitochondrial DNA, Myobatrachidae Received 24 March 2008; revision received 6 May 2008; accepted 20 May 2008 Griffiths 2005). Australia has a highly diverse and Introduction species-rich frog fauna and a number of species have The recent decline of frogs worldwide is now well declined significantly over the last 30 years. Two iconic established but the causes of this decline remain unclear species in particular have been the subject of considerable (Alford & Richards 1999; Stuart et al. 2004; Beebee & concern because their decline has been so rapid and extreme. The northern corroboree frog, Pseudophryne pengilleyi, and the southern corroboree frog, Pseudophryne Correspondence: A/Prof. J. Scott Keogh, Fax: 61-2-6125-5573; E-mail: [email protected] corroboree (Family Myobatrachidae), are small, pond- §Present Address: Section of Integrative Biology, University of breeding and very brightly coloured terrestrial frogs Texas, Austin, TX 78712-1100, USA restricted parts of the alpine and subalpine regions of the © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd CONSERVATION GENETICS OF THE ENDANGERED CORROBOREE FROGS 3449 Fig. 1 Map showing disjunct distributions of P. pengilleyi and P. corroboree and the locations of historical records of now extinct populations (white dots) and the 41 sample sites used in the genetic analyses (black dots). Site names and haplotypes correspond to the codes in Table 1. southern highlands of New South Wales and the Australian monitoring as part of a recovery programme has documented Capital Territory in Australia (Woodruff 1975; Osborne further declines in both species. A recent census, taken in 1989) (Fig. 1). Pseudophryne pengilleyi is known from sites the summer of 2006, found the number of calling male within three disjunct geographical regions; the Fiery Ranges, P. corroboree to have fallen from 450 frogs at 79 sites in 1999 the Northern Brindabella Ranges and Southern Brindabella to just 39 frogs at 14 sites in 2006 (Hunter et al. 2006), making Ranges, whereas P. corroboree is known only from sites within this species possibly the most critically endangered ver- the Snowy Mountains (Fig. 1). Once considered a single tebrate in Australia. Declines in P. pengilleyi numbers have species, populations now considered P. pengilleyi have been been equally severe in several parts of this species’ range taxonomically distinguished from P. corroboree based on with the most recent census data finding fewer than 450 hybridization experiments (Osborne & Norman 1991), calling males across this species’ known distribution (Hunter morphology (Pengilley 1966; Woodruff 1975; Osborne et al. et al. 2006). The disease chytridomycosis, caused by infection 1996), call structure (Osborne et al. 1996) and genetic data with the Amphibian Chytrid Fungus (Batrachochytrium including allozymes and immunological distance (Roberts dendrobatidis), has been identified as the primary causal & Maxson 1989; Daly et al. 1990; Osborne & Norman 1991). factor in the decline of both corroboree frog species The rapid decline of both species of corroboree frog is (Hunter 2007). The initial decline of these species coincided noteworthy because it has been so well documented. with the first appearance of this disease in populations Observations until 1966 indicated that both species were (Hunter 2007), and also with the decline of other frog abundant and occurred in large numbers during the species in the Australian Alps (Osborne et al. 1999) and breeding season (Colefax 1956; Pengilley 1966), but numbers elsewhere along the eastern ranges of Australia which have of P. corroboree in the Snowy Mountains and P. pengilleyi been attributed to chytridomycosis (Berger et al. 1998). in the Brindabella Ranges had declined markedly by the In response to the continued decline and critically low 1980s (Osborne 1989; Osborne et al. 1999). Population population size, a captive husbandry programme has © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd 3450 M. J. MORGAN ET AL. commenced. This recovery action currently includes (Woodworth et al. 2002; Gilligan & Frankham 2003). In (i) collection of eggs from the wild each summer with particular, the maintenance of high genetic diversity in captive rearing of tadpoles through to metamorphosis captivity is important to the long-term viability of popu- and subsequent re-introduction of frogs to the wild, and (ii) lations because it may enable populations to adapt in establishment of a captive breeding colony for producing response to environmental changes, and low heterozygosity frogs for re-introduction. Saving species from extinction is directly linked to reduced population fitness in many through captive breeding and re-introduction is a highly con- species, including frogs (Reed & Frankham 2003; Halverson troversial conservation tool (Fischer & Lindenmayer 2000; et al. 2006; Kraaijeveld-Smit et al. 2006). Seigel & Dodd 2002), although the introduction of cap- A previous allozyme study of seven wild corroboree tive-bred individuals has been successful in the Mallorcan frog populations (Osborne & Norman 1991) found: (i) no midwife toad, Alytes muletensis (Bloxan & Tonge 1995; private alleles, but significant differences in allele frequencies Kraaijeveld-Smit et al. 2005, 2006). between frogs in the Snowy Mountains region and those in An understanding of the magnitude and patterns of the northern regions at five of seven loci; (ii) some evidence genetic diversity among populations is critical for conser- of genetic differentiation between populations in allopatric vation efforts attempting to maintain evolutionarily viable geographical regions, that is, those the Fiery Ranges and species (Avise 2004). Pond-breeding amphibians such as those in the Brindabella Ranges; (iii) little genetic differen- corroboree frogs often employ distinct environments tiation between populations within geographical regions; for breeding, development and adult survival. Breeding and (iv) levels of genetic diversity (number of polymorphic habitat is typically distributed patchily throughout a species loci, heterozygosity, average number of alleles per locus) range, and landscape features that affect the genetic in P. corroboree within the Snowy Mountains region to be connectivity of populations, as well as life-history charac- lower than P. pengilleyi in the Fiery Ranges and Brindabella teristics of the individual species, can have a major effect on Ranges. population structure. Recent studies of genetic structure in Osborne & Norman’s (1991) study encompassed only pond-breeding amphibians have shown that some species seven populations: two in the Fiery Ranges (Cromwell Hill may show significant genetic structure across small geo- and Broken Cart), two in the Brindabellas (Coree Flats and graphical distances whereas others show little structure Ginini Flats), and three in the Snowy Mountains (Maragale across similar scales (Rowe et al. 2000; Newman & Squire Range, Ogilive’s Creek and Mount Jagungal). Here we 2001; Burns et