Greta J. Frankham

Novel insights into the phylogenetic relationships of the endangered marsupial genus Potorous

The three extant potoroo species of the marsupial genus Potorous -Potorous tridactylus, P. longipes and P. gilbertii - are all of conservation concern due to introduced predators and habitat loss associated with the European settlement of Australia. Robust phylogenies can be useful to inform conservation management, but past phylogenetic studies on potoroos have been unable to fully resolve relationships within the genus. Here, a multi-locus approach was employed, using three mitochondrial DNA (mtDNA): NADH dehydrogenase subunit 2, cytochrome c oxidase subunit 1 and 12S rRNA and four nuclear DNA (nuDNA) gene regions: breast and ovarian cancer susceptibility gene, recombination activating gene-1, apolipoprotein B and omega globin. This was coupled with widespread geographic sampling of the broadly distributed P. tridactylus, to investigate the phylogenetic relationships within this genus. Analyses of the mtDNA identified five distinct and highly divergent lineages including, P. longipes, P. gilbertii and three distinct lineages within P. tridactylus (northern mainland, southern mainland and Tasmanian). P. tridactylus was paraphyletic with the P. gilbertii lineage, suggesting that cryptic taxa may exist within P. tridactylus. NuDNA sequences lacked the resolution of mtDNA. Although they resolved the three currently recognised species, they were unable to differentiate lineages within P. tridactylus. Current management of P. tridactylus as two sub-species (mainland and Tasmania) does not recognise the full scope of genetic diversity within this species, especially that of the mainland populations. Until data from more informative nuDNA markers are available, we recommend this species be managed as the following three subspecies: Potorous tridactylus tridactylus (southern Queensland and northern New South Wales); Potorous tridactylus trisulcatus (southern New South Wales and Victoria) Potorous tridactylus apicalis (Tasmania). Molecular dating estimated that divergences within Potorous occurred in the late Miocene through to the early Pliocene.

Phylogeography of the Koala, (Phascolarctos cinereus), and Harmonising Data to Inform Conservation

The Australian continent exhibits complex biogeographic patterns but studies of the impacts of Pleistocene climatic oscillation on the mesic environments of the Southern Hemisphere are limited. The koala (Phascolarctos cinereus), one of Australia’s most iconic species, was historically widely distributed throughout much of eastern Australia but currently represents a complex conservation challenge. To better understand the challenges to koala genetic health, we assessed the phylogeographic history of the koala. Variation in the maternally inherited mitochondrial DNA (mtDNA) Control Region (CR) was examined in 662 koalas sampled throughout their distribution. In addition, koala CR haplotypes accessioned to Genbank were evaluated and consolidated. A total of 53 unique CR haplotypes have been isolated from koalas to date (including 15 haplotypes novel to this study). The relationships among koala CR haplotypes were indicative of a single Evolutionary Significant Unit and do not support the recognition of subspecies, but were separated into four weakly differentiated lineages which correspond to three geographic clusters: a central lineage, a southern lineage and two northern lineages co-occurring north of Brisbane. The three geographic clusters were separated by known Pleistocene biogeographic barriers: the Brisbane River Valley and Clarence River Valley, although there was evidence of mixing amongst clusters. While there is evidence for historical connectivity, current koala populations exhibit greater structure, suggesting habitat fragmentation may have restricted female-mediated gene flow. Since mtDNA data informs conservation planning, we provide a summary of existing CR haplotypes, standardise nomenclature and make recommendations for future studies to harmonise existing datasets. This holistic approach is critical to ensuring management is effective and small scale local population studies can be integrated into a wider species context.

Undiagnosed cryptic diversity in small, microendemic frogs (Leptolalax) from the Central Highlands of Vietnam.

A major obstacle in prioritizing species or habitats for conservation is the degree of unrecognized diversity hidden within complexes of morphologically similar, “cryptic” species. Given that amphibians are one of the most threatened groups of organisms on the planet, our inability to diagnose their true diversity is likely to have significant conservation consequences. This is particularly true in areas undergoing rapid deforestation, such as Southeast Asia. The Southeast Asian genus Leptolalax is a group of small-bodied, morphologically conserved frogs that inhabit the forest-floor. We examined a particularly small-bodied and morphologically conserved subset, the Leptolalax applebyi group, using a combination of molecular, morphometric, and acoustic data to identify previously unknown diversity within. In order to predict the geographic distribution of the group, estimate the effects of habitat loss and assess the degree of habitat protection, we used our locality data to perform ecological niche modelling using MaxEnt. Molecular (mtDNA and nuDNA), acoustic and subtle morphometric differences revealed a significant underestimation of diversity in the L. applebyi group; at least two-thirds of the diversity may be unrecognised. Patterns of diversification and microendemism in the group appear driven by limited dispersal, likely due to their small body size, with several lineages restricted to watershed basins. The L. applebyi group is predicted to have historically occurred over a large area of the Central Highlands of Vietnam, a considerable portion of which has already been deforested. Less than a quarter of the remaining forest predicted to be suitable for the group falls within current protected areas. The predicted distribution of the L. applebyi group extends into unsurveyed watershed basins, each potentially containing unsampled diversity, some of which may have already been lost due to deforestation. Current estimates of amphibian diversity based on morphology alone are misleading, and accurate alpha taxonomy is essential to accurately prioritize conservation efforts.

Population genetics of the koala (Phascolarctos cinereus) in north-eastern New South Wales and south-eastern Queensland

Abstract. Habitat loss and fragmentation are key threats to local koala (Phascolarctos cinereus) populations. Broad-scale management is suboptimal for koalas because distribution models are not easily generalised across regions. Therefore, it is imperative that data relevant to local management bodies are available. Genetic data provides important information on gene flow and potential habitat barriers, including anthropogenic disturbances. Little genetic data are available for nationally significant koala populations in north-eastern New South Wales, despite reported declines due to urbanisation and habitat loss. In this study, we develop 14 novel microsatellite loci to investigate koala populations in north-eastern New South Wales (Port Macquarie, Coffs Harbour, Tyagarah, Ballina) and south-eastern Queensland (Coomera). All locations were significantly differentiated (FST = 0.096–0.213; F′ ST = 0.282–0.582), and this pattern was not consistent with isolation by distance (R2 = 0.228, P = 0.058). Population assignment clustered the more northern populations (Ballina, Tyagarah and Coomera), suggesting contemporary gene flow among these sites. For all locations, low molecular variation among (16%) rather than within (84%) sites suggests historical connectivity. These results suggest that koala populations in north-eastern New South Wales and south-eastern Queensland are experiencing contemporary impediments to gene flow, and highlight the importance of maintaining habitat connectivity across this region.

Adaptation and conservation insights from the koala genome

The koala, the only extant species of the marsupial family Phascolarctidae, is classified as ‘vulnerable’ due to habitat loss and widespread disease. We sequenced the koala genome, producing a complete and contiguous marsupial reference genome, including centromeres. We reveal that the koala’s ability to detoxify eucalypt foliage may be due to expansions within a cytochrome P450 gene family, and its ability to smell, taste and moderate ingestion of plant secondary metabolites may be due to expansions in the vomeronasal and taste receptors. We characterized novel lactation proteins that protect young in the pouch and annotated immune genes important for response to chlamydial disease. Historical demography showed a substantial population crash coincident with the decline of Australian megafauna, while contemporary populations had biogeographic boundaries and increased inbreeding in populations affected by historic translocations. We identified genetically diverse populations that require habitat corridors and instituting of translocation programs to aid the koala’s survival in the wild.The assembly of the genome of the koala provides insights into its adaptive biology and identifies gene expansions that contribute to its ability to detoxify eucalyptus-derived compounds and perceive plant secondary metabolites.

Population ecology of the long-nosed potoroo (Potorous tridactylus) on French Island, Victoria

The elusive nature of the long-nosed potoroo (Potorous tridactylus) has hindered the collection of long-term data for this threatened species. Between June 2005 and May 2009, data on the ecology of a wild population of long-nosed potoroos located on French Island, Victoria, were collected during a series of research projects. Over this period, 33 individual potoroos were trapped a total of 251 times. Up to nine individuals were known to be alive at once on the 15-ha study site of mature remnant native forest. Adult potoroos showed high site fidelity and significant sexual size dimorphism, with males heavier and having longer head and pes lengths than females. Congruent with other studies, we found no evidence of seasonality in breeding. Births occurred in every month of the year and the testis volume of males did not vary throughout the year. In contrast to previous studies, however, we did not observe peaks in breeding activity. Our research and review of existing literature suggests that the ecology of the long-nosed potoroo is strongly influenced by local environmental conditions and emphasises the need to consider long-term and site-specific data when developing management strategies to conserve this ecologically important species.

SNP Marker Discovery in Koala TLR Genes

Toll-like receptors (TLRs) play a crucial role in the early defence against invading pathogens, yet our understanding of TLRs in marsupial immunity is limited. Here, we describe the characterisation of nine TLRs from a koala immune tissue transcriptome and one TLR from a draft sequence of the koala genome and the subsequent development of an assay to study genetic diversity in these genes. We surveyed genetic diversity in 20 koalas from New South Wales, Australia and showed that one gene, TLR10 is monomorphic, while the other nine TLR genes have between two and 12 alleles. 40 SNPs (16 non-synonymous) were identified across the ten TLR genes. These markers provide a springboard to future studies on innate immunity in the koala, a species under threat from two major infectious diseases.

Absence of mammary development in male Dromiciops gliroides: another link to the Australian marsupial fauna

Abstract The South American microbiotherian marsupial Dromiciops gliroides is regarded as a sister taxon to the monophyletic Australasian marsupial clade. Mammary anlagen have been documented in male American marsupials but have not been observed in males of any Australian marsupials. The aim of this study was to determine if mammary anlagen were present in developing pouch young (PY) of Dromiciops. The developmental anatomy, particularly the presence of mammary anlagen, was investigated in 3 neonatal male (n  =  2) and female (n  =  1) Dromiciops PY (∼15–20 days postpartum). External and internal structures were examined to confirm PY gender and to provide basic information on development, particularly of the genitourinary system. In the female PY early pouch development was observed as well as 4 clearly defined abdominal mammary anlagen. No external teat or mammary anlagen development was seen in the 2 male PY and serial sections from the external genitalia to upper thorax failed to reveal any histological structures similar to mammary anlagen seen in the female. Absence of mammary anlagen in male Dromiciops PY provides further evidence that Dromiciops shares a closer relationship with the Australasian marsupial clade than with the American marsupial fauna.

The genetic mating system of the long-nosed potoroo (Potorous tridactylus) with notes on male strategies for securing paternity

Abstract. The potoroids are a small group of cryptic macropodoid marsupials that are difficult to directly monitor in the wild. Consequently, information regarding their social and mating systems is limited. A population of long-nosed potoroos (Potorous tridactylus) on French Island, Victoria, was monitored from June 2005 to August 2010. Tissue samples were collected from 32 (19 ♂, 13 ♀) independent potoroos and 17 pouch young. We aimed to determine the genetic mating system and identify patterns of paternity through genotyping individuals at 10 microsatellite loci. Additionally, we investigated the importance of body mass and site residency as strategies in securing paternity. Twelve of the 17 pouch young sampled were assigned paternity with confidence to five males. Multiple pouch young were sampled from two long-term resident females, one of which had 10 pouch young sired by multiple partners, with some repeat paternity, while the other had three young sired by one male, suggesting that the mating system is not entirely promiscuous. Sires were recorded on site for significantly longer periods than non-sires but were not significantly larger than non-sires at conception. This suggests that sires employ strategies other than direct competition, such as scramble competition, to secure paternity in P. tridactylus.

Does the ‘extinct’ Eastern Quoll (Dasyurus viverrinus) persist in Barrington Tops, New South Wales

The eastern quoll (Dasyurus viverrinus) is believed to be extinct on the Australian mainland, with the last confirmed record in 1963. Recently an eastern quoll specimen was located that had been found in northern Barrington Tops National Park (200 km north of Sydney) in 1989. Partial sequences (~200 bp) of the mitochondrial DNA gene Cytochrome b were obtained from the Barrington Tops specimen and compared with sequences from known mainland and Tasmanian eastern quolls. The genetic data, while limited, are most consistent with the Barrington Tops specimen being derived from the ‘extinct’ mainland eastern quoll population. This suggests that eastern quolls survived for decades longer on the Australian mainland than previously thought and raises the possibility that they may still persist in remote areas such as Barrington Tops.