Bronwyn A. Houlden

Low genetic variability of the koala Phascolarctos cinereus in south‐eastern Australia following a severe population bottleneck

Genotyping of koalas at CA‐repeat microsatellite loci has revealed significant differences in the levels of allelic diversity (A) and expected heterozygosity (HE) between populations from north‐eastern and south‐eastern Australia. In the 10 populations studied, allelic diversity ranged from 8.0 in the Nowendoc population to 1.7 in the Kangaroo Is. population, and values of HE ranged from 0.831 in the Nowendoc population to 0.331 in the Kangaroo Is. population. Data from pooled populations revealed koalas from the northeastern region had significantly higher levels of allelic diversity (A= 11.5 ± 1.4) than those from south‐eastern Australia (A= 5.3 ± 1.0). Furthermore significantly higher heterozygosity levels were found in the north‐eastern (HE= 0.851) vs. the south‐eastern (HE= 0.436) regions of Australia. Following a near‐extinction bottleneck in the 1920s, mainland Victorian and Kangaroo Is. koalas have been involved in an extensive program of relocations. The source populations of the relocated animals were islands in Westernport Bay, which were founded by very few individuals in the late 1800s and early 1900s. The significantly lower levels of variation between south‐eastern Australian populations suggests that human intervention has had a severe effect on levels of genetic diversity in this region, and this may have long‐term genetic consequences.

Phylogeographic differentiation in the mitochondrial control region in the koala, Phascolarctos cinereus (Goldfuss 1817)

The koala, Phascolarctos cinereus, is a geographically widespread species endemic to Australia, with three currently recognized subspecies: P.c. adustus, P.c. cinereus, and P.c. victor. Intraspecific variation in the mitochondrial DNA (mtDNA) control region was examined in over 200 animals from 16 representative populations throughout the species’ range. Eighteen different haplotypes were defined in the ≈ 860 bp mtDNA control region, as determined by heteroduplex analysis/temperature gradient gel electrophoresis (HDA/TGGE). Any single population typically possessed only one or two haplotypes yielding an average within‐population haplotypic diversity of 0.180 ± 0.003, and nucleotide diversity of 0.16%. Overall, mtDNA control region sequence diversity between populations averaged 0.67%, and ranged from 0% to 1.56%. Nucleotide divergence between populations averaged 0.51%, and ranged from 0% to 1.53%. Neighbour‐joining methods revealed limited phylogenetic distinction between geographically distant populations of koalas, and tentative support for a single evolutionarily significant unit (ESU). This is consistent with previous suggestions that the morphological differences formalized by subspecific taxonomy may be interpreted as clinal variation. Significant differentiation in mtDNA‐haplotype frequencies between localities suggested that little gene flow currently exists among populations. When combined with microsatellite analysis, which has revealed substantial differentiation among koala populations, we conclude that the appropriate short‐term management unit (MU) for koalas is the local population.

Phylogeographical population structure of tiger quolls Dasyurus maculatus (Dasyuridae: Marsupialia), an endangered carnivorous marsupial

Tiger quolls, Dasyurus maculatus, are the largest carnivorous marsupials still extant on the mainland of Australia, and occupy an important ecological niche as top predators and scavengers. Two allopatric subspecies are recognized, D.m. gracilis in north Queensland, and D.m. maculatus in the southeast of the mainland and Tasmania. D.m. gracilis is considered endangered while D.m. maculatus is listed as vulnerable to extinction; both subspecies are still in decline. Phylogeographical subdivision was examined to determine evolutionarily significant units (ESUs) and management units (MUs) among populations of tiger quolls to assist in the conservation of these taxa. Ninety‐three tiger quolls from nine representative populations were sampled from throughout the species range. Six nuclear microsatellite loci and the mitochondrial DNA (mtDNA) control region (471 bp) were used to examine ESUs and MUs in this species. We demonstrated that Tasmanian tiger quolls are reciprocally monophyletic to those from the mainland using mtDNA analysis, but D.m. gracilis was not monophyletic with respect to mainland D.m. maculatus. Analysis of microsatellite loci also revealed significant differences between the Tasmanian and mainland tiger quolls, and between D.m. gracilis and mainland D.m. maculatus. These results indicate that Tasmanian and mainland tiger quolls form two distinct evolutionary units but that D.m. gracilis and mainland D.m. maculatus are different MUs within the same ESU. The two marker types used in this study revealed different male and female dispersal patterns and indicate that the most appropriate units for short‐term management are local populations. A revised classification and management plan are needed for tiger quolls, particularly in relation to conservation of the Tasmanian and Queensland populations.

Microsatellite variation and population structure in a declining Australian Hylid Litoria aurea

The green and golden bell frog (Litoria aurea) was once a common Australian Hylid. Today, many populations are small and fragmented as a result of dramatic declines in distribution and abundance. We undertook a large‐scale assessment of genetic structure and diversity in L. aurea using four species‐specific microsatellite markers. Twenty‐one locations were sampled from throughout the species range covering 1000 km of the east coast of Australia. Levels of allelic diversity and heterozygosity were high (uncorrected mean alleles/locus and HE were 4.8–8.8 and 0.43–0.8, respectively) compared to other amphibian species and significant differences among sampled sites were recorded. Despite recent population declines, no sites displayed a genetic signature indicative of a population bottleneck. Significant genetic structuring (overall FST 0.172) was detected throughout the species range, but was relatively low compared to previous amphibian studies employing microsatellites. In addition we found that some areas sampled within continuous habitat showed evidence of weak genetic structuring (data subset FST 0.034). We conclude that maintaining areas of continuous habitat is critical to the conservation of the species and argue that population recovery and/or persistence in all areas sampled is possible if appropriate protection and management are afforded.

High effective inbreeding coefficients correlate with morphological abnormalities in populations of South Australian koalas (Phascolarctos cinereus)

Koalas have undergone a series of sequential founding events on islands in south-eastern Australia in recent times. Populations in South Australia at the Eyre Peninsula and Mt Lofty Ranges were founded in the 1960s from a colony on Kangaroo Island. The Kangaroo Is. colony was derived from animals introduced to French Island from mainland Victoria over a century ago. In this study, we first use microsatellite markers to quantify levels of genetic variation within the South Australian koala populations and the relatively unperturbed Strzelecki Ranges population from mainland Victoria. This analysis revealed low levels of allelic diversity (1.7 ± 0.2 to 2.7 ± 0.5) and heterozygosity (0.208 ± 0.088 to 0.340 ± 0.110) in the three South Australian koala populations relative to the Strzelecki Ranges population, which has the highest levels of allelic diversity (4.7 ± 1.1) and heterozygosity (0.476 ± 0.122) in Victoria. Second, we measured the incidence of testicular aplasia, a unilateral or bilateral failure in testicular development, in the Eyre Peninsula and Kangaroo Is. populations, and in the ultimate founding population at French Is. Testicular aplasia was present at a frequency of 4.3% in French Is., 12.8% in Kangaroo Is. and 23.9% in the Eyre Peninsula, but was undetectable in the non-bottlenecked Pilliga State Forest population of New South Wales. The incidence of testicular aplasia correlated positively with effective inbreeding coefficients derived from heterozygosity values (0.13 ± 0.06 in the Pilliga State Forest, 0.57 ± 0.17 in French Is., 0.63 ± 0.12 on Kangaroo Is. and 0.77 ± 0.12 in the Eyre Peninsula), which may indicate inbreeding depression. These findings are of concern when evaluating the long-term conservation and viability of the South Australian koala populations, which may benefit from genetic augmentation in the future. Finally, unconfirmed reports suggested that animals from other states in Australia were introduced into the Mt Lofty Ranges population. Therefore, we quantified differentiation between the three South Australian populations and the Strzelecki Ranges and French Is. populations, based on microsatellites and mtDNA d-loop region variation. R -statistics and Goldsteins delta mu square distance revealed that differentiation at nuclear loci between populations paralleled known recent population history, except for the close relationship between Mt Lofty Ranges and French Is. This suggested a recent contribution to the Mt Lofty Ranges populations of animals derived from the French Is. translocation program. Furthermore, mtDNA d-loop analysis found no evidence of contributions to the gene pool from animals of New South Wales or Queensland stock, implying that the population was derived exclusively from Victorian stock.

Low Phylogeographic Structure in a Wide Spread Endangered Australian Frog Litoria aurea (Anura: Hylidae)

The green and golden bell frog (Litoria aurea) has a widespread distribution along the south-east coast of Australia. The species range, however, is highly fragmented and remaining populations are predominately isolated and restricted to the coastline. Previously, the range extended further inland and the species was considered common. Here we report a study designed to identify the phylogeographic and conservation genetic parameters of L. aurea. Mitochondrial DNA sequences were examined from 263 individuals sampled from 26 locations using both phylogenetic and population analyses. Despite a general consensus that amphibians are highly structured we found no phylogeographic divisions within the species, however, there was significant structure amongst extant populations (FST=0.385). Patterns of haplotype relatedness, high haplotypic diversity (mean h=0.547) relative to low nucleotide diversity (mean π=0.003) and mismatch distribution analysis supported a Pleistocene expansion hypothesis with continued restricted dispersal and gene flow. We conclude that the genetic structure of the species may permit ‘well managed’ intervention to mediate gene flow amongst isolated populations and provide some guidelines for the implementation of such conservation strategies.

Genetic diversity and gene flow among southeastern Queensland koalas (Phascolarctos cinereus)

Habitat fragmentation and destruction associated with the rapid urban and rural development of southeast Queensland presents an immediate threat to the survival of koala populations within this region. A sensitive method combining heteroduplex analysis (HDA) with temperature gradient gel electrophoresis (TGGE) was optimized to detect within‐species variation in a mitochondrial DNA (mtDNA) control‐region fragment, ≈ 670 bp in length, from the koala. Eight different haplotypes were characterized in koalas, of which four were novel. Analysis of mtDNA diversity in 96 koalas from five populations in southeast Queensland revealed that the number of haplotypes in a single population ranged from one to five, with an average within‐population haplotype diversity of 0.379 ± 0.016, and nucleotide diversity of 0.22 ± 0.001%. Nucleotide divergence between populations averaged 0.09 ± 0.001% and ranged from 0.00 to 0.14%. Significant genetic heterogeneity was observed among most populations, suggesting that koala populations may be spatially structured along matrilines, although this may not be universal. The limited distribution of the central phylogenetic haplotype suggested the possibility of historical population bottlenecks north of the Gold Coast, while the presence of two highly divergent haplotypes at the Moreton site may indicate the occurrence of one or more undocumented translocation events into this area.

Conservation genetics of the black rhinoceros (Diceros bicornis)

We analysed genetic variation in mitochondrial DNA (mtDNA) control region in the Diceros bicornis minor and D. b. michaelianimals at the Western Plains Zoo, which form part of an international ex-situ breeding program. Six of the nine D. b. minor animals were wild-caught from Zimbabwe during the 1990s, and our study revealed five distinct mtDNA haplotypes, and a haplotype diversity of 0.86 in the colony. Phylogenetic relationships between mtDNA haplotypes analyzed using the neighbor joining method reveal that for the small sample available, D. b. minor and D. b. michaeli are reciprocally monophyletic and represent separate ancestral lineages. Nucleotide divergence between the black and and white rhinoceros(Ceratotherium simum) was 14.0%, and nucleotide divergencebetween the D. b. minor and the D. b. michaeli subspecies was 2.6%. This suggests a divergence time for the two blackrhinoceros subspecies of between 0.93 MY and 1.3 MY.

Variability and differentiation of microsatellites in the genus Dasyurus and conservation implications for the large Australian carnivorous marsupials

All four species of Australian quolls (Dasyurusspecies) have declined since European settlement in terms of bothrange and population numbers. Six highly polymorphic simplesequence repeats (CAn microsatellites) were used to estimate thegenetic variability and population differentiation within andamong twenty populations (including museum specimens from sixpopulations), as a preliminary means of assessing populationconservation status and relative levels of variability withinmembers of the genus. Overall mean expected heterozygosity (HE)and corrected allelic diversity (A′) were highest among westernquolls. Northern quolls, eastern quolls, and tiger quolls werenot significantly different from each other in either measure. There were also significant differences in diversity amongpopulations within species. Genetic differentiation wasestimated by a number of methods and showed that themicrosatellites used here were useful for defining differencesboth among species and populations. Allele frequency data weresummarised by two-dimensional MDS, which was able to partitionpopulations into distinct species clusters. Similarly, theassignment test was able to assign most individuals to both thecorrect species and population levels. Results of MDS and theassignment test may prove useful in forensic applications. Genetic distance and subdivision between pairs of populationswere assessed by two means based on different mutation models formicrosatellites: infinite alleles model (Neis D, FST) andstepwise mutation model (Goldsteins δ mu;2, RST). Pairwisemeasures of population subdivision indicate that most populationsshould be conserved as separate management units. We discussresults of these analyses in terms of applications toconservation for each of the four Australian species of quoll andprovide a genetic basis for future population monitoring in thesespecies.

Genetic Variation in Captive Koalas (Phascolarctos cinereus): Parentage Determination and Individual Identification

Highly repeatable randomly amplified polymorphicDNA (RAPD) markers were developed for parentage studiesin the koala (Phascolarctos cinereus). Of the 25 RAPDprimers screened, 5 (20.0%) produced 32 repeatable polymorphic RAPD bands (average/primer = 6.4± 4.2). A high level of polymorphism was observedfor each group of koalas (Featherdale, 71.9%; Lone Pine,84.4%). All 25 koalas could be uniquely identified using either RAPD or microsatellite markers. Of the32 RAPD markers generated in koalas, 25 were informativefor parentage analyses. These RAPD markers successfullydetermined both parents to three offspring and a male parent to a fourth offspring.Paternity analysis (where the female parent is known)succeeded in assigning the correct male parent to sevenoffspring. Our RAPD–PCR method generatesinformative genetic markers that are useful for parentagedetermination and individual identification of captivekoalas. This would provide genetic analysis to zoos andwildlife parks as a low-cost alternative to the more expensive microsatellite markers.