Kathryn M. Real

Mogurnda adspersa microsatellite markers: multiplexing and multi-tailed primer tagging

A set of twelve microsatellite DNA loci were developed for the threatened Australian freshwater fish Mogurnda adspersa (Eleotridae). Primers were tailed with one of four 20-mer oligonucleotides for use in four-colour fluorescent detection and optimised for multiplex PCR. The loci were used to genotype individuals from two populations in the Pioneer River catchment of central Queensland, eastern Australia. Number of alleles per locus ranged from 2 to 33 and per locus heterozygosity ranged from 0.06 to 0.81. Successful cross-species amplification of all loci was achieved in the congener M. mogurnda. These markers will be used to estimate effective population size and to examine the relationship between flow regime and population demographic parameters.

Extreme Genetic Structure in a Small-Bodied Freshwater Fish, the Purple Spotted Gudgeon, Mogurnda adspersa (Eleotridae)

Freshwater fish are a group that is especially susceptible to biodiversity loss as they often exist naturally in small, fragmented populations that are vulnerable to habitat degradation, pollution and introduction of exotic species. Relatively little is known about spatial dynamics of unperturbed populations of small-bodied freshwater fish species. This study examined population genetic structure of the purple spotted gudgeon (Mogurnda adspersa, Eleotridae), a small-bodied freshwater fish that is widely distributed in eastern Australia. The species is threatened in parts of its range but is common in coastal streams of central Queensland where this study took place. Microsatellite (msat) and mitochondrial DNA (mtDNA) variation was assessed for nine sites from four stream sections in two drainage basins. Very high levels of among population structure were observed (msat F ST = 0.18; mtDNA ΦST = 0.85) and evidence for contemporary migration among populations was rare and limited to sites within the same section of stream. Hierarchical structuring of variation was best explained by stream section rather than by drainage basin. Estimates of contemporary effective population size for each site was low (range 28 – 63, Sibship method), but compared favorably with similar estimates for other freshwater fish species, and there was no genetic evidence for inbreeding or recent population bottlenecks. In conclusion, within a stable part of its range, M adspersa exists as a series of small, demographically stable populations that are highly isolated from one another. Complimentary patterns in microsatellites and mtDNA indicate this structuring is the result of long-term processes that have developed over a remarkably small spatial scale. High population structure and limited dispersal mean that recolonisation of locally extinct populations is only likely to occur from closely situated populations within stream sections. Limited potential for recolonisation should be considered as an important factor in conservation and management of this species.

Extremely Low Microsatellite Diversity but Distinct Population Structure in a Long-Lived Threatened Species, the Australian Lungfish Neoceratodus forsteri (Dipnoi)

The Australian lungfish is a unique living representative of an ancient dipnoan lineage, listed as ‘vulnerable’ to extinction under Australia’s Environment Protection and Biodiversity Conservation Act 1999. Historical accounts indicate this species occurred naturally in two adjacent river systems in Australia, the Burnett and Mary. Current day populations in other rivers are thought to have arisen by translocation from these source populations. Early genetic work detected very little variation and so had limited power to answer questions relevant for management including how genetic variation is partitioned within and among sub-populations. In this study, we use newly developed microsatellite markers to examine samples from the Burnett and Mary Rivers, as well as from two populations thought to be of translocated origin, Brisbane and North Pine. We test whether there is significant genetic structure among and within river drainages; assign putatively translocated populations to potential source populations; and estimate effective population sizes. Eleven polymorphic microsatellite loci genotyped in 218 individuals gave an average within-population heterozygosity of 0.39 which is low relative to other threatened taxa and for freshwater fishes in general. Based on F ST values (average over loci = 0.11) and STRUCTURE analyses, we identify three distinct populations in the natural range, one in the Burnett and two distinct populations in the Mary. These analyses also support the hypothesis that the Mary River is the likely source of translocated populations in the Brisbane and North Pine rivers, which agrees with historical published records of a translocation event giving rise to these populations. We were unable to obtain bounded estimates of effective population size, as we have too few genotype combinations, although point estimates were low, ranging from 29 - 129. We recommend that, in order to preserve any local adaptation in the three distinct populations that they be managed separately.

Allegory of a cave crustacean: systematic and biogeographic reality of Halosbaena (Peracarida: Thermosbaenacea) sought with molecular data at multiple scales

Halosbaena Stock, 1976 are small crustaceans found in a number of distant, isolated subterranean locations in the Northern (Caribbean and Canary Islands) and Southern Hemispheres (Christmas Island and north-western Australia in Cape Range, Barrow Island and Pilbara regions). This distribution is surprising for an animal that produces few eggs, has no free-living larval stage, and succours their young in a dorsal brood pouch. It is usually explained by the passive movement of ancestral populations on tectonic plates as the ancient Tethys Ocean spread. We used molecular data (one mitochondrial and three nuclear genes) to reconstruct phylogenies and time-trees to understand their biogeography at the global scale and at four diminishing scales within the Southern Hemisphere. We found: (1) a basal split between species in the Northern and Southern Hemispheres, but the inferred ages of divergences between species are not old enough to be associated with the spread of the Tethys; (2) a recently discovered species from Christmas Island which is the sister to Australian mainland taxa; (3) the one described species from mainland Australia, H. tulki, probably constitutes at least five separate species that reflect local geography (Cape Range west, Cape Range east, Barrow Island, Pilbara low elevation, Pilbara high elevation); (4) the Pilbara high elevation taxon is likely not old enough to have been stranded high inland during an Eocene marine transgression; and (5) phylogeographic breaks within Cape Range west are congruent with other breaks in sympatric cave species.

Isolation and characterization of 21 polymorphic microsatellite loci in the iconic Australian lungfish, Neoceratodus forsteri, using the Ion Torrent next-generation sequencing platform

We isolated and characterized 21 microsatellite loci in the vulnerable and iconic Australian lungfish, Neoceratodus forsteri. Loci were screened across eight individuals from the Burnett River and 40 individuals from the Pine River. Genetic diversity was low with between one and six alleles per locus within populations and a maximum expected heterozygosity of 0.774. These loci will now be available to assess effective population sizes and genetic structure in N. forsteri across its natural range in South East Queensland, Australia.

Genetic structure and effective population size of the most northern population of the Australian River Blackfish, Gadopsis marmoratus (Richardson 1848): implications for long-term population viability

Upland freshwater habitats support populations that are especially susceptible to anthropogenic change. Furthermore, their isolation from other suitable habitats, and the fragmented, dendritic structure of headwaters make dispersal an unlikely response to change. We investigated genetic structure and variation in the northern-most population of Gadopsis marmoratus, which is isolated in a tiny area in the headwaters of the Condamine River catchment, in the Murray–Darling Basin, Australia. Strong genetic structure was detected among subpopulations based on microsatellites (FST = 0.173, p < 0.0001) and mitochondrial (mt)DNA (FST = 0.369, p < 0.05). Effective population size was low, ranging between 18.8 and 48.2, depending on the estimation method used. Bayesian clustering revealed 3 genetic clusters, but they were not congruent with drainage patterns, suggesting a complex history of dispersal among headwaters that are isolated by waterfalls. Overall, these results suggest that G. marmoratus is unlikely to disperse into new habitats if local conditions become unsuitable. Low effective population size and genetic diversity also suggest that local adaptation would be unlikely.

Microsatellite markers for Australian temperate diadromous fishes Pseudaphritis urvillii (Bovichtidae) and Lovettia sealii (Galaxiidae)

Thirteen microsatellite loci were developed and characterised for two fishes from temperate Australia that exhibit atypical diadromous migration strategies. Cloning and sequencing of an enriched partial genomic library was used to develop seven highly polymorphic loci for the catadromous species Pseudaphritis urvillii (known as tupong or congolli). Mean number of alleles per locus was 16.5, and average observed and expected heterozygosity was between 0.90 and 0.87, respectively. Six polymorphic markers characterised for the anadromous species Lovettia sealii (known as Tasmanian whitebait) included a mean of 12.3 alleles per locus and average observed and expected heterozygosity of 0.71–0.77, respectively. These microsatellites will be employed to understand regional patterns of recruitment, migration and stock structure.

Characterisation of polymorphic microsatellite loci in Hephaestus fuliginosus and cross-amplification in closely related Hephaestus tulliensis

Eleven polymorphic microsatellite loci were isolated and characterised from sooty grunter (Hephaestus fuliginosus) and Tully grunter (H. tulliensis) from Northern Australia. Each primer pair amplified good quality and polymorphic products. The versatilities of these markers were tested using different H. fuliginosus populations and cross-amplified to a closely related species, H. tulliensis, found within the northern Australian wet tropics. The number of alleles ranged from 5 to 20 per locus and expected heterozygosities ranged from 0.043 to 0.926. All loci conformed to Hardy–Weinberg expectations, with the exception of 2Hf44 and 2Hf53 for Daly R. and Mitchell R. populations of H. fuliginosus, respectively. No evidence of linkage disequilibrium was detected between any pair of loci. The markers reported here would be very useful for population genetic studies, evolution and conservation of these species.

Tracking and tracing central Queensland’s Macroderma – determining the size of the Mount Etna ghost bat population and potential threats

The ghost bat, Macroderma gigas, colony at Mount Etna was at the centre of Australia’s longest-running conservation campaign. To protect the colony the Queensland Government removed recreational facilities and gated caves. The size and genetic diversity of the Mount Etna M. gigas population were estimated using cave searches, direct captures and molecular analysis to determine whether these actions have benefitted the species. In addition, telemetry was undertaken and red fox, Vulpes vulpes, scats analysed to identify possible threats. Results suggest that the population has declined by 79% since the late 1990s, has low microsatellite diversity, low effective population size (Ne) and is undergoing a population bottleneck. VHF- and GPS-collared animals were found to forage over agricultural land up to 11.8 km from their daytime roost, suggesting that poor land management and barbed-wire fences could be potential threats. No ghost bat remains were found in fox scats. We recommend that compliance be increased around Johansen’s Cave to reduce disturbance during the maternity season and landholders be encouraged to undertake management that is sympathetic to ghost bats.