Marion Hoehn

The tales of two geckos: does dispersal prevent extinction in recently fragmented populations?

Although habitat loss and fragmentation threaten species throughout the world and are a major threat to biodiversity, it is apparent that some species are at greater risk of extinction in fragmented landscapes than others. Identification of these species and the characteristics that make them sensitive to habitat fragmentation has important implications for conservation management. Here, we present a comparative study of the population genetic structure of two arboreal gecko species (Oedura reticulata and Gehyra variegata) in fragmented and continuous woodlands. The species differ in their level of persistence in remnant vegetation patches (the former exhibiting a higher extinction rate than the latter). Previous demographic and modelling studies of these two species have suggested that their difference in persistence levels may be due, in part, to differences in dispersal abilities with G. variegata expected to have higher dispersal rates than O. reticulata. We tested this hypothesis and genotyped a total of 345 O. reticulata from 12 sites and 353 G. variegata from 13 sites at nine microsatellite loci. We showed that O. reticulata exhibits elevated levels of structure (FST = 0.102 vs. 0.044), lower levels of genetic diversity (HE = 0.79 vs. 0.88), and fewer misassignments (20% vs. 30%) than similarly fragmented populations of G. variegata, while all these parameters were fairly similar for the two species in the continuous forest populations (FST = 0.003 vs. 0.004, HE = 0.89 vs. 0.89, misassignments: 58% vs. 53%, respectively). For both species, genetic structure was higher and genetic diversity was lower among fragmented populations than among those in the nature reserves. In addition, assignment tests and spatial autocorrelation revealed that small distances of about 500 m through fragmented landscapes are a barrier to O. reticulata but not for G. variegata. These data support our hypothesis that G. variegata disperse more readily and more frequently than O. reticulata and that dispersal and habitat specialization are critical factors in the persistence of species in habitat remnants.

An optimisation approach to increase DNA amplification success of otter faeces

Faeces have proved to be a suitable non-invasive DNA source for microsatellite analysis in wildlife research. For the success of such studies it is essential to obtain the highest possible PCR amplification success rate. These rates are still relatively low in most carnivorous species, especially in the otter (Lutra lutra). We therefore optimised the entire microsatellite genotyping process by combining our findings with results from previous studies to gain a high rate of reliable genotypes. We investigated the influence of otter faecal quality in relation to the quantity of slimy secretions and three levels of storage periods at −20°C on amplification success. Further, we tested the cost-effective and time-saving Chelex extraction method against the profitable QIAamp® DNA Stool Kit (Qiagen), and compared three PCR methods - a standard single-step PCR protocol, a single-locus two-step PCR procedure and a multiplex two-step PCR procedure - regarding success rate and genotyping errors. The highest amplification success rate (median: 94%; mean: 78%) was achieved using faecal samples consisting only of jelly extracted with the QIAamp® DNA Stool Mini Kit (Qiagen) immediately after collection and amplified following the time and cost efficient multiplex two-step PCR protocol. The two-step procedure, also referred to as pre-amplification approach, turned out to be the main improvement as it increases amplification success about 11% and reduces genotyping errors about 53%, most notably allelic dropouts.

Can Genetic Estimators Provide Robust Estimates of the Effective Number of Breeders in Small Populations

The effective population size (Ne) is proportional to the loss of genetic diversity and the rate of inbreeding, and its accurate estimation is crucial for the monitoring of small populations. Here, we integrate temporal studies of the gecko Oedura reticulata, to compare genetic and demographic estimators of Ne. Because geckos have overlapping generations, our goal was to demographically estimate NbI, the inbreeding effective number of breeders and to calculate the NbI/Na ratio (Na = number of adults) for four populations. Demographically estimated NbI ranged from 1 to 65 individuals. The mean reduction in the effective number of breeders relative to census size (NbI/Na) was 0.1 to 1.1. We identified the variance in reproductive success as the most important variable contributing to reduction of this ratio. We used four methods to estimate the genetic based inbreeding effective number of breeders NbI(gen) and the variance effective populations size NeV(gen) estimates from the genotype data. Two of these methods - a temporal moment-based (MBT) and a likelihood-based approach (TM3) require at least two samples in time, while the other two were single-sample estimators - the linkage disequilibrium method with bias correction LDNe and the program ONeSAMP. The genetic based estimates were fairly similar across methods and also similar to the demographic estimates excluding those estimates, in which upper confidence interval boundaries were uninformative. For example, LDNe and ONeSAMP estimates ranged from 14–55 and 24–48 individuals, respectively. However, temporal methods suffered from a large variation in confidence intervals and concerns about the prior information. We conclude that the single-sample estimators are an acceptable short-cut to estimate NbI for species such as geckos and will be of great importance for the monitoring of species in fragmented landscapes.

Microsatellite DNA markers for Australian geckos

Nine highly polymorphic microsatellite markers were developed for Gehyra variegata from an enriched library screened for tetranucleotide repeats. An average of 19.44 alleles per locus and an average observed heterozygosity of 0.86 were found in 81 samples from fragmented populations in the Western Australian wheatbelt. Different gecko species exhibit varying rates of extinction in fragmented landscapes and the genetic markers were developed to test whether these difference are due to different rates of dispersal. The loci were tested for their ability to cross amplify in 12 species of Australian Gekkonidae and Pygopodidae, in an attempt to identify species which might be amenable to population genetic analysis using these microsatellite markers.

In situ hybridisation assay for localisation of rabbit calicivirus Australia-1 (RCV-A1) in European rabbit (Oryctolagus cuniculus) tissues.

Recently, a new lagovirus enzootic in Australian wild rabbits was identified and described as rabbit calicivirus Australia-1 (RCV-A1). Unlike the closely related Rabbit Haemorrhagic Disease Virus (RHDV), which causes fulminant hepatitis and rabbit death, RCV-A1 does not appear to induce any clinical disease. RCV-A1 has been postulated to act as an imperfect natural vaccine to RHDV thus reducing RHDV-induced rabbit mortality, which is detrimental for bio-control of rabbits in Australia. This study was carried out to determine in which cells RCV-A1 replication occurs. An in situ hybridisation (ISH) protocol was developed using a RCV-A1 specific probe to localise the virus in rabbit tissues. The results were compared to those obtained with a quantitative RT-PCR assay that had previously been developed to measure RCV-A1 RNA in rabbit tissues. The histology of the tissues was also examined. ISH showed that virus replication, inferred by the presence of detectable RNA, was limited to a small number of epithelial cells towards the tip of the villi in the duodenum. Quantitative RT-PCR detected RCV-A1 RNA in jejunum, ileum and lymphoid tissue at day 3, 4 and 7 post-infection, but no hybridisation was detected in these tissues.

Isolation and characterization of 10 tetranucleotide microsatellites in the Grassland Earless Dragon (Tympanocryptis pinguicolla)

The endangered Grassland Earless Dragon (Tympanocryptis pinguicolla) exists only in fragmented populations separated by agricultural and urban landscapes. To provide the tools for estimating the genetic structure and dispersal characteristics critical for the proper management of this species, we isolated and tested 10 tetranucleotide polymorphic microsatellite markers. The loci were highly polymorphic when tested on 32 individuals from one population showing seven to 18 alleles. The values of both the observed and the expected heterozygosities ranged from 0.350 to 0.938 and from 0.710 to 0.880, respectively. We detected no linkage disequilibrium among these loci following Bonferroni correction, however, one locus (ED2G3) consistently violated Hardy–Weinberg expectations when tested on natural populations and is expected to have null alleles. A second locus (ED2H5) was found to violate Hardy–Weinberg expectations in one population but not in others and will require evaluation more broadly. In summary, we have developed eight or nine markers to study the fine-scale population genetics of Tympanocryptis pinguicolla in the ACT.

A model‐derived short‐term estimation method of effective size for small populations with overlapping generations

Summary If not actively managed, small and isolated populations lose their genetic variability and the inbreeding rate increases. Combined, these factors limit the ability of populations to adapt to environmental changes, increasing their risk of extinction. The effective population size (Ne) is proportional to the loss of genetic diversity and therefore of considerable conservation relevance. However, estimators of Ne that account for demographic parameters in species with overlapping generations require sampling of populations across generations, which is often not feasible in long-lived species. We created an individual-based model that allows calculation of Ne based on demographic parameters that can be obtained in a time period much shorter than a generation. It can be adapted to every life-history parameter combination. The model is freely available as an r-package NEff. The model was first used in a simulation experiment observing changes in Ne in response to different degrees of generational overlap. Results showed that increased generational overlap slowed annual rates of heterozygosity loss, resulting in higher annual effective sizes (Ny) but decreased Ne per generation. Adding the effect of different recruitment rates only affected Ne for populations with low generational overlap. The model was further tested using real population data of the Australian arboreal gecko Gehyra variegata. Simulation results were compared to genetic analyses and matched estimates of the real population very well. Unlike other estimation methods of Ne, NEff neither requires long time series of population monitoring nor genetic analyses of changes in gene frequencies. Thus, it seems to be the first method for calculating Ne within short time periods and comparably low costs facilitating the use of Ne in applied conservation and management.

Robust innate immunity of young rabbits mediates resistance to rabbit haemorrhagic disease caused by Lagovirus europaeus GI.1 but not GI.2

The rabbit caliciviruses Lagovirus europaeus GI.1 and GI.2 both cause acute necrotizing hepatitis in European rabbits (Oryctolagus cuniculus). Whilst GI.2 is highly virulent in both young and adult rabbits, rabbits younger than eight weeks of age are highly resistant to disease caused by GI.1, although they are still permissive to infection and viral replication. To investigate the underlying mechanism(s) of this age related resistance to GI.1, we compared liver transcriptomes of young rabbits infected with GI.1 to those of adult rabbits infected with GI.1 and young rabbits infected with GI.2. Our data suggest that kittens have constitutively heightened innate immune responses compared to adult rabbits, particularly associated with increased expression of major histocompatibility class II molecules and activity of natural killer cells, macrophages, and cholangiocytes. This enables them to respond more rapidly to GI.1 infection than adult rabbits and thus limit virus-induced pathology. In contrast, these responses were not fully developed during GI.2 infection. We speculate that the observed downregulation of multiple genes associated with innate immunity in kittens during GI.2 infection may be due to virally-mediated immunomodulation, permitting fatal disease to develop. Our study provides insight into the fundamental host–pathogen interactions responsible for the differences in age-related susceptibility, which likely plays a critical role in defining the success of GI.2 in outcompeting GI.1 in the field.