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, approximately 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.     

Genetic variation and structuring in the threatened koala populations of Southeast Queensland

Habitat fragmentation can act to cause reproductive isolation between conspecifics and undermine species’ persistence, though most studies have reported the genetic condition of populations that have already declined to a very small size. We examined genetic diversity within the vulnerable, declining koala (Phascolarctos cinereus) population in Southeast Queensland, Australia to determine the genetic impact of ongoing threatening processes. Five hundred and twelve koalas from ten Southeast Queensland Local Government Areas on the mainland and one island were genotyped at six polymorphic microsatellite loci. Based on Bayesian cluster analysis incorporating spatial data, the regional koala population was subdivided into six clusters, with location of major roads and rivers appearing to be consistent with being barriers to gene flow. The distribution of mtDNA control region haplotypes identified distinct coastal and inland clades suggesting that historically there was gene flow between koalas along the coast (though little interchange between coastal and inland animals). In contrast, koalas from the Koala Coast (Brisbane City, Logan City and Redland Shire) were shown by microsatellite analysis to be genetically distinct from adjacent areas. It is likely, therefore, that more recent reductions in population size and restricted gene flow through urbanisation have contributed to the genetic differentiation of koalas in the Koala Coast region.     

Anthropogenic changes to the landscape resulted in colonization of koalas in north‐east New South Wales,Australia

Significant changes in the distribution and persistence of species have been driven by Pleistocene cyclical climate changes and, more recently, by human modification of the environment. In eastern Australia, Pleistocene cyclical patterns in temperature and aridity led to the expansion and retraction of rainforest and likely affected the distribution of the koala (Phascolarctos cinereus, family Phascolarctidae), a species preferring dry or open woodland. More recently, anthropogenic landscape modification has led to a large‐scale change in distribution of the koala following the destruction of approx. 75 000 ha of subtropical rainforest in north‐east New South Wales termed the ‘Big Scrub’. Sharing of the control region haplotypes to the north and south of this region indicate historical connectivity of coastal koala populations. However, the majority (110/115) of sampled koalas from this region shared a single mitochondrial control region haplotype, suggesting that koalas did not persist in multiple refugial pockets within a heterogenous rainforest but expanded into the region following deforestation. Bayesian cluster analysis of microsatellite data consistently identified two clusters of koalas. One cluster, in the north of the area, had high microsatellite diversity (10 alleles per locus, He = 0.79) and clustered with koalas further to the north, thus suggesting a southerly expansion into the cleared area. To the south was a cluster with significantly lower diversity (six alleles per locus, He = 0.59, P < 0.001). It is possible there has been restricted or filtered movement of koalas between these clusters, which coincides with a cleared river valley and associated roads or immigration from populations both to the north and to the south. This study gives an insight into the timescale of changes in species distribution following rapid alterations to suitable habitat.     

Genetic structure and diversity of the koala population in South Gippsland,Victoria: a remnant population of high conservation significance

In the Australian state of Victoria, the history of koalas and their management has resulted in the homogenisation and reduction of genetic diversity in many contemporary populations. Decreased genetic diversity may reduce a species’ ability to adapt to future environmental pressures such as climate change or disease. The South Gippsland koala population is considered to be unique in Victoria, as it is believed to be a remnant population, not originating from managed populations that have low genetic variation. This study investigated genetic structure and diversity of koalas in South Gippsland, with comparison to other populations in Victoria (French Island/Cape Otway, FI and Raymond Island, RI), New South Wales and south east Queensland. Population analyses were undertaken using both microsatellite genotype and mitochondrial DNA sequence data. Non-invasive sampling of koala scats was used to source koala DNA, allowing 222 South Gippsland koalas to be genotyped. Using nuclear data the South Gippsland koala population was found to be significantly differentiated (D_jost 95% CI SG–RI?=?0.03–0.06 and SG–FI?=?0.08–012) and more diverse (A_R 95% CI SG?=?4.7–5.6, RI?=?3.1–3.3, FI?=?3.0–3.3; p?=?0.001) than other Victorian koala populations, supporting the premise that koalas in the South Gippsland region are part of a remnant population, not derived from translocated island stock. These results were also supported by mitochondrial data where eight haplotypes (Pc4, Pc17, Pc26, Pc27, and Pc56–Pc59) were identified in South Gippsland while a single haplotype (Pc27) was found in all island koalas tested. Compared to other Victorian koala populations, greater genetic diversity found in South Gippsland koalas, may provide this population with a greater chance of survival in the face of future environmental pressures. The South Gippsland koala population is, therefore, of high conservation significance, warranting the implementation of strategies to conserve this population and its diversity into the future.     

Quantifying the impacts of bushfire on populations of wild koalas (Phascolarctos cinereus): Insights from the 2019/20 fire season

The impact of bushfire events on wild Koala (Phascolarctos cinereus) populations is poorly understood. Following the 2019/2020 bushfire season in eastern Australia, we resurveyed 123 field sites for which contemporaneous (current koala generation) pre‐fire survey data were available. Field sites were distributed across six fire grounds between Foster and Ballina on the north coast of New South Wales. At these sites, pre‐fire naïve occupancy levels by koalas ranged from 25% to 71% of the sampled habitat, while post‐fire naïve occupancy levels ranged from 0% to 47%. The median reduction in the naïve occupancy rate by koalas when considered across all six fire grounds was 71% when standardized against pre‐fire occupancy levels. Field data provided strong corroboration between site‐based, post‐fire foliage canopy cover estimates and modelled Google Earth Engine Burnt Area Map (GEEBAM) fire‐severity categories. In terms of GEEBAM fire‐severity categories, koala survival was five times more likely in areas where forest canopies were modelled as Unburnt or Partially burnt, compared to areas where forest canopies were Fully burnt. The capacity of bushfire‐affected koala populations to recover from the 2019/20 fire season will be conditional upon size of the original population in each fire‐affected area, the enactment and implementation of supportive, recovery‐themed management regimes, future inter‐fire intervals and associated intensities. Management actions necessary to assist recovery actions are discussed.     

Genome-wide SNP loci reveal novel insights into koala (<Emphasis Type="Italic">Phascolarctos cinereus</Emphasis>) population variability across its range

The koala (Phascolarctos cinereus) is an iconic Australian species that is currently undergoing a number of threatening processes, including disease and habitat loss. A thorough understanding of population genetic structuring and genomic variability of this species is essential to effectively manage populations across the species range. Using a reduced representation genome sequencing method known as double digest restriction-associated sequencing, this study has provided the first genome-wide SNP marker panel in the koala. In this study, 33,019 loci were identified in the koala and a filtered panel of 3060 high-utility SNP markers, including 95 sex-linked markers, were used to provide key insights into population variability and genomic variation in 171 koalas from eight populations across their geographic range. Broad-scale genetic differentiation between geographically separated populations (including sub-species) was assessed and revealed significant differentiation between all populations (F_ST range = 0.01–0.28), with the largest divergence observed between the three geographically distant subgroups (QLD, NSW and VIC) along the east coast of Australia (average F_ST range = 0.17–0.23). Sub-group divergence appears to be a reflection of an isolation by distance effect and sampling strategy rather than true evidence of sub-speciation. This is further supported by low proportions of AMOVA variation between sub-species groups (11.19 %). Fine-scale analysis using genome-wide SNP loci and the NETVIEW pipeline revealed cryptic genetic sub-structuring within localised geographic regions, which corresponded to the hierarchical mating system of the species. High levels of genome-wide SNP heterozygosity were observed amongst all populations (He = 0.25–0.35), and when evaluating across the species to other vertebrate taxa were amongst the highest values observed. This illustrates that the species as a whole still retains high levels of diversity which is comparable to other outbred vertebrate taxa for genome-wide SNPs. Insights into the potential for adaptive variation in the koala were also gained using outlier analysis of genome-wide SNPs. A total of 10 putative outlier SNPs were identified indicating the high likelihood of local adaptations within populations and regions. This is the first use of genome-wide markers to assess population differentiation at a broad-scale in the koala and the first time that sex-linked SNPs have been identified in this species. The application of this novel genomic resource to populations across the species range will provide in-depth information allowing informed conservation priorities and management plans for in situ koalas across Australia and ex situ around the world.     

Low genetic diversity and significant structuring of the common hamster populations <Emphasis Type="Italic">Cricetus cricetus</Emphasis> in Poland revealed by the mtDNA control region sequence variation

The genetic diversity of 12 populations in the present range of the common hamster Cricetus cricetus (Linnaeus, 1758) in Poland was established. The 366 bp of the mtDNA control region was sequenced for 195 individuals. As few as seven haplotypes were found and their distribution was geographically structured. The large geographic areas were fixed or almost fixed for a single haplotype and three groups of populations, that do not share any haplotypes, have been defined. Proportions of genetic diversity attributable to variation between groups of populations, between populations within groups and within populations were 93.64, 1.92 and 4.45% (SAMOVA: p < 0.001 for all estimates), respectively. Such pattern of variation is most probably the result of historical, postglacial bottlenecks and present genetic drift after the population decline in the last few decades.     

Conservation genetics of the koala (Phascolarctos cinereus) II. Limited variability in minisatellite DNA sequences

We have examined variability inTaqI andEcoRI restriction fragment sizes of DNA from the koala (Phascolarctos cinereus) using six HVR (hypervariable region) probes which reveal complex, individual-specific restriction patterns in humans and other species. Frequency of band-sharing among unrelated koalas was extremely high. This result is likely to be a consequence of the history of near-extinction and artificial recolonization of the populations we have studied, rather than a general marsupial or koala-wide phenomenon.This work was funded by the Australian Research Council.     

Estimating koala density from incidental koala sightings in South‐East Queensland,Australia (1997–2013), using a self‐exciting spatio‐temporal point process model

The koala, Phascolarctos cinereus, is an iconic Australian wildlife species facing a rapid decline in South‐East Queensland (SEQLD). For conservation planning, the ability to estimate the size of koala populations is crucial. Systematic surveys are the most common approach to estimate koala populations but because of their cost they are often restricted to small geographic areas and are conducted infrequently. Public interest and participation in the collection of koala sighting data is increasing in popularity, but such data are generally not used for population estimation. We modeled monthly sightings of koalas reported by members of the public from 1997 to 2013 in SEQLD by developing a self‐exciting spatio‐temporal point process model. This allowed us to account for characteristics that are associated with koala presence (which vary over both space and time) while accounting for detection bias in the koala sighting process and addressing spatial clustering of observations. The density of koalas varied spatially due to the heterogeneous nature of koala habitat in SEQLD, with a mean density of 0.0019 koalas per km² over the study period. The percentage of land areas with very low densities (0–0.0005 koalas per km²) remained similar throughout the study period representing, on average, 66% of the total study area. The approach described in this paper provides a useful starting point to allow greater use to be made of incidental koala sighting data. We propose that the model presented here could be used to combine systematic koala survey data (which is spatially restricted, but more precise) with koala sighting data (which is incidental and often biased by nature, but often collected over large geographical areas). Our approach could also be adopted for modeling the density of other wildlife species where data is collected in the same manner.     

Influences on koala habitat selection across four local government areas on the far north coast of NSW

Conserving habitats crucial for threatened koala (Phascolarctos cinereus) populations requires rating habitat quality from a fine spatial scale to patches, landscapes and then regions. The koala has a specialized diet focused on the leaves of a suite of Eucalyptus species. We asked: what are the key regional influences on habitat selection by koalas in the far north coast of New South Wales? We addressed this question by investigating the multi-scale factors, and within-scale and cross-scale interactions, that influence koala habitat selection and distribution across four local government areas on the far north coast of New South Wales. We assembled and analysed a large data set of tree selection, identified by the presence of scats, in a wide range of randomly selected 5 × 5 km grids across the region. This resulted in more than 9000 trees surveyed for evidence of koala use from 302 field sites, together with associated biophysical site features. The dominant factor influencing habitat use and koala occurrence was the distribution of five Eucalyptus species. Koalas were more likely to use medium-sized trees of these species where they occurred on soils with high levels of Colwell phosphorous. We also identified new interactions among the distribution of preferred tree species and soil phosphorous, and their distribution with the amount of suitable habitat in the surrounding landscape. Our study confirmed that non-preferred species of eucalypts and non-eucalypts are extensively used by koalas and form important components of koala habitat. This finding lends support to restoring a mosaic of koala-preferred tree species and other species recognized for their value as shelter. Our study has provided the ecological foundation for developing a novel regional-scale approach to the conservation of koalas, with adaptability to other wildlife species.     

MHC class II diversity of koala (Phascolarctos cinereus) populations across their range

Major histocompatibility complex class II (MHCII) genes code for proteins that bind and present antigenic peptides and trigger the adaptive immune response. We present a broad geographical study of MHCII DA β1 (DAB) and DB β1 (DBB) variants of the koala (Phascolarctos cinereus; n=191) from 12 populations across eastern Australia, with a total of 13 DAB and 7 DBB variants found. We identified greater MHCII variation and, possibly, additional gene copies in koala populations in the north (Queensland and New South Wales) relative to the south (Victoria), confirmed by STRUCTURE analyses and genetic differentiation using analysis of molecular variance. The higher MHCII diversity in the north relative to south could potentially be attributed to (i) significant founder effect in Victorian populations linked to historical translocation of bottlenecked koala populations and (ii) increased pathogen-driven balancing selection and/or local genetic drift in the north. Low MHCII genetic diversity in koalas from the south could reduce their potential response to disease, although the three DAB variants found in the south had substantial sequence divergence between variants. This study assessing MHCII diversity in the koala with historical translocations in some populations contributes to understanding the effects of population translocations on functional genetic diversity.     

Randomly Amplified Polymorphic DNA Variation in Populations of Eastern Australian Koalas, Phascolarctos cinereus

Randomly amplified polymorphic DNA (RAPD) variation in populations of the koala, Phascolarctos cinereus, was investigated, revealing significant differences in the level of diversity between southern and northern regions of eastern Australia. Of the 20 polymorphic RAPD markers identified in koalas, 4-7 were polymorphic in southern populations, while 12-17 were polymorphic in northern populations. Analysis of molecular variance revealed a significant difference in the estimated variance between koalas from northern and those from southern regions (P < 0.001), where populations from the north were greater than twice as variable as their southern cousins. The total genetic diversity observed was attributed to regional differences (30.91%), population differences within a region (11.77%), and differences among individuals within a population (57.32%). For the within-region analyses, a large proportion of the genetic diversity was attributable to individual differences within a population, 80.34% for the north and 91.23% for the south. These results demonstrate that RAPD markers are useful for determining population structure among koalas.     

Mitochondrial DNA diversity of <Emphasis Type="Italic">Cleruchoides noackae</Emphasis> (Hymenoptera: Mymaridae): a potential biological control agent for <Emphasis Type="Italic">Thaumastocoris peregrinus</Emphasis> (Hemiptera: Thaumastocoridae)

Thaumastocoris peregrinus Carpintero and Dellapé (Hemiptera: Thaumastocoridae) is a native Australian Eucalyptus sap-feeding insect that has become invasive and seriously damaging to commercially grown Eucalyptus in the Southern Hemisphere. Cleruchoides noackae Lin and Huber (Hymenoptera: Mymaridae) was recently discovered as an egg parasitoid of the Thaumastocoridae in Australia. Mitochondrial DNA (mtDNA; cytochrome c oxidase subunit I, COI) sequence diversity amongst 104 individuals from these native C. noackae populations revealed 24 sequence haplotypes. The COI haplotypes of individuals collected from the Sydney and Southeast Queensland clustered in distinct groups, indicating limited spread of the insect between the regions. Individuals collected from Perth in Western Australia were represented by four COI haplotypes. Although this population is geographically more isolated from other populations, two COI haplotypes were identical to haplotypes found in the Sydney region. The results suggest that C. noackae has recently been introduced into Perth, possibly from the Sydney area. The high mtDNA diversity and limited spread that is suggested for C. noackae is in contrast to the lack of geographic associated mtDNA diversity and extensive spread of T. peregrinus. If implemented as a biological control agent, this factor will need to be considered in collecting and releasing C. noackae.     

Genetic diversity and population structure of silver pomfret (Pampus argenteus) in the Indo-West Pacific revealed by mitochondrial control region sequences

Sequence variation of the mitochondrial control region in silver pomfret (Pampus argenteus) among six populations from the Indo-West Pacific region was analyzed. A total of 309 polymorphic sites were detected in the 464–466 bp segment of the control region among 165 individuals which defined 72 haplotypes. Haplotype diversity was ranged from 0.800 to 0.943 and nucleotide diversity ranged from 0.333% to 1.886%. Genetic distance and the fixation index indicated significant differences among populations in different geographical groups (P < 0.05). Median-joining network analysis data showed distinct patterns of phylogeographic structure. The present results suggested that P. argenteus populations have developed significant genetic structure, especially among geographically isolated groups.     

Climate‐driven shifts in the distribution of koala‐browse species from the Last Interglacial to the near future

The koala's Phascolarctos cinereus distribution is currently restricted to eastern and south‐eastern Australia. However, fossil records dating from 70 ± 4 ka (ka = 10³ yr) from south‐western Australia and the Nullarbor Plain are evidence of subpopulation extinctions in the southwest at least after the Last Interglacial (~128–116 ka). We hypothesize that koala sub‐population extinctions resulted from the eastward retraction of the koala's main browse species in response to unsuitable climatic conditions. We further posit a general reduction in the distribution of main koala‐browse trees in the near future in response climate change. We modelled 60 koala‐browse species and constructed a set of correlative species distribution models for five time periods: Last Interglacial (~128–116 ka), Last Glacial Maximum (~23–19 ka), Mid‐Holocene (~7–5 ka), present (interpolations of observed data, representative of 1960–1990), and 2070. We based our projections on five hindcasts and one forecast of climatic variables extracted from WorldClim based on two general circulation models (considering the most pessimistic scenario of high greenhouse‐gas emissions) and topsoil clay fraction. We used 17 dates of koala fossil specimens identified as reliable from 70 (± 4) to 535 (± 49) ka, with the last appearance of koalas at 70 ka in the southwest. The main simulated koala‐browse species were at their greatest modelled extent of suitability during the Last Glacial Maximum, with the greatest loss of koala habitat occurring between the Mid‐Holocene and the present. We predict a similar habitat loss between the present and 2070. The spatial patterns of habitat change support our hypothesis that koala extinctions in the southwest, Nullarbor Plain and central South Australia resulted from the eastward retraction of the dominant koala‐browse species in response to long‐term climate changes. Future climate patterns will likely increase the extinction risk of koalas in their remaining eastern ranges.     

Population genetics of shortnose sturgeon Acipenser brevirostrum based on mitochondrial DNA control region sequences

Shortnose sturgeon is an anadromous North American acipenserid that since 1973 has been designated as federally endangered in US waters. Historically, shortnose sturgeon occurred in as many as 19 rivers from the St. John River, NB, to the St. Johns River, FL, and these populations ranged in census size from 10(1) to 10(4), but little is known of their population structure or levels of gene flow. We used the polymerase chain reaction (PCR) and direct sequence analysis of a 440 bp portion of the mitochondrial DNA (mtDNA) control region to address these issues and to compare haplotype diversity with population size. Twenty-nine mtDNA nucleotide-substitution haplotypes were revealed among 275 specimens from 11 rivers and estuaries. Additionally, mtDNA length variation (6 haplotypes) and heteroplasmy (2-5 haplotypes for some individuals) were found. Significant genetic differentiation (P < 0.05) of mtDNA nucleotide-substitution haplotypes and length-variant haplotypes was observed among populations from all rivers and estuaries surveyed with the exception of the Delaware River and Chesapeake Bay collections. Significant haplotype differentiation was even observed between samples from two rivers (Kennebec and Androscoggin) within the Kennebec River drainage. The absence of haplotype frequency differences between samples from the Delaware River and Chesapeake Bay reflects a probable current absence of spawning within the Chesapeake Bay system and immigration of fish from the adjoining Delaware River. Haplotypic diversity indices ranged between 0.817 and 0.641; no relationship (P > 0.05) was found between haplotype diversity and census size. Gene flow estimates among populations were often low (< 2.0), but were generally higher at the latitudinal extremes of their distribution. A moderate level of haplotype diversity and a high percentage (37.9%) of haplotypes unique to the northern, once-glaciated region suggests that northern populations survived the Pleistocene in a northern refugium. Analysis of molecular variance best supported a five-region hierarchical grouping of populations, but our results indicate that in almost all cases populations of shortnose sturgeon should be managed as separate units.     

Intraspecific mitochondrial DNA variation in the Colorado potato beetle,Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)

Restriction endonuclease analyses of mitochondrial DNA (mtDNA) were used to examine genetic variability and population structure inLeptinotarsa decemlineata (Say). A group of three enzymes, EcoRI, HpaI, and PstI, was used to reveal polymorphism both within and among some of the 10 populations tested, yielding 16 haplotypes in combination. The frequencies of these 16 haplotypes differed significantly across geographic regions, indicating some partitioning of mtDNA haplotypes. Estimates of mtDNA sequence divergence (δ) between haplotypes ranged from 0.016 to 0.135%, suggesting local differentiation of mtDNA in some populations. Analysis of these data suggests that Texas was colonized by more than one mtDNA lineage, most likely originating in Mexico. We hypothesize that a larger founder size for the initial introductions or high levels of variability in the parent population at the edge of the CPB expanding range led to the initial partitioning of haplotypes observed in samples from Texas.     

Mitochondrial DNA variation in Indo-Pacific populations of the giant tiger prawn,Penaeus monodon

Surveys of mitochondrial DNA (mtDNA) variation in the giant tiger prawn, Penaeus monodon, using restriction fragment length polymorphisms have provided the first clear evidence that the Indo-West Pacific region is a site of accumulation of genetic diversity rather than a site of origin of genetic diversity. No haplotyes were found in common between a group of five southeast African populations and a group of five Australian (including Western Australia) and three southeast Asian populations. The dominant haplotype was different in the Australian and southeast Asian population groups. Genetic diversity (pi) was greatest in Indonesia (pi averaged 0.05), less in the Philippines and Australia (pi averaged 0.01), and markedly less in the southeast African and the West Australian populations (pi averaged 0.003). The high diversity of the southeast Asian populations resulted from the occurrence in those populations of a set of haplotypes found only in southeast Asia but derived from the southeast African haplotypes. These genetic variants therefore evolved in the Indian Ocean and later migrated into the Indo-West Pacific region. Low genetic variation in the geographically marginal populations in southeast Africa and Western Australia is considered to be the result of bottlenecks, but mismatch distributions suggest that large population sizes have been maintained in Indonesian populations for long periods.