Reversing the decline of threatened koala (Phascolarctos cinereus) populations in New South Wales: Using genomics to enhance conservation outcomes.

Genetic management is a critical component of threatened species conservation. Understanding spatial patterns of genetic diversity is essential for evaluating the resilience of fragmented populations to accelerating anthropogenic threats. Nowhere is this more relevant than on the Australian continent, which is experiencing an ongoing loss of biodiversity that exceeds any other developed nation. Using a proprietary genome complexity reduction-based method (DArTSeq), we generated a data set of 3239 high quality Single Nucleotide Polymorphisms (SNPs) to investigate spatial patterns and indices of genetic diversity in the koala (Phascolarctos cinereus), a highly specialised folivorous marsupial that is experiencing rapid and widespread population declines across much of its former range. Our findings demonstrate that current management divisions across the state of New South Wales (NSW) do not fully represent the distribution of genetic diversity among extant koala populations, and that care must be taken to ensure that translocation paradigms based on these frameworks do not inadvertently restrict gene flow between populations and regions that were historically interconnected. We also recommend that koala populations should be prioritised for conservation action based on the scale and severity of the threatening processes that they are currently faced with, rather than placing too much emphasis on their perceived value (e.g., as reservoirs of potentially adaptive alleles), as our data indicate that existing genetic variation in koalas is primarily partitioned among individual animals. As such, the extirpation of koalas from any part of their range represents a potentially critical reduction of genetic diversity for this iconic Australian species.

Correction to "Insights into the Effects of Violating Statistical Assumptions for Dimensionality Reduction for Chemical "-omics" Data with Multiple Explanatory Variables".

[This corrects the article DOI: 10.1021/acsomega.3c01613.].

Insights into the Effects of Violating Statistical Assumptions for Dimensionality Reduction for Chemical "-omics" Data with Multiple Explanatory Variables.

Biological volatilome analysis is inherently complex due to the considerable number of compounds (i.e., dimensions) and differences in peak areas by orders of magnitude, between and within compounds found within datasets. Traditional volatilome analysis relies on dimensionality reduction techniques which aid in the selection of compounds that are considered relevant to respective research questions prior to further analysis. Currently, compounds of interest are identified using either supervised or unsupervised statistical methods which assume the data residuals are normally distributed and exhibit linearity. However, biological data often violate the statistical assumptions of these models related to normality and the presence of multiple explanatory variables which are innate to biological samples. In an attempt to address deviations from normality, volatilome data can be log transformed. However, whether the effects of each assessed variable are additive or multiplicative should be considered prior to transformation, as this will impact the effect of each variable on the data. If assumptions of normality and variable effects are not investigated prior to dimensionality reduction, ineffective or erroneous compound dimensionality reduction can impact downstream analyses. It is the aim of this manuscript to assess the impact of single and multivariable statistical models with and without the log transformation to volatilome dimensionality reduction prior to any supervised or unsupervised classification analysis. As a proof of concept, Shingleback lizard (Tiliqua rugosa) volatilomes were collected across their species distribution and from captivity and were assessed. Shingleback volatilomes are suspected to be influenced by multiple explanatory variables related to habitat (Bioregion), sex, parasite presence, total body volume, and captive status. This work determined that the exclusion of relevant multiple explanatory variables from analysis overestimates the effect of Bioregion and the identification of significant compounds. The log transformation increased the number of compounds that were identified as significant, as did analyses that assumed that residuals were normally distributed. Among the methods considered in this work, the most conservative form of dimensionality reduction was achieved through analyzing untransformed data using Monte Carlo tests with multiple explanatory variables.

A forensically validated genetic toolkit for the species and lineage identification of the highly trafficked shingleback lizard (Tiliqua rugosa).

Shingleback lizards (Tiliqua rugosa) are among the most trafficked native fauna from Australia in the illegal pet trade. There are four morphologically recognised subspecies of shinglebacks, all with differing overseas market values. Shinglebacks from different geographic locales are often trafficked and housed together, which may complicate identifying the State jurisdiction where the poaching event occurred. Additionally, shinglebacks can be housed and trafficked with other species within the same genus, which may complicate DNA analysis, especially in scenarios where indirect evidence (e.g. swabs, faeces) is taken for analysis. In this study, a forensic genetic toolkit was designed and validated to target shingleback DNA for species identification and geographic origin. To do this, field sampling across Australia was conducted to expand the phylogeographic sampling of shinglebacks across their species range and include populations suspected to be poaching hotspots. A commonly used universal reptile primer set (ND4/LEU) was then validated for use in forensic casework related to the genus Tiliqua. Two additional ND4 primer sets were designed and validated. The first primer set was designed and demonstrated to preferentially amplify an ∼510 bp region of the genus Tiliqua over other reptiles and builds on existing data to expand the available phylogeographic database. The second primer set was designed and demonstrated to solely amplify an ∼220 bp region of T. rugosa ND4 over any other reptile species. Through the validation process, all primers were demonstrated to amplify T. rugosa DNA from a variety of sample types (e.g. degraded, low quality and mixed). Two of the primer sets were able to distinguish the genetic lineage of T. rugosa from the phylogeographic database. This work provides the first forensically validated toolkit and phylogeographic genetic database for Squatmate lizards.

Future-proofing the koala: Synergising genomic and environmental data for effective species management.

Climatic and evolutionary processes are inextricably linked to conservation. Avoiding extinction in rapidly changing environments often depends upon a species' capacity to adapt in the face of extreme selective pressures. Here, we employed exon capture and high-throughput next-generation sequencing to investigate the mechanisms underlying population structure and adaptive genetic variation in the koala (Phascolarctos cinereus), an iconic Australian marsupial that represents a unique conservation challenge because it is not uniformly threatened across its range. An examination of 250 specimens representing 91 wild source locations revealed that five major genetic clusters currently exist on a continental scale. The initial divergence of these clusters appears to have been concordant with the Mid-Brunhes Transition (~430 to 300 kya), a major climatic reorganisation that increased the amplitude of Pleistocene glacial-interglacial cycles. While signatures of polygenic selection and environmental adaptation were detected, strong evidence for repeated, climate-associated range contractions and demographic bottleneck events suggests that geographically isolated refugia may have played a more significant role in the survival of the koala through the Pleistocene glaciation than in situ adaptation. Consequently, the conservation of genome-wide genetic variation must be aligned with the protection of core koala habitat to increase the resilience of vulnerable populations to accelerating anthropogenic threats. Finally, we propose that the five major genetic clusters identified in this study should be accounted for in future koala conservation efforts (e.g., guiding translocations), as existing management divisions in the states of Queensland and New South Wales do not reflect historic or contemporary population structure.

Correction: Phylogeography of the iconic Australian red-tailed black-cockatoo (Calyptorhynchus banksii) and implications for its conservation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Phylogeography of the iconic Australian red-tailed black-cockatoo (Calyptorhynchus banksii) and implications for its conservation.

Advances in sequencing technologies have revolutionized wildlife conservation genetics. Analysis of genomic data sets can provide high-resolution estimates of genetic structure, genetic diversity, gene flow, and evolutionary history. These data can be used to characterize conservation units and to effectively manage the genetic health of species in a broad evolutionary context. Here we utilize thousands of genome-wide single-nucleotide polymorphisms (SNPs) and mitochondrial DNA to provide the first genetic assessment of the Australian red-tailed black-cockatoo (Calyptorhynchus banksii), a widespread bird species comprising populations of varying conservation concern. We identified five evolutionarily significant units, which are estimated to have diverged during the Pleistocene. These units are only partially congruent with the existing morphology-based subspecies taxonomy. Genetic clusters inferred from mitochondrial DNA differed from those based on SNPs and were less resolved. Our study has a range of conservation and taxonomic implications for this species. In particular, we provide advice on the potential genetic rescue of the Endangered and restricted-range subspecies C. b. graptogyne, and propose that the western C. b. samueli population is diagnosable as a separate subspecies. The results of our study highlight the utility of considering the phylogeographic relationships inferred from genome-wide SNPs when characterizing conservation units and management priorities, which is particularly relevant as genomic data sets become increasingly accessible.

Museum specimens provide reliable SNP data for population genomic analysis of a widely distributed but threatened cockatoo species.

Natural history museums harbour a plethora of biological specimens which are of potential use in population and conservation genetic studies. Although technical advancements in museum genomics have enabled genome-wide markers to be generated from aged museum specimens, the suitability of these data for robust biological inference is not well characterized. The aim of this study was to test the utility of museum specimens in population and conservation genomics by assessing the biological and technical validity of single nucleotide polymorphism (SNP) data derived from such samples. To achieve this, we generated thousands of SNPs from 47 red-tailed black cockatoo (Calyptorhychus banksii) traditional museum samples (i.e. samples that were not collected with the primary intent of DNA analysis) and 113 fresh tissue samples (cryopreserved liver/muscle) using a restriction site-associated DNA marker approach (DArTseq™ ). Thousands of SNPs were successfully generated from most of the traditional museum samples (with a mean age of 44 years, ranging from 5 to 123 years), although 38% did not provide useful data. These SNPs exhibited higher error rates and contained significantly more missing data compared with SNPs from fresh tissue samples, likely due to considerable DNA fragmentation. However, based on simulation results, the level of genotyping error had a negligible effect on inference of population structure in this species. We did identify a bias towards low diversity SNPs in older samples that appears to compromise temporal inferences of genetic diversity. This study demonstrates the utility of a RADseq-based method to produce reliable genome-wide SNP data from traditional museum specimens.

Correction: A rapid multiplex PCR assay for presumptive species identification of rhinoceros horns and its implementation in Vietnam.

[This corrects the article DOI: 10.1371/journal.pone.0198565.].

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.

A rapid multiplex PCR assay for presumptive species identification of rhinoceros horns and its implementation in Vietnam.

Rhinoceros (rhinos) have suffered a dramatic increase in poaching over the past decade due to the growing demand for rhino horn products in Asia. One way to reverse this trend is to enhance enforcement and intelligence gathering tools used for species identification of horns, in particular making them fast, inexpensive and accurate. Traditionally, species identification tests are based on DNA sequence data, which, depending on laboratory resources, can be either time or cost prohibitive. This study presents a rapid rhino species identification test, utilizing species-specific primers within the cytochrome b gene multiplexed in a single reaction, with a presumptive species identification based on the length of the resultant amplicon. This multiplex PCR assay can provide a presumptive species identification result in less than 24 hours. Sequence-based definitive testing can be conducted if/when required (e.g. court purposes). This work also presents an actual casework scenario in which the presumptive test was successfully utlitised, in concert with sequence-based definitive testing. The test was carried out on seized suspected rhino horns tested at the Institute of Ecology and Biological Resources, the CITES mandated laboratory in Vietnam, a country that is known to be a major source of demand for rhino horns. This test represents the basis for which future 'rapid species identification tests' can be trialed.

Unmasking the complexity of species identification in Australasian flying-foxes.

Pteropus (flying-foxes) are a speciose group of non-echolocating large bats, with five extant Australian species and 24 additional species distributed amongst the Pacific Islands. In 2015, an injured flying-fox with unusual facial markings was found in Sydney, Australia, following severe and widespread storms. Based on an initial assessment, the individual belonged to Pteropus but could not be readily identified to species. As a consequence, four hypotheses for its identification/origin were posited: the specimen represented (1) an undescribed Australian species; or (2) a morphological variant of a recognised Australian species; or (3) a hybrid individual; or (4) a vagrant from the nearby Southwest Pacific Islands. We used a combination of morphological and both mitochondrial- and nuclear DNA-based identification methods to assess these hypotheses. Based on the results, we propose that this morphologically unique Pteropus most likely represents an unusual P. alecto (black flying-fox) potentially resulting from introgression from another Pteropus species. Unexpectedly, this individual, and the addition of reference sequence data from newly vouchered specimens, revealed a previously unreported P. alecto mitochondrial DNA lineage. This lineage was distinct from currently available haplotypes. It also suggests long-term hybridisation commonly occurs between P. alecto and P. conspicillatus (spectacled flying-fox). This highlights the importance of extensive reference data, and the inclusion of multiple vouchered specimens for each species to encompass both intraspecific and interspecific variation to provide accurate and robust species identification. Moreover, our additional reference data further demonstrates the complexity of Pteropus species relationships, including hybridisation, and potential intraspecific biogeographical structure that may impact on their management and conservation.

An internationally standardized species identification test for use on suspected seized rhinoceros horn in the illegal wildlife trade.

Rhinoceros (rhino) numbers have dwindled substantially over the past century. As a result, three of the five species are now considered to be critically endangered, one species is vulnerable and one species is near-threatened. Poaching has increased dramatically over the past decade due to a growing demand for rhino horn products, primarily in Asia. Improved wildlife forensic techniques, such as validated tests for species identification of seized horns, are critical to aid current enforcement and prosecution efforts and provide a deterrent to future rhino horn trafficking. Here, we present an internationally standardized species identification test based on a 230 base pair cytochrome-b region. This test improves on previous nested PCR protocols and can be used for the discrimination of samples with <20pg of template DNA, thus suitable for DNA extracted from horn products. The assay was designed to amplify water buffalo samples, a common 'rhino horn' substitute, but to exclude human DNA, a common contaminant. Phylogenetic analyses using this partial cytochrome-b region resolved the five extant rhino species. Testing successfully returned a sequence and correct identification for all of the known rhino horn samples and vouchered rhino samples from museum and zoo collections, and provided species level identification for 47 out of 52 unknown samples from seizures. Validation and standardization was carried out across five different laboratories, in four different countries, demonstrating it to be an effective and reproducible test, robust to inter laboratory variation in equipment and consumables (such as PCR reagents). This is one of the first species identification tests to be internationally standardized to produce data for evidential proceedings and the first published validated test for rhinos, one of the flagship species groups of the illegal wildlife trade and for which forensic tools are urgently required. This study serves as a model for how species identification tests should be standardized and disseminated for wildlife forensic testing.

A new species of treefrog (Litoria) from Cape York Peninsula, Australia.

We describe a new species of treefrog from northern Australia. Litoria bella sp. nov. is morphologically and genetically most similar to frogs in the L. gracilenta and L. chloris groups but is distinguished from all members in these groups by a combination of a moderately large male body size (34.5-41.8 mm SVL), near-immaculate green dorsum, orange venter, bright orange digits and webbing, bluish purple lateral surfaces of the thighs, no pale canthal stripe, white bones, and a highly-pulsed, single-note, male advertisement call with a pulse rate of 56-64 pulses/s and dominant frequency of 2.6-2.8 kHz. Litoria bella sp. nov. has a patchy distribution across the Cape York Peninsula, inhabiting rainforest and monsoon vine thicket in close association with watercourses. The new species' affinities lie with L. auae from southern New Guinea rather than with L. gracilenta from eastern Australia. Molecular data suggest that the L. gracilenta group should be expanded to include L. chloris and L. xanthomera, two moderately large green treefrogs from eastern Australia.

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.

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.

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.