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Groundwater is a vital ecosystem of the global water cycle, hosting unique biodiversity and providing essential services to societies. Despite being the largest unfrozen freshwater resource, in a period of depletion by extraction and pollution, groundwater environments have been repeatedly overlooked in global biodiversity conservation agendas. Disregarding the importance of groundwater as an ecosystem ignores its critical role in preserving surface biomes. To foster timely global conservation of groundwater, we propose elevating the concept of keystone species into the realm of ecosystems, claiming groundwater as a keystone ecosystem that influences the integrity of many dependent ecosystems. Our global analysis shows that over half of land surface areas (52.6%) has a medium-to-high interaction with groundwater, reaching up to 74.9% when deserts and high mountains are excluded. We postulate that the intrinsic transboundary features of groundwater are critical for shifting perspectives towards more holistic approaches in aquatic ecology and beyond. Furthermore, we propose eight key themes to develop a science-policy integrated groundwater conservation agenda. Given ecosystems above and below the ground intersect at many levels, considering groundwater as an essential component of planetary health is pivotal to reduce biodiversity loss and buffer against climate change.

Globally, there has been an increase in the use of herbal remedies including traditional Chinese medicine (TCM). There is a perception that products are natural, safe and effectively regulated, however, regulatory agencies are hampered by a lack of a toolkit to audit ingredient lists, adulterants and constituent active compounds. Here, for the first time, a multidisciplinary approach to assessing the molecular content of 26 TCMs is described. Next generation DNA sequencing is combined with toxicological and heavy metal screening by separation techniques and mass spectrometry (MS) to provide a comprehensive audit. Genetic analysis revealed that 50% of samples contained DNA of undeclared plant or animal taxa, including an endangered species of Panthera (snow leopard). In 50% of the TCMs, an undeclared pharmaceutical agent was detected including warfarin, dexamethasone, diclofenac, cyproheptadine and paracetamol. Mass spectrometry revealed heavy metals including arsenic, lead and cadmium, one with a level of arsenic >10 times the acceptable limit. The study showed 92% of the TCMs examined were found to have some form of contamination and/or substitution. This study demonstrates that a combination of molecular methodologies can provide an effective means by which to audit complementary and alternative medicines.

Aim: We examined how the threatened and endemic white-tailed black cockatoos of Western Australia have responded genetically to recent and comprehensive habitat loss with the ultimate aim of identifying units for conservation. We assessed the population structure, connectivity and genetic diversity at spatial and temporal scales for Calyptorhynchus baudinii and C. latirostris, which have undergone dramatic population declines. Genetic comparisons of pre- and post-population decline were carried out by including historical samples dating back to 1920. We examined samples collected from across 700 km of their distribution and sampled approximately 1% of the current population census size to produce significant insights into the population genetics of white-tailed black cockatoos and generate genetic information crucial for conservation management. Location: Southwest corner of Western Australia. Methods: Six hundred and eighty-four cockatoo samples were collected from 1920 to 2010 and profiled with 19 microsatellites to identify spatial population structure and loss of genetic diversity. Results: The temporal and spatial microsatellite data illustrated that the geographically defined genetic structuring in white-tailed black cockatoos is likely to represent a recent phenomenon. We identified: (1) spatial population substructure east and west of extensively cleared habitat (>95,800 km2), but the historical samples clustered with the current western population, regardless of origin, (2) a regional loss of allelic diversity over 3-4 generations for the current eastern population, (3) a lack of a genetic signal of the recent population decline, but perhaps a mid-Holocene population collapse and lastly, (4) limited genetic differentiation between the two currently recognized white-tailed black-cockatoo species suggests a review of taxonomy and/or management units should be undertaken. Main conclusion: Based on extensive spatio-temporal sampling, we have demonstrated that r

Fossils and sediments preserved in caves are an excellent source of information for investigating impacts of past environmental changes on biodiversity. Until recently studies have relied on morphology-based palaeontological approaches, but recent advances in molecular analytical methods offer excellent potential for extracting a greater array of biological information from these sites. This study presents a thorough assessment of DNA preservation from late Pleistocene-Holocene vertebrate fossils and sediments from Kelly Hill Cave Kangaroo Island, South Australia. Using a combination of extraction techniques and sequencing technologies, ancient DNA was characterised from over 70 bones and 20 sediment samples from 15 stratigraphic layers ranging in age from >20ka to ~6.8ka. A combination of primers targeting marsupial and placental mammals, reptiles and two universal plant primers were used to reveal genetic biodiversity for comparison with the mainland and with the morphological fossil record for Kelly Hill Cave. We demonstrate that Kelly Hill Cave has excellent long-term DNA preservation, back to at least 20ka. This contrasts with the majority of Australian cave sites thus far explored for ancient DNA preservation, and highlights the great promise Kangaroo Island caves hold for yielding the hitherto-elusive DNA of extinct Australian Pleistocene species

Background: Wildlife collisions with aircraft cost the airline industry billions ofdollars per annum and represent a public safety risk. Clearly, adaptingaerodrome habitats to become less attractive to hazardous wildlife willreduce the incidence of collisions. Formulating effective habitat managementstrategies relies on accurate species identification of high-risk species.This can be successfully achieved for all strikes either through morphologyand/or DNA-based identifications. Beyond species identification, dietaryanalysis of birdstrike gut contents can provide valuable intelligence forairport hazard management practices in regards to what food is attractingwhich species to aerodromes. Here, we present birdstrike identification anddietary data from Perth Airport, Western Australia, an aerodrome that sawapproximately 140,000 aircraft movements in 2012. Next-generation highthroughput DNA sequencing was employed to investigate 77 carcasses from 16bird species collected over a 12-month period. Five DNA markers, whichbroadly characterize vertebrates, invertebrates and plants, were used totarget three animal mitochondrial genes (12S rRNA, 16S rRNA, and COI) and aplastid gene (trnL) from DNA extracted from birdstrike carcassgastrointestinal tracts.Results: Over 151,000 DNA sequences were generated, filtered and analyzed by afusion-tag amplicon sequencing approach. Across the 77 carcasses, the mostcommonly identified vertebrate was Mus musculus (house mouse).Acrididae (grasshoppers) was the most common invertebrate family identified,and Poaceae (grasses) the most commonly identified plant family. TheDNA-based dietary data has the potential to provide some key insights intofeeding ecologies within and around the aerodrome.Conclusions: The data generated here, together with the methodological approach, willgreatly assist in the development of hazard management plans and, incombination with existing observational studies, provide an improved way tomonitor the effectiveness o

Highly fragmented and morphologically indistinct fossil bone is common in archaeological and paleontological deposits but unfortunately it is of little use in compiling faunal assemblages. The development of a cost-effective methodology to taxonomically identify bulk bone is therefore a key challenge. Here, an ancient DNA methodology using high-throughput sequencing is developed to survey and analyse thousands of archaeological bones from southwest Australia. Fossils were collectively ground together depending on which of fifteen stratigraphical layers they were excavated from. By generating fifteen synthetic blends of bulk bone powder, each corresponding to a chronologically distinct layer, samples could be collectively analysed in an efficient manner. A diverse range of taxa, including endemic, extirpated and hitherto unrecorded taxa, dating back to c.46,000 years BP was characterized. The method is a novel, cost-effective use for unidentifiable bone fragments and a powerful molecular tool for surveying fossils that otherwise end up on the taxonomic “scrapheap”.

The study of arid palaeoenvironments is often frustrated by the poor or non-existent preservation of plant and animal material, yet these environments are of considerable environmental importance. The analysis of pollen and macrofossils isolated from herbivore middens has been an invaluable source of information regarding past environments and the nature of ecological fluctuations within arid zones. The application of ancient DNA (aDNA) techniques to hot, arid zone middens remains unexplored. This paper attempts to retrieve and characterise aDNA from four Southern Hemisphere fossil middens; three located in hot, arid regions of Australia and one sample from South Africa's Western Cape province. The middens are dated to between 30,490 (±380) and 710 (±70) cal yr BP. The Brockman Ridge midden in this study is potentially the oldest sample from which aDNA has been successfully extracted in Australia. The application of high-throughput sequencing approaches to profile the biotic remains preserved in midden material has not been attempted to date and this study clearly demonstrates the potential of such a methodology. In addition to the taxa previously detected via macrofossil and palynological analyses, aDNA analysis identified unreported plant and animal taxa, some of which are locally extinct or endemic. The survival and preservation of DNA in hot, arid environments is a complex and poorly understood process that is both sporadic and rare, but the survival of DNA through desiccation may be important. Herbivore middens now present an important source of material for DNA metabarcoding studies of hot, arid palaeoenvironments and can potentially be used to analyse middens in these environments throughout Australia, Africa, the Americas and the Middle East.

Traditional Chinese medicine (TCM) has been practiced for thousands of years, but only within the last few decades has its use become more widespread outside of Asia. Concerns continue to be raised about the efficacy, legality, and safety of many popular complementary alternative medicines, including TCMs. Ingredients of some TCMs are known to include derivatives of endangered, trade-restricted species of plants and animals, and therefore contravene the Convention on International Trade in Endangered Species (CITES) legislation. Chromatographic studies have detected the presence of heavy metals and plant toxins within some TCMs, and there are numerous cases of adverse reactions. It is in the interests of both biodiversity conservation and public safety that techniques are developed to screen medicinals like TCMs. Targeting both the p-loop region of the plastid trnL gene and the mitochondrial 16S ribosomal RNA gene, over 49,000 amplicon sequence reads were generated from 15 TCM samples presented in the form of powders, tablets, capsules, bile flakes, and herbal teas. Here we show that second-generation, high-throughput sequencing (HTS) of DNA represents an effective means to genetically audit organic ingredients within complex TCMs. Comparison of DNA sequence data to reference databases revealed the presence of 68 different plant families and included genera, such as Ephedra and Asarum, that are potentially toxic. Similarly, animal families were identified that include genera that are classified as vulnerable, endangered, or critically endangered, including Asiatic black bear (Ursus thibetanus) and Saiga antelope (Saiga tatarica). Bovidae, Cervidae, and Bufonidae DNA were also detected in many of the TCM samples and were rarely declared on the product packaging. This study demonstrates that deep sequencing via HTS is an efficient and cost-effective way to audit highly processed TCM products and will assist in monitoring their legality and safety especially when plant

Parrots and cockatoos are highly prized aviary birds and the demands for such species has fuelled their illegal trade and harvest from the wild. Here we report on three forensic case studies involving black-cockatoos (Calyptorhynchus spp.) endemic to Australia. These cases involve suspected poaching and illegal killing of endangered red- and white-tailed black-cockatoos. Through the prior development of 20 polymorphic microsatellite loci and population databases for white- and red-tailed black-cockatoos, the tools are available to conduct high-resolution paternity and individual identity testing. In one case, we matched a red-tailed black-cockatoo nestling to a tree hollow from which it was poached through the use of DNA from eggshell recovered from the nest. For the second case, we utilized our provenance population database (nest sites), and identified the kinship and geographic origin of a white-tailed black-cockatoo, which was illegally harvested from the wild. The third case determined the number individual white-tailed black-cockatoos allegedly shot at a fruit grower's orchard from body part remains. These genetic investigations highlight the significance and statistical confidence of DNA profiling and associated databases for endangered taxa, such as exotic birds. Our cockatoo population databases are the first of their kind in Australia, and demonstrate the efficacy of such approaches to identify such illegal activity. With a robust set of genetic markers and methodologies in place, we aim to broaden our population databases to include other cockatoo species of conservation concern.

Psittaciformes (parrots and cockatoos) are charismatic birds, their plumage and capacity for learning make them highly sought after pets. The illegal trade in parrots and cockatoos poses a serious threat to the viability of native populations; in addition, species transported to non-endemic areas may potentially vector disease and genetically 'pollute' local native avifauna. To reduce the logistical difficulties associated with trafficking live birds, smugglers often transport eggs. This creates a problem for authorities in elucidating accurate species identification without the laborious task of incubation and hand rearing until a morphological identification can be made. Here, we use 99 avian eggs seized from carriers coming into and within Australia, as a result of suspected illegal trade. We investigate and evaluate the use of mitochondrial DNA (mtDNA) to accurately identify eggs to family, genus or species level. However, Identification of a species based on percentage mtDNA similarities is difficult without good representations of the inter- and intra-levels of species variation. Based on the available reference database, we were able to identify 52% of the eggs to species level. Of those, 10 species from eight genera were detected, all of which belong to the parrot (Psittacidae) and cockatoo (Cacatuidae) families. Of the remaining 48%, a further 36% of eggs were identified to genus level, and 12% identified to family level using our assignment criteria. Clearly the lack of validated DNA reference sequences is hindering our ability to accurately assign a species identity, and accordingly, we advocate that more attention needs to be paid to establishing validated, multi locus mtDNA reference databases for exotic birds that can both assist in genetic identifications and withstand legal scrutiny.

The genetic analysis of faecal material represents a relatively non-invasive way to study animal diet and has been widely adopted in ecological research. Due to the heterogeneous nature of faecal material the primary obstacle, common to all genetic approaches, is a means to dissect the constituent DNA sequences. Traditionally, bacterial cloning of PCR amplified products was employed; less common has been the use of species-specific quantitative PCR (qPCR) assays. Currently, with the advent of High-Throughput Sequencing (HTS) technologies and indexed primers it has become possible to conduct genetic audits of faecal material to a much greater depth than previously possible. To date, no studies have systematically compared the estimates obtained by HTS with that of qPCR. What are the relative strengths and weaknesses of each technique and how quantitative are deep-sequencing approaches that employ universal primers? Using the locally threatened Little Penguin (Eudyptula minor) as a model organism, it is shown here that both qPCR and HTS techniques are highly correlated and produce strikingly similar quantitative estimates of fish DNA in faecal material, with no statistical difference. By designing four species-specific fish qPCR assays and comparing the data to the same four fish in the HTS data it was possible to directly compare the strengths and weaknesses of both techniques. To obtain reproducible quantitative data one of the key, and often overlooked, steps common to both approaches is ensuring that efficient DNA isolation methods are employed and that extracts are free of inhibitors. Taken together, the methodology chosen for long-term faecal monitoring programs is largely dependent on the complexity of the prey species present and the level of accuracy that is desired. Importantly, these methods should not be thought of as mutually exclusive, as the use of both HTS and qPCR in tandem will generate datasets with the highest fidelity.

Cockatoos are the distinctive family Cacatuidae, a major lineage of the order of parrots (Psittaciformes) and distributed throughout the Australasian region of the world. However, the evolutionary history of cockatoos is not well understood. We investigated the phylogeny of cockatoos based on three mitochondrial and three nuclear DNA genes obtained from 16 of 21 species of Cacatuidae. In addition, five novel mitochondrial genomes were used to estimate time of divergence and our estimates indicate Cacatuidae diverged from Psittacidae approximately 40.7. million years ago (95% CI 51.6-30.3. Ma) during the Eocene. Our data shows Cacatuidae began to diversify approximately 27.9. Ma (95% CI 38.1-18.3. Ma) during the Oligocene. The early to middle Miocene (20-10. Ma) was a significant period in the evolution of modern Australian environments and vegetation, in which a transformation from mainly mesic to xeric habitats (e.g., fire-adapted sclerophyll vegetation and grasslands) occurred. We hypothesize that this environmental transformation was a driving force behind the diversification of cockatoos. A detailed multi-locus molecular phylogeny enabled us to resolve the phylogenetic placements of the Palm Cockatoo (Probosciger aterrimus), Galah (Eolophus roseicapillus), Gang-gang Cockatoo (Callocephalon fimbriatum) and Cockatiel (Nymphicus hollandicus), which have historically been difficult to place within Cacatuidae. When the molecular evidence is analysed in concert with morphology, it is clear that many of the cockatoo species' diagnostic phenotypic traits such as plumage colour, body size, wing shape and bill morphology have evolved in parallel or convergently across lineages.

Genetic tools are an effective, non-invasive method for gaining an understanding into taxonomic structure, genetic structure, movement and biodiversity of endangered populations. Nuclear and mitochondrial markers are being developed with the following aims; (1) gain an understanding of the underlying diversity and taxonomic structure of Black-cockatoos in Western Australia; (2) discriminate cockatoo species and their provenance; (3) calibrate current levels of genetic structure within present day populations using historical museum specimens; and (4) contribute towards the prosecution of illegal wildlife traffickers. This research will involve cockatoo and parrot species from across Australia to facilitate the construction of a comprehensive DNA database.