Analysing the effects of distance, taxon and biomass on vertebrate detections using bulk-collected carrion fly iDNA.

Invertebrate-derived DNA (iDNA) metabarcoding from carrion flies is a powerful, non-invasive tool that has value for assessing vertebrate diversity. However, unknowns exist around the factors that influence vertebrate detections, such as spatial limits to iDNA signals or if detections are influenced by taxonomic class or estimated biomass of the vertebrates of interest. Using a bulk-collection method, we captured flies from within a zoo and along transects extending 4 km away from this location. From 920 flies, we detected 28 vertebrate species. Of the 28 detected species, we identified 9 species kept at the zoo, 8 mammals and 1 bird, but no reptiles. iDNA detections were highly geographically localized, and only a few zoo animals were detected outside the zoo setting. However, due to the low number of detections in our dataset, we found no influence of the taxonomic group or the estimated biomass of animals on their detectability. Our data suggest that iDNA detections from bulk-collected carrion flies, at least in urban settings in Australia, are predominantly determined by geographic proximity to the sampling location. This study presents an important step in understanding how iDNA techniques can be used in biodiversity monitoring.

Spider webs capture environmental DNA from terrestrial vertebrates.

Environmental DNA holds significant promise as a non-invasive tool for tracking terrestrial biodiversity. However, in non-homogenous terrestrial environments, the continual exploration of new substrates is crucial. Here we test the hypothesis that spider webs can act as passive biofilters, capturing eDNA from vertebrates present in the local environment. Using a metabarcoding approach, we detected vertebrate eDNA from all analyzed spider webs (N = 49). Spider webs obtained from an Australian woodland locality yielded vertebrate eDNA from 32 different species, including native mammals and birds. In contrast, webs from Perth Zoo, less than 50 km away, yielded eDNA from 61 different vertebrates and produced a highly distinct species composition, largely reflecting exotic species hosted in the zoo. We show that higher animal biomass and proximity to animal enclosures increased eDNA detection probability in the zoo. Our results indicate a tremendous potential for using spider webs as a cost-effective means to monitor terrestrial vertebrates.

On a roll: a direct comparison of extraction methods for the recovery of eDNA from roller swabbing of surfaces.

OBJECTIVE: Roller swabbing of surfaces is an effective way to obtain environmental DNA, but the current DNA extraction method for these samples is equipment heavy, time consuming, and increases potential contamination through multiple handling. Here, we used rollers to swab a dog kennel and compared three DNA extraction approaches (water filtration, roller trimming and direct buffer) using two different platforms (Qiacube, Kingfisher). DNA extraction methods were evaluated based on cost, effort, DNA concentration and PCR result. RESULTS: The roller trim method emerged as the optimal method with the best PCR results, DNA concentration and cost efficiency, while the buffer-based methods were the least labour intensive but produced mediocre PCR results and DNA concentrations. Additionally, the Kingfisher magnetic bead extractions generally ranked higher in all categories over the Qiacube column-based DNA extractions. Ultimately, the ideal DNA extraction method for a particular study is influenced by logistical constraints in the field such as the size of the roller, the availability of cold storage, and time constraints on the project. Our results demonstrate the strengths and weaknesses of each approach, allowing for informed decision making by researchers.

Use of carrion fly iDNA metabarcoding to monitor invasive and native mammals.

Severely fragmented habitats increase the risk of extirpation of native mammal populations through isolation, increased edge effects, and predation. Therefore, monitoring the movement of mammal populations through anthropogenically altered landscapes can inform conservation. We used metabarcoding of invertebrate-derived DNA (iDNA) from carrion flies (Calliphoridae and Sarcophagidae) to track mammal populations in the wheat belt of southwestern Australia, where widespread clearing for agriculture has removed most of the native perennial vegetation and replaced it with an agricultural system. We investigated whether the localization of the iDNA signal reflected the predicted distribution of 4 native species-echidna (Tachyglossus aculeatus), numbat (Myrmecobius fasciatus), woylie (Bettongia penicillata), and chuditch (Dasyurus geoffroii)-and 2 non-native, invasive mammal species-fox (Vulpes vulpes) and feral cat (Felis catus). We collected bulk iDNA samples (n = 150 samples from 3428 carrion flies) at 3 time points from 3 conservation reserves and 35 road edges between them. We detected 14 of the 40 mammal species known from the region, including our target species. Most detections of target taxa were in conservation reserves. There were a few detections from road edges. We detected foxes and feral cats throughout the study area, including all conservation reserves. There was a significant difference between the diversity (F3, 98  = 5.91, p < 0.001) and composition (F3, 43  = 1.72, p < 0.01) of taxa detections on road edges and conservation reserves. Conservation reserves hosted more native biodiversity than road edges. Our results suggest that the signals from iDNA reflect the known distribution of target mammals in this region. The development of iDNA methods shows promise for future noninvasive monitoring of mammals. With further development, iDNA metabarcoding could inform decision-making related to conservation of endangered taxa, invasive species management, and impacts of habitat fragmentation.

Caracterización genética del ADNi de la mosca carroñera para monitorear mamíferos invasores y nativos Resumen Los hábitats con mucha fragmentación aumentan el riesgo de extirpación de las poblaciones de mamíferos nativos debido al aislamiento, el aumento de los efectos de borde y la depredación. Por lo tanto, el monitoreo del movimiento de las poblaciones de mamíferos a través de paisajes alterados antropogénicamente puede guiar a la conservación. Utilizamos la caracterización genética del ADN derivado de invertebrados (ADNi) de moscas de la carroña (Calliphoridae y Sarcophagidae) para rastrear poblaciones de mamíferos en la región de Wheatbelt del suroeste de Australia, en donde la tala generalizada ha sustituido la mayor parte de la vegetación perenne nativa por un sistema agrícola. Investigamos si la localización de la señal de ADNi reflejaba la distribución prevista de cuatro especies autóctonas: equidna (Tachyglossus aculeatus), numbat (Myrmecobius fasciatus), rata canguro (Bettongia penicillata) y cuol occidental (Dasyurus geoffroii), y dos especies de mamíferos invasores no autóctonos: el zorro (Vulpes vulpes) y el gato feral (Felis catus). Recogimos muestras masivas de ADNi (n = 150 muestras de 3,428 moscas de la carroña) en tres puntos temporales de tres reservas ecológicas y 35 bordes de carreteras entre ellas. Detectamos 14 de las 40 especies de mamíferos conocidas en la región, incluidas nuestras especies objetivo. La mayoría de las detecciones de los taxones objetivo se produjeron en las reservas ecológicas. Pocas detecciones ocurrieron en los bordes de las carreteras. Detectamos zorros y gatos ferales en toda la zona de estudio, incluidas todas las reservas ecológicas. Hubo una diferencia significativa entre la diversidad (F3, 98 = 5.91, p<0.001) y la composición (F3, 43 = 1.72, p<0.01) de los taxones detectados en los bordes de las carreteras y en las reservas ecológicas. Las reservas ecológicas albergaron más biodiversidad nativa que los bordes de las carreteras. Nuestros resultados sugieren que las señales de ADNi reflejan la distribución conocida de los mamíferos objetivo en esta región. El desarrollo de métodos de ADNi es prometedor para el futuro monitoreo no invasivo de mamíferos. Con un mayor desarrollo, la caracterización genética del ADNi podría servir de base para decidir sobre la conservación de taxones amenazados, la gestión de especies invasoras y los impactos de la fragmentación del hábitat.

eDNA metabarcoding of avocado flowers: 'Hass' it got potential to survey arthropods in food production systems?

In the face of global biodiversity declines, surveys of beneficial and antagonistic arthropod diversity as well as the ecological services that they provide are increasingly important in both natural and agro-ecosystems. Conventional survey methods used to monitor these communities often require extensive taxonomic expertise and are time-intensive, potentially limiting their application in industries such as agriculture, where arthropods often play a critical role in productivity (e.g. pollinators, pests and predators). Environmental DNA (eDNA) metabarcoding of a novel substrate, crop flowers, may offer an accurate and high throughput alternative to aid in the detection of these managed and unmanaged taxa. Here, we compared the arthropod communities detected with eDNA metabarcoding of flowers, from an agricultural species (Persea americana-'Hass' avocado), with two conventional survey techniques: digital video recording (DVR) devices and pan traps. In total, 80 eDNA flower samples, 96 h of DVRs and 48 pan trap samples were collected. Across the three methods, 49 arthropod families were identified, of which 12 were unique to the eDNA dataset. Environmental DNA metabarcoding from flowers revealed potential arthropod pollinators, as well as plant pests and parasites. Alpha diversity levels did not differ across the three survey methods although taxonomic composition varied significantly, with only 12% of arthropod families found to be common across all three methods. eDNA metabarcoding of flowers has the potential to revolutionize the way arthropod communities are monitored in natural and agro-ecosystems, potentially detecting the response of pollinators and pests to climate change, diseases, habitat loss and other disturbances.

Detection of vertebrates from natural and artificial inland water bodies in a semi-arid habitat using eDNA from filtered, swept, and sediment samples.

Biomonitoring is vital for establishing baseline data that is needed to identify and quantify ecological change and to inform management and conservation activities. However, biomonitoring and biodiversity assessment in arid environments, which are predicted to cover 56% of the Earth's land surface by 2100, can be prohibitively time consuming, expensive, and logistically challenging due to their often remote and inhospitable nature. Sampling of environmental DNA (eDNA) coupled with high-throughput sequencing is an emerging biodiversity assessment method. Here we explore the application of eDNA metabarcoding and various sampling approaches to estimate vertebrate richness and assemblage at human-constructed and natural water sources in a semi-arid region of Western Australia. Three sampling methods: sediment samples, filtering through a membrane with a pump, and membrane sweeping in the water body, were compared using two eDNA metabarcoding assays, 12S-V5 and 16smam, for 120 eDNA samples collected from four gnammas (gnamma: Australian Indigenous Noongar language term-granite rock pools) and four cattle troughs in the Great Western Woodlands, Western Australia. We detected higher vertebrate richness in samples from cattle troughs and found differences between assemblages detected in gnammas (more birds and amphibians) and cattle troughs (more mammals, including feral taxa). Total vertebrate richness was not different between swept and filtered samples, but all sampling methods yielded different assemblages. Our findings indicate that eDNA surveys in arid lands will benefit from collecting multiple samples at multiple water sources to avoid underestimating vertebrate richness. The high concentration of eDNA in small, isolated water bodies permits the use of sweep sampling that simplifies sample collection, processing, and storage, particularly when assessing vertebrate biodiversity across large spatial scales.

Evolution of cox2 introns in angiosperm mitochondria and efficient splicing of an elongated cox2i691 intron.

In angiosperms, the mitochondrial cox2 gene harbors up to two introns, commonly referred to as cox2i373 and cox2i691. We studied the cox2 from 222 fully-sequenced mitogenomes from 30 angiosperm orders and analyzed the evolution of their introns. Unlike cox2i373, cox2i691 shows a distribution among plants that is shaped by frequent intron loss events driven by localized retroprocessing. In addition, cox2i691 exhibits sporadic elongations, frequently in domain IV of introns. Such elongations are poorly related to repeat content and two of them showed the presence of LINE transposons, suggesting that increasing intron size is very likely due to nuclear intracelular DNA transfer followed by incorporation into the mitochondrial DNA. Surprisingly, we found that cox2i691 is erroneously annotated as absent in 30 mitogenomes deposited in public databases. Although each of the cox2 introns is âˆ¼1.5 kb in length, a cox2i691 of 4.2 kb has been reported in Acacia ligulata (Fabaceae). It is still unclear whether its unusual length is due to a trans-splicing arrangement or the loss of functionality of the interrupted cox2. Through analyzing short-read RNA sequencing of Acacia with a multi-step computational strategy, we found that the Acacia cox2 is functional and its long intron is spliced in cis in a very efficient manner despite its length.

Taking eDNA underground: Factors affecting eDNA detection of subterranean fauna in groundwater.

Stygofauna are aquatic fauna that have evolved to live underground. The impacts of anthropogenic climate change, extraction and pollution on groundwater pose major threats to groundwater health, prompting the need for efficient and reliable means to detect and monitor stygofaunal communities. Conventional survey techniques for these species rely on morphological identification and can be biased, labour-intensive and often indeterminate to lower taxonomic levels. By contrast, environmental DNA (eDNA)-based methods have the potential to dramatically improve on existing stygofaunal survey methods in a large range of habitats and for all life stages, reducing the need for the destructive manual collection of often critically endangered species or for specialized taxonomic expertise. We compared eDNA and haul-net samples collected in 2020 and 2021 from 19 groundwater bores and a cave on Barrow Island, northwest Western Australia, and assessed how sampling factors influenced the quality of eDNA detection of stygofauna. The two detection methods were complementary; eDNA metabarcoding was able to detect soft-bodied taxa and fish often missed by nets, but only detected seven of the nine stygofaunal crustacean orders identified from haul-net specimens. Our results also indicated that eDNA metabarcoding could detect 54%-100% of stygofauna from shallow-water samples and 82%-90% from sediment samples. However, there was significant variation in stygofaunal diversity between sample years and sampling types. The findings of this study demonstrate that haul-net sampling has a tendency to underestimate stygofaunal diversity and that eDNA metabarcoding of groundwater can substantially improve the efficiency of stygofaunal surveys.

Aquatic environmental DNA: A review of the macro-organismal biomonitoring revolution.

Environmental DNA (eDNA) is the fastest growing biomonitoring tool fuelled by two key features: time efficiency and sensitivity. Technological advancements allow rapid biodiversity detection at both species and community levels with increasing accuracy. Concurrently, there has been a global demand to standardise eDNA methods, but this is only possible with an in-depth overview of the technological advancements and a discussion of the pros and cons of available methods. We therefore conducted a systematic literature review of 407 peer-reviewed papers on aquatic eDNA published between 2012 and 2021. We observed a gradual increase in the annual number of publications from four (2012) to 28 (2018), followed by a rapid growth to 124 publications in 2021. This was mirrored by a tremendous diversification of methods in all aspects of the eDNA workflow. For example, in 2012 only freezing was applied to preserve filter samples, whereas we recorded 12 different preservation methods in the 2021 literature. Despite an ongoing standardisation debate in the eDNA community, the field is seemingly moving fast in the opposite direction and we discuss the reasons and implications. Moreover, by compiling the largest PCR-primer database to date, we provide information on 522 and 141 published species-specific and metabarcoding primers targeting a wide range of aquatic organisms. This works as a user-friendly 'distillation' of primer information that was hitherto scattered across hundreds of papers, but the list also reflects which taxa are commonly studied with eDNA technology in aquatic environments such as fish and amphibians, and reveals that groups such as corals, plankton and algae are under-studied. Efforts to improve sampling and extraction methods, primer specificity and reference databases are crucial to capture these ecologically important taxa in future eDNA biomonitoring surveys. In a rapidly diversifying field, this review synthetises aquatic eDNA procedures and can guide eDNA users towards best practice.

DNA metabarcoding identifies urban foraging patterns of oligolectic and polylectic cavity-nesting bees.

Urbanisation modifies natural landscapes resulting in built-up space that is covered by buildings or hard surfaces and managed green spaces that often substitute native plant species with exotics. Some native bee species have been able to adapt to urban environments, foraging and reproducing in these highly modified areas. However, little is known on how the foraging ecology of native bees is affected by urbanised environments, and whether impacts vary among species with different degrees of specialisation for pollen collection. Here, we aim to investigate the responses of native bee foraging behaviour to urbanisation, using DNA metabarcoding to identify the resources within nesting tubes. We targeted oligolectic (specialist) and polylectic (generalist) cavity-nesting bee species in residential gardens and remnant bushland habitats. We were able to identify 40 families, 50 genera, and 23 species of plants, including exotic species, from the contents of nesting tubes. Oligolectic bee species had higher diversity of plant pollen in their nesting tubes in residential gardens compared to bushland habitats, along with significantly different forage composition between the two habitats. This result implies a greater degree of forage flexibility for oligolectic bee species than previously thought. In contrast, the diversity and composition of plant forage in polylectic bee nesting tubes did not vary between the two habitat types. Our results suggest a complex response of cavity-nesting bees to urbanisation and support the need for additional research to understand how the shifts in foraging resources impact overall bee health.

Key factors to consider in the use of environmental DNA metabarcoding to monitor terrestrial ecological restoration.

Ecological restoration of terrestrial environments is a globally important process to combat the loss of biodiversity and ecosystem services. Holistic monitoring of restored biota and active management of restoration is necessary to improve restoration processes and outcomes, and provide evidence to stakeholders that targets are being achieved. Increasingly, environmental DNA (eDNA) metabarcoding is used as a restoration monitoring tool because it is able to generate biodiversity data rapidly, accurately, non-destructively, and reliably, on a wide breadth of organisms from soil microbes to mammals. The overall objective of this review is to discuss the key factors to consider in the use of environmental DNA for monitoring of restored terrestrial ecosystems, hopefully improving monitoring, and ultimately, restoration outcomes. We identified that the majority of eDNA based studies of ecosystem restoration are currently conducted in Europe, North America, and Australia, and that almost half of total studies were published in 2021-22. Soil was the most popular sample substrate, soil microbial communities the most targeted taxa, and forests the most studied ecosystem. We suggest there is no 'one size fits all' approach to restoration monitoring using eDNA, and discuss survey design. Factors to consider include substrate selection, sample collection and storage, assay selection, and data interpretation, all of which require careful planning to obtain reliable, and accurate information that can be used for restoration monitoring and decision making. We explore future directions for research and argue that eDNA metabarcoding can be a useful tool in the restoration monitoring 'toolkit', but requires informed application and greater accessibility to data by a wide spectrum of stakeholders.

Applications of environmental DNA (eDNA) in agricultural systems: Current uses, limitations and future prospects.

Global food production, food supply chains and food security are increasingly stressed by human population growth and loss of arable land, becoming more vulnerable to anthropogenic and environmental perturbations. Numerous mutualistic and antagonistic species are interconnected with the cultivation of crops and livestock and these can be challenging to identify on the large scales of food production systems. Accurate identifications to capture this diversity and rapid scalable monitoring are necessary to identify emerging threats (i.e. pests and pathogens), inform on ecosystem health (i.e. soil and pollinator diversity), and provide evidence for new management practices (i.e. fertiliser and pesticide applications). Increasingly, environmental DNA (eDNA) is providing rapid and accurate classifications for specific organisms and entire species assemblages in substrates ranging from soil to air. Here, we aim to discuss how eDNA is being used for monitoring of agricultural ecosystems, what current limitations exist, and how these could be managed to expand applications into the future. In a systematic review we identify that eDNA-based monitoring in food production systems accounts for only 4 % of all eDNA studies. We found that the majority of these eDNA studies target soil and plant substrates (60 %), predominantly to identify microbes and insects (60 %) and are biased towards Europe (42 %). While eDNA-based monitoring studies are uncommon in many of the world's food production systems, the trend is most pronounced in emerging economies often where food security is most at risk. We suggest that the biggest limitations to eDNA for agriculture are false negatives resulting from DNA degradation and assay biases, as well as incomplete databases and the interpretation of abundance data. These require in silico, in vitro, and in vivo approaches to carefully design, test and apply eDNA monitoring for reliable and accurate taxonomic identifications. We explore future opportunities for eDNA research which could further develop this useful tool for food production system monitoring in both emerging and developed economies, hopefully improving monitoring, and ultimately food security.

eDNA in subterranean ecosystems: Applications, technical aspects, and future prospects.

Monitoring of biota is pivotal for the assessment and conservation of ecosystems. Environments worldwide are being continuously and increasingly exposed to multiple adverse impacts, and the accuracy and reliability of the biomonitoring tools that can be employed shape not only the present, but more importantly, the future of entire habitats. The analysis of environmental DNA (eDNA) metabarcoding data provides a quick, affordable, and reliable molecular approach for biodiversity assessments. However, while extensively employed in aquatic and terrestrial surface environments, eDNA-based studies targeting subterranean ecosystems are still uncommon due to the lack of accessibility and the cryptic nature of these environments and their species. Recent advances in genetic and genomic analyses have established a promising framework for shedding new light on subterranean biodiversity and ecology. To address current knowledge and the future use of eDNA methods in groundwaters and caves, this review explores conceptual and technical aspects of the application and its potential in subterranean systems. We briefly introduce subterranean biota and describe the most used traditional sampling techniques. Next, eDNA characteristics, application, and limitations in the subsurface environment are outlined. Last, we provide suggestions on how to overcome caveats and delineate some of the research avenues that will likely shape this field in the near future. We advocate that eDNA analyses, when carefully conducted and ideally combined with conventional sampling techniques, will substantially increase understanding and enable crucial expansion of subterranean community characterisation. Given the importance of groundwater and cave ecosystems for nature and humans, eDNA can bring to the surface essential insights, such as study of ecosystem assemblages and rare species detection, which are critical for the preservation of life below, as well as above, the ground.

Evaluating restoration trajectories using DNA metabarcoding of ground-dwelling and airborne invertebrates and associated plant communities.

Invertebrates are important for restoration processes as they are key drivers of many landscape-scale ecosystem functions; including pollination, nutrient cycling and soil formation. However, invertebrates are often overlooked in restoration monitoring because they are highly diverse, poorly described, and time-consuming to survey, and require increasingly scarce taxonomic expertise to enable identification. DNA metabarcoding is a relatively new tool for rapid survey that is able to address some of these concerns, and provide information about the taxa with which invertebrates are interacting via food webs and habitat. Here, we evaluate how invertebrate communities may be used to determine ecosystem trajectories during restoration. We collected ground-dwelling and airborne invertebrates across chronosequences of mine-site restoration in three ecologically disparate locations in Western Australia and identified invertebrate and plant communities using DNA metabarcoding. Ground-dwelling invertebrates showed the clearest restoration signals, with communities becoming more similar to reference communities over time. These patterns were weaker in airborne invertebrates, which have higher dispersal abilities and therefore less local fidelity to environmental conditions. Although we detected directional changes in community composition indicative of invertebrate recovery, patterns observed were inconsistent between study locations. The inclusion of plant assays allowed identification of plant species, as well as potential food sources and habitat. We demonstrate that DNA metabarcoding of invertebrate communities can be used to evaluate restoration trajectories. Testing and incorporating new monitoring techniques such as DNA metabarcoding is critical to improving restoration outcomes.

Association of putatively adaptive genetic variation with climatic variables differs between a parasite and its host.

Parasitism is a pervasive phenomenon in nature with the relationship between species driving evolution in both parasite and host. Due to their host-dependent lifestyle, parasites may adapt to the abiotic environment in ways that differ from their hosts or from free-living relatives; yet rarely has this been assessed. Here, we test two competing hypotheses related to whether putatively adaptive genetic variation in a specialist mistletoe associates with the same, or different, climatic variables as its host species. We sampled 11 populations of the specialist mistletoe Amyema gibberula var. tatei (n = 154) and 10 populations of its associated host Hakea recurva subsp. recurva (n = 160). Reduced-representation sequencing was used to obtain genome-wide markers and putatively adaptive variation detected using genome scan methods. Climate associations were identified using generalized dissimilarity modelling, and these were mapped geographically to visualize the spatial patterns of genetic composition. Our results supported the hypothesis of parasites and host species responding differently to climatic variables. Temperature was relatively more important in predicting allelic turnover in the specialist mistletoe while precipitation was more important for the host. This suggests that parasitic plants and host species may respond differently to selective pressures, potentially as a result of differing nutrient acquisition strategies. Specifically, mistletoes acquire water from hosts (rather than the abiotic environment), which may provide a buffer to precipitation as a selective pressure. This work deepens and complements the physiological and other ecological studies of adaptation and provides a window into the evolutionary processes that underlie previously observed phenomena. Applying these methods to a comparative study in a host-parasite system has also highlighted factors that affect the study of selection pressure on nonmodel organisms, such as differing adaptation rates and lack of reference genomes.

Contrasting patterns of local adaptation along climatic gradients between a sympatric parasitic and autotrophic tree species.

Sympatric tree species are subject to similar climatic drivers, posing a question as to whether they display comparable adaptive responses. However, no study has explicitly examined local adaptation of co-occurring parasitic and autotrophic plant species to the abiotic environment. Here we test the hypotheses that a generalist parasitic tree would display a weaker signal of selection and that genomic variation would associate with fewer climatic variables (particularly precipitation) but have similar spatial patterns to a sympatric autotrophic tree species. To test these hypotheses, we collected samples from 17 sites across the range of two tree species, the hemiparasite Nuytsia floribunda (n = 264) and sympatric autotroph Melaleuca rhaphiophylla (n = 272). We obtained 5,531 high-quality genome-wide single nucleotide polymorphisms (SNPs) for M. rhaphiophylla and 6,727 SNPs for N. floribunda using DArTseq genome scan technology. Population differentiation and environmental association approaches were used to identify signals of selection. Generalized dissimilarly modelling was used to detect climatic and spatial patterns of local adaptation across climatic gradients. Overall, 322 SNPs were identified as putatively adaptive for the autotroph, while only 57 SNPs were identified for the parasitic species. We found genomic variation to associate with different sets of bioclimatic variables for each species, with precipitation relatively less important for the parasite. Spatial patterns of predicted adaptive variability were different and indicate that co-occurring species with disparate life history traits may not respond equally to selective pressures (i.e., temperature and precipitation). Together, these findings provide insight into local adaptation of sympatric parasitic and autotrophic tree species to abiotic environments.

Testing multiple substrates for terrestrial biodiversity monitoring using environmental DNA metabarcoding.

Biological surveys based on visual identification of the biota are challenging, expensive and time consuming, yet crucial for effective biomonitoring. DNA metabarcoding is a rapidly developing technology that can also facilitate biological surveys. This method involves the use of next generation sequencing technology to determine the community composition of a sample. However, it is uncertain as to what biological substrate should be the primary focus of metabarcoding surveys. This study aims to test multiple sample substrates (soil, scat, plant material and bulk arthropods) to determine what organisms can be detected from each and where they overlap. Samples (n = 200) were collected in the Pilbara (hot desert climate) and Swan Coastal Plain (hot Mediterranean climate) regions of Western Australia. Soil samples yielded little plant or animal DNA, especially in the Pilbara, probably due to conditions not conducive to long-term preservation. In contrast, scat samples contained the highest overall diversity with 131 plant, vertebrate and invertebrate families detected. Invertebrate and plant sequences were detected in the plant (86 families), pitfall (127 families) and vane trap (126 families) samples. In total, 278 families were recovered from the survey, 217 in the Swan Coastal Plain and 156 in the Pilbara. Aside from soil, 22%-43% of the families detected were unique to the particular substrate, and community composition varied significantly between substrates. These results demonstrate the importance of selecting appropriate metabarcoding substrates when undertaking terrestrial surveys. If the aim is to broadly capture all biota then multiple substrates will be required.

Large scale genome skimming from herbarium material for accurate plant identification and phylogenomics.

BACKGROUND: Herbaria are valuable sources of extensive curated plant material that are now accessible to genetic studies because of advances in high-throughput, next-generation sequencing methods. As an applied assessment of large-scale recovery of plastid and ribosomal genome sequences from herbarium material for plant identification and phylogenomics, we sequenced 672 samples covering 21 families, 142 genera and 530 named and proposed named species. We explored the impact of parameters such as sample age, DNA concentration and quality, read depth and fragment length on plastid assembly error. We also tested the efficacy of DNA sequence information for identifying plant samples using 45 specimens recently collected in the Pilbara. RESULTS: Genome skimming was effective at producing genomic information at large scale. Substantial sequence information on the chloroplast genome was obtained from 96.1% of samples, and complete or near-complete sequences of the nuclear ribosomal RNA gene repeat were obtained from 93.3% of samples. We were able to extract sequences for the core DNA barcode regions rbcL and matK from 96 to 93.3% of samples, respectively. Read quality and DNA fragment length had significant effects on sequencing outcomes and error correction of reads proved essential. Assembly problems were specific to certain taxa with low GC and high repeat content (Goodenia, Scaevola, Cyperus, Bulbostylis, Fimbristylis) suggesting biological rather than technical explanations. The structure of related genomes was needed to guide the assembly of repeats that exceeded the read length. DNA-based matching proved highly effective and showed that the efficacy for species identification declined in the order cpDNA >> rDNA > matK >> rbcL. CONCLUSIONS: We showed that a large-scale approach to genome sequencing using herbarium specimens produces high-quality complete cpDNA and rDNA sequences as a source of data for DNA barcoding and phylogenomics.

The Bacterial Microbiome Associated With Arid Biocrusts and the Biogeochemical Influence of Biocrusts Upon the Underlying Soil.

Biocrusts are aggregated crusts that exist on the soil surface of arid environments. They are complex microbial communities comprised of cyanobacteria, lichens, mosses, algae and fungi. Recently, biocrusts have gained significant attention due to their ubiquitous distribution and likely important ecological roles, including soil stabilization, soil moisture retention, carbon (C) and nitrogen (N) fixation, as well as microbial engineers for semi-arid ecosystem restoration. Here, we collected three co-occurring types of biocrust (Cyanobacterial crust, Crustose lichen, and Foliose lichen) and their underlying soil from arid zones within Western Australia. Bacterial microbiome composition was determined through 16S rRNA gene amplicon sequencing to assess the extent of microbiome selection within the crusts versus underlying soil and biogeochemical measures performed to determine whether the crusts had significant impact upon the underlying soil for nutrient input. We determined that the bacterial communities of native biocrusts are distinct from those in their underlying soil, where dominant bacterial taxa differed according to crust morphologies. δ15N revealed that N-fixation appeared most evident in Foliose lichen crust (1.73 ± 1.04‰). Consequently, depending upon the crust type, biocrusts contained higher concentrations of organic C (2 to 50 times), total N (4 to 16 times) and available ammonium (2 to 4 times), though this enrichment did not extend to the soils underneath them. These findings demonstrate that biocrust communities are seemingly islands of biological activity in an arid landscape, uniquely different from their surrounding and underlying soil.

Recent range expansion in Australian hummock grasses (Triodia) inferred using genotyping-by-sequencing.

The Australian arid zone (AAZ) has undergone aridification and the formation of vast sandy deserts since the mid-Miocene. Studies on AAZ organisms, particularly animals, have shown patterns of mesic ancestry, persistence in rocky refugia and range expansions in arid lineages. There has been limited molecular investigation of plants in the AAZ, particularly of taxa that arrived in Australia after the onset of aridification. Here we investigate populations of the widespread AAZ grass Triodia basedowii to determine whether there is evidence for a recent range expansion, and if so, its source and direction. We also undertake a dating analysis for the species complex to which T. basedowii belongs, in order to place its diversification in relation to changes in AAZ climate and landscapes. We analyse a genomic single nucleotide polymorphism data set from 17 populations of T. basedowii in a recently developed approach for detecting the signal and likely origin of a range expansion. We also use alignments from existing and newly sequenced plastomes from across Poaceae for analysis in BEAST to construct fossil-calibrated phylogenies. Across a range of sampling parameters and outgroups, we detected a consistent signal of westward expansion for T. basedowii, originating in central or eastern Australia. Divergence time estimation indicates that Triodia began to diversify in the late Miocene (crown 7.0-8.8 million years (Ma)), and the T. basedowii complex began to radiate during the Pleistocene (crown 1.4-2.0 Ma). This evidence for range expansion in an arid-adapted plant is consistent with similar patterns in AAZ animals and likely reflects a general response to the opening of new habitat during aridification. Radiation of the T. basedowii complex through the Pleistocene has been associated with preferences for different substrates, providing an explanation why only one lineage is widespread across sandy deserts.