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.

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.

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.

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.

Genome-scale transfer of mitochondrial DNA from legume hosts to the holoparasite Lophophytum mirabile (Balanophoraceae).

Angiosperm mitochondrial horizontal gene transfer (HGT) has been widely reported during the past decades. With a few exceptions, foreign sequences are mitochondrial genes or intronic regions from other plants, indicating that HGT has played a major role in shaping mitochondrial genome evolution. Host-parasite relationships are a valuable system to study this phenomenon due to the high frequency of HGT. In particular, the interaction between mimosoid legumes and holoparasites of the genus Lophophytum represents an outstanding opportunity to discern HGT events. The mitochondrial genome of the holoparasite L. mirabile has remarkable properties, the most extraordinary of which is the presence of 34 out of 43 mitochondrial protein genes acquired from its legume host, with the stunning replacement of up to 26 native homologs. However, the origin of the intergenic sequences that represent the majority (>90%) of the L. mirabile mtDNA remains largely unknown. The lack of mitochondrial sequences available from the donor angiosperm lineage (mimosoid legumes) precluded a large-scale evolutionary study. We sequenced and assembled the mitochondrial genome of the mimosoid Acacia ligulata and performed genome wide comparisons with L. mirabile. The A. ligulata mitochondrial genome is almost 700 kb in size, encoding 60 genes. About 60% of the L. mirabile mtDNA had greatest affinity to members of the family Fabaceae (∼49% to mimosoids in particular) with an average sequence identity of ∼96%, including genes but mostly intergenic regions. These findings strengthen the mitochondrial fusion compatibility model for angiosperm mitochondrion-to-mitochondrion HGT.

Wide outcrossing provides functional connectivity for new and old Banksia populations within a fragmented landscape.

Habitat fragmentation affects landscape connectivity, the extent of which is influenced by the movement capacity of the vectors of seed and pollen dispersal for plants. Negative impacts of reduced connectivity can include reduced fecundity, increased inbreeding, genetic erosion and decreased long-term viability. These are issues for not only old (remnant) populations, but also new (restored) populations. We assessed reproductive and connective functionality within and among remnant and restored populations of a common tree, Banksia menziesii R.Br. (Proteaceae), in a fragmented urban landscape, utilising a genetic and graph theoretical approach. Adult trees and seed cohorts from five remnants and two restored populations were genotyped using microsatellite markers. Genetic variation and pollen dispersal were assessed using direct (paternity assignment) and indirect (pollination graphs and mating system characterisation) methods. Restored populations had greater allelic diversity (Ar = 8.08; 8.34) than remnant populations (Ar range = 6.49-7.41). Genetic differentiation was greater between restored and adjacent remnants (FST = 0.03 and 0.10) than all other pairwise comparisons of remnant populations (mean FST = 0.01 ± 0.01; n = 16 P = 0.001). All populations displayed low correlated paternity (rp = 0.06-0.16) with wide-ranging realised pollen dispersal distances (< 1.7 km) and well-connected pollen networks. Here, we demonstrate reproductive and connective functionality of old and new populations of B. menziesii within a fragmented landscape. Due to long-distance pollination events, the physical size of these sites underestimates their effective population size. Thus, they are functionally equivalent to large populations, integrated into a larger landscape matrix.

Evaluating the diversity and composition of bacterial communities associated with Vachellia pachyceras - the only existing native tree species in the Kuwait desert.

We investigated the diversity and composition of bacterial communities in rhizospheric and non-rhizospheric bulk soils as well as root nodule bacterial communities of Vachellia pachyceras - the only native tree species existing in the Kuwait desert. Community fingerprinting comparisons and 16S rDNA sequence identifications were used for characterization of the bacterial population using specific primers. The bacterial characterization of soil samples revealed four major phyla: Acidobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. In situ (desert) samples of both rhizospheric and non-rhizospheric bulk soil were dominated by the bacterial phyla Firmicutes and Bacteroidetes, whereas the phylum Betaproteobacteria was present only in non-rhizospheric bulk soil. Ex situ (nursery growing condition) V. pachyceras resulted in restricted bacterial communities dominated by members of a single phylum, Bacteroidetes. Results indicated that the soil organic matter and rhizospheric environments might drive the bacterial community. Despite harsh climatic conditions, data demonstrated that V. pachyceras roots harbor endophytic bacterial populations. Our findings on bacterial community composition and structure have major significance for evaluating how Kuwait's extreme climatic conditions affect bacterial communities. The baseline data obtained in this study will be useful and assist in formulating strategies in ecological restoration programs, including the application of inoculation technologies.

What can we do with 1000 plastid genomes?

The plastid genome of plants is the smallest and most gene-rich of the three genomes in each cell and the one generally present in the highest copy number. As a result, obtaining plastid DNA sequence is a particularly cost-effective way of discovering genetic information about a plant. Until recently, the sequence information gathered in this way was generally limited to small portions of the genome amplified by polymerase chain reaction, but recent advances in sequencing technology have stimulated a substantial rate of increase in the sequencing of complete plastid genomes. Within the last year, the number of complete plastid genomes accessible in public sequence repositories has exceeded 1000. This sudden flood of data raises numerous challenges in data analysis and interpretation, but also offers the keys to potential insights across large swathes of plant biology. We examine what has been learnt so far, what more could be learnt if we look at the data in the right way, and what we might gain from the tens of thousands more genome sequences that will surely arrive in the next few years. The most exciting new discoveries are likely to be made at the interdisciplinary interfaces between molecular biology and ecology.

Integration of complete chloroplast genome sequences with small amplicon datasets improves phylogenetic resolution in Acacia.

Combining whole genome data with previously obtained amplicon sequences has the potential to increase the resolution of phylogenetic analyses, particularly at low taxonomic levels or where recent divergence, rapid speciation or slow genome evolution has resulted in limited sequence variation. However, the integration of these types of data for large scale phylogenetic studies has rarely been investigated. Here we conduct a phylogenetic analysis of the whole chloroplast genome and two nuclear ribosomal loci for 65 Acacia species from across the most recent Acacia phylogeny. We then combine this data with previously generated amplicon sequences (four chloroplast loci and two nuclear ribosomal loci) for 508 Acacia species. We use several phylogenetic methods, including maximum likelihood bootstrapping (with and without constraint) and ExaBayes, in order to determine the success of combining a dataset of 4000bp with one of 189,000bp. The results of our study indicate that the inclusion of whole genome data gave a far better resolved and well supported representation of the phylogenetic relationships within Acacia than using only amplicon sequences, with the greatest support observed when using a whole genome phylogeny as a constraint on the amplicon sequences. Our study therefore provides methods for optimal integration of genomic and amplicon sequences.

The critical role of ants in the extensive dispersal of Acacia seeds revealed by genetic parentage assignment.

Ants are prominent seed dispersal agents in many ecosystems, and dispersal distances are small in comparison with vertebrate dispersal agents. However, the distance and distribution of ant-mediated dispersal in arid/semi-arid environments remains poorly explored. We used microsatellite markers and parentage assignment to quantify the distance and distribution of dispersed seeds of Acacia karina, retrieved from the middens of Iridomyrmex agilis and Melophorus turneri perthensis. From parentage assignment, we could not distinguish the maternal from each parent pair assigned to each seed, so we applied two approaches to estimate dispersal distances, one conservative (CONS), where the parent closest to the ant midden was considered to be maternal, and the second where both parents were deemed equally likely (EL) to be maternal, and used both distances. Parentage was assigned to 124 seeds from eight middens. Maximum seed dispersal distances detected were 417 m (CONS) and 423 m (EL), more than double the estimated global maximum. Mean seed dispersal distances of 40 m (±5.8 SE) (CONS) and 79 m (±6.4 SE) (EL) exceeded the published global average of 2.24 m (±7.19 SD) by at least one order of magnitude. For both approaches and both ant species, seed dispersal was predominantly (44-84% of all seeds) within 50 m from the maternal source, with fewer dispersal events at longer distances. Ants in this semi-arid environment have demonstrated a greater capacity to disperse seeds than estimated elsewhere, which highlights their important role in this system, and suggests significant novel ecological and evolutionary consequences for myrmecochorous species in arid/semi-arid Australia.

Genetic and palaeo-climatic evidence for widespread persistence of the coastal tree species Eucalyptus gomphocephala (Myrtaceae) during the Last Glacial Maximum.

BACKGROUND AND AIMS: Few phylogeographic studies have been undertaken of species confined to narrow, linear coastal systems where past sea level and geomorphological changes may have had a profound effect on species population sizes and distributions. In this study, a phylogeographic analysis was conducted of Eucalyptus gomphocephala (tuart), a tree species restricted to a 400 × 10 km band of coastal sand-plain in south west Australia. Here, there is little known about the response of coastal vegetation to glacial/interglacial climate change, and a test was made as to whether this species was likely to have persisted widely through the Last Glacial Maximum (LGM), or conforms to a post-LGM dispersal model of recovery from few refugia. METHODS: The genetic structure over the entire range of tuart was assessed using seven nuclear (21 populations; n = 595) and four chloroplast (24 populations; n = 238) microsatellite markers designed for eucalypt species. Correlative palaeodistribution modelling was also conducted based on five climatic variables, within two LGM models. KEY RESULTS: The chloroplast markers generated six haplotypes, which were strongly geographically structured (GST = 0·86 and RST = 0·75). Nuclear microsatellite diversity was high (overall mean HE 0·75) and uniformly distributed (FST = 0·05), with a strong pattern of isolation by distance (r(2) = 0·362, P = 0·001). Distribution models of E. gomphocephala during the LGM showed a wide distribution that extended at least 30 km westward from the current distribution to the palaeo-coastline. CONCLUSIONS: The chloroplast and nuclear data suggest wide persistence of E. gomphocephala during the LGM. Palaeodistribution modelling supports the conclusions drawn from genetic data and indicates a widespread westward shift of E. gomphocephala onto the exposed continental shelf during the LGM. This study highlights the importance of the inclusion of complementary, non-genetic data (information on geomorphology and palaeoclimate) to interpret phylogeographic patterns.

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.

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.

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.

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.

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.

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.

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.