Single metabarcoding multiplex captures community‐level freshwater biodiversity and beyond

Cost‐effective and accurate quantification of biodiversity is important for biodiversity conservation, resource management, and forecasting. Traditional monitoring approaches have relied on direct observations, remote sensing, and mark‐recapture techniques, providing insights into species ecology and the impact of pollution and climate change on indicator species. However, these techniques are typically low throughput, expensive, and can be invasive. In addition, they cannot detect cryptic diversity and are biased toward species that leave identifiable remains. DNA‐based methods, such as metabarcoding or single marker gene assays, have enabled high throughput screening of a wide range of taxonomic groups, including ones without well‐preserved remains. When compared with traditional techniques, these approaches have high throughput, can resolve cryptic diversity, do not require taxonomic specialist skills, and are non‐invasive. However, although they are comparatively cheaper than traditional approaches, they are expensive when applied at the community‐level as single marker assays are amplified and sequenced independently. Multilocus approaches in which multiple gene markers are amplified in a single reaction are desirable to deliver community‐level assessments in a cost‐effective manner. Yet, they are uncommon because of technical challenges that may lead to biases in downstream analyses, such as index hopping and unbalanced representation of taxonomic groups. Here, we developed a highly multiplexed protocol that combines the early pooling of marker genes that target broad taxonomic groups and taxon‐specific markers in a single tube reaction. This step is followed by the pooling of up to 384 samples per locus (N = 15,636 samples) with unique dual‐indexed sequencing adapters in a single sequencing run. This approach dramatically reduces the costs of community‐level biodiversity quantification and lowers the need for input DNA without compromising output quality. We optimized the multiplex assay on lake freshwater sediment samples and benchmarked the assay on samples from other environmental matrices, demonstrating its direct application to the river and marine communities

Using molecular diet analysis to inform invasive species management: A case study of introduced rats consuming endemic New Zealand frogs

The decline of amphibians has been of international concern for more than two decades, and the global spread of introduced fauna is a major factor in this decline. Conservation management decisions to implement control of introduced fauna are often based on diet studies. One of the most common metrics to report in diet studies is Frequency of Occurrence (FO), but this can be difficult to interpret, as it does not include a temporal perspective. Here, we examine the potential for FO data derived from molecular diet analysis to inform invasive species management, using invasive ship rats (Rattus rattus) and endemic frogs (Leiopelma spp.) in New Zealand as a case study. Only two endemic frog species persist on the mainland. One of these, Leiopelma archeyi, is Critically Endangered (IUCN 2017) and ranked as the world\u27s most evolutionarily distinct and globally endangered amphibian (EDGE, 2018). Ship rat stomach contents were collected by kill-trapping and subjected to three methods of diet analysis (one morphological and two DNA-based). A new primer pair was developed targeting all anuran species that exhibits good coverage, high taxonomic resolution, and reasonable specificity. Incorporating a temporal parameter allowed us to calculate the minimum number of ingestion events per rat per night, providing a more intuitive metric than the more commonly reported FO. We are not aware of other DNA-based diet studies that have incorporated a temporal parameter into FO data. The usefulness of such a metric will depend on the study system, in particular the feeding ecology of the predator. Ship rats are consuming both species of native frogs present on mainland New Zealand, and this study provides the first detections of remains of these species in mammalian stomach contents

Advancing the use of molecular methods for routine freshwater macroinvertebrate biomonitoring : the need for calibration experiments

Over the last decade, steady advancements have been made in the use of DNA-based methods for detection of species in a wide range of ecosystems. This progress has culminated in molecular monitoring methods being employed for the detection of several species for enforceable management purposes of endangered, invasive, and illegally harvested species worldwide. However, the routine application of DNA-based methods to monitor whole communities (typically a metabarcoding approach) in order to assess the status of ecosystems continues to be limited. In aquatic ecosystems, the limited use is particularly true for macroinvertebrate communities. As part of the DNAqua-Net consortium, a structured discussion was initiated with the aim to identify potential molecular methods for freshwater macroinvertebrate community assessment and identify important knowledge gaps for their routine application. We focus on three complementary DNA sources that can be metabarcoded: 1) DNA from homogenised samples (bulk DNA), 2) DNA extracted from sample preservative (fixative DNA), and 3) environmental DNA (eDNA) from water or sediment. We provide a brief overview of metabarcoding macroinvertebrate communities from each DNA source and identify challenges for their application to routine monitoring. To advance the utilisation of DNA-based monitoring for macroinvertebrates, we propose an experimental design template for a series of methodological calibration tests. The template compares sources of DNA with the goal of identifying the effects of molecular processing steps on precision and accuracy. Furthermore, the same samples will be morphologically analysed, which will enable the benchmarking of molecular to traditional processing approaches. In doing so we hope to highlight pathways for the development of DNA-based methods for the monitoring of freshwater macroinvertebrates

Detecting Frogs as Prey in the Diets of Introduced Mammals

Amphibians are currently the most threatened group of vertebrates, with an estimated 40 % of amphibian species currently in danger of extinction. The global spread of introduced fauna has been accepted as a major factor in the decline of amphibians, as native amphibians often have little or no evolutionary history with introduced predators. In particular, introduced small mammals such as rodents pose a major threat, as they are generalist predators that together have an almost global distribution. Wildlife management decisions regarding the control of introduced predators to protect endangered species are often based on predation rates derived from diet studies of the predators concerned. However, many studies have reported difficulties in identifying amphibians as prey in diet analysis, due to the degraded nature of any diagnostic prey components surviving the mastication and digestion processes of the predator. The aim of the research presented in this thesis was to aid global amphibian conservation by assessing the available tools, and developing further tools, to detect frogs as prey of introduced mammals. I investigated the feeding behaviour of introduced mammals (ship rats Rattus rattus, Norway rats Rattus norvegicus, house mice Mus musculus and hedgehogs Erinaceus europaeus) when presented with frogs (southern bell frogs Litoria raniformis) as prey and explored the usefulness of two diet analysis methods: traditional morphological analysis and novel DNA-based methods. Using these methods I estimated the impact of ship rats on two endangered New Zealand native frogs (Hochstetter’s frog Leiopelma hochstetteri and Archey’s frog Leiopelma archeyi). The results show that, using morphological diet analysis, 11 % of stomach and faecal samples collected after small mammals had ingested frogs could be classed as containing frogs as prey, but less than 1 % could be identified to species level. Although very small bones can be used to identify frogs as prey, the odds of successful identification dramatically increase as prey bone length in predator stomach and faecal contents increases. The use of DNA-based methods increased prey identification (to species level) from less than 1 % to 58 %. Prey detection periods exceeded known gastrointestinal transit times for the small mammal species concerned, indicating that it is primarily the passage of prey material through the gastrointestinal tract that limits the successful detection of DNA, rather than the degradative effects of ingestion or digestion. The DNA-based methods were validated for field-collected samples and were successful where morphological analysis was not. Both Hochstetter’s frogs and Archey’s frogs were identified in the stomach contents of wild ship rats and estimates of predation rates at the study sites ranged from 0.01 – 0.9 frogs/ha/night, but it remains unclear whether ship rats alone threaten them with extinction. This is the first study to investigate the usefulness of DNA-based diet analysis for detecting amphibians as prey, the first to estimate predation rates on New Zealand’s native frogs, and the first to compare stomach versus faecal prey DNA detection over time in any vertebrate. DNA-based diet analyses are highly adaptable and they offer a reliable and cost-effective approach to conservation managers hoping to make assessments of the impacts of introduced fauna on native amphibians, which are necessary to make informed decisions on the implementation of predator control

eDNA is a useful tool to evaluate the success of the eradication program of Xenopus laevis in Portugal

Biological invasions are widely recognized as a major driver of global biodiversity loss. The most cost-effective answer is often population eradication, while the number of individuals is still limited. The detection of the invasive species at low densities is essential for eradication success, which can be difficult using traditional methods. Environmental DNA (eDNA) can facilitate the detection and monitoring of invasive aquatic species at low densities and can be more sensitive than traditional sampling. The African clawed frog (Xenopus laevis) is a highly invasive species and has been recorded in several European countries. In Portugal, the species was discovered in 2006 and since 2010, streams of the Lisbon area, suspected or at risk of invasion, are being monitored every year and removal is ongoing. The main method of detection is electrofishing but methods like draining artificial ponds have also been used. This program so far succeeded in containing the spread and reduced frog abundance, although total eradication has not yet been accomplished. To evaluate the success of this program, we collected water samples from fifteen sites. In each site we sampled both pools and riifles to test if lotic microhabitat influences X. laevis eDNA detection and concentration. Surface water velocity in each sampling point was also measured. We filtered the samples, extracted DNA from filters, and assayed the extracted DNA for X. laevis DNA using quantitative polymerase chain reaction (qPCR). From the fifteen sites sampled, five were positive for X. laevis eDNA: four lotic sites and one lentic site. Local eradication success was evident mainly in lentic habitats, and three potential failures were also identified. Finding X. laevis at one of these sites only after X. laevis eDNA detection was an important contribution of this technique for the success of the control and eradication program. No significative differences were found between lotic microhabitat (riffles and pools) regarding X. laevis eDNA concentration and detection but velocity affected the concentration of X. laevis eDNA captured. Our results corroborate other studies that recommend eDNA as a complementary sampling approach to other traditional methods and suggests its suitability as a tool for the detection of invasive amphibians.To be presented as a flash oral presentation at the DNAQUA International Conference - First international conference on the use of DNA for water biomonitoring

Species detection from aquatic eDNA: assessing the importance of capture methods

Original ResearchEnvironmental DNA (eDNA) is increasingly used for biodiversity monitoring, particularly in aquatic systems. However, each step, from sample collection to bioinformatic analysis, can introduce biases and influence the reliability of results. While much effort has been put into the optimization of laboratory methods, less attention has been devoted to estimate the impacts of eDNA capture methods. To address this issue, water samples were collected at nine small ponds and puddles where up to 10 amphibian species occur, using precipitation, disc filters, and capsules. We focused on targeted detection of an amphibian species, Salamandra salamandra, and on the composition of the whole amphibian community. Species detection was performed using a novel qPCR assay for S. salamandra and high-throughput sequencing, combined with stringent versus relaxed PCR replication thresholds. Filtration techniques (disc filters and capsules) outperformed precipitation, generating a higher number of detections of S. salamandra and higher amounts of captured eDNA, while species detection was identical between disc filters and capsules. There were no significant differences between capture methods regarding amphibian community composition. The variation in detection success associated with capture methods was far higher than that associated with PCR replication, regardless of the detection method used. Our results highlight the importance of choosing a suitable capture method for eDNA studies and suggest that the choice of capture method outweighs the choice of detection method used. To the best of our knowledge, this is the first study to compare high-capacity capsules with common eDNA methods for water samples, such as precipitation and standard disc filtersinfo:eu-repo/semantics/publishedVersio

A web-based tool to standardise reporting and interpret results of eDNA qPCR assays

Environmental DNA, or eDNA, methodologies can enable the rapid detection of a target species without either visual or physical confirmation of the species presence. Over the last decade, targeted quantitative PCR (qPCR) assays have become an increasingly useful method employed by government and non-government agencies alike for purposes such as protecting and preserving ecosystems from invasive species, or for the conservation of endangered species. As the application of eDNA to answer ecological questions pushes the limits of qPCR-based detection, there is a pressing need to standardise the way qPCR results are reported and interpreted, as well as the way qPCR assays are evaluated for use outside of the remit of the original study.Natural England is one such government agency who have begun to use eDNA methodologies more widely to answer ecological questions. However, while some qPCR assays available for detecting the presence or absence of species such as the great crested newt (Triturus cristatus) have been specified, validated and quality assured to a high degree (Biggs et al. 2014), existing qPCR assays for other species are generally less well developed and validated. Additionally, for some species there are multiple qPCR assays available, with each being developed and validated to different stages. As such, Natural England identified a need to understand how the data derived from eDNA should be interpreted dependent on the level of qPCR assay development, and ultimately the confidence they can have in the accuracy of resulting data, including the associated risk of false positives or false negatives.NatureMetrics has developed a prototype web-based tool and protocol (European Technical Readiness Level 5) which would enable end users such as Natural England to inform their interpretation of qPCR results. The prototype is currently in the beta testing stage and is expected to be available in the coming months. The web-based tool will simplify qPCR assay evaluation, enabling end users to select the most appropriate qPCR assay for their needs, as well as standardise the reporting and interpretation of their qPCR results by generating a report at both the sample and site level from the inputted qPCR data

Using meta-barcoding tools to monitor primate meat consumption at dedicated establishments in Guinea-Bissau, West Africa

Guinea-Bissau (GB) is a regional stronghold for primate conservation. Ten primates occur in the country, including the Western chimpanzee (Pan troglodytes verus) and two colobus monkeys (Colobus polykomos and Piliocolobus badius temminckii). Primate meat is consumed at households and bushmeat-dedicated establishments, locally named "Abafatório". Such establishments are mentioned to be common in urban areas since the 1980s and to be specialized in serving primate meat while drinking alcoholic beverages. The meat is typically cooked in a stew and eaten with bread. However, as the trade and consumption of primate meat are illegal activities, the location of Abafatório establishments and details of the trade, namely species being consumed, are usually hidden from outsiders. Here, we characterize illicit bushmeat commerce and consumption at six Abafatórios of a small town. Our team visited the establishments every week for 15 months (2015-2017) and collected data on the type and prices of meals and gathered tissue samples taken from carcasses by establishment owners. A meta-barcoding approach (cytb and 12S mitochondrial DNA regions and Illumina MiSeq next-generation sequencing technology) was used to identify tissue samples to the species level. Two types of establishments can be distinguished – “restaurants” and “snack-bars”. Restaurants are similar to the ones found by previous works in the capital city where primate meat is sold as a dish containing few pieces of stewed meat. Snack-bars are smaller and the meat is sold inexpensively and by the piece. In the present study, 249 tissue samples were identified to be from four primates (Cercopithecus campbelli, Chlorocebus sabaeus, Papio papio, and Erythrocebus patas) and four Artiodactyla (Philantomba maxwellii, Tragelaphus scriptus, Potamochoerus porcus and Phacochoerus africanus). Primates represented approximately 92% of all species consumed across establishments, and C. campbelli was the most traded species. Our work suggests that primate meat is monetarily accessible for locals in rural areas and that the trade at Abafatórios may have extensive negative consequences to primate conservation, in particular, the reduction of primates' populations in the southern part of GB. Our work quantifies and identifies the species consumed in Abafatório establishments for the first time and highlights the need to improve regulation and law enforcement in Guinea-Bissau

Challenges for assessing vertebrate diversity in turbid Saharan water-bodies using environmental DNA

The Sahara desert is the largest warm desert in the world and a poorly explored area. Small water-bodies occur across the desert and are crucial habitats for vertebrate biodiversity. Environmental DNA (eDNA) is a powerful tool for species detection and is being increasingly used to conduct biodiversity assessments. However, there are a number of difficulties with sampling eDNA from such turbid water-bodies and it is often not feasible to rely on electrical tools in remote desert environments. We trialled a manually powered filtering method in Mauritania, using pre-filtration to circumvent problems posed by turbid water in remote arid areas. From nine vertebrate species expected in the water-bodies, four were detected visually, two via metabarcoding, and one via both methods. Difficulties filtering turbid water led to severe constraints, limiting the sampling protocol to only one sampling point per study site, which alone may largely explain why many of the expected vertebrate species were not detected. The amplification of human DNA using general vertebrate primers is also likely to have contributed to the low number of taxa identified. Here we highlight a number of challenges that need to be overcome to successfully conduct metabarcoding eDNA studies for vertebrates in desert environments in Africa

Short-term impact of recycling-derived fertilizers on their P supply for perennial ryegrass (Lolium perenne)

Various nutrient recycling technologies are currently under development in order to alleviate the dependency of non-renewable raw material for the production of mineral phosphorus fertilizers commonly used in agriculture. The resulting products, such as struvites and ashes, need to be assessed for their application as so-called recycling-derived fertilizers (RDFs) in the agricultural sector prior to commercialization. Here, we conducted a short-term (54 days) trial to investigate the impact of different phosphorus fertilizers on plant growth and the soil P cycling microbiota. Lolium perenne was grown with application of superphosphate (SP) as inorganic fertilizer, two ashes (poultry litter ash (PLA) and sewage sludge ash (SSA)), and two struvites (municipal wastewater struvite (MWS) and commercial CrystalGreen® (CGS)) applied at 20 and 60 kg P ha−1 in four replicates. A P-free control (SP0) was also included in the trial. Struvite application increased plant dry weights, and available P acid phosphatase activity was significantly improved for struvites at the high P application rate. The ash RDFs showed a liming effect at 60 kg P ha−1 , and PLA60 negatively affected acid phosphatase activity, while PLA20 had significantly lower phoD copy numbers. P mobilization from phosphonates and phytates was not affected. TCP solubilization was negatively affected by mineral SP fertilizer application at both P concentrations. The bacterial (16S and phoD) communities were only marginally affected by the tested P fertilizers. Overall, struvites appeared to be a suitable substitute for superphosphate fertilization for Irish L. perenne pastures.</p