Defining a High Throughput Sequencing identification framework for freshwater ecosystem biomonitoring

Freshwater ecosystems are currently amongst the most threatened habitats due to high levels of anthropogenic stress and increasing efforts are required to monitor their status and assess aquatic biodiversity. Biomonitoring, which is the systematic measurement of the responses of aquatic biota to environmental stressors, is used to evaluate ecosystem status. Macroinvertebrates are commonly used organisms for ecosystem assessment, due to their numerous biomonitoring qualities, which qualify them as ecological indicators. Traditional taxonomy-based monitoring is labour intensive, which limits the throughput, and is often inefficient in providing species level identification, which limits the accuracy of detections. The introduction of molecular based methods for biomonitoring, especially when coupled with High Throughput Sequencing (HTS) applications, offers a step change in ecosystem monitoring. Here I tested the utility of DNA based applications for increasing the efficiency of freshwater ecosystem biomonitoring, using benthic macroinvertebrates as a target group. For the first part of this work, I used DNA barcoding of the Cytochrome Oxidase Subunit I (COI), from individual specimens, to populate a barcode reference library for 94 species of Trichoptera, Gastropoda and Chironomidae from the UK. Then, I used High Throughput Sequencing (HTS) methods to characterise diversity from complex environmental samples. First, I used metabarcoding of aqueous environmental DNA (eDNA) and community invertebrate samples (Chironomidae pupal exuviae), collected on regular intervals throughout a year, to identify diversity levels and temporal patterns of community variation on ecosystem-wide and group specific scales. Finally, I used a structured design of mock macroinvertebrate communities, of known biomass content, to perform a comparison between PCR-based metabarcoding of the COI gene and PCR-free shotgun sequencing of mitochondrial genomes (mito-metagenomics), and evaluate their efficiency for accurate characterisation of biomass content of bulk samples. Overall, HTS has demonstrated great potential for advancing biomonitoring efforts, allowing ecosystem scale diversity detection from non-invasive types of samples, such as eDNA, whilst moving into mito-metagenomic work could improve the field even further by improving quantitative abundance results on the community composition level

Annual time-series analysis of aqueous eDNA reveals ecologically relevant dynamics of lake ecosystem biodiversity

The use of environmental DNA (eDNA) in biodiversity assessments offers a step-change in sensitivity, throughput and simultaneous measures of ecosystem diversity and function. There remains, however, a need to examine eDNA persistence in the wild through simultaneous temporal measures of eDNA and biota. Here, we use metabarcoding of two markers of different lengths, derived from an annual time series of aqueous lake eDNA to examine temporal shifts in ecosystem biodiversity and in an ecologically important group of macroinvertebrates (Diptera: Chironomidae). The analyses allow different levels of detection and validation of taxon richness and community composition (β-diversity) through time, with shorter eDNA fragments dominating the eDNA community. Comparisons between eDNA, community DNA, taxonomy and UK species abundance data further show significant relationships between diversity estimates derived across the disparate methodologies. Our results reveal the temporal dynamics of eDNA and validate the utility of eDNA metabarcoding for tracking seasonal diversity at the ecosystem scale

Environmental DNA provides higher resolution assessment of riverine biodiversity and ecosystem function via spatio-temporal nestedness and turnover partitioning.

Rapidly assessing biodiversity is essential for environmental monitoring; however, traditional approaches are limited in the scope needed for most ecological systems. Environmental DNA (eDNA) based assessment offers enhanced scope for assessing biodiversity, while also increasing sampling efficiency and reducing processing time, compared to traditional methods. Here we investigated the effects of landuse and seasonality on headwater community richness and functional diversity, via spatio-temporal dynamics, using both eDNA and traditional sampling. We found that eDNA provided greater resolution in assessing biodiversity dynamics in time and space, compared to traditional sampling. Community richness was seasonally linked, peaking in spring and summer, with temporal turnover having a greater effect on community composition compared to localized nestedness. Overall, our assessment of ecosystem function shows that community formation is driven by regional resource availability, implying regional management requirements should be considered. Our findings show that eDNA based ecological assessment is a powerful, rapid and effective assessment strategy that enables complex spatio-temporal studies of community diversity and ecosystem function, previously infeasible using traditional methods

Environmental DNA metabarcoding:Transforming how we survey animal and plant communities

The genomic revolution has fundamentally changed how we survey biodiversity on earth. High-throughput sequencing (?HTS?) platforms now enable the rapid sequencing of DNA from diverse kinds of environmental samples (termed ?environmental DNA? or ?eDNA?). Coupling HTS with our ability to associate sequences from eDNA with a taxonomic name is called ?eDNA metabarcoding? and offers a powerful molecular tool capable of noninvasively surveying species richness from many ecosystems. Here, we review the use of eDNA metabarcoding for surveying animal and plant richness, and the challenges in using eDNA approaches to estimate relative abundance. We highlight eDNA applications in freshwater, marine and terrestrial environments, and in this broad context, we distill what is known about the ability of different eDNA sample types to approximate richness in space and across time. We provide guiding questions for study design and discuss the eDNA metabarcoding workflow with a focus on primers and library preparation methods. We additionally discuss important criteria for consideration of bioinformatic filtering of data sets, with recommendations for increasing transparency. Finally, looking to the future, we discuss emerging applications of eDNA metabarcoding in ecology, conservation, invasion biology, biomonitoring, and how eDNA metabarcoding can empower citizen science and biodiversity educationpublishersversionPeer reviewe

Strategies for sample labelling and library preparation in DNA metabarcoding studies.

Metabarcoding of DNA extracted from environmental or bulk specimen samples is increasingly used to profile biota in basic and applied biodiversity research because of its targeted nature that allows sequencing of genetic markers from many samples in parallel. To achieve this, PCR amplification is carried out with primers designed to target a taxonomically informative marker within a taxonomic group, and sample-specific nucleotide identifiers are added to the amplicons prior to sequencing. The latter enables assignment of the sequences back to the samples they originated from. Nucleotide identifiers can be added during the metabarcoding PCR and during "library preparation", that is, when amplicons are prepared for sequencing. Different strategies to achieve this labelling exist. All have advantages, challenges and limitations, some of which can lead to misleading results, and in the worst case compromise the fidelity of the metabarcoding data. Given the range of questions addressed using metabarcoding, ensuring that data generation is robust and fit for the chosen purpose is critically important for practitioners seeking to employ metabarcoding for biodiversity assessments. Here, we present an overview of the three main workflows for sample-specific labelling and library preparation in metabarcoding studies on Illumina sequencing platforms; one-step PCR, two-step PCR, and tagged PCR. Further, we distill the key considerations for researchers seeking to select an appropriate metabarcoding strategy for their specific study. Ultimately, by gaining insights into the consequences of different metabarcoding workflows, we hope to further consolidate the power of metabarcoding as a tool to assess biodiversity across a range of applications

An integrated spatio-temporal view of riverine biodiversity using environmental DNA metabarcoding 2

Anthropogenically forced changes in global freshwater biodiversity demands better monitoring approaches. Consequently, environmental DNA (eDNA) analysis is enabling ecosystem-scale biodiversity assessment, yet the accurate spatiotemporal resolution at which robust biodiversity information can be detected remains ambiguous. Here, using intensive, annual spatiotemporal eDNA sampling across space (five rivers in the USA and Europe, with an upper range of 20-35 km between samples), time (19 timepoints across 2017 to 2018) and environmental conditions (river flow, pH, conductivity, temperature and rainfall), we characterise the resolution at which information on diversity across the animal kingdom can be gathered from rivers. In space, beta diversity was mainly dictated by turnover, on a scale of tens of kilometres, highlighting that diversity measures are not confounded by eDNA from upstream. Fish communities showed nested assemblages along some rivers, coinciding with habitat use. Across time, seasonal life history events, including salmon and eel migration, were detected. Finally, effects of abiotic factors were taxon-specific, reflecting habitat filtering of communities rather than environmental effects on DNA molecules. We conclude that riverine eDNA metabarcoding can measure biodiversity at spatiotemporal scales relevant to species and community ecology, demonstrating its utility in delivering insights into river ecology during an epoch of environmental change

The era of reference genomes in conservation genomics

Progress in genome sequencing now enables the large-scale generation of reference genomes. Various international initiatives aim to generate reference genomes representing global biodiversity. These genomes provide unique insights into genomic diversity and architecture, thereby enabling comprehensive analyses of population and functional genomics, and are expected to revolutionize conservation genomics

Genomics of cold adaptations in the Antarctic notothenioid fish radiation

Numerous novel adaptations characterise the radiation of notothenioids, the dominant fish group in the freezing seas of the Southern Ocean. To improve understanding of the evolution of this iconic fish group, here we generate and analyse new genome assemblies for 24 species covering all major subgroups of the radiation, including five long-read assemblies. We present a new estimate for the onset of the radiation at 10.7 million years ago, based on a time-calibrated phylogeny derived from genome-wide sequence data. We identify a two-fold variation in genome size, driven by expansion of multiple transposable element families, and use the long-read data to reconstruct two evolutionarily important, highly repetitive gene family loci. First, we present the most complete reconstruction to date of the antifreeze glycoprotein gene family, whose emergence enabled survival in sub-zero temperatures, showing the expansion of the antifreeze gene locus from the ancestral to the derived state. Second, we trace the loss of haemoglobin genes in icefishes, the only vertebrates lacking functional haemoglobins, through complete reconstruction of the two haemoglobin gene clusters across notothenioid families. Both the haemoglobin and antifreeze genomic loci are characterised by multiple transposon expansions that may have driven the evolutionary history of these genes