Combining environmental DNA and remote sensing for efficient, fine-scale mapping of arthropod biodiversity.

Arthropods contribute importantly to ecosystem functioning but remain understudied. This undermines the validity of conservation decisions. Modern methods are now making arthropods easier to study, since arthropods can be mass-trapped, mass-identified, and semi-mass-quantified into 'many-row (observation), many-column (species)' datasets, with homogeneous error, high resolution, and copious environmental-covariate information. These 'novel community datasets' let us efficiently generate information on arthropod species distributions, conservation values, uncertainty, and the magnitude and direction of human impacts. We use a DNA-based method (barcode mapping) to produce an arthropod-community dataset from 121 Malaise-trap samples, and combine it with 29 remote-imagery layers using a deep neural net in a joint species distribution model. With this approach, we generate distribution maps for 76 arthropod species across a 225 km2 temperate-zone forested landscape. We combine the maps to visualize the fine-scale spatial distributions of species richness, community composition, and site irreplaceability. Old-growth forests show distinct community composition and higher species richness, and stream courses have the highest site-irreplaceability values. With this 'sideways biodiversity modelling' method, we demonstrate the feasibility of biodiversity mapping at sufficient spatial resolution to inform local management choices, while also being efficient enough to scale up to thousands of square kilometres. This article is part of the theme issue 'Towards a toolkit for global insect biodiversity monitoring'.

Novel community data in ecology-properties and prospects.

New technologies for monitoring biodiversity such as environmental (e)DNA, passive acoustic monitoring, and optical sensors promise to generate automated spatiotemporal community observations at unprecedented scales and resolutions. Here, we introduce 'novel community data' as an umbrella term for these data. We review the emerging field around novel community data, focusing on new ecological questions that could be addressed; the analytical tools available or needed to make best use of these data; and the potential implications of these developments for policy and conservation. We conclude that novel community data offer many opportunities to advance our understanding of fundamental ecological processes, including community assembly, biotic interactions, micro- and macroevolution, and overall ecosystem functioning.

Phylogenetic diversity only weakly mitigates climate-change-driven biodiversity loss in insect communities.

To help address the underrepresentation of arthropods and Asian biodiversity from climate-change assessments, we carried out year-long, weekly sampling campaigns with Malaise traps at different elevations and latitudes in Gaoligongshan National Park in southwestern China. From these 623 samples, we barcoded 10,524 beetles and compared scenarios of climate-change-induced biodiversity loss, by designating seasonal, elevational, and latitudinal subsets of beetles as communities that plausibly could go extinct as a group, which we call "loss sets". The availability of a published mitochondrial-genome-based phylogeny of the Coleoptera allowed us to compare the loss of species diversity with and without accounting for phylogenetic relatedness. We hypothesised that phylogenetic relatedness would mitigate extinction, since the extinction of any loss set would result in the disappearance of all its species but only part of its evolutionary history, which is still extant in the remaining loss sets. We found different patterns of community clustering by season and latitude, depending on whether phylogenetic information was incorporated. However, accounting for phylogeny only slightly mitigated the amount of biodiversity loss under climate change scenarios, against our expectations: there is no phylogenetic "escape clause" for biodiversity conservation. We achieve the same results whether phylogenetic information was derived from the mitogenome phylogeny or from a de novo barcode-gene tree. We encourage interested researchers to use this data set to study lineage-specific community assembly patterns in conjunction with life-history traits and environmental covariates.

Extracting abundance information from DNA-based data.

The accurate extraction of species-abundance information from DNA-based data (metabarcoding, metagenomics) could contribute usefully to diet analysis and food-web reconstruction, the inference of species interactions, the modelling of population dynamics and species distributions, the biomonitoring of environmental state and change, and the inference of false positives and negatives. However, multiple sources of bias and noise in sampling and processing combine to inject error into DNA-based data sets. To understand how to extract abundance information, it is useful to distinguish two concepts. (i) Within-sample across-species quantification describes relative species abundances in one sample. (ii) Across-sample within-species quantification describes how the abundance of each individual species varies from sample to sample, such as over a time series, an environmental gradient or different experimental treatments. First, we review the literature on methods to recover across-species abundance information (by removing what we call "species pipeline biases") and within-species abundance information (by removing what we call "pipeline noise"). We argue that many ecological questions can be answered with just within-species quantification, and we therefore demonstrate how to use a "DNA spike-in" to correct for pipeline noise and recover within-species abundance information. We also introduce a model-based estimator that can be used on data sets without a physical spike-in to approximate and correct for pipeline noise.

Measuring protected-area effectiveness using vertebrate distributions from leech iDNA.

Protected areas are key to meeting biodiversity conservation goals, but direct measures of effectiveness have proven difficult to obtain. We address this challenge by using environmental DNA from leech-ingested bloodmeals to estimate spatially-resolved vertebrate occupancies across the 677 km2 Ailaoshan reserve in Yunnan, China. From 30,468 leeches collected by 163 park rangers across 172 patrol areas, we identify 86 vertebrate species, including amphibians, mammals, birds and squamates. Multi-species occupancy modelling shows that species richness increases with elevation and distance to reserve edge. Most large mammals (e.g. sambar, black bear, serow, tufted deer) follow this pattern; the exceptions are the three domestic mammal species (cows, sheep, goats) and muntjak deer, which are more common at lower elevations. Vertebrate occupancies are a direct measure of conservation outcomes that can help guide protected-area management and improve the contributions that protected areas make towards global biodiversity goals. Here, we show the feasibility of using invertebrate-derived DNA to estimate spatially-resolved vertebrate occupancies across entire protected areas.

Climate-induced forest dieback drives compositional changes in insect communities that are more pronounced for rare species.

Species richness, abundance and biomass of insects have recently undergone marked declines in Europe. We metabarcoded 211 Malaise-trap samples to investigate whether drought-induced forest dieback and subsequent salvage logging had an impact on ca. 3000 species of flying insects in silver fir Pyrenean forests. While forest dieback had no measurable impact on species richness, there were significant changes in community composition that were consistent with those observed during natural forest succession. Importantly, most observed changes were driven by rare species. Variation was explained primarily by canopy openness at the local scale, and the tree-related microhabitat diversity and deadwood amount at landscape scales. The levels of salvage logging in our study did not explain compositional changes. We conclude that forest dieback drives changes in species assemblages that mimic natural forest succession, and markedly increases the risk of catastrophic loss of rare species through homogenization of environmental conditions.

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.

Competition-based screening helps to secure the evolutionary stability of a defensive microbiome.

BACKGROUND: The cuticular microbiomes of Acromyrmex leaf-cutting ants pose a conundrum in microbiome biology because they are freely colonisable, and yet the prevalence of the vertically transmitted bacteria Pseudonocardia, which contributes to the control of Escovopsis fungus garden disease, is never compromised by the secondary acquisition of other bacterial strains. Game theory suggests that competition-based screening can allow the selective recruitment of antibiotic-producing bacteria from the environment, by providing abundant resources to foment interference competition between bacterial species and by using Pseudonocardia to bias the outcome of competition in favour of antibiotic producers. RESULTS: Here, we use RNA-stable isotope probing (RNA-SIP) to confirm that Acromyrmex ants can maintain a range of microbial symbionts on their cuticle by supplying public resources. We then used RNA sequencing, bioassays, and competition experiments to show that vertically transmitted Pseudonocardia strains produce antibacterials that differentially reduce the growth rates of other microbes, ultimately biassing the bacterial competition to allow the selective establishment of secondary antibiotic-producing strains while excluding non-antibiotic-producing strains that would parasitise the symbiosis. CONCLUSIONS: Our findings are consistent with the hypothesis that competition-based screening is a plausible mechanism for maintaining the integrity of the co-adapted mutualism between the leaf-cutting ant farming symbiosis and its defensive microbiome. Our results have broader implications for explaining the stability of other complex symbioses involving horizontal acquisition.

Connecting high-throughput biodiversity inventories: Opportunities for a site-based genomic framework for global integration and synthesis.

High-throughput sequencing (HTS) is increasingly being used for the characterization and monitoring of biodiversity. If applied in a structured way, across broad geographical scales, it offers the potential for a much deeper understanding of global biodiversity through the integration of massive quantities of molecular inventory data generated independently at local, regional and global scales. The universality, reliability and efficiency of HTS data can potentially facilitate the seamless linking of data among species assemblages from different sites, at different hierarchical levels of diversity, for any taxonomic group and regardless of prior taxonomic knowledge. However, collective international efforts are required to optimally exploit the potential of site-based HTS data for global integration and synthesis, efforts that at present are limited to the microbial domain. To contribute to the development of an analogous strategy for the nonmicrobial terrestrial domain, an international symposium entitled "Next Generation Biodiversity Monitoring" was held in November 2019 in Nicosia (Cyprus). The symposium brought together evolutionary geneticists, ecologists and biodiversity scientists involved in diverse regional and global initiatives using HTS as a core tool for biodiversity assessment. In this review, we summarize the consensus that emerged from the 3-day symposium. We converged on the opinion that an effective terrestrial Genomic Observatories network for global biodiversity integration and synthesis should be spatially led and strategically united under the umbrella of the metabarcoding approach. Subsequently, we outline an HTS-based strategy to collectively build an integrative framework for site-based biodiversity data generation.

Horizon Scan of the Belt and Road Initiative.

The Belt and Road Initiative (BRI) represents the largest infrastructure and development project in human history, and presents risks and opportunities for ecosystems, economies, and communities. Some risks (habitat fragmentation, roadkill) are obvious, however, many of the BRI's largest challenges for development and conservation are not obvious and require extensive consideration to identify. In this first BRI Horizon Scan, we identify 11 frontier issues that may have large environmental and social impacts but are not yet recognised. More generally, the BRI will increase China's participation in international environmental governance. Thus, new cooperative modes of governance are needed to balance geopolitical, societal, and environmental interests. Upgrading and standardising global environmental standards is essential to safeguard ecological systems and human societies.

SPIKEPIPE: A metagenomic pipeline for the accurate quantification of eukaryotic species occurrences and intraspecific abundance change using DNA barcodes or mitogenomes.

The accurate quantification of eukaryotic species abundances from bulk samples remains a key challenge for community ecology and environmental biomonitoring. We resolve this challenge by combining shotgun sequencing, mapping to reference DNA barcodes or to mitogenomes, and three correction factors: (a) a percent-coverage threshold to filter out false positives, (b) an internal-standard DNA spike-in to correct for stochasticity during sequencing, and (c) technical replicates to correct for stochasticity across sequencing runs. The SPIKEPIPE pipeline achieves a strikingly high accuracy of intraspecific abundance estimates (in terms of DNA mass) from samples of known composition (mapping to barcodes R2  = .93, mitogenomes R2  = .95) and a high repeatability across environmental-sample replicates (barcodes R2  = .94, mitogenomes R2  = .93). As proof of concept, we sequence arthropod samples from the High Arctic, systematically collected over 17 years, detecting changes in species richness, species-specific abundances, and phenology. SPIKEPIPE provides cost-efficient and reliable quantification of eukaryotic communities.

Environmental DNA facilitates accurate, inexpensive, and multiyear population estimates of millions of anadromous fish.

Although environmental DNA shed from an organism is now widely used for species detection in a wide variety of contexts, mobilizing environmental DNA for management requires estimation of population size and trends in addition to assessing presence or absence. However, the efficacy of environmental-DNA-based indices of abundance for long-term population monitoring have not yet been assessed. Here we report on the relationship between six years of mark-recapture population estimates for eulachon (Thaleichthys pacificus) and "eDNA rates" which are calculated from the product of stream flow and DNA concentration. Eulachon are a culturally and biologically important anadromous fish that have significantly declined in the southern part of their range but were historically rendered into oil and traded. Both the peak eDNA rate and the area under the curve of the daily eDNA rate were highly predictive of the mark-recapture population estimate, explaining 84.96% and 92.53% of the deviance, respectively. Even in the absence of flow correction, the peak of the daily eDNA concentration explained an astonishing 89.53% while the area under the curve explained 90.74% of the deviance. These results support the use of eDNA to monitor eulachon population trends and represent a >80% cost savings over mark-recapture, which could be further increased with automated water sampling, reduced replication, and focused temporal sampling. Due to its logistical ease and affordability, eDNA sampling can facilitate monitoring a larger number of rivers and in remote locations where mark-recapture is infeasible.

Efficient assembly and long-term stability of defensive microbiomes via private resources and community bistability.

Understanding the mechanisms that promote the assembly and maintenance of host-beneficial microbiomes is an open problem. Empirical evidence supports the idea that animal and plant hosts can combine 'private resources' with the ecological phenomenon known as 'community bistability' to favour some microbial strains over others. We briefly review evidence showing that hosts can: (i) protect the growth of beneficial strains in an isolated habitat, (ii) use antibiotics to suppress non-beneficial, competitor strains, and (iii) provide resources that only beneficial strains are able to translate into an increased rate of growth, reproduction, or antibiotic production. We then demonstrate in a spatially explicit, individual-based model that these three mechanisms act similarly by selectively promoting the initial proliferation of preferred strains, that is, by acting as a private resource. The faster early growth of preferred strains, combined with the phenomenon of 'community bistability,' allows those strains to continue to dominate the microbiome even after the private resource is withdrawn or made public. This is because after a beneficial colony reaches a sufficiently large size, it can resist invasion by parasites without further private support from the host. We further explicitly model localized microbial interactions and diffusion dynamics, and we show that an intermediate level of antibiotic diffusion is the most efficient mechanism in promoting preferred strains and that there is a wide range of parameters under which hosts can promote the assembly of a self-sustaining defensive microbiome. This in turn supports the idea that hosts readily evolve to promote host-beneficial defensive microbiomes.

An efficient and robust laboratory workflow and tetrapod database for larger scale environmental DNA studies.

BACKGROUND: The use of environmental DNA for species detection via metabarcoding is growing rapidly. We present a co-designed lab workflow and bioinformatic pipeline to mitigate the 2 most important risks of environmental DNA use: sample contamination and taxonomic misassignment. These risks arise from the need for polymerase chain reaction (PCR) amplification to detect the trace amounts of DNA combined with the necessity of using short target regions due to DNA degradation. FINDINGS: Our high-throughput workflow minimizes these risks via a 4-step strategy: (i) technical replication with 2 PCR replicates and 2 extraction replicates; (ii) using multi-markers (12S,16S,CytB); (iii) a "twin-tagging," 2-step PCR protocol; and (iv) use of the probabilistic taxonomic assignment method PROTAX, which can account for incomplete reference databases. Because annotation errors in the reference sequences can result in taxonomic misassignment, we supply a protocol for curating sequence datasets. For some taxonomic groups and some markers, curation resulted in >50% of sequences being deleted from public reference databases, owing to (i) limited overlap between our target amplicon and reference sequences, (ii) mislabelling of reference sequences, and (iii) redundancy. Finally, we provide a bioinformatic pipeline to process amplicons and conduct PROTAX assignment and tested it on an invertebrate-derived DNA dataset from 1,532 leeches from Sabah, Malaysia. Twin-tagging allowed us to detect and exclude sequences with non-matching tags. The smallest DNA fragment (16S) amplified most frequently for all samples but was less powerful for discriminating at species rank. Using a stringent and lax acceptance criterion we found 162 (stringent) and 190 (lax) vertebrate detections of 95 (stringent) and 109 (lax) leech samples. CONCLUSIONS: Our metabarcoding workflow should help research groups increase the robustness of their results and therefore facilitate wider use of environmental and invertebrate-derived DNA, which is turning into a valuable source of ecological and conservation information on tetrapods.

Environmental DNA for the enumeration and management of Pacific salmon.

Pacific salmon are a keystone resource in Alaska, generating annual revenues of well over ~US$500 million/year. Due to their anadromous life history, adult spawners distribute amongst thousands of streams, posing a huge management challenge. Currently, spawners are enumerated at just a few streams because of reliance on human counters and, rarely, sonar. The ability to detect organisms by shed tissue (environmental DNA, eDNA) promises a more efficient counting method. However, although eDNA correlates generally with local fish abundances, we do not know if eDNA can accurately enumerate salmon. Here we show that daily, and near-daily, flow-corrected eDNA rate closely tracks daily numbers of returning sockeye and coho spawners and outmigrating sockeye smolts. eDNA thus promises accurate and efficient enumeration, but to deliver the most robust numbers will need higher-resolution stream-flow data, at-least-daily sampling, and a focus on species with simple life histories, since shedding rate varies amongst jacks, juveniles, and adults.

The genetic basis of a social polymorphism in halictid bees.

The emergence of eusociality represents a major evolutionary transition from solitary to group reproduction. The most commonly studied eusocial species, honey bees and ants, represent the behavioral extremes of social evolution but lack close relatives that are non-social. Unlike these species, the halictid bee Lasioglossum albipes produces both solitary and eusocial nests and this intraspecific variation has a genetic basis. Here, we identify genetic variants associated with this polymorphism, including one located in the intron of syntaxin 1a (syx1a), a gene that mediates synaptic vesicle release. We show that this variant can alter gene expression in a pattern consistent with differences between social and solitary bees. Surprisingly, syx1a and several other genes associated with sociality in L. albipes have also been implicated in autism spectrum disorder in humans. Thus, genes underlying behavioral variation in L. albipes may also shape social behaviors across a wide range of taxa, including humans.

Why the COI barcode should be the community DNA metabarcode for the metazoa.

Metabarcoding of complex metazoan communities is increasingly being used to measure biodiversity in terrestrial, freshwater and marine ecosystems, revolutionizing our ability to observe patterns and infer processes regarding the origin and conservation of biodiversity. A fundamentally important question is which genetic marker to amplify, and although the mitochondrial cytochrome oxidase subunit I (COI) gene is one of the more widely used markers in metabarcoding for the Metazoa, doubts have recently been raised about its suitability. We argue that (a) the extensive coverage of reference sequence databases for COI; (b) the variation it presents; (c) the comparative advantages for denoising protein-coding genes; and (d) recent advances in DNA sequencing protocols argue in favour of standardizing for the use of COI for metazoan community samples. We also highlight where research efforts should focus to maximize the utility of metabarcoding.

Chemical warfare between leafcutter ant symbionts and a co-evolved pathogen.

Acromyrmex leafcutter ants form a mutually beneficial symbiosis with the fungus Leucoagaricus gongylophorus and with Pseudonocardia bacteria. Both are vertically transmitted and actively maintained by the ants. The fungus garden is manured with freshly cut leaves and provides the sole food for the ant larvae, while Pseudonocardia cultures are reared on the ant-cuticle and make antifungal metabolites to help protect the cultivar against disease. If left unchecked, specialized parasitic Escovopsis fungi can overrun the fungus garden and lead to colony collapse. We report that Escovopsis upregulates the production of two specialized metabolites when it infects the cultivar. These compounds inhibit Pseudonocardia and one, shearinine D, also reduces worker behavioral defenses and is ultimately lethal when it accumulates in ant tissues. Our results are consistent with an active evolutionary arms race between Pseudonocardia and Escovopsis, which modifies both bacterial and behavioral defenses such that colony collapse is unavoidable once Escovopsis infections escalate.

Debugging diversity - a pan-continental exploration of the potential of terrestrial blood-feeding leeches as a vertebrate monitoring tool.

The use of environmental DNA (eDNA) has become an applicable noninvasive tool with which to obtain information about biodiversity. A subdiscipline of eDNA is iDNA (invertebrate-derived DNA), where genetic material ingested by invertebrates is used to characterize the biodiversity of the species that served as hosts. While promising, these techniques are still in their infancy, as they have only been explored on limited numbers of samples from only a single or a few different locations. In this study, we investigate the suitability of iDNA extracted from more than 3,000 haematophagous terrestrial leeches as a tool for detecting a wide range of terrestrial vertebrates across five different geographical regions on three different continents. These regions cover almost the full geographical range of haematophagous terrestrial leeches, thus representing all parts of the world where this method might apply. We identify host taxa through metabarcoding coupled with high-throughput sequencing on Illumina and IonTorrent sequencing platforms to decrease economic costs and workload and thereby make the approach attractive for practitioners in conservation management. We identified hosts in four different taxonomic vertebrate classes: mammals, birds, reptiles and amphibians, belonging to at least 42 different taxonomic families. We find that vertebrate blood ingested by haematophagous terrestrial leeches throughout their distribution is a viable source of DNA with which to examine a wide range of vertebrates. Thus, this study provides encouraging support for the potential of haematophagous terrestrial leeches as a tool for detecting and monitoring terrestrial vertebrate biodiversity.

Metabarcoding of freshwater invertebrates to detect the effects of a pesticide spill.

Biomonitoring underpins the environmental assessment of freshwater ecosystems and guides management and conservation. Current methodology for surveys of (macro)invertebrates uses coarse taxonomic identification where species-level resolution is difficult to obtain. Next-generation sequencing of entire assemblages (metabarcoding) provides a new approach for species detection, but requires further validation. We used metabarcoding of invertebrate assemblages with two fragments of the cox1 "barcode" and partial nuclear ribosomal (SSU) genes, to assess the effects of a pesticide spill in the River Kennet (southern England). Operational taxonomic unit (OTU) recovery was tested under 72 parameters (read denoising, filtering, pair merging and clustering). Similar taxonomic profiles were obtained under a broad range of parameters. The SSU marker recovered Platyhelminthes and Nematoda, missed by cox1, while Rotifera were only amplified with cox1. A reference set was created from all available barcode entries for Arthropoda in the BOLD database and clustered into OTUs. The River Kennet metabarcoding produced matches to 207 of these reference OTUs, five times the number of species recognized with morphological monitoring. The increase was due to the following: greater taxonomic resolution (e.g., splitting a single morphotaxon "Chironomidae" into 55 named OTUs); splitting of Linnaean binomials into multiple molecular OTUs; and the use of a filtration-flotation protocol for extraction of minute specimens (meiofauna). Community analyses revealed strong differences between "impacted" vs. "control" samples, detectable with each gene marker, for each major taxonomic group, and for meio- and macrofaunal samples separately. Thus, highly resolved taxonomic data can be extracted at a fraction of the time and cost of traditional nonmolecular methods, opening new avenues for freshwater invertebrate biodiversity monitoring and molecular ecology.