Environmental DNA

Environmental nucleic acid (eRNA and eDNA) metabarcoding is a promising tool for biodiversity assessment as it supports low-cost, noninvasive biological surveys. A critical challenge for eRNA/eDNA analyses is accounting for nucleic acid contamination that can compromise the accuracy of biomonitoring results. The primary aim of the current study was to examine carcasses as potential sources of eRNA/eDNA contamination, a contribution that remains poorly understood. Using Japanese medaka (Oryzias latipes), we quantified the detectability and persistence of carcass-derived eRNA/eDNA in water through two complementary experiments: (i) a shedding study comparing eRNA/eDNA release from live and dead individuals and (ii) a decay experiment measuring temperature-dependent degradation from 4 to 34 {degrees}C. Dead fish released detectable amounts of both eDNA and eRNA, indicating that eRNA-based surveys cannot fully eliminate carcass-derived signals. Arrhenius-based analysis showed that eRNA degradation was more temperature-dependent than eDNA degradation. These results suggest that eRNA may improve temporal resolution for recent organismal presence under warm conditions, whereas carcass-derived eRNA may persist under cold conditions and complicate interpretation of positive detections. Overall, eRNA may help reduce carcass-related false positives, particularly in warm conditions, but should be paired with temperature-aware sampling strategies and temporal replication during colder periods.

Freshwater biodiversity is declining at alarming rates globally, yet our capacity to monitor it depends heavily on the methods used for biodiversity assessment. Stream benthic macroinvertebrates are widely used as bioindicators, but traditional morphological identification is labor-intensive and limited by taxonomic expertise, while DNA metabarcoding offers a promising but incompletely evaluated alternative. Here, we compared biodiversity patterns detected by four macroinvertebrate sampling and identification methods (morphological identification of benthic samples, morphological identification of combined benthic and stream-edge samples, eDNA metabarcoding of bulk benthic samples, and eDNA metabarcoding of streamwater) across 62 sites in seven watersheds spanning the southern continental United States. We compared methods across three facets of biodiversity (gamma diversity, alpha diversity, and taxonomic composition) and three taxonomic levels (family, genus, and species). We found that which method detected more taxa shifted with taxonomic resolution: morphological methods detected more families and genera, while metabarcoding methods detected more species. Bulk benthic metabarcoding detected more families and genera than water eDNA metabarcoding, consistent with the "watered-down biodiversity" effect of DNA dilution in water samples, but the two metabarcoding methods did not differ significantly in species richness. All four methods detected largely distinct suites of taxa, with metabarcoding methods yielding more unique taxa than morphological methods. Notably, taxa uniquely detected by water eDNA were dominated by soft-bodied organisms (oligochaetes, leeches, earthworms) and water-column-associated taxa, while benthic eDNA uniquely detected hard-bodied EPT insects. We attribute this pattern to fundamental differences in eDNA shedding, transport, and deposition dynamics across organism types. These results demonstrate that no single method captures a complete picture of stream macroinvertebrate biodiversity, and that method choice should be guided by the taxonomic resolution and community components most relevant to the goals of the bioassessment program.

Environmental DNA (eDNA) offers a non-invasive alternative to traditional, more destructive sampling methods for determining species occupancy at ecological sites of interest. Aquatic eDNA sampling entails filtering a known volume of water to capture and detect genetic material shed by organisms. While the influence of individual environmental properties on the presence of target eDNA has been widely studied, it remains unclear how variables like temperature, pH, flow rate and conductivity correlate collectively with site electrofishing counts and eDNA concentrations. Resolving this question is important for two reasons: (1) typically, only eDNA, not physical specimens, is collected and measured, and (2) when methods like electrofishing and eDNA sampling are used in tandem, results often differ. Here unsupervised association rule-based machine learning is employed to discover interesting relationships among sampled covariates within a previously published case study of native brook trout (Salvelinus fontinalis) collected from Hanlon Creek (Guelph, Ontario, Canada) in September 2019. From a dataset of only 126 observations, the mining process revealed over 12000 plausible association rules linking covariates to eDNA concentrations (low/high) and electrofishing outcomes (absence/presence of brook trout). A strict pruning strategy reduced this ruleset to a manageable size of 153 associations, some of which were corroborated by existing literature, and some of which were novel (such as those potentially relating electrical conductivity to microbial and enzymatic activity). The entire workflow is included as a new R package called RulesTools. These results highlight the promise of association rule mining as a tool for guiding eDNA metadata collection, complementing statistical modelling, and informing conservation and management decision-making.

Freshwater bioassessment relies on assessing aquatic assemblages to infer ecological conditions, yet conventional surveys require extensive field sampling, specimen processing, and specialized taxonomic expertise. Existing environmental DNA (eDNA) methods have not yet provided a practical alternative to conventional macroinvertebrate assays in part because current approaches cannot feasibly recover broad taxonomic diversity at sufficient taxonomic resolution. Here, we evaluated targeted hybridization capture of mitochondrial cytochrome oxidase I (COI) target sequences as a unified molecular approach for cross-phylum freshwater bioassessment. Environmental DNA was collected at 18 sites along 63 km of Boulder Creek spanning nearly 1,500 m of elevation from forested headwaters to agricultural plains. COI targets were enriched using custom RNA bait panels designed to target regional freshwater arthropods, annelids, and molluscs. Hybridization capture increased recovery of COI sequences [~]1,760-fold relative to unenriched shotgun libraries, generating Folmer-region COI contigs that averaged [~]400 bp. Across the watershed, we recovered sequences for approximately 450 macroinvertebrate genera across 8 phyla. Detected macroinvertebrate richness averaged 56 genera per site and increased down Boulder Canyon before declining downstream of the city. Macroinvertebrate assemblage composition from hybridization capture paralleled patterns observed with past conventional bioassessment. These results demonstrate that targeted hybridization capture enables robust, cross-phylum detection of species used for freshwater bioassessment from environmental DNA.

Inshore coastal waters are almost invariably supersaturated with respect to methane and are thereby sources of methane to the atmosphere. We investigated floating algal mats and surface waters of four contrasting inshore habitats and quantified methane concentrations, sea-to-air emissions, and microbial community composition of surface waters over a seasonal cycle to determine the potential for in-situ microbial methane production in shallow oxygen-saturated surface waters with floating algal biomass. 16S rDNA sequencing indicated that Archaea belonging to the genera Methanocorpusculum, Methanosarcina, Candidatus Methanomethylophilus, and some genera from order Methanobacteriales occurred in the floating algal mats. qPCR of the genes encoding the methyl coenzyme M reductase mcrA revealed the highest expression levels during the warmest sampling periods supporting active methane production directly in surface water. Co-occurrence of the Archaea sequences and sequences belonging to the cyanobacterium strain Nodularia PCC 9350 suggests a structural relationship. Our study underscores the significant, yet underexplored impact of methane production on the surface in aggregates of floating algal material. While Nodularia and methanogens can exist independently in surface waters, their co-occurrence in algal mats reveals where the layered mat structure creates distinct microenvironments that facilitate direct metabolic exchange and provide physical stability for both groups, thereby potentially enhancing methane production in these shallow coastal systems.

O_LIEnvironmental DNA (eDNA) methods are developing rapidly for ecological surveys, and passive eDNA sampling has emerged as a promising approach for integrating DNA signals over deployment time. However, how deployment duration affects the amount of detectable DNA retained by a sampler remains poorly understood. C_LIO_LIHere, an analytical model was developed to examine how DNA input, degradation, finite substrate capacity and residual retention of degraded DNA shape passive eDNA accumulation. The model distinguishes detectable adsorbed DNA from degraded, non-detectable DNA that may remain on the substrate and continue to occupy capacity. The residual-retention parameter,{theta} , represents the fraction of degraded DNA that remains capacity-occupying, with{theta} = 0 corresponding to complete replacement and{theta} = 1 to complete non-replacement. C_LIO_LIThe model predicts three key behaviours. First, when degraded DNA does not occupy substrate capacity ({theta} = 0), detectable eDNA accumulates monotonically towards equilibrium, but equilibrium recovery increases less than proportionally with DNA input. Thus, passive-sampler measurements can compress quantitative differences in environmental DNA supply. Second, when degraded DNA remains capacity-occupying ({theta} > 0), detectable eDNA can reach a finite peak and subsequently decline. Higher DNA input increases peak yield but shifts the peak earlier, whereas greater substrate capacity increases peak yield and delays the peak. Third, under prolonged deployment with{theta} > 0, a higher-input condition can yield less detectable eDNA than a lower-input condition, reversing the expected input-rate ranking. C_LIO_LIThese results show that passive eDNA recovery can follow saturating, unimodal or intermediate dynamics depending on substrate capacity and post-adsorption DNA fate. Thus, retrieval time cannot be optimised by adjusting deployment duration alone. Although investigators can choose deployment duration and sampler design, including substrate capacity, optimisation also requires calibration or explicit assumptions about ambient DNA supply, DNA degradation rate and residual retention of degraded DNA. C_LI

Ranges of small benthic fauna are notoriously difficult to assess. In some of these cases, modern eDNA methods can shed light on species occurrence. Here we conduct an exploratory study on the fish eDNA recoverable from the gastrovascular cavities of the easy-to-sample pore water siphoning benthic invertebrate, Cassiopea, across six sites within the Florida Keys. Twenty-seven fish 12S identities were recovered from water samples, two from sediment samples, and seventeen from Cassiopea gut swabs. In total, thirty-two different species were identified from nineteen families, including one shark species (Ginglymostoma cirratum), and five species of cryptobenthic reef fishes (f: Gobiidae, Labrisomidae). Additionally, five species were identified from medusae samples that were not recovered in water or sediment samples. The species identities recovered may provide insight into the fish in direct proximity to Cassiopea assemblages, as well as indicate that Cassiopea may accrue disproportionate eDNA from cryptobenthic reef fish compared to surrounding environmental samples. The unorthodox sampling technique of using eDNA recovered from jellyfish stomachs yields another avenue for epibenthic community data acquisition.

Environmental DNA (eDNA) metabarcoding has become a powerful approach for biodiversity assessment, yet whether different environmental substrates provide equivalent or complementary representations of terrestrial biodiversity remains unresolved. Here we addressed this question by comparing insect assemblages recovered from Malaise traps and six environmental substrates (water, sediment, soil, spiderwebs, deadwood, and anthills) across 45 forest locations using a standardized COI metabarcoding workflow. Substrate identity consistently shaped insect detection, diversity, taxonomic representation, and community composition, with each substrate recovering distinct components of insect biodiversity rather than a common biodiversity signal. Malaise traps recovered the greatest overall richness, while water and spiderwebs contributed the largest proportion of additional taxa, demonstrating that no single substrate represented the full spectrum of insect biodiversity. These findings show that environmental substrates capture complementary representations of biodiversity through distinct ecological processes of eDNA deposition, transport, accumulation, and persistence. Different substrates should therefore be interpreted as complementary sources of biodiversity observations instead of interchangeable sampling media, and their integration can support more comprehensive ecological inference. Our study identifies substrate complementarity as a key consideration for interpreting terrestrial eDNA observations and for designing future biodiversity surveys.

Environmental DNA (eDNA) has emerged as a transformative tool for biodiversity monitoring, offering a non-invasive means to detect and identify species across various ecosystems. However, despite its potential, current eDNA methodologies have significant limitations, including limited sensitivity, restriction to a specific taxonomic group, lengthy processing times, the need for specialized laboratory conditions and equipment, as well as computational bottlenecks in equipment, connectivity, and algorithms. Overcoming these limitations is crucial for the real-time monitoring of a high number of taxa in field-based scenarios. Here we present an integrated eDNA workflow from extraction to data analysis that mitigates several of these challenges through several technical innovations. Our approach adapts the Tagsteady protocol to Nanopore sequencing, integrates multiplex PCR for simultaneous plant and animal detection, and introduces RTBioScan, a real-time analysis pipeline employing hierarchical basecalling and OTU-level consensus generation to achieve high-accuracy taxonomic assignments during sequencing. Using a single set of universal primers, we integrate metabarcoding and barcoding into a cohesive protocol, utilizing universal markers for both animals and plants (cytochrome oxidase 1 region, COI, and Internal Transcribed Spacer 2, ITS2). This comprehensive approach can detect and identify a broad spectrum of species, from individual organisms to entire communities in a single workflow.

Soil nematodes play key roles in regulating organic matter decomposition, nutrient cycling and trophic interactions in the soil ecosystem, and are very useful bioindicators of soil condition and soil health. But to characterise them, we need reliable measurements of nematode biodiversity in soils. DNA-based techniques such as metabarcoding have recently explored their biodiversity, with successful results. However, there are many methodological steps that can modify the outcome considerably and require informed decisions. In this study, we tested the effect on nematode alpha and beta diversity metrics of three methodological steps, namely i) type of extraction of genetic material from the matrix, either from soil directly with different starting amount of material or extracting nematodes first via the Baermann or Oostenbrink methods, ii) the choice of taxonomically-relevant DNA segment for amplification, with a nematode specific primer (NemF/18Sr2b) and a universal eukaryotic primer (3NDf/1132rmod), both within 18S rDNA, and iii) the selection of appropriate reference library for taxonomic assignment (comparing three nematode specialised databases and three broad taxonomic databases). Observed differences in alpha diversity were more pronounced when comparing the nematode extraction vs soil extractions. In particular, direct soil extractions co-extracted many non-nematode taxa, especially if amplified with a universal primer, which can significantly reduce the number of nematode reads obtained and therefore the accuracy of nematode community indexes and other nematode-based metrics. The choice of the reference database strongly affected the taxonomic identification, which calls for extreme caution when making this selection. Notably, specialised databases tended to over assign reads to nematodes that were detected as non-nematodes by broad taxonomic databases. However, beta diversity metrics were consistent across methods, highlighting these metrics as robust against methodological choices. Our study clearly demonstrates that the choices of methods for nematode biodiversity studies using molecular tools can have a big impact on the results, and decisions should be made consciously and carefully.

Hydropower development has emerged as a major driver of global environmental change, causing significant ecological damage to freshwater ecosystems and threatening aquatic biodiversity. However, the impact of cascade dams on fish communities in large river systems remains poorly understood, particularly in biodiversity hotspots such as China. Here, we used environmental DNA (eDNA) metabarcoding to assess the spatiotemporal patterns of fish diversity and community structure in the Ganjiang River, the largest tributary of the Yangtze River, in relation to cascade hydropower development. We collected water samples from 19 sites along the main stem and major tributaries in both wet and dry seasons, and analyzed the eDNA data using high-throughput sequencing and advanced bioinformatics pipelines. Our results revealed significant seasonal and spatial variations in fish diversity and community composition, with lower species richness and distinct assemblages in the impounded sections upstream of the dams compared to the downstream reaches. The fish communities were strongly influenced by environmental factors such as water temperature, pH, dissolved oxygen, and nutrients, which exhibited complex interactions with dam operation and land use practices. Spatial scale analyses further indicated that the impact of dams on fish diversity was most pronounced at the local scale, but also detectable at the regional scale. Our findings provide novel insights into the ecological consequences of cascade hydro-power development on fish communities and highlight the importance of considering spatial scales and environmental heterogeneity in assessing the impacts of human activities on freshwater biodiversity. Our study demonstrates the power of eDNA metabarcoding as a cost-effective and non-invasive tool for biodiversity assessment and conservation planning in large river systems facing multiple stressors and knowledge gaps.

Passive sampling is the deployment of a collection material in the environment to continuously capture environmental DNA (eDNA) over time, offering the potential to integrate biodiversity signals while reducing the need for repeated active water collection. However, the mechanisms governing eDNA capture and retention on passive samplers remain poorly understood, limiting the interpretation of passive eDNA signals and their broader application. Here, we investigated the mechanistic performance of glass fibre passive samplers using controlled mesocosm experiments with three invasive freshwater bivalves: zebra mussels (Dreissena polymorpha), quagga mussels (Dreissena bugensis), and Asian clams (Corbicula fluminea). Specifically, we quantified eDNA accumulation dynamics, evaluated the contribution of different eDNA states, tested the persistence of captured eDNA, and compared passive sampler signals with conventional active sampling. Passive samplers rapidly accumulated target eDNA within hours of deployment, after which concentrations either plateaued or continued to increase depending on species. Sequential transfer of passive samplers between mesocosms containing different species showed that previously captured eDNA declined while new target eDNA accumulated to concentrations comparable to freshly deployed samplers, demonstrating continual turnover rather than permanent retention. Dissolved eDNA showed little evidence of accumulation beyond the concentration retained in the pore water within the membrane, suggesting that it is unlikely to be the dominant contributor to long-term passive sampler signals. Instead, the observed variability among replicate samplers, together with the physical properties of glass fibre membranes, suggests that membrane-bound and particulate eDNA are the primary contributors to passive eDNA capture. Collectively, these findings support a model in which glass fibre passive sampler signals reflect a dynamic equilibrium between ongoing eDNA capture and concurrent loss processes rather than cumulative accumulation over time. This mechanistic framework provides a foundation for interpreting passive eDNA data and informs the future development of passive sampling materials, deployment strategies, and biodiversity monitoring applications.

Environmental DNA (eDNA) metabarcoding enables sensitive, non-invasive assessment of fish communities, but highly multiplexed analyses using Oxford Nanopore Technologies (ONT) platforms require stringent control of sample-index misassignment and sequencing errors. We developed a MiFish experimental workflow combining unique dual indexes, a library protocol that omitted end preparation and used 5'-phosphorylated primers, BLAST-based demultiplexing, quality-dependent clustering, consensus generation, and haplotype partitioning by SNP/INDEL patterns. Omitting pooled end-prep reduced the mean index-chimera rate from 0.0674% to 0.000420%, a 160-fold reduction. We applied the workflow to an archive of seawater samples collected weekly off Monbetsu, Hokkaido, Japan, from 2012 to 2022. Relative read abundance (RRA) data were obtained for 274 samples, and eight taxa showed significant seasonality. For six of these taxa, the three-year mean RRA peaks coincided with reported spawning periods in Hokkaido. The workflow substantially reduced index misassignment and enabled cost-efficient, highly multiplexed analysis of a decadal fish eDNA time series.

Effective management of invasive species requires surveillance methods that reliably detect populations while minimizing field effort. Environmental DNA (eDNA) offers a potentially rapid alternative to conventional monitoring, but direct comparisons with other non-invasive methods remain limited for invasive mammals. Here, we compared eDNA water sampling and camera trapping for detecting established populations of coypu (Myocastor coypus), an invasive semi-aquatic rodent. Using replicated data collected across Mediterranean river systems in southern France, we estimated detection probabilities conditional on coypu presence using Bayesian generalized linear mixed models and quantified the sampling effort required to achieve high cumulative detection probability. Detection probability was 93.0% (95% credible interval: 72.1-98.9%) for a single eDNA water sample, compared with 52.8% (22.6-91.1%) for 30 camera-trap days, with a 96.9% posterior probability that eDNA detection was higher. Two eDNA samples were sufficient to achieve a median cumulative detection probability above 95%, compared with four 30-day camera-trapping periods. Detection also varied at different spatial scales, with camera-trap detectability being heterogeneous among local camera locations and eDNA detectability varying more among catchments. These results show that eDNA provides a rapid and reliable approach for confirming the presence of established coypu populations, while camera traps provide complementary information on activity, behaviour and habitat use. Combining rapid eDNA screening with targeted camera trapping may therefore provide an efficient surveillance strategy for invasive semi-aquatic mammals.

Remote and vast ecosystems present a challenge for biodiversity monitoring. The Central Kalahari Game Reserve in Botswana is one such ecosystem spanning 52,800 km2, a mosaic of fossilized rivers, clay pans, semi-arid sand dunes, scrub, and woodland. It is one of the last remaining untouched wildernesses in the world and hosts unique biodiversity, including populations of threatened mammals. Due to its extreme arid environment, size, and inaccessibility, surveying biodiversity across the reserve is both logistically and technically difficult. Here we demonstrate that a low-effort approach of sampling soil across the reserve is an efficient way to rapidly assess the terrestrial mammal biodiversity using eDNA metabarcoding. We found that eDNA from soil near waterholes performed better at capturing local biodiversity than soil from roads. eDNA metabarcoding detected 38 mammal species, 15 bird species and two amphibians and a simultaneously conducted camera trapping study detected 29 mammal species. eDNA was particularly powerful for detecting small-bodied taxa, including murids, mongooses and bats, which were largely missed by camera traps, while camera trapping was aimed at and performed well for detecting felids. Mammal species that had a higher activity nearby eDNA sampling points (i.e., a higher number of camera trapping recordings nearby) had a higher probability of being detected via eDNA. We propose that simple soil collection for eDNA metabarcoding, particularly close to animal congregation points such as savanna waterholes, offers a practical, scalable and cost-effective strategy for DNA-based biomonitoring in large, remote areas such those in Southern Africa.

Molecular identification of cetacean bone remains can be limited by DNA degradation and the presence of PCR inhibitors. Here, we present an optimized DNA extraction protocol based on a total demineralization method for environmentally exposed cetacean bones. The protocol uses 100 mg of bone powder, 24 h digestion with EDTA, N-lauroylsarcosine, and proteinase K, followed by a modified silica-column purification. Nine environmentally degraded bone samples representing eight individuals were processed. DNA concentrations ranged from 7.3 to 57.1 ng/uL (mean SD = 25.91- 13.91 ng/uL). The mitochondrial cytochrome b gene was successfully amplified from all samples using conventional PCR, and five samples (55.6%) yielded sequences suitable for downstream analysis. BLASTn identified Balaenoptera physalus as the closest database match for all recovered sequences, and phylogenetic analysis further supported their association with B. physalus reference sequences. These results demonstrate that the proposed protocol provides a practical approach for recovering amplifiable and molecularly informative mitochondrial DNA from environmentally degraded cetacean bone material, facilitating molecular identification from challenging skeletal remains.

Benthic algae integrate catchment-and reach-scale pressures in streams. We assessed land-use effects on benthic algal assemblage structure, chlorophyll-a, ash-free dry mass (AFDM), and autotrophic index across 24 headwater streams in the Sondu-Miriu River basin, Kenya, and explored wet-dry patterns in two monitoring streams. We sampled across four land-use categories: natural forest (NF), tea and tree plantations (TTP), smallholder agriculture (SHA), and smallholder tea (SHT). Synoptic sampling occurred in August 2025, and one NF stream and one SHA stream were sampled five times between November 2024 and May 2025. Shannon and Simpson diversity were highest in NF streams and lowest in SHT streams. Chlorophyll-a and AFDM were highest in SHA streams; chlorophyll-a was lowest in SHT, while AFDM and autotrophic index were lowest in NF. Relative abundance patterns showed that SHA streams were dominated by Lyngbya and Stigeoclonium, whereas SHT streams were dominated mainly by Stigeoclonium; NF streams supported persistent diatom-rich assemblages. Non-metric multidimensional scaling showed land-use separation of benthic algal assemblages, while redundancy analysis indicated weak, non-significant evidence of environmental associations in the synoptic dataset. Composition and biomass metrics tracked land-use gradients and wet-dry patterns, supporting periphyton-based bioassessment for monitoring disturbance in Afrotropical headwater streams.

The marine biodiversity inhabiting rocky shores in the Colombian Pacific remains largely undocumented, primarily due to geographic isolation, logistical challenges, and socio-political constraints. To address the existing knowledge gap, we conducted an expedition to enhance baseline biodiversity knowledge in rocky shores by integrating multiple complementary approaches, including visual censuses, specimen collection with morphological identification, environmental DNA (eDNA) metabarcoding and DNA barcodes. eDNA samples were collected at four coastal sites adjacent to rocky substrates, along with biological specimens obtained from fourteen locations through SCUBA diving at depths ranging from 1 to 25 meters. Tissue samples were subjected to genomic DNA isolation, followed by the generation and validation of cytochrome c oxidase subunit I (COI) barcode sequences, which were subsequently corroborated through taxonomic assessment to ensure accurate species identification. eDNA metabarcoding analyses yielded over 7 million high-quality sequence reads. Although taxonomic resolution at the species level was constrained by the limited completeness of reference sequence databases, a total of 106 species and 83 families were successfully identified, predominantly within the classes Actinopteri, Chondrichthyes, and marine mammals. From the 769 specimens obtained we generated 871 sequences, including 414 validated COI barcodes representing 76 species across 64 families. The integration of DNA barcoding and eDNA approaches resulted in over 1,400 taxonomic detections spanning five phyla, with only six species shared between methodologies. Richness and diversity varied among sites, and revealed significant differences along the coastline between Jurado and Cupica Gulf. All sequences were deposited in BOLDsystems database under the CCBIO project and were visualized through OBIS and GBIF databases. These findings provide the first molecular-based baseline for rocky shore biodiversity in the Colombian Pacific, highlighting the value of integrative approaches for monitoring and conservation.

Ecological forecasts often rely on established relationships between the ecological process of interest and predictor variables that are more easily measured. Proliferations of benthic (i.e., bottom-dwelling) cyanobacteria have been increasingly observed in rivers globally and are an emerging ecological forecasting issue as they pose a public health threat due to the production of potent neurotoxins known as anatoxins. Controls on these benthic cyanobacteria are poorly understood, thus predicting or forecasting their extent and anatoxin production is a significant challenge. Here, we measured benthic cyanobacterial cover and anatoxin concentrations for two common taxa associated with anatoxins (Microcoleus and Anabaena) at biweekly to weekly intervals during June to September in 2022 and 2023 in three northern Californian rivers. We then built predictive models to test how incorporation of a biotic predictor (river reach-scale gross primary productivity [GPP]) affected predictive accuracy in addition to widely measured abiotic predictors (i.e., nutrients, discharge, and temperature). Temporal patterns in taxon-specific benthic cyanobacterial cover and anatoxin concentrations were highly variable among rivers and between taxa. While Microcoleus cover peaked in rivers during periods of relatively low GPP, there were no clear relationships between GPP and Anabaena cover nor anatoxin concentrations of either taxon among rivers. Among multiple reaches sampled weekly in the South Fork Eel River, magnitudes of taxon-specific benthic cyanobacterial cover and anatoxin concentrations differed, but the timing of peak taxon-specific cover and anatoxin concentrations were generally consistent. Furthermore, Anabaena displayed a hysteresis relationship where increases in cover were followed by increases in anatoxin concentrations while Microcoleus lacked such relationship. While incorporating GPP as a covariate improved our predictions of Anabaena cover, we had more success predicting Microcoleus cover than Anabaena cover due to its strong negative relationship with discharge. In contrast, our models predicting Anabaena anatoxin concentrations outperformed those predicting Microcoleus anatoxin concentrations due to the hysteresis relationship between Anabaena cover and anatoxins. Overall, our predictive modeling results highlight the application of ecological forecasting for benthic cyanobacterial cover and anatoxin concentrations in rivers and demonstrate the importance of incorporating taxon-specific predictors into future forecasts of benthic cyanobacteria.

Replication is central to most experimental and sampling designs, increasing inferential power and capturing fine-scale data heterogeneity. However, its importance remains poorly evaluated in some ecological and evolutionary settings. This is the case of metabarcoding studies using DNA recovered from sedimentary archives, in which biological signals may integrate ecological information through depositional and burial processes, and are often inferred from a single sediment core per site. Here, we evaluated the effect of different types of replication using sedimentary DNA (sedaDNA) metabarcoding data from two genetic markers (mitochondrial COI and nuclear 18S), under a nested sampling design. The design included three intertidal sites, three spatially separated sediment cores per site (biological replicates), two sediment depth horizons per core, and eight PCR (technical) replicates per sediment sample. Variance partitioning showed that site identity and sediment age group together explained >70% of the variation in beta diversity, indicating that among-site spatial variation and stratigraphic variation were the dominant drivers of community composition. In contrast, variation among different cores within sites was small and non-significant (<5%). Among PCR replicates from the same sediment sample, richness varied substantially, whereas Shannon diversity was more consistent. Despite this variability, differences in community composition among technical replicates remained smaller than among biological replicates and site identity, indicating limited influence on broader ecological patterns. Community composition was highly similar among replicate cores within sites, consistent with stratigraphic coherence. These results indicate limited within-site heterogeneity and suggest that, under stratigraphically coherent conditions, increasing biological replication may yield limited additional information, whereas enhancing technical replication and stratigraphic resolution can improve ecological inference from sedaDNA metabarcoding datasets.