Metabarcoding and Metagenomics

Bottom trawling is the most widespread human disturbance on the seafloor, known to affect seabed structure and benthic biota. Yet, its impact on benthic communities remains documented mainly for taxa identifiable through morphology, leaving microscopic benthic communities largely overlooked. The Gulf of Lion continental shelf has been trawled for decades by French and Spanish fleets, to the point that encountering untrawled reference areas became elusive. Here, we examined prokaryote, protist and Animalia benthic communities at eight stations, using eDNA metabarcoding of core sediment samples, with five primer-pairs. Benthic communities were cross-referenced with contrasted trawling impacts, integrated across three periods: over one month, three years or ten years preceding the sampling. The community composition was primarily structured by ecological habitats, yet, trawling pressure showed association with prokaryotes and Animalia diversity. Diversity was higher at sites trawled more intensively in the preceding month, whereas long-term trawling was associated with a significant erosion of Animalia alpha diversity. Unexpectedly, communities did not display consistent vertical structure and the relative contributions of trawling and hydrodynamic reworking remain unresolved due to the absence of untrawled sites. Overall, our results show that trawling effects extend to prokaryotic compartments rarely considered in impact assessments, providing a proof of concept for eDNA-based assessment, and underline a pervasive long-term effect on Animalia communities. This study also highlights the importance of the timescale used to quantify fishing pressure and underscores the complexity of impact assessment when monitoring begins too late to establish a clear baseline, particularly when no reference site remains.

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.

Background: Amplicon sequencing of the 16S rRNA gene is widely used to characterise microbial communities, but the performance of commonly used clustering and denoising pipelines has not been systematically evaluated for highly diverse environmental datasets. We therefore benchmarked four established clustering and denoising pipelines, VSEARCH cluster_size, UNOISE as implemented in VSEARCH, Swarm, and DADA2, using simulated microbial communities with known ground-truth compositions and evaluated how performance varied with species richness, abundance unevenness, sequencing depth, and minimum abundance threshold. Results: Differences among pipelines were small at low species richness but became greater as species richness increased. High average cluster purity did not necessarily correspond to accurate species-level recovery, as species could be split across multiple clusters or recovered incompletely. Among the pipelines, UNOISE may be preferable when high species representation and cluster purity are prioritised, while also showing strong reconstruction of relative abundance profiles, albeit with extensive species splitting and many unclustered reads. DADA2 showed similarly strong reconstruction of relative abundance profiles and less species splitting but represented fewer species and had lower cluster purity at higher richness. Swarm provided a balanced compromise between species representation and splitting, whereas cluster_size may be useful when limiting species splitting is a priority, despite weaker reconstruction of relative abundance profiles. Increasing the minimum abundance threshold reduced species splitting but also reduced the number of clusters and perfect clusters and, at higher thresholds, decreased concordance with ground-truth relative abundance profiles. These effects were more pronounced at lower sequencing depths. Analysis of seafloor sediment samples also showed threshold-dependent sequence loss, including the loss of sequences repeatedly detected across replicate samples. Conclusions: Pipeline performance in highly diverse 16S rRNA amplicon datasets varies with dataset characteristics, evaluation criteria, and parameter settings. Pipeline selection should therefore reflect dataset characteristics and the analytical objective rather than rely on a single measure of performance, and minimum abundance thresholds should be evaluated in relation to sequencing depth rather than applied as fixed defaults. These findings emphasise the importance of reconsidering established analytical practices and transparently reporting bioinformatic settings as 16S rRNA amplicon sequencing is increasingly applied to highly diverse environmental microbial communities.

Detecting dugongs at low densities remains a major challenge for conservation monitoring, particularly in remote or turbid coastal habitats where conventional surveys are difficult. Environmental DNA (eDNA) offers a complementary approach, but robust species-specific tools are needed before it can be confidently applied to dugong surveillance. We developed and analytically validated a quantitative PCR TaqMan assay for detecting dugong (Dugong dugon) eDNA and evaluated its performance across controlled aquarium conditions in Australia and Abu Dhabi, and wild populations in northern Australia and the Arabian/Persian Gulf. The assay detected dugong eDNA in aquarium and field samples, with Sanger sequencing confirming amplification of dugong mitochondrial DNA from positive field detections. Our assay provides a robust and geographically transferable tool for targeted dugong eDNA detection, performing reliably across contrasting coastal environments and widely separated populations. This approach can strengthen local-scale surveillance of low-density or difficult-to-detect dugong populations.

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.

International wildlife trade represents one of the major drivers of biodiversity exploitation worldwide. However, the true taxonomic diversity embedded within commercial wildlife products often remains unknown because processing removes diagnostic morphological characteristics, preventing reliable species identification. Consequently, biodiversity assessments based solely on product labels may substantially underestimate the diversity of species involved in trade networks. To investigate hidden biodiversity within wildlife trade products, we applied DNA barcoding based on the mitochondrial cytochrome c oxidase subunit I (COI) gene to 77 products commercialized as fish swim bladders and seized at Guarulhos International Airport, Brazil. Molecular analyses successfully identified all samples and revealed the presence of four species: Plagioscion auratus (n = 38), Cynoscion acoupa (n = 7), Melanosuchus niger (n = 17), and Caiman crocodilus (n = 15). Fish species accounted for 71.4% of all samples, whereas crocodilians represented 28.6%, demonstrating that products marketed under a single commercial category may conceal substantial taxonomic diversity. Notably, the occurrence of two Amazonian crocodilian species within a trade chain traditionally associated with fish products reveals a previously undocumented component of the international wildlife trade. Our findings demonstrate that DNA barcoding is an effective tool for uncovering hidden biodiversity within processed wildlife products and provide evidence that wildlife trade networks may involve a broader spectrum of species than suggested by commercial labels. These results highlight the importance of molecular surveillance for biodiversity monitoring, wildlife trade regulation, and conservation planning.

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 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.

Heavy metal contamination of aquatic environments poses persistent risks to ecosystems and human health. Aquatic macrophytes such as Pistia stratiotes are increasingly used for phytoremediation due to their capacity to accumulate contaminants without displaying symptoms of abiotic stress. However, the molecular mechanisms underlying their metal tolerance remain poorly understood. Metallothioneins (MTs) are cysteine-rich proteins involved in metal binding and detoxification in plants. In this study, genome-wide analysis was performed to identify and characterise candidate MT genes in P. stratiotes and to assess their transcriptional response to copper exposure. Six novel MT genes (PsMT2f, PsMT3a-d, and PsMT4) were identified and classified into MT2, MT3, and MT4 subfamilies based on conserved cysteine motifs and domain architecture. Quantitative real-time PCR showed differential and tissue-specific expression of MT genes in roots and leaves following exposure to copper for up to 4 h. These findings provide the first comprehensive characterisation of the MT gene family in P. stratiotes and demonstrate their potential role in copper tolerance, supporting the application of this species in the phytoremediation of metal-contaminated aquatic systems.

Ground-nesting wild bees require suitable soil conditions for their below-ground development and overwintering life stages. Understanding how soil translocation and disturbance affect individuals and the following generations of bees is important for identifying conservation measures for ground-nesting wild bees. We investigated the effects of soil translocation and disturbance on the ground-nesting solitary spring mining bee Colletes cunicularius. In a replicated controlled design, we compared the effects of soil translocation with different degrees of disturbance on the number of surviving bees and the emergence time. We found that soil translocation and disturbance reduced bee survival but did not result in complete mortality, with some individuals surviving even under severe soil disturbance. There was no evidence that soil disturbance affected the timing of spring emergence, suggesting that disturbance is unlikely to alter the timing of nest construction and, consequently, the production of the next generation. Our results indicate that translocating soil containing overwintering bees could be a promising conservation measure to compensate for habitat loss caused by land conversion.

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.

The Olympia oyster (Ostrea lurida) was once a major food source and a key component of coastal marine ecosystems and culture on the US West Coast. Overfishing and habitat contamination in the early to mid-20th century severely depleted populations of Olympia oysters, and the species is currently considered a species of concern. Although multiple restoration efforts are underway, progress understanding the species biology and recovery potential has been limited by the historical lack of genomic resources. Here, we report a chromosome-level assembly of the Olympia oyster. The assembly was built using Oxford Nanopore reads and Hi-C to obtain a highly contiguous 1.03 Gb-assembly consisting of 10 chromosomes. RNA from gills, mantle, adductor muscle, and a mix of brooding eggs and larvae were also sequenced as 150 nt long paired-end reads which were used to guide the genome annotation. Over 52 K genes were predicted in the genome, of which 34K were protein-coding. A BUSCO analysis on the assembly showed 98.9% of the mollusca_odb12 gene set were complete (with 2.1% duplicated, and 0.8% missing). The short RNA reads were also used to identify tissue-level gene expression signatures in adductor muscles, egg and brooding larvae, and mantle. Development-related genes were highly expressed in eggs and brooding larvae whereas many xenobiotic metabolism genes were present in mantle tissues, highlighting mantles role in the interaction of the oyster with external environments. This new genomic resource will greatly advance our understanding of this keystone mollusk and provide a critical tool for guiding restoration efforts and the development of sustainable commercial production.

Abstract Background Many ecologically important small invertebrates are preserved in ethanol, which compromises DNA integrity and severely limits their potential to be utilized in further genomic research. The lack of soil arthropod representation ultimately results in limited availability of reference genomes and population genomic inference from pooled sequencing (Pool-seq). Results We introduce PoLoCo (Pooled Low-Coverage), a reproducible workflow integrating pooled specimen draft genome assembly from ethanol-preserved specimens with population-level allele-frequency analysis. We assembled a 411 Mb draft genome of Entomobrya nivalis from pooled short reads, achieving 97.7% BUSCO completeness despite notable fragmentation. Comparison with the published reference genome showed partial genomic correspondence while also revealing reference-dependent differences in the retained SNP datasets. The application of PoLoCo to 82 retained pooled populations produced a final distance-thinned allele-frequency dataset suitable for population-scale genomic inference. Case-study execution documented the computational requirements of the major workflow components on standard HPC infrastructure, with the workflow, documentation, and software environments openly available in Zenodo. Conclusions PoLoCo demonstrates that draft genome assemblies and reproducible Pool-seq allele-frequency analyses can be performed with long-preserved sample material from the field. PoLoCo integrates reference construction, sync-based allele-frequency analysis, and benchmarking into a free and open workflow. It establishes a reproducibility benchmark for biodiversity genomics and yields a transferable workflow for ecological and evolutionary initiatives where high-quality DNA or long-read sequencing is not feasible.

Recent years have witnessed the discovery of an immense diversity of protists in almost all ecosystems on Earth. However, a major challenge consists in identifying diversity patterns and the drivers that explain the distribution of this diversity. While several works have focused on local parameters, only a few studies have addressed this question at regional scales. In this study, we characterize the diversity and distribution of Arcellinida (testate amoebae) at regional scale of a climatically contrasted country, Bulgaria. We collected 75 samples geographically evenly distributed and representative of the country's natural ecosystems and used metabarcoding to determine their diversity. Communities composition differed markedly among habitats, revealing sharp beta diversity patterns. "Mesophilic" environments such as forests and ponds were most diverse, while acidic and oligotrophic Sphagnum peatlands were poorest and hosted the highest proportion of unique taxa. Our analyses of richness-environment based on a Random Forest approach applied to all soil systems indicated that stable humidity (low silt content, low precipitation seasonality) and intermediate soil carbon stocks had a positive effect on richness. The predicted increase in continentality in the next decades in Bulgaria calls for testing climatic models to evaluate the potential risk of declining Arcellinida biodiversity.

Background: Long non protein coding (lnc)RNAs are ubiquitous transcripts known to be major regulators of cellular processes in eukaryotes. In diatoms, specific lncRNA expression is tightly correlated to nutrient fluctuations, which suggests function, but their biological relevance remains to be established. In order to fill this knowledge gap, we sought to design a protocol to generate diatom lncRNA knockout mutants by CRISPR-Cas9 enabling subsequent functional studies to shed light into their mechanistic roles. Since lncRNAs do not contain open reading frames that can be targeted by point mutations for the generation of functional knockouts, we designed a multiplexed CRISPR-Cas9 plasmid harboring two independent gRNA for the specific ablation of lncRNA loci in P. tricornutum. As proof of concept, we used our plasmid-based system to target three phosphate depletion responsive lncRNAs. Results: Using the golden gate based uLoop system, we assembled a single plasmid containing all the necessary components to generate CRISPR-Cas9 large genomic ablations of lncRNAs in P. tricornutum using bacterial conjugation. Three intergenic lncRNAs (Linc91, Linc77 and Linc24) responsive to phosphate fluctuations were selected and independently targeted. Around 32% of the exconjugants presented a monoallelic ablation of the targeted loci and 11% presented a biallelic ablation on the first PCR-screening round. Mutated lines, harboring biallelic lncRNAs ablations for each lncRNA targeted, were successfully generated for each of the targeted lncRNAs. Additionally, a first line of phenotypic analysis revealed significant growth rate differences for one of the mutants ({Delta}Lnc24) under both full and phosphate depleted conditions, suggesting its involvement in cell division regulation under nutrient fluctuations. Conclusions: This work provides a simple and efficient method for the generation of lncRNA knock-out mutants in P. tricornutum. Furthermore, this approach can be easily adapted to other microalgae amenable to transformation, enabling the enhancement of our understanding of these important cellular regulatory transcripts in the marine environment.

Traditional veterinary pathology investigations on wild animals generally conclude a primary cause of death based on a combination of observed clinical signs, gross and microscopic lesions, microbial culture and PCR. However, these methods may fail to capture the broader microbial community of the host. We employed metatranscriptomic sequencing to characterise the potential pathobiome of necropsied marine turtles, comparing these findings with corresponding veterinary pathology reports. Tissue samples were collected from 76 individuals spanning three species of marine turtles -- the green turtle (Chelonia mydas), the hawksbill turtle (Eretmochelys imbricata), and the loggerhead turtle (Caretta caretta) -- across the coastline of New South Wales, Australia. Reported causes of mortality ranged from infectious disease to trauma. We identified a diverse range of parasites within the genera Neospirochis, Hapalotrema, Paraorgymatobothrium, Eimeria and Callitetrarhynchus, and bacteria including the genera Aeromonas, Clostridium, and Vibrio. Novel viruses of the families Astroviridae, Caliciviridae, Hepaciviridae, Hepeviridae, Nyamivirdae, Orthomyxoviridae, Picobirnaviridae and Rhabdoviridae were also identified. In cases where infectious disease was noted in the pathology report, we frequently observed high pathogen read counts consistent with the listed primary diagnoses. However, we also detected a range of incidental infections involving eukaryotic parasites, bacteria, and viruses. The diversity of infectious agents detected in marine turtles suggests that the observed disease outcomes are unlikely to be driven by a single causative agent, but rather by complex interactions between host health, the pathobiome, and environmental stressors. We discuss the potential implications for turtle health and conservation.

Cyclosporiasis is a foodborne illness caused by the parasite Cyclospora cayetanensis. Clinical surveillance often lags infection by weeks because diagnosis depends on a clinician ordering a test that is not part of routine parasite panels. Wastewater metagenomic sequencing does not depend on care seeking, which may be useful for early detection and community monitoring. We designed 1,670 31-mers from C. cayetanensis mature ribosomal RNA and retained 1,464 without an exact non-target match in the 28 July 2025 NCBI core_nt release. By analyzing 293 public wastewater metagenomes, we selected a threshold of 24 distinct diagnostic 31-mers on one read. Applying that threshold to 2,333 public runs from BioProject PRJNA1247874 yielded putative C. cayetanensis reads in 81 runs, or 3.5%, and in 24 of the 30 longitudinally sampled sewersheds. Detections rose through the summer in two consecutive years, matching the known seasonality of cyclosporiasis. The most parsimonious explanation is that these reads come from parasites shed into wastewater, although undescribed Cyclospora species absent from reference databases cannot be excluded. The same approach can be applied to other pathogens in existing wastewater sequencing data.

Global arthropod decline demands effective biodiversity monitoring strategies. However, most current monitoring approaches do not provide an exhaustive picture of arthropod community structure. In particular, biotic interactions and temporal patterns of biodiversity change are still poorly understood. Here we explore the possibility of addressing these two shortfalls using spiders, one of the most important predators of terrestrial arthropods, as natural samplers for arthropod community DNA. We conducted several experiments comparing the recovered community composition between spider gut contents and traditional monitoring methods. Additionally, we used archived spiders from long-term collections to assess the preservation of prey DNA over time. Spiders proved to be highly efficient natural DNA samplers, with gut content metabarcoding revealing similar community composition and alpha- and beta-diversity like traditional methods. Unique arthropod taxa were detected by spider gut contents and traditional methods respectively, indicating that these two approaches can serve as valuable complements to each other. Well-preserved archived spiders effectively reconstructed historical diets up to 20 years back in time, showing that historical spider collections can act as time capsules of the wider diversity that occurred in the spider's habitat. Spiders as natural samplers overcome critical shortfalls in biodiversity monitoring and contribute to our future understanding of community assembly across space and time.

Management of marine protected areas (MPAs) require long-term monitoring and evaluation that is often limited by the cost and effort of conventional visual based surveys. We evaluated a community science-based eDNA approach to MPA monitoring over two years in the Point Dume State Marine Reserve, the Point Dume State Marine Conservation Area, and two paired unprotected reference sites. Across the sampling period, we detected 119 species of fish and marine mammals, including 19 of the 22 regional management-priority species for Southern CA MPAs. Protected and unprotected comparison sites were broadly similar in overall assemblage with 93 species shared across all sites. Multivariate analyses revealed site-level differences, although season and year explained more variation than protection status. Species presence also shifted during a La-Nina transition, highlighting the importance of long-term monitoring under different climatological contexts. Community scientists greatly expanded the temporal and spatial scope of eDNA sampling and generated high-quality data. Subsampling analysis showed that quarterly sampling, with three sample replicates and sequencing depths between 700k-990k, recovered 95% of observed biodiversity of the full time series. These results demonstrate that a community science-based eDNA program could provide a standardized, scalable, low-cost, and management-relevant approach for long-term monitoring across California's MPA network.

Introduction: Despite decades of investment, stream restoration often fails to recover diverse benthic macroinvertebrate communities, as restored reaches tend to remain dominated by pollution-tolerant taxa. This shortfall in recovery is commonly attributed to either poor habitat quality or dispersal limitation of aquatic macroinvertebrate taxa, yet these hypotheses are rarely tested directly. Objectives: We conducted a manipulative field experiment in the eastern Piedmont region (Maryland, USA) within the Chesapeake Bay Watershed to evaluate whether restored streams can support pollution-sensitive taxa when dispersal barriers are removed. Methods: We translocated whole macroinvertebrate communities from three nearby reference streams into three restored streams using standardized habitat substrates and quantified macroinvertebrate survival after 28 days. We also assessed how effective leaf vs rock substrates were for accumulating diverse and abundant macroinvertebrate communities for translocation. Results: Across all sites, 77% of translocated individuals and 16 of 22 taxa sensitive and moderately sensitive to pollution persisted under restored stream conditions, demonstrating that restored reaches can support short-term survival of pollution-sensitive taxa that typically fail to recover. Substrate comparisons revealed that leaf packs accumulated higher macroinvertebrate abundances, whereas both substrates accumulated macroinvertebrate communities with similar taxa richness, EPT richness, and Shannon Diversity. Conclusions: This work suggests that dispersal limitation, rather than unsuitable habitat, may be an underappreciated constraint limiting macroinvertebrate recovery in restored streams. These findings challenge the assumption that habitat restoration alone is sufficient for biological recovery and highlight the potential for whole-community translocations to accelerate recovery and ecological uplift as part of stream restoration practices. Implication for Practice: Our study highlights the need to consider habitat connectivity and dispersal pathways during stream restoration designs. Restoring streams to meet habitat requirements of pollution-sensitive macroinvertebrate taxa may not be sufficient for their populations to recover if these taxa are unable to reach the restored stream. In scenarios where restoring connectivity to source populations is infeasible, whole community translocations may offer a novel restoration tool to overcome dispersal barriers and accelerate biological recovery in restored streams.