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) 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.

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.

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.

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.

Understanding floral visitor dynamics along elevational gradients is essential for assessing potential implications for pollen limitation and population genetic processes in fragmented populations of cliff-dwelling alpine endemics. Physoplexis comosa (Campanulaceae) is a chasmophytic paleoendemic restricted to vertical rock faces on dolomitic and limestone bedrock from 200 to 2,800 m a.s.l. The species exhibits a specific floral morphology with fused corolla apices, basal openings for nectar access, and secondary pollen presentation, yet its functional floral visitor assemblage has remained poorly characterized across the species' elevational range. In this pilot study, we tested a two-tier camera-monitoring setup combining action cameras for diurnal monitoring with Raspberry Pi-based systems equipped with IR illumination for combined diurnal and nocturnal recording. We conducted monitoring across five elevational sites (1,480 to 2,400 m a.s.l.) in the Eastern Alps. Recorded video material was downsampled and annotated using the Addax platform alongside manual cross-validation to record physical contact events. Preliminary results confirm the technical feasibility of continuous monitoring in steep terrain and suggest possible elevational variation in diurnal visitors, alongside nocturnal activity by Geometridae moths. Camera-based observations provided higher functional resolution and revealed a different visitor assemblage than previous eDNA metabarcoding trials. This proof-of-concept establishes a methodological foundation for forthcoming multi-day monitoring campaigns and complements ongoing studies of the mating system and population-genetic analyses.

Soil is one of the most biodiverse habitats on the planet, yet the extent of soil biodiversity knowledge in Canada is unknown. To better understand our knowledge, we systematically mapped soil invertebrate biodiversity knowledge from peer-reviewed literature to identify spatial and taxonomic gaps and to assess whether areas projected to experience different magnitudes of climate change are adequately sampled. A total of 1,176 Canadian sampling locations were identified from 427 articles. We found biases in sampling coverage spatially and in relation to current and projected climatic conditions. The geographic region above 60 degrees North is sparsely sampled. Colder regions (< -0.12 C) and regions receiving the least (< 646.47 mm), moderately high (2268.67 to 2809.40 mm), and high (2809.40 to 3350.14 mm) amounts of precipitation were under sampled. Regions projected to have the smallest (< 0.86 C) and greatest (> 1.59 C) magnitudes in temperature change and regions projected to have moderate (-2.06 to +2.69 mm) to large changes (+2.69 to +7.44 mm) in precipitation were underrepresented. The sampled locations also suffer from taxonomic biases which were skewed toward Nematoda (19%), Annelida (18%), Acari (15%), and Collembola (14%). Our results suggest that methods such as stratified sampling is essential to capture underrepresented regions, taxonomic groups, and an array of current and projected climatic conditions. Additionally, shifting to a multispecies sampling approach is essential for broader soil ecosystem knowledge. Importantly, data from well-sampled areas should be examined to determine how it can be leveraged to advance conservation management of soil invertebrates.

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.

Animal movements can transport materials across ecosystem boundaries, while simultaneously modifying habitats through physical engineering. We integrated GPS telemetry, camera trapping, sediment measurements and physicochemical sampling of ponds on Santa Cruz Island, Galapagos, to evaluate how giant tortoises connect and shape terrestrial and freshwater ecosystems. Telemetry revealed 2,254 visits by 21 tortoises to 12 ponds, with visits lasting up to 21.38 h and showing strong seasonal and diel variation, while camera traps showed peak pond use in January and during early morning and late afternoon. Greater tortoise abundance was associated with higher ammonium, dissolved organic carbon and total phosphorus concentrations, while ponds with persistently greater tortoise abundance had higher total suspended solids. Departing tortoises carried a mean 505.9 g of sediment, yielding a conservative potential export of 8.20 kg per tortoise per year and tens of tonnes annually. These findings identify giant tortoises as mobile zoogeochemical engineers linking terrestrial and freshwater ecosystems through physical and biogeochemical pathways.

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.

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.

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.

The rapid global expansion of dams has significantly reshaped freshwater ecosystems, yet long-term trophic dynamics and food-web stability within reservoirs remain underexplored. This study examines the reorganization of fish communities induced by the trophic surge following dam construction and river impoundment. We used the predator-to-consumer mass ratio (PCMR) as a proxy for trophic pyramid structure to track changes in fish biomass distribution across three large boreal reservoirs in Quebec, Canada, over 20 years, using data from before and after impoundment. We found transient top-heaviness events (i.e., PCMR values > 1; predator accumulation), characterized by a hump-shaped trajectory of the PCMR values in two reservoirs (Opinaca and Robert Bourassa). The third reservoir (Caniapiscau) showed a gradual increase in top-heaviness but did not exhibit a clear transient pattern. The magnitude and duration of top-heaviness varied across reservoirs, potentially due to differences in the system's capacity to receive and accumulate nutrients, which are driven by water residence time and reservoir size. This research provides a conceptual framework for understanding the long-term trophic dynamics of reservoirs, which can help inform the development of more effective management strategies to preserve ecological health in these ecosystems.

Tropical forests contain much of Earth's biodiversity, yet community-wide responses to seasonal climate transitions remain poorly resolved. We used weekly airborne environmental DNA sampling, supplemented by spatially intensive dry- and wet-season campaigns, to characterize fungi, plants, arthropods, and vertebrates across the dry-to-wet-season transition in a lowland tropical forest. All four assemblages shifted abruptly and nearly synchronously following rainfall late in the dry season, before the meteorological wet season began. These compositional transitions were largely independent of taxonomic richness and airborne DNA concentrations, indicating coordinated changes in detection patterns rather than simple seasonal gains in richness. Across all samples, we recovered more than 5,400 operational taxonomic units, including 2,148 fungi, 857 plants, 2,157 arthropods, and 239 vertebrates. We also recovered 949 full-length insect DNA barcodes, demonstrating compatibility with standard barcoding approaches and expanding opportunities for species discovery. Because the onset of the meteorological wet season showed no long-term trend at Barro Colorado Island, predicting future phenological and compositional shifts will require greater attention to dry-season rainfall variability and the cues that organisms track during this transition. Airborne environmental DNA offers a scalable, noninvasive framework for monitoring biodiversity and phenology across taxonomic groups.

Mangrove reforestation is expanding across the Caribbean, but restoration success is still judged almost exclusively by tree survival and canopy cover. Whether the sediment microbiome, which governs carbon burial and nutrient cycling, follows the recovery of the vegetation remains largely unknown. We combined 16S rRNA gene metabarcoding (V4, Illumina MiSeq) with CHNS elemental analysis, pH and salinity measurements to compare sediment prokaryotic communities along 0-100 cm profiles in a reference, a reforested (Rhizophora mangle plantings since 2019) and an altered mangrove within the Las Dunas de Las Calderas Natural Monument, Dominican Republic (39 cores pooled into 28 depth-resolved composite samples). Reference sediments contained 2.5-5.5-fold more organic carbon (median 7.5% vs 3.0% in altered and 1.3% in reforested sediments), had higher C/N ratios and were less alkaline (pH 8.2 vs 8.8). Reference communities were more diverse and even, and differed in composition from both disturbed stands (pairwise PERMANOVA, R^2 ~ 0.09, adjusted P = 0.0015), whereas reforested and altered communities were statistically indistinguishable (R^2 = 0.04, P = 0.25). Disturbed sediments were dominated by Cyanobacteriota (~41-45% of reads), which tracked high pH, whereas reference sediments were enriched in Chloroflexota, Thermodesulfobacteriota and Thermoproteota, lineages linked to anaerobic degradation of complex organic matter and sulfate reduction. Depth structured communities by separating surface from subsurface layers, and sediment organic carbon and nitrogen were the only significant constraints in distance-based redundancy analysis. These results indicate that, within a few years of the first plantings (2019), reforestation had not restored the carbon-rich, anaerobic sediment habitat of the reference forest. We propose sediment organic carbon and microbial community structure as complementary indicators for mangrove restoration monitoring in the Caribbean.

Deadwood is a common ephemeral resource in boreal forests, but arthropod succession across the full decay gradient and transition into soil remains poorly understood. We analyzed the successional trajectory of arthropods in deadwood to soil. Further, we investigated whether the succession pattern differed in previously clear-cut and near-natural forests in southeastern Norway. This was done by DNA metabarcoding analyses of arthropod communities in collected deadwood and soil samples. We detected 741 arthropod OTUs, with 565 OTUs in deadwood and 291 in organic soil layers, of which 115 were found in both deadwood and organic soil layers. Arthropod richness increased with deadwood decay stage and organic soil layers supported higher richness than all deadwood decay stages. Community composition of arthropods changed significantly along the decay gradient, with late-decay stages sharing more OTUs with organic soil layers than with earlier decay stages. This suggests a gradual transition between communities in highly decayed deadwood and organic soil layers. Forest type (previously clear-cut vs. near-natural forest) had limited effects on overall richness and community composition, although previously clear-cut and near-natural forests supported partially distinct species pools and differed in co-occurrence patterns in late decay and organic soil layers. Our results show that arthropod communities are strongly structured along the decay gradient from freshly dead wood to organic soil layers. This highlights the importance of retaining deadwood across different decay stages for maintaining arthropod diversity and community convergence between deadwood and soil in boreal forests.