Environmental DNA

Environmental DNA (eDNA) methodology is widely applied in the biomonitoring of organisms, but it requires the target DNA to be detected in a simple, stable, and highly sensitive manner. Detection sensitivity of eDNA measurement becomes particularly critical when monitoring species present at low abundance. In this study, we aimed to improve the detection sensitivity through a method of DNA-extract reconcentration. This approach involves reconcentrating eDNA samples that were originally extracted using the widely adopted DNeasy Blood and Tissue Kit (Qiagen), utilizing the same kits reagents, and does not require any additional equipment or reagents. We evaluated the ability of this DNA reconcentration method using field samples including river, lake and costal marine habitats. Evaluation of this DNA reconcentration method showed that when ten conventionally extracted samples were pooled, the DNA concentration increased by approximately sevenfold, as confirmed by DNA quantification and quantitative PCR analyses, demonstrating enhanced detection sensitivity.

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.

Chinese Bahaba (Bahaba taipingensis, Sciaenidae) is a Critically Endangered, Grade I state-protected marine fish in China. Reliable, non-invasive monitoring tools are urgently needed to inform conservation and restocking efforts. We developed and validated three TaqMan probe-based qPCR assays targeting mitochondrial 12S rRNA, ND5, and control region (D-loop) loci for species-specific detection of B. taipingensis. Primer-probe sets were designed from complete mitochondrial genomes and evaluated in silico against GenBank and in vitro against tissue DNA from B. taipingensis and closely related sciaenids, plus positive eDNA samples; sensitivity was quantified using serial dilutions of synthetic target DNA. All three assays showed high specificity and consistent amplification; 12S rRNA assay (Bhb_12S) was selected for field screening based on superior low-copy sensitivity and minimal by-products. We applied the Bhb_12S assay to 414 seawater samples collected from 23 sites in western Hong Kong during 2025 surveys, detecting B. taipingensis eDNA in the September samples (0.71-2.85 copies per L; mean 2.05), with positive detections confirmed by Sanger sequencing. Overall, these validated qPCR assays provide a robust molecular toolkit for non-invasive monitoring of B. taipingensis and will aid conservation planning, restocking evaluation, and long-term biodiversity assessments in the Pearl River Estuary and adjacent coastal waters.

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

O_LIInformation on diet composition at both individual and population levels is fundamental to understanding resource use and availability, which influence individual fitness and the population dynamics of both predators and their prey. DNA diet metabarcoding offers a powerful molecular approach to diet analysis, but its application can be limited by methodological biases, particularly in generalist species with highly diverse diets. C_LIO_LIHere, we evaluate the performance of DNA metabarcoding for diet analysis in the lesser black-backed gull (Larus fuscus), a highly generalist mesopredator whose populations have shown complex responses to changes in natural and anthropogenic food resources over the past half-century. We collected faecal and regurgitate samples from pre-fledging chicks at two coastal and inland breeding colonies in northwest England, alongside pharyngeal, stomach, intestinal, and cloacal swabs from adult gull carcasses. Samples were analysed using COI (targeting animal DNA) and 12S (targeting vertebrate DNA) metabarcoding markers, and three blocking primers were developed to reduce host amplification in 12S libraries. C_LIO_LIMetabarcoding performance varied substantially among sample types and primer combinations. Without blocking primers, usable dietary information was recovered from regurgitate and stomach samples but not from intestinal or faecal samples. Blocking primers improved recovery of dietary DNA from faecal samples, but also increased the amplification of contaminants, elevating the risk of false-positive detections. C_LIO_LIAcross all sample types, metabarcoding identified 71 unique species belonging to 61 genera, including earthworms, small mammals, commercial and non-commercial fish species, lapwing, and urban-derived food products originating from livestock species. Dietary profiles revealed distinct clusters of consumed species associated with urban, agricultural, and marine foraging strategies, and demonstrated differences in dietary niche between age and colony cohorts. C_LIO_LIOverall, DNA metabarcoding enabled the detection of a highly diverse diet and revealed differences in resource use among colonies and age cohorts. These findings demonstrate the potential of DNA metabarcoding to advance our understanding of diet in highly generalist species and contribute to ongoing efforts to understand how dietary variation may shape demographic processes in highly dynamic populations. 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.

DNA metabarcoding of faeces is a powerful, non-invasive method for assessing predator diets. However, when studying the diet of generalist predators, broad PCR primers are used to amplify the wide range of potential prey species and metabarcoding outputs are often dominated by sequences from the predator. While blocking primers can be used to reduce PCR amplification of predator DNA, they frequently cause partial predator suppression and unintended prey blocking. Peptide nucleic acid (PNA) clamps, offer a promising, underutilised alternative by binding strongly and selectively to predator DNA to block its PCR amplification. In this study we designed and validated a novel PNA clamp targeting the 18S rRNA gene to suppress bird and mammal predator DNA in dietary samples. We tested this clamp on tissue mixtures and faecal samples from three seabird and two seal species across temperate, subantarctic, and Antarctic regions. The PNA clamp substantially increased the proportion of prey reads recovered while maintaining consistent prey community composition across all predator species. Our results demonstrate not only the general effectiveness of PNA clamps over standard blocking primers, but also provide a powerful, broadly applicable new tool to improve the accuracy in DNA diet metabarcoding studies.

Forest-floor leaf litter is a dynamic and structurally complex ecological transition zone and thus a promising substrate for terrestrial eDNA metabarcoding. Yet, extraction workflows for this heterogeneous matrix remain poorly standardized, especially in tropical systems, making it largely impossible to compare ecological functions across space, time, and taxa. To guide workflow selection across a series of selection criteria, including biological target, research question and practical considerations, we compared DNA extraction workflows for leaf-litter eDNA collected from 42 biological samples across seven different forest sites on Oahu, Hawaii. We evaluated four DNA extraction workflows: (1) Two low-volume approaches, with DNA extracted directly from 200 mg of homogenized litter using (i) CTAB or (ii) DNeasy PowerSoil(R); and (2) two high-volume approaches using PBS wash-based from 10 g of litter followed by (i) Centrifugation or (ii) Filtration. Taxonomic recovery from each workflow was evaluated with two COI primer sets targeting arthropods (ANML and shorter NoPlant), and one ITS marker targeting fungi. Results show that eDNA workflows tested here recovered site-level differences among forest-floor communities, but biodiversity recovery depended strongly on extraction workflow and marker. For low volumes, PowerSoil recovered the highest fungal richness (with ITS marker), and produced the most reproducible PCR-replicate profiles across markers, and required the least hands-on time, while CTAB was less expensive but required handling hazardous chemicals. For high volumes workflow, Centrifugation recovered higher arthropod diversity with ANML primer. Differences in community composition were nonetheless recovered by each method. At the same time, sampling sites explained more ASV-level compositional variation than extraction workflow across markers, showing that all workflows retained site-level ecological signals. Together, these results support a workflow framework in which extraction choice depends on target organism group, DNA state, reproducibility needs, and practical constraints.

Here, we report the first genetic confirmation of golden mussels (Limnoperna fortunei) in North America, and the subsequent development, optimization, and deployment of golden mussel eDNA monitoring procedures. Aquatic species invasions are economically costly, disrupt ecosystem functionality, and impact native aquatic communities. Early detection of new invasive species enables rapid response via implementation of effective eradication or control measures and is key for reducing harmful outcomes. Initial species detection and taxonomic identification can be aided by genetic methods that have high detection sensitivity and accuracy. Genetic methods such as environmental DNA (eDNA) sampling can be used to detect invasive species before they become established in new systems, providing an early alert system to inform resource managers. Golden mussels were first detected in North America in October 2024 near the Port of Stockton in the San Francisco Estuary (SFE). The SFE is particularly vulnerable to invasion due to the access and connectivity provided by the presence of engineering infrastructure and shipping lanes. Collaborative efforts between public agencies and academic institutions are underway to develop a coordinated detection and response plan. Early detection followed by a rapid response is the best defense against prolific invasive species, such as the golden mussel.

1. Detecting environmental DNA (eDNA) from rare or low-abundance aquatic species remains a major challenge, particularly when it is highly degraded, present at low concentrations, and dominated by DNA from non-target taxa. These challenges are further amplified in sedimentary ancient DNA (sedaDNA) studies, where thousands of years can degrade eDNA further, making the detection and quantitative interpretation of weak biological signals difficult. 2. Metabarcoding is commonly used to produce high-throughput community-level data from eDNA but is inherently compositional and influenced by amplification biases. Nonetheless, metabarcoding read abundance or PCR replicate detection frequency are increasingly used as proxies for relative DNA concentration, but their quantitative interpretation has rarely been evaluated against independent measures of absolute DNA abundance. 3. We used droplet digital PCR (ddPCR) to quantify mitochondrial DNA from Atlantic cod (Gadus morhua) and Atlantic herring (Clupea harengus) in 136 ancient eDNA extracts from Icelandic marine sediment cores spanning the last three millennia. We compared ddPCR copy number estimates with metabarcoding (18S) derived relative abundance and detection frequency, and evaluated whether temporal DNA trends corresponded with proxy reconstructed sea surface temperature (SST) variability. 4. We found that ddPCR-measured fish sedaDNA abundance was positively correlated with the proportion of metabarcoding PCR replicates for both Atlantic cod and Atlantic herring. Moreover, temporal trends in Atlantic herring DNA abundance were consistent with proxy reconstructed SST variability, supporting the ecological relevance of the molecular signal. 5. Overall, our results show that ddPCR-derived DNA concentrations and metabarcoding PCR replicate detection frequency capture consistent patterns in low-abundance fish sedaDNA from marine sediments. The observed agreement between approaches supports the use of PCR replicate detection frequency as a semi-quantitative proxy for low-abundance sedaDNA.

Offshore industry still largely relies on traditional approaches for regulatory compliance to environmental impact on the water column. Implementing environmental DNA (eDNA) workflow can offer several advantages, but early stages such as sampling and conservation of the samples require standardization and simplification before they can be routinely applied in offshore monitoring programs. In this study, we assessed the effect of several filter types (Durapore disc, Sterivex capsule and Wattera high-capacity capsule; all with 0.22 {micro}m pore size) allowing for different volume of filtration used for sampling eDNA. We also evaluated the effect of 25 days conservation of unfiltered water samples with different preservative solutions (Benzalkonium chloride -BAC, Longmires solution LONGI and a modified Longmires solution without SDS, LNoSDS) as a viable option when immediate filtration and cold storage are not possible. For downstream eDNA evaluation of filter types and preservation, we used quantitative digital PCR on selected target DNA and metabarcoding for qualitative assessment of marine prokaryotic and eukaryotic communities. Overall, filter choice had relatively less effects on quantitative and qualitative information from eDNA compared with water preservation. Sterivex and Durapore were better filter choices for biodiversity assessment. While the Wattera filter allowed processing of larger water volumes and improved quantification of metazoan DNA, handling and processing were more challenging. For water conservation, LNoSDS was the best option. Chemical agents of LONGI and BAC may provide favourable substrates for some tolerant bacterial strains, altering the microbial community composition, with consequences for the overall qualitative evaluation of conserved eDNA. For targeted metazoan eDNA, however, chemical preservation showed clear benefits. This research highlights key considerations and viable options for eDNA sampling and simple preservation workflows without cold storage for implementation in offshore water column monitoring. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/733101v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@22a175org.highwire.dtl.DTLVardef@1960864org.highwire.dtl.DTLVardef@1010f49org.highwire.dtl.DTLVardef@92a2f6_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LINeed for standardization of eDNA workflow for offshore water column monitoring C_LIO_LIImportance of eDNA sampling (filters) and eDNA conservation (preservatives) C_LIO_LIFilter choice does not affect drastically the dominant eDNA communities C_LIO_LIConservation outside cold storage challenging for eDNA-based biodiversity evaluation C_LIO_LIViable options: Sterivex filter for sampling; Longmires (no SDS) for conservation C_LI

Sinking marine particles is a key process regulating carbon export through the biological carbon pump, yet direct measurements of sinking dynamics remain limited in many coastal environments. One barrier is that most existing approaches require expensive instrumentation and large research platforms. Here, we present a low-cost, modular method for concentrating fast-sinking particles and measuring their individual sinking velocities under controlled conditions. This combines large settling tanks (110 L) for field-based particle fractionation with a video-based tracking system that quantifies the sinking behavior of natural marine particles. The particle sinking speed chamber is surrounded on three sides by a temperature-controlled water chamber, minimizing the problem of advection during measurements. The post-processing Python script delivers sinking velocity, particle size, circularity, and RGB-based properties for large numbers of particles. The method accuracy was validated using reference beads with known theoretical sinking velocities derived from Stokes law. Field deployments in the Baltic Sea demonstrated successful enrichment of fast-sinking particles and stable operation from both a research vessel and a small boat. Compared to existing methods, the approach substantially reduces logistical and financial barriers while maintaining particle-resolved measurements and compatibility with complementary biogeochemical analyses. This enables a broader observational coverage of sinking particle processes across environments that are currently underrepresented in carbon export studies.

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 has become a cornerstone of marine biodiversity monitoring, yet it recovers genetic material irrespective of organism viability and may therefore conflate historical and contemporary community signals. Environmental RNA (eRNA), derived from less stable ribonucleic acid, is hypothesized to be biased toward metabolically active organisms and may provide a more temporally resolved snapshot of living communities. Here we present a paired eDNA/eRNA metabarcoding comparison across a tropical marine seascape, analyzing 19 co-sampled sites spanning coral reefs, mangroves, a seagrass bed, shipwrecks, a cenote, and coastal infrastructure around San Andres Island, Colombia. To our knowledge this is the first in situ, ecosystem-scale paired eDNA/eRNA survey of the broad eukaryotic community across multiple natural habitat types in a tropical marine system, extending mesocosm and freshwater work (e.g., Giroux et al., 2022) to a field setting. Using COI-region amplicon sequencing processed by NatureMetrics, we recovered 1,944 operational taxonomic units (OTUs) across the 19 paired sites. Of these, 1,015 (52.2%) were detected by both approaches, 305 (15.7%) were unique to eDNA, and 624 (32.1%) were unique to eRNA. The eRNA-unique fraction was taxonomically enriched for groups including diatoms (class Bacillariophyceae, phylum Ochrophyta), ciliates, and other protists. Paired Wilcoxon signed-rank tests showed that eRNA recovered significantly higher OTU richness (median 239 vs. 207; W = 36, p = 0.016) and Shannon diversity (median 3.64 vs. 3.38; W = 40, p = 0.026) than eDNA. The mean per-site Jaccard similarity between paired samples was 0.40, indicating substantial turnover in the rare-taxon composition recovered by each method. Principal coordinates analysis of Bray-Curtis dissimilarity showed that habitat type structured abundance-weighted community composition (PERMANOVA F = 2.49, p = 0.001) whereas molecular method did not (F = 1.37, p = 0.107). A PERMDISP test found homogeneous multivariate dispersion between methods (F = 0.01, p = 0.92), reinforcing the absence of a method effect, but significant dispersion heterogeneity among habitats (F = 24.0, p < 0.01), so the habitat result is interpreted with caution. Indicator species analysis identified 73 OTUs significantly associated with one template: eDNA indicators were dominated by dinoflagellates (Dinophyceae) and eRNA indicators by diatoms (Bacillariophyceae) and fungi, consistent with an eRNA bias toward metabolically active microbial eukaryotes. A read-weighted overlap analysis showed that although eRNA-unique OTUs outnumbered eDNA-unique OTUs roughly two to one, the large majority of reads (>95%) fell in shared OTUs, so method-unique detections are predominantly rare taxa. We discuss the complementary value of eRNA for marine monitoring, with the seagrass habitat -- where eRNA reduced masking by terrestrial plant material -- as the clearest use case, and propose, rather than prescribe, the integration of eRNA into routine programs.

Environmental DNA (eDNA) has emerged as a promising tool for estimating fish abundance, yet linking eDNA concentration to true density remains a significant challenge in seasonal systems, where the signal is strongly influenced by temperature. We investigated whether eDNA can serve as an abundance index for three-spined stickleback (Gasterosteus aculeatus) in four coastal bays of the Baltic Sea (5.7-20.5{degrees}C, April-July 2023), by pairing eDNA sampling with two trap types of contrasting catchability. Light traps capture fish by phototactic attraction during darkness, so their catchability is driven primarily by night duration rather than temperature, while benthic traps respond to temperature through the same activity-driven mechanism as eDNA production. The temperature sensitivity of eDNA estimated from field data was far higher than physiological expectation (Q10 = 12.4, against a maximum metabolic rate benchmark of Q10 = 3.5), indicating that the field temperature signal reflects ecological change in addition to metabolism. We then compared how well three eDNA predictors tracked a combined trap-based abundance index: uncorrected eDNA, eDNA corrected with the temperature response constrained to the laboratory metabolic rate (a first-principles correction), and eDNA corrected with the response estimated from the field data. Uncorrected and first-principles-corrected eDNA were both strong predictors of abundance (standardised slopes of 0.45 and 0.43), whereas the field-corrected predictor was not (0.08). Uncorrected and first-principles-corrected eDNA performed comparably because temperature and abundance increased together over the season; the first-principles correction is nonetheless preferable, as it remains reliable when this covariation is unknown a priori. We conclude that estimating a temperature correction from field data should be avoided in seasonal eDNA monitoring, because it removes the abundance signal together with the temperature effect and assumes a stability in abundance that cannot be verified without independent reference data.

As we face the sixth mass extinction, understanding how ecosystems have persisted--or collapsed--through millennia of changing climates and human activity is critical for preventing biodiversity loss. We bolstered the past 24,000 years of plant and mammal records using targeted capture of ancient sedimentary DNA (sedaDNA) from Southern Californias Lake Elsinore, a cultural center for the Payomkawichum (Luiseno), Cahuilla, and other Peoples. Our sedaDNA approach generated a diverse dataset that included 18 plant orders not previously documented from Lake Elsinore. We paired these records with local measurements and paleo evidence of fire regimes, climate, demographic history, and ethnobotanical knowledge. We find that ecological stability persisted for 10,000 years of continuous human presence, reflecting ecosystem resilience through major climatic shifts, altered fire regimes, and varying intensities of Indigenous land use. SedaDNA revealed increased availability of food, medicinal, and utilitarian plant taxa during this period of botanical stability, shedding light on ancient fire-environment-human interactions that can inform contemporary management strategies.

Among eukaryotes, insects are by far the most diverse organisms on Earth, yet their global decline threatens ecosystem stability. Understanding local and regional biodiversity patterns is critical for conservation planning, ecosystem management, and predicting responses to environmental change, but traditional surveys for assessing insect diversity (e.g., manual collection, morphological identification, and counting) are highly labor-intensive, time-consuming, and often require rare or simply unavailable dedicated taxonomic expertise. DNA metabarcoding offers an efficient, high-resolution alternative to assess insect communities. Here, we report on the first insect metabarcoding survey on Crete that spans two years of sample collection between 2021 and 2023 from a small area in Southern Central Crete in the context of a citizen science project. A total of 29 samples yielded 10,865 Exact Sequence Variants (ESVs), 10,516 of which were assigned to insects, covering 988 species, 900 genera, and 227 families across 14 orders. A comparison with the existing observation records reveals 406 potential newly-observed species and an estimated 690 unclassified species, indicating substantial cryptic diversity. Our results demonstrate that even small-scale sampling can unravel substantial insect diversity and highlight critical gaps in barcode reference databases. Our study demonstrates how DNA metabarcoding can accelerate biodiversity discovery and monitoring in understudied regions.

Animal COI (mitochondrial cytochrome oxidase I) metabarcoding of environmental DNA (eDNA) is increasingly used to assess biodiversity in complex substrates such as soil. However, due to read-length constraints of second-generation sequencing platforms, mini-barcodes have been used instead of the full barcode region. Long-read sequencing technologies now enable the recovery of full-length barcode sequences, and are more commonly applied for studying microbes, but their use for metabarcoding the full-length standard COI barcoding region in animals remains limited. In this study, we compared three COI amplicon sets -- 313 bp, 660 bp, and 1,256 bp -- amplified from soil eDNA samples and sequenced using Illumina and PacBio platforms to evaluate their overall concurrence, the effectiveness of identifying nuclear mitochondrial DNA segments (NUMTs) and chimeras, as well as their respective taxonomic resolution. The long-read datasets exhibited a higher identification rate of NUMTs and true chimeras, suggesting that longer sequences improve the detection of noise in COI metabarcoding data, thereby reducing the occurrence of spurious taxa. Taxonomy assignment confidence was similar between the 313 bp and 660 bp datasets, whereas extending the amplicon beyond the standard COI barcode region (1,256 bp) reduced confidence, likely because longer reads extend into regions poorly represented in barcode reference databases. Despite substantially lower sequencing depth in the 660 bp dataset, per-sample OTU richness did not differ significantly from that recovered with the Illumina 313 bp amplicon set. Similarly, the relationships between samples were strongly correlated across the detected OTU communities, indicating consistent ecological interpretations between short and long amplicons. We conclude that the standard ~658 bp COI barcode is an optimal marker for soil animal metabarcoding from eDNA, balancing target recovery, artifact detection, taxonomic assignment and ecological interpretability. As COI eDNA metabarcoding becomes increasingly used in biodiversity assessment and is increasingly adopted in large-scale monitoring initiatives, this study provides methodological guidance for improving the robustness of soil animal community biomonitoring.

Littoral mysids facilitate benthic-pelagic coupling through horizontal migration, yet quantitative monitoring in structurally complex habitats remains methodologically challenged where traditional active gears fail. We evaluated the efficacy of standardized light traps for monitoring littoral mysids (Neomysis integer, Praunus flexuosus) and mesopredatory three-spined sticklebacks (Gasterosteus aculeatus) in the northern Baltic proper, Baltic Sea. Using a paired experimental design with predator-exclusion and unmodified traps, alongside concurrent passive benthic trapping, we assessed abiotic drivers affecting catchability, biotic interactions, and statistical power to monitor changes in population size over time. Results indicated significant biotic interference: unmodified traps attracted high densities of sticklebacks, which reduced mysid catches by approximately 85% through predation or behavioural avoidance. Consequently, physical predator exclusion is mandatory for accurate mysid sampling. Generalized Linear Mixed Models (GLMMs) confirmed that catch rates for all taxa were primarily driven by night duration rather than water temperature. While passive benthic trap catches tracked metabolic activity (peaking in warm summer months), light trap efficiency peaked in spring and collapsed during summer, confirming that sampling efficiency was strictly limited by the short duration of the night. Simulation-based power analysis revealed a stark contrast in monitoring utility based on spatial aggregation. For highly aggregated mysids, the method demonstrated low precision (Power < 0.25 to detect a 50% decline), rendering it suitable primarily for detecting substantial population collapses (>90%). In contrast, for less aggregated sticklebacks, the method achieved a more robust statistical power (>0.80 for a 60% decline), validating light traps as a precise tool for monitoring these abundant mesopredators. We conclude that light traps fill a critical methodological gap for winter and early spring monitoring when traditional passive gears underperform. Appropriate abundance indices should be based on statistical models accounting for night duration and strictly employ physical exclusion barriers when targeting mysids.