Environmental DNA最新文献

{"title":"CRISPR-Dx and Metabarcoding Perform Similarly for Monitoring Mammals With eDNA on the Catchment Level in High-Alpine Ecosystems","authors":"Flurin Leugger,&nbsp;Martina Lüthi,&nbsp;Michel Schmidlin,&nbsp;Sarah Thurnheer,&nbsp;Zacharias Kontarakis,&nbsp;Loïc Pellissier","doi":"10.1002/edn3.70161","DOIUrl":"https://doi.org/10.1002/edn3.70161","url":null,"abstract":"<p>Biodiversity monitoring in difficult-to-access areas, such as rugged mountain ranges, is currently challenging and thus often absent. Environmental DNA (eDNA) offers new opportunities to monitor remote or strictly protected areas, as rivers integrate the biodiversity information of entire catchments. Environmental samples can be analyzed either with metabarcoding or using species-specific assays. Species-specific assays like quantitative polymerase chain reaction assays do not require a fully-equipped laboratory and thus can be used in settings with limited resources and are especially suited to monitoring elusive or threatened species of management concern. Recently developed molecular tools, such as CRISPR-based diagnostic systems (CRISPR-Dx), provide new avenues to facilitate eDNA analysis through species-specific assays. Here, we combine multispecies primers with CRISPR-Dx to detect terrestrial mammal species in parallel with one amplification to detect multiple species with CRISPR-Dx. Given the short length of metabarcoding amplicons, designing species-specific assays within them can be challenging. We designed species-specific CRISPR-Dx for eight terrestrial mammals within a commonly used metabarcoding amplicon ~59 base pairs in length and tested the assays on eDNA samples collected in high-alpine catchments. Additionally, we compared the detections from CRISPR-Dx with metabarcoding results of the same samples and with catchment-based species inventories obtained through traditional monitoring. First, we show that designing species-specific CRISPR-Dx within a short amplicon allows terrestrial mammal detection in eDNA. Second, we demonstrate that CRISPR-Dx assays combined with multispecies primers are comparable in sensitivity to metabarcoding and thus can bridge a gap between species-specific assays and community analysis without requiring fully equipped laboratories. Third, we highlight that catchment-based eDNA sampling can be used to monitor terrestrial mammals in remote or protected areas. Overall, we demonstrate that eDNA and particularly CRISPR-Dx are a promising tool to monitor inaccessible and/or protected areas and to detect rare species across large spatiotemporal scales, thereby promoting biodiversity conservation.</p>","PeriodicalId":52828,"journal":{"name":"Environmental DNA","volume":"7 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/edn3.70161","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144647300","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Inland Saline Lakes as Hotspots of Specialized, Nonmarine Protist Diversity: The Case of Arcellinida (Amoebozoa)","authors":"Fernando Useros,&nbsp;Leonardo D. Fernández,&nbsp;Rubén González-Miguéns,&nbsp;Carmen Soler-Zamora,&nbsp;Enrique Lara","doi":"10.1002/edn3.70133","DOIUrl":"https://doi.org/10.1002/edn3.70133","url":null,"abstract":"<p>Inland saline lakes are dynamic environments with fluctuating salinity levels, sometimes reaching values higher than those of seawater. Their abiotic conditions are often extreme and more unpredictable than in marine systems, which could imply lower biotic pressures. This could have allowed nonmarine organisms to cross the salinity barrier and subsequently specialize and diversify there. Alternatively, inland saline lakes could host generalist species that are also found in other environments. In order to answer this question, we focused on a group of protists characterized by their narrow ecological tolerance, Arcellinida testate amoebae. We studied their diversity using a metabarcoding approach in different thalassic and athalassic saline systems from Spain and Chile. The majority of operational taxonomic units (OTUs) were exclusive to athalassohaline systems. Three clades included most diversity in athalassohaline systems, in addition to several specific colonization events. Inland saline systems have been colonized by freshwater species, which diversified there, turning these athalassohaline environments into hotspots of specialized Arcellinida biodiversity.</p>","PeriodicalId":52828,"journal":{"name":"Environmental DNA","volume":"7 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/edn3.70133","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144624455","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Inferring Plant Community Phenology via Bee-Collected Pollen","authors":"Sydney B. Wizenberg,&nbsp;Mateus Pepinelli,&nbsp;Bao Ngoc Do,&nbsp;Mashaba Moubony,&nbsp;Darya Tamashekan,&nbsp;Lewis Nguyen,&nbsp;Ida M. Conflitti,&nbsp;Amro Zayed","doi":"10.1002/edn3.70149","DOIUrl":"https://doi.org/10.1002/edn3.70149","url":null,"abstract":"<p>Global climate change is producing novel biospheric conditions, presenting a threat to the stability of ecological systems and the health of the organisms that reside within them. Variation in climatic conditions is expected to facilitate phenological reshuffling within plant communities, impacting the plant-pollinator interface and the release of allergenic pollen into the atmosphere. Impacts on plant, invertebrate, and human health remain unclear largely due to the variable nature of phenological reshuffling and insufficient monitoring of these trends. Large-scale temporal surveillance of plant community flowering has been difficult in the past due to logistical constraints. To address this, we set out to test if metabarcoding (<i>ITS2</i> and <i>rbcL1</i>) of pollen collected by honey bees could be used to infer the phenology of plant communities via comparison to in situ field monitoring at our urban apiary in Toronto, Canada. We found that pooled pollen samples from the five honey bee colonies used in our pilot project could accurately indicate the onset of anthesis, but not its duration, in the wide variety of plant genera they forage on. Increasing the number of colonies used to monitor and employing a multi-locus approach for metabarcoding of pollen substantially increased the genus detection power of our approach. Here, we demonstrate that metabarcoding of bee-collected pollen could streamline the establishment of long-term phenological monitoring programs to document the consequences of global climate change and its impact on the temporal aspects of plant-pollinator relationships.</p>","PeriodicalId":52828,"journal":{"name":"Environmental DNA","volume":"7 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/edn3.70149","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144624454","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Impact of CO2-Induced Aquatic Acidification on Environmental DNA and RNA Shedding and Persistence","authors":"Mark Louie D. Lopez,&nbsp;Kate C. Rolheiser,&nbsp;Jacob Etzkorn,&nbsp;Jacob J. Imbery,&nbsp;Matthew A. Lemay,&nbsp;Iria Gimenez,&nbsp;Caren C. Helbing","doi":"10.1002/edn3.70158","DOIUrl":"https://doi.org/10.1002/edn3.70158","url":null,"abstract":"<p>Anticipated future increases in CO₂ levels are predicted to have a diverse array of lethal and non-lethal effects on the marine ecosystem. While there has been extensive research on the physiological impacts of ocean acidification on marine species, our understanding of how increasing levels of carbon dioxide affect the shedding and decay of environmental DNA and RNA (eDNA/eRNA) in marine habitats is limited. This may impede the effective adoption of environmental nucleic acid–based molecular tools for monitoring marine biodiversity and detecting rare or invasive species. In the present study, we conducted mesocosm experiments to determine the shedding and decay rate constants of eDNA and eRNA in <i>M. gigas</i> (<i>Magallana</i> [<i>Crassostrea</i>] <i>gigas</i>) using mitochondrially encoded tRNA leucine 1 (<i>mt-tl1</i>) marker at various partial pressures of CO₂ in seawater. To our knowledge, this is the first study manipulating seawater pH using CO₂. We developed a sensitive and specific quantitative PCR-based assay to detect <i>M. gigas</i> eDNA and eRNA. Higher CO₂ levels increased shedding rates, indicating greater organism stress and biological effects on oysters. Additionally, increased CO₂ accelerates DNA and RNA decay, suggesting that ocean acidification may impact the reliability of eDNA-based biodiversity monitoring. Furthermore, eRNA displayed lower steady-state concentrations and a shorter persistence time in comparison to eDNA, as is consistent with known biochemical properties of the molecules. These findings are presented in the context of previous work that adjusted pH through acid–base adjustment and temperature and highlight the importance of considering ocean acidification caused by differing CO₂ levels when using molecular tools for marine conservation and fisheries management.</p>","PeriodicalId":52828,"journal":{"name":"Environmental DNA","volume":"7 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/edn3.70158","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144624456","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of Rapid DNA Extraction Methods to Better Enable Point-of-Use Environmental DNA Detection","authors":"Melisa E. Kozaczek,&nbsp;Stephen F. Spear,&nbsp;Tyler J. Untiedt,&nbsp;Paul Albosta,&nbsp;Caden Jungbluth,&nbsp;Jared J. Homola,&nbsp;Elliott P. Barnhart,&nbsp;Christopher M. Merkes","doi":"10.1002/edn3.70159","DOIUrl":"https://doi.org/10.1002/edn3.70159","url":null,"abstract":"<p>Recent developments in molecular testing have created the opportunity for biologists and managers to detect environmental DNA (eDNA) of target species rapidly and without the requirement of a laboratory. These point-of-use protocols may be especially useful for early detection and rapid response for invasive species or surveillance for at-risk native species, where timely management decisions are critical. Point-of-use eDNA protocols also facilitate wider and less expensive implementation of eDNA methods. One of the key components to an effective point-of-use protocol is a rapid DNA extraction method. Several rapid extraction protocols are suitable for implementation in the field, but information regarding their relative effectiveness is lacking. We evaluated extraction efficiency of four DNA rapid extraction protocols using filters spiked with primary cultured grass carp (<i>Ctenopharyngodon idella</i>) gill cells. The extraction methods included two syringe-based column extractions, a lysis and extraction solution, and a divalent cation chelation resin (Chelex) extraction protocol alongside a laboratory-based control kit. We estimated DNA yield using a newly designed quantitative polymerase chain reaction (qPCR) assay targeting the grass carp nuclear genome. We evaluated two additional factors, filter type (mixed cellulose ester [MCE] and polyethersulfone [PES]) and background eDNA source (aquaculture or river). The lysis and extraction solution and Chelex extraction both had the highest overall yield, with MCE filters further increasing Chelex yield while the enzyme extraction yield was dependent on interaction with both filter and eDNA source. Our results indicate that rapid extraction protocols, such as solutions with short heating steps, are effective for DNA isolation and help to increase the overall accessibility of eDNA analyses.</p>","PeriodicalId":52828,"journal":{"name":"Environmental DNA","volume":"7 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/edn3.70159","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144624457","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Winds of Change: Charting a Pathway to Ecosystem Monitoring Using Airborne Environmental DNA","authors":"Rachel L. Tulloch,&nbsp;Clare I. M. Adams,&nbsp;Matthew A. Barnes,&nbsp;Elizabeth L. Clare,&nbsp;Henrik C. van de Ven,&nbsp;Andrew Cridge,&nbsp;Francisco Encinas-Viso,&nbsp;Kristen Fernandes,&nbsp;Dianne M. Gleeson,&nbsp;Erin Hill,&nbsp;Anna J. M. Hopkins,&nbsp;Anna M. Kearns,&nbsp;Gracie C. Kroos,&nbsp;Anna J. MacDonald,&nbsp;Francesco Martoni,&nbsp;Angela McGaughran,&nbsp;Todd G. B. McLay,&nbsp;Linda E. Neaves,&nbsp;Paul Nevill,&nbsp;Andrew Pugh,&nbsp;Kye J. Robinson,&nbsp;Fabian Roger,&nbsp;Tracey V. Steinrucken,&nbsp;Mieke van der Heyde,&nbsp;Cecilia Villacorta-Rath,&nbsp;Jenny Vivian,&nbsp;Erin E. Hahn","doi":"10.1002/edn3.70134","DOIUrl":"https://doi.org/10.1002/edn3.70134","url":null,"abstract":"<p>Airborne environmental DNA (airborne eDNA) analysis leverages the globally ubiquitous medium of air to deliver broad species distribution data and support ecosystem monitoring across diverse environments. As this emerging technology matures, addressing critical challenges and seizing key opportunities will be essential to fully realize its potentially transformative impact. In June 2024, the Southern eDNA Society convened over 100 researchers, industry leaders, and biodiversity management stakeholders in a landmark workshop to evaluate the current state of airborne eDNA research and chart a course for future development. Participants explored opportunities for integrating airborne eDNA into existing monitoring systems, but they unanimously agreed that research must first be applied to improving understanding of airborne eDNA ecology. The workshop emphasized the importance of collaborative engagement with stakeholders—including government agencies, Indigenous communities, and citizen scientists—to ensure practical and ethical implementation. This summary highlights current challenges and actionable recommendations, including improving our understanding of airborne eDNA ecology, harmonizing sampling methodology (e.g., devices, materials, sampling density, duration), identifying and mitigating sources of error, and fostering early, sustained stakeholder collaboration. By addressing these challenges, airborne eDNA analysis can become a transformative tool for biodiversity, biosecurity, and conservation monitoring on a global scale. Its ability to detect diverse taxonomic groups—including fungi, plants, arthropods, microbes, and vertebrates—positions airborne eDNA as a pivotal technology for holistic terrestrial biodiversity assessments that transcend traditional, species-focused monitoring approaches.</p>","PeriodicalId":52828,"journal":{"name":"Environmental DNA","volume":"7 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/edn3.70134","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144624458","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Assessment of Environmental DNA Survey Design for the Detection of Freshwater Unionid Mussels","authors":"Nathaniel T. Marshall,&nbsp;W. Cody Fleece","doi":"10.1002/edn3.70152","DOIUrl":"https://doi.org/10.1002/edn3.70152","url":null,"abstract":"<p>Implementation of environmental DNA (eDNA) for environmental consultation in association with permitting purposes has been rare within the United States. In part, this is due to the lack of developed standards and guidelines needed to design robust eDNA surveys. This study provides a descriptive analysis for assessing freshwater mussel eDNA detection compared to an exhaustive visual mussel search. We evaluated an eDNA survey at two different levels of sampling effort: (1) at the transect level assessing the collection of eDNA along transects and (2) at the water sample replicate level assessing species detections obtained from subsamples within a transect. Logistic regression assessed eDNA detection probability against the visually observed abundance for each mussel species, informing the level of effort required to detect rare mussel species. This study offers critical insight into survey design guidelines that will be instrumental for building confidence for the implementation of eDNA into freshwater mussel assessments.</p>","PeriodicalId":52828,"journal":{"name":"Environmental DNA","volume":"7 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/edn3.70152","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144624523","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Navigating the Murky Waters: Variation in Low Abundance Sequence Filtration in Fish eDNA Metabarcoding","authors":"Mariana Blanco,&nbsp;Brooke Walker,&nbsp;Mark Duchene,&nbsp;Rachel Morris,&nbsp;Lauren Weeks,&nbsp;Evelyn Denomme-Brown,&nbsp;Robert Hanner","doi":"10.1002/edn3.70106","DOIUrl":"https://doi.org/10.1002/edn3.70106","url":null,"abstract":"<p>Environmental DNA (eDNA) metabarcoding is a valuable tool for assessing fish communities and informing environmental management strategies. Well-defined and informed methodologies are necessary to increase the repeatability and accuracy of eDNA results. This review evaluates 87 fish eDNA metabarcoding studies with a focus on low abundance sequence filtration methods used. This study aims to reveal the variety of methodological approaches used in eDNA metabarcoding and to provide recommendations based on these findings. A rubric of 32 criteria was developed to standardize the evaluation process, focusing not only on low abundance sequence filtration methods, but also on the incorporation of replicates, controls, primer validations, data availability, and other best practice criteria. We found diverse approaches to low abundance sequence filtering which showed little justification for threshold selection. While most studies incorporated some form of negative control or replicate, their implementation and reporting were inconsistent. There was also limited use of positive controls and primer validation throughout the studies. We recommend the adoption of various practices: (1) increasing the use of controls and replicates, (2) providing rationale behind low abundance filtering criteria or omitting it entirely, (3) completing analyses to validate primers, (4) improving the completion and appropriate communication of methods and results, and (5) making raw sequence data publicly available. Refined methodologies in eDNA metabarcoding research are imperative to ensure the reproducibility and accuracy of fish community assessment and environmental management practices. This is especially important as this tool continues to integrate into conservation and management efforts.</p>","PeriodicalId":52828,"journal":{"name":"Environmental DNA","volume":"7 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/edn3.70106","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144558108","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A qPCR Assay for the Threatened Pilbara Olive Python (Liasis olivaceus barroni)","authors":"Kathryn L. Dawkins,&nbsp;Benjamin L. Heyward,&nbsp;Mahsa Mousavi-Derazmahalleh,&nbsp;Nicole E. White,&nbsp;Morten E. Allentoft","doi":"10.1002/edn3.70114","DOIUrl":"https://doi.org/10.1002/edn3.70114","url":null,"abstract":"<p>The Pilbara olive python (<i>Liasis olivaceus barroni</i>) is endemic to the Pilbara region of Western Australia and listed as threatened. Despite being a priority conservation target, they are difficult to monitor as their cryptic nature complicates conventional surveying methods, constraining effective conservation in a region that is heavily impacted by the natural resource industry. As part of ongoing efforts to improve monitoring and conservation of this endangered top predator, we developed a subspecies-specific probe-based qPCR assay to allow for the detection of <i>L. o. barroni</i> DNA in environmental samples. The assay was validated in silico and in vitro against closely related non-target species, with synthetic DNA used for assessing assay sensitivity and the effect of inhibition. Field validation was performed using water samples collected from rock pools across the Pilbara (<i>n</i> = 21) that had previously been investigated with eDNA metabarcoding methods. Our validation showed that the assay is specific to <i>L. o. barroni</i> and highly sensitive, with modeled limits of detection and quantification of 4 copies/reaction and 26 copies/reaction, respectively. We successfully detected <i>L. o. barroni</i> DNA in 10/10 field samples that had previously shown the presence of <i>L. o. barroni</i> based on metabarcoding. Additionally, 2/11 samples reported as negative through metabarcoding were observed to amplify using our qPCR assay, a discrepancy we ascribe to improved sensitivity resulting from the shorter amplicon length of our assay and less competition for primers in the qPCR reactions. This qPCR assay will be a valuable tool for the eDNA biomonitoring of <i>L. o. barroni</i>, furthering our understanding of its distribution, ecology, and conservation status.</p>","PeriodicalId":52828,"journal":{"name":"Environmental DNA","volume":"7 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/edn3.70114","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144520080","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of Extraction Methods for the Recovery of Vertebrate DNA From Spider Webs","authors":"Joshua P. Newton,&nbsp;Morten E. Allentoft,&nbsp;Philip W. Bateman,&nbsp;Matthew A. Campbell,&nbsp;Mahsa Mousavi-Derazmahalleh,&nbsp;Paul Nevill","doi":"10.1002/edn3.70122","DOIUrl":"https://doi.org/10.1002/edn3.70122","url":null,"abstract":"<p>Spider webs have recently been recognized as an excellent source of vertebrate eDNA. Here we compare four sample preparation methods and three commercially available DNA extraction kits to optimize vertebrate eDNA recovery. We assessed DNA yield, purity, and fragment length before amplification, as well as vertebrate ZOTU richness and community composition after sequencing. Our results demonstrate that both sample preparation and extraction kit significantly influence DNA recovery from spider webs. Digestion of webs in ATL buffer with proteinase K for 72 h, followed by extraction with a column-based Blood &amp; Tissue kit, yielded the highest mean DNA concentration (10.6 ng/μL) with a mean ZOTU richness of 11.8 ZOTUs per sample. The same digestion method paired with magnetic bead-based extraction produced lower mean DNA concentrations (6.19 ng/μL) but achieved the highest mean ZOTU richness (14.8 ZOTUs per sample). Increased proteinase K concentrations and longer digest times did not significantly improve DNA yield or species richness. Washing webs with phosphate buffer solution combined with PowerLyzer PowerSoil column-based extraction kit produced the lowest total DNA yield (0.16 ng/μL) and richness per sample (1 ZOTU). Although the bead-beating preparation method produced high total DNA yield, it often recovered the lowest mean ZOTU richness, indicating the presence of DNA from non-target taxa such as bacteria. Overall, digestion with ATL buffer and proteinase K (irrespective of digest time), combined with either the Blood &amp; Tissue or MagMAX Microbiome kits, increased vertebrate species detections from spider webs.</p>","PeriodicalId":52828,"journal":{"name":"Environmental DNA","volume":"7 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/edn3.70122","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144520065","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}