Environmental DNA for endangered freshwater mussel monitoring: a review on global synthesis of eDNA methods, challenges, and innovative strategies

1 Introduction

Freshwater mussels (family Unionidae) are among the most diverse and ecologically important groups of benthic macroinvertebrates inhabiting rivers and lakes worldwide. Globally, they are experiencing some of the highest rates of decline among freshwater fauna, with numerous species classified as threatened, endangered, or extinct (Lopes‐Lima et al., 2017). North America is a global hotspot of freshwater mussel diversity, supporting approximately 300 species, more than two-thirds of which are considered imperiled (Haag and Williams, 2014; U.S. Geological Survey, 2015). Similar declines have been reported across Europe and Asia, largely driven by habitat degradation, pollution, altered flow regimes, and invasive species such as the zebra mussel (Dreissena polymorpha), which displaces native unionids (Karatayev and Burlakova, 2025).

Freshwater mussels are not only important components of aquatic biodiversity but also function as ecosystem engineers. An individual mussel can filter liters of water per day, removing suspended particles, bacteria, and phytoplankton, thereby improving water clarity (Vaughn, 2018). Their biodeposits enhance nutrient cycling and support benthic communities, while their shells stabilize sediments and provide habitat for numerous aquatic organisms (Gaston et al., 2013). Consequently, the loss of mussel beds can have cascading effects throughout freshwater ecosystems, reducing water quality, altering food webs, affecting fish communities, and diminishing ecosystem services. Despite their ecological and economic importance, freshwater mussels remain comparatively understudied relative to fish and other charismatic aquatic taxa.

Conventional freshwater mussel surveys primarily rely on qualitative searches, tactile surveys, and quantitative quadrat sampling conducted by trained malacologists and divers (Harvey et al., 2020). Although these approaches provide valuable demographic information, including size-frequency distributions, recruitment, and population structure (Kersting and García-March, 2017), they are labor-intensive, time-consuming, and logistically challenging, particularly in large or turbid rivers. Moreover, survey efficiency may be reduced for deeply burrowed, cryptic, or low-density populations, resulting in false-negative detections that can have important conservation and regulatory consequences (Buckland and Johnston, 2017; Livore et al., 2021). Budgetary and logistical constraints further limit the spatial and temporal coverage of conventional monitoring programs.

In recent years, environmental DNA (eDNA) has transformed aquatic biodiversity monitoring by providing a sensitive and non-invasive approach for species detection. Initially developed for amphibians and invasive fishes (Ficetola et al., 2008; Jerde et al., 2011), eDNA applications to freshwater mussels have progressed more slowly due to low DNA shedding rates, sediment-associated habitats, and PCR inhibition by humic substances (Klymus et al., 2015). However, advances in DNA extraction protocols, Unionidae-specific primers, droplet digital PCR (ddPCR), high-throughput sequencing, and bioinformatic analyses have substantially improved detection sensitivity and analytical reliability (Prié et al., 2021; Wood et al., 2020). Multiple studies have demonstrated that eDNA can improve the detection of freshwater mussels, including rare or endangered species that may be missed by conventional tactile surveys (Sansom and Sassoubre, 2017). Consequently, agencies such as the U.S. Fish and Wildlife Service and organizations implementing the European Water Framework Directive are evaluating eDNA as a complementary tool for biodiversity monitoring and conservation (U.S. Fish and Wildlife Service, 2022; Hering et al., 2018). However, key challenges include contamination risks, DNA transport and persistence, methodological heterogeneity, difficulties linking eDNA concentration to population abundance, incomplete reference databases, and lack of routine monitoring protocols (Hauck et al., 2023; Wu et al., 2024; Chen et al., 2025).

Although previous reviews have addressed eDNA methods or broad freshwater biodiversity issues (Goldberg et al., 2016; Harper et al., 2019; Ruppert et al., 2019), none have comprehensively synthesized global freshwater mussel eDNA research. Previous reviews have focused on general eDNA techniques, broad aquatic biodiversity assessment or freshwater applications but have not comprehensively synthesized the research history, methodological innovation, ecological context and conservation status relating to the use of eDNA for freshwater mussels (Rees et al., 2014; Thomsen and Willerslev, 2015; Deiner et al., 2017; Taberlet et al., 2018). With rapid growth in this field, a timely review that highlighting methodological advances, knowledge gaps, and future research directions is essential for informing scientific research and conservation efforts.

This review systematically examines peer-reviewed literature on freshwater mussel eDNA, focusing on research trends, methodological innovations, ecological interpretation, and regulatory potential of freshwater mussel eDNA monitoring. Specifically, we (i) analyze research characteristics, geographical distribution, study goals, and detection methods; (ii) review sampling strategies, laboratory workflows, molecular markers, and quality controls; (iii) evaluate ecological factors affecting eDNA detection and interpretation; (iv) assess conservation and regulatory evidence; and (v) identify gaps and future research priorities to enhance the role of eDNA in freshwater mussel monitoring, conservation, and regulation.

2 Materials and methods

Our literature search primarily used Google Scholar for broad coverage, supplemented by Web of Science and Scopus to verify references, identify additional relevant studies, and confirm that no major eligible peer-reviewed studies meeting the inclusion criteria were overlooked. The search focused on publications from 2010 to 2025, reflecting eDNA’s rapid development. We conducted three Boolean searches of increasing specificity, rather than successive filtering steps, to comprehensively identify studies relevant to freshwater mussel eDNA. The initial broad search, (“environmental DNA” OR “eDNA”) AND (“freshwater mussel” OR “Unionidae” OR “bivalve”) AND (monitoring OR survey OR conservation OR detection OR metabarcoding OR qPCR OR ddPCR), identified 3,760 records. The search was then refined to (“environmental DNA” OR “eDNA”) AND (“freshwater mussel” OR “Unionidae”) AND (metabarcoding OR qPCR OR ddPCR), yielding 665 records. A final, methodology-focused search, (“environmental DNA” OR “eDNA”) AND (“Unionidae”) AND (metabarcoding OR qPCR OR ddPCR) AND (primer OR filtration OR extraction OR assay), identified 285 records. Reference list screening identified an additional 15 records, bringing the total to 300 records for screening. After applying inclusion/exclusion criteria, which excluded non-peer-reviewed literature (e.g., conference abstracts, reports, and theses) from the systematic review. More detailed inclusion and exclusion criteria are provided in Supplementary Table 1. However, peer-reviewed studies on non-unionid freshwater bivalves were retained only if they generated transferable eDNA methodologies or analytical approaches relevant to freshwater mussel monitoring. Government reports and technical documents were included only to provide regulatory and management context and were not synthesized systematically. After screening titles and abstracts, 234 records were excluded for not meeting the previously set eligibility criteria, and 66 articles were retained for full-text assessment. Of these, 17 studies were excluded for failing to meet the prespecified inclusion criteria, resulting in a final qualitative synthesis of 49 studies (Figure 1). To provide an overview of the temporal distribution of the selected studies, publication years were recorded and summarized (Figure 2). The characteristics of all 49 included studies were summarized in Table 1, including the author and publication year, country or region, target species or group, primary study objective, and detection approach. This table provides an overview of the geographical distribution, taxonomic coverage, research objectives, and methodological focus of the available freshwater mussel eDNA literature.

Table 1. Characteristics of the 49 freshwater mussel environmental DNA (eDNA) studies included in this review.

For each included study, we extracted the study objective, geographical location, target species or group, sampling strategy, laboratory methodology, detection approach, and principal findings. Detailed methodological variables, including sampling matrix, water volume, filtration method, filter pore size, sample preservation, DNA extraction protocol, molecular marker, analytical platform, and quality assurance/quality control (QA/QC) procedures, were synthesized across studies and are presented in Table 2. Evidence on validation against conventional surveys, detection performance, ecological interpretation, methodological limitations, conservation applications, and future research priorities was subsequently synthesized and critically evaluated (Table 3). Because many studies addressed multiple methodological and ecological themes, individual studies could contribute to more than one synthesis category. This final synthesis encompasses research from North America, Europe, and Asia, covering regional case studies (Pont et al., 2018; Doi et al., 2017; Stoeckle et al., 2016) and broader methodological reviews (Rees et al., 2014; Thomsen and Willerslev, 2015). Although not a comprehensive meta-analysis, this systematic approach provides a structured qualitative assessment of the current state of eDNA technology for monitoring and conserving freshwater mussels.

Table 2. Methodological synthesis of environmental DNA (eDNA) approaches used for freshwater mussel and bivalve detection, including sampling strategies, laboratory workflows, molecular methods, sequencing platforms, and quality assurance practices.

Studies (n) are not mutually exclusive because a single study may contribute to multiple methodological categories. The Studies (n) column indicates the total number of studies assigned to each category based on the coded master database. Representative studies are provided as illustrative examples rather than an exhaustive list of all included studies. Counts were derived only from studies for which sufficient methodological information was available.

Table 3. Evidence synthesis of environmental DNA (eDNA) studies on freshwater mussels highlighting major findings, conservation applications, methodological limitations, and future research priorities.

Supporting studies (n) are not mutually exclusive because a single study may contribute to multiple evidence themes. The Supporting studies (n) column indicates the total number of studies assigned to each evidence category based on the coded master database. Representative studies are provided as illustrative examples rather than an exhaustive list of all included studies. Counts were derived only from studies for which sufficient information was available.

2.1 Qualitative assessment of methodological quality

Because the included studies comprised heterogeneous methodological and observational investigations rather than intervention studies, no single established numerical risk-of-bias tool was considered suitable for all studies. Instead, each study was evaluated using a structured qualitative appraisal of methodological attributes relevant to freshwater mussel eDNA monitoring, including sampling design, filtration and preservation methods, DNA extraction protocols, molecular detection methods, QA/QC procedures, ecological validation, and acknowledgment of study limitations. These assessments, presented in Sections 4–7, and the method comparisons in Tables 1-3 are based on these evaluations.

3 Conventional freshwater mussel survey methods: strengths, limitations, and regulatory considerations

3.1 Strengths of conventional freshwater mussel surveys

Field surveys provide the only direct evidence of live mussels. They can be used to collect morphometric data (e.g., age class and size) and to estimate recruitment, mortality, and size-frequency distributions (Simmons et al., 2021; Dolson et al., 2023). Since these methods involve physically locating individuals, they are useful for translocation and reintroduction efforts and provide detailed population information that eDNA cannot yet provide (Hart et al., 2016; U.S. Department of the Interior, 2023). Conventional surveys also provide density estimates, occupancy information, demographic structure, and individual health assessments, which remain difficult to infer reliably from eDNA data alone. Additionally, tactile surveys enable habitat evaluation by comparing mussel distribution with substrate type, flow conditions, and water quality (Haag, 2012), all of which are crucial for recovery planning and regulatory compliance.

3.2 Practical and logistical challenges

However, tactile surveys are costly and resource intensive. Large-river diver-based surveys often involve trained divers, boats, and safety precautions, they can be costly and resource-intensive and may involve disturbance associated with diver-based specimen searches (Peters et al., 2019). Safety concerns arise due to strong currents, poor visibility, and the risk of entanglement (Kurth et al., 2007). Detection can be low due to mussels burrowing into sediments or occurring at low densities, leading to false absences despite extensive effort. Meador et al. (2011) reported capture probabilities ranging from 8–20% for Alasmidonta arcula, Lampsilis dolabraeformis, Lampsilis splendida, and Pyganodon gibbosa in a large lowland river, while Reid (2016) reported particularly low detection probabilities for Triangle Floater (Alasmidonta undulata) during short-duration timed-search surveys. These limitations restrict survey coverage and can compromise the representativeness of results. Recent advances, including underwater imaging systems, side-scan sonar, remotely operated vehicles (ROVs), occupancy modelling, and geographic information system (GIS)-assisted survey planning, have improved survey efficiency in some aquatic environments (Dolson et al., 2023). Despite these advances, these methods still rely on visual or specimen verification and are generally labor-intensive, especially for deep, turbid or large river systems.

3.3 Regulatory and conservation implications

The outcomes of traditional surveys carry legal significance. Even detecting a single protected species can delay or alter infrastructure projects, such as dredging, bridge construction, and pipeline installation. Conversely, false negatives pose the risk of habitat destruction if a species is actually present but not observed (Akani et al., 2022). Survey results are used to inform regulatory decisions, including conservation planning, environmental impact assessments, species recovery programs, and endangered species-related legislation (Dokai et al., 2023). As a result, false negatives can either postpone conservation measures or risk habitat disturbance. This dual risk highlights the importance of accurate survey methods, yet imperfect detection probabilities may still introduce uncertainty into conservation planning, permitting decisions, and species recovery programs (Buckland and Johnston, 2017; Akani et al., 2022). Overall, conventional freshwater mussel surveys remain indispensable for estimating population size, demographic structure, recruitment, and habitat condition (Harvey et al., 2020; Kersting and García-March, 2017); however, their relatively high cost, logistical constraints, and imperfect detection highlight the need for complementary molecular approaches (Peters et al., 2019; Meador et al., 2011; Reid, 2016). A comparison of the strengths, limitations, and conservation applications of conventional survey methods and eDNA-based approaches is presented in Table 4. This comparison provides the foundation for the following sections, which examine how advances in eDNA technologies can address many of the limitations associated with traditional freshwater mussel surveys while complementing, rather than replacing, conventional monitoring methods.

Table 4. Comparison of conventional freshwater mussel survey methods and environmental DNA (eDNA) approaches for monitoring, conservation, and regulatory applications.

4 Advances in eDNA approaches for freshwater mussel monitoring

The use of eDNA to study mussels is rapidly evolving from single-species proof-of-concept studies to a mature tool capable of surveying entire communities for conservation management. Advances in molecular methods, such as sequencing and bioinformatics, have greatly increased the efficiency of eDNA detection and enabled broader ecological insights (Deiner et al., 2017; Ruppert et al., 2019).

4.1 Development of freshwater mussel eDNA research

Freshwater mussel eDNA research has expanded rapidly over the past decade, reflecting the increasing adoption of molecular approaches for biodiversity assessment and conservation (Figure 2). The 49 studies included in this review (Table 1) were published between 2015 and 2025, with publication frequency increasing markedly after 2020 and peaking in 2025. The geographic distribution of the included studies is summarized in Figure 3. Geographically, research has been dominated by North America, with the United States accounting for 38.8% of the reviewed studies, followed by Canada (12.2%), China (8.2%), Japan (8.2%), France (6.1%), and New Zealand (4.1%), while the remaining studies were distributed across Europe and Asia (Table 1). This geographic distribution echoes the strong research focus on freshwater mussel conservation in areas with imperiled unionids that are prime management targets, reinforcing the need for future validation in under-represented regions. The characteristics of the studies included in this review also highlighted three key stages in the development of eDNA research with freshwater mussels. Early research focused on assay development and proof-of-concept experiments to identify single threatened or invasive taxa. As freshwater mussel eDNA methods became more reliable, research expanded beyond assay development to examine ecological applications, including abundance estimation, downstream DNA transport, seasonal variation, and habitat-specific detection. More recent studies have increasingly used high-throughput metabarcoding and community-level analyses to support biodiversity assessment, conservation planning, and ecosystem monitoring. This trajectory highlights the evolution of freshwater mussel eDNA work from a simple species-detection assay to a fundamental platform for ecological monitoring and conservation management.

The literature reviewed also corroborates a discernible evolution in both scientific aims and molecular technologies. The initial studies mainly focused on the development of species-specific assays for individual endangered or invasive freshwater mussels; by contrast, recent studies more frequently used metabarcoding approaches to characterize entire communities of freshwater mollusks to support biodiversity assessments and conservation planning, transport modeling, abundance estimation, and ecosystem-level monitoring. Consistent with this transition, qPCR-based methods represented the most frequently used detection approach (46.0% of reviewed studies), followed by metabarcoding approaches (32.0%), whereas conventional PCR and digital PCR/ddPCR were applied less frequently, mainly for assay development, validation, and rare-species detection (Table 1). Collectively, these findings demonstrate a progressive shift from targeted species detection toward community-level ecological assessment, establishing eDNA as an increasingly mature and versatile tool for freshwater mussel research and conservation. These research trends provide the foundation for the technological developments discussed below and the methodological and ecological syntheses presented in subsequent sections.

4.2 Evolution of molecular detection technologies

eDNA detection for freshwater mussels has evolved from conventional PCR-based assays to qPCR, ddPCR, and high-throughput metabarcoding approaches, each providing distinct analytical advantages and limitations depending on study objectives (Valentini et al., 2016; Taberlet et al., 2018). Conventional PCR is supported for primary development and confirmation of presence or absence, yet it offers lower analytical sensitivity than quantitative methods. qPCR has emerged as the dominant platform due to its high sensitivity, quantitative nature, and relatively low cost (Wood et al., 2020) and utility for regular monitoring of endangered freshwater mussels. Consistent with this trend, qPCR-based approaches accounted for 46% of the reviewed studies (Table 1). ddPCR further improves sensitivity by partitioning reactions into thousands of droplets, enabling absolute DNA quantification without standard curves and increasing robustness when target DNA concentrations are extremely low or PCR inhibitors are present (Wacker et al., 2019). Consequently, ddPCR is particularly valuable for detecting rare or highly threatened mussel populations. In contrast, metabarcoding enables simultaneous detection of multiple taxa from a single environmental sample (Prié et al., 2023; Marshall et al., 2022). Although metabarcoding generally provides broader ecological information than species-specific assays, its performance is influenced by primer bias, sequencing depth, and the completeness of reference databases (Klymus et al., 2020b; Prié et al., 2021). No single molecular approach is optimal for all monitoring objectives. Platform selection should therefore be guided by the study goal: species-specific assays generally provide greater sensitivity for detecting rare or threatened target species, whereas metabarcoding is more efficient for community-level biodiversity assessments and simultaneous detection of multiple taxa. These approaches are increasingly regarded as complementary components of integrated freshwater mussel monitoring programs rather than as competing alternatives. From a practical perspective, qPCR is commonly used for routine monitoring of target species because it generally offers relatively high throughput and lower analytical costs. ddPCR typically requires more specialized equipment and may involve higher per-sample costs and longer processing times, but it can provide more precise quantification at low target concentrations and greater tolerance to PCR inhibition under some conditions (Doi et al., 2015; Wood et al., 2019; Guri et al., 2024). Metabarcoding requires sequencing and bioinformatic analysis and may be less sensitive than a well-validated species-specific assay for individual rare taxa; however, it can efficiently characterize multiple taxa simultaneously and is therefore particularly useful for community-level biodiversity assessments (Miya et al., 2015; McColl-Gausden et al., 2023).

4.3 Advances in bioinformatics

Advances in bioinformatics have substantially improved the accuracy and reproducibility of freshwater mussel eDNA analyses. Amplicon sequence variant approaches, including Divisive Amplicon Denoising Algorithm 2 (DADA2), UNOISE denoising algorithm, and Deblur, are now widely used as alternatives to conventional operational taxonomic unit (OTU) clustering. These methods model or remove sequencing errors to infer exact sequence variants, potentially providing single-nucleotide resolution and improving the reusability and comparability of results across datasets generated from the same marker region, and facilitating direct sequence comparison among studies and against reference databases (Callahan et al., 2016, Callahan et al., 2017). However, OTU-based approaches remain in use, and the choice of bioinformatic method can substantially affect estimates of diversity and community composition; therefore, amplicon sequence variants (ASV)-based analyses should not be assumed to outperform OTU clustering in every application (Joos et al., 2020; Chiarello et al., 2022; Nilsen et al., 2024). Despite advancements, bioinformatics performance remains heavily reliant on the quality and taxonomic coverage of reference sequence databases. This parameter is especially relevant to freshwater mussels, given their long evolutionary histories and the fact that many rare, endemic or geologically restricted species are not yet included in public databases, resulting in lower taxonomic resolution, leading to higher rates of unassigned sequences (Klymus et al., 2020a; Prié et al., 2021).

5 From field to laboratory: sampling, preservation, and molecular approaches in unionid mussel eDNA studies

Field protocols are crucial to the success of any eDNA study; sampling choices influence detection probabilities and repeatability. For freshwater mussels, which can have patchy distributions and release DNA in low amounts, the design of the sampling strategy, filtration method, and preservation protocol is particularly important (Figure 4). Table 2 synthesizes methodological information from the 49 reviewed studies and confirms substantial variation in sampling matrixes, water volumes, filtration strategies, pore sizes, preservation methods, DNA extraction protocols, molecular markers, analytical platforms, and quality assurance/quality control (QA/QC) procedures. This methodological heterogeneity remains one of the principal barriers to protocol harmonization and direct comparison among freshwater mussel eDNA studies. Despite this variability, several consistent methodological trends emerged across the reviewed literature (Table 2). Water was the dominant sampling matrix (34 studies), moderate- to high-volume sampling (>1–5 L) represented the most common field strategy, freezing remained the primary preservation approach, silica-based commercial extraction kits were the predominant DNA extraction method, and targeted qPCR assays were the most frequently used molecular approach, followed by metabarcoding. Although these findings suggest increasing convergence toward common laboratory workflows, considerable variation persists in filtration materials, pore sizes, molecular markers, sequencing platforms, and QA/QC procedures, underscoring the need for standardized methodologies before widespread regulatory implementation.

5.1 Sampling design and replication

Site selection should account for both hydrological conditions and freshwater mussel ecology. Sampling upstream and downstream of known or suspected mussel beds can help evaluate local occurrence while accounting for downstream eDNA transport (Sansom and Sassoubre, 2017; Preece et al., 2021). Spatial replication, including multiple sampling locations within a river reach and multiple water samples per location, can improve detection probability and capture variation associated with habitat type, flow conditions, and the patchy distribution of unionid populations. Temporal replication is also important because reproductive activity, discharge, temperature, and season can influence eDNA shedding, transport, degradation, and detection (Stoeckle et al., 2017; Marshall et al., 2025). Consistent with Table 2, the reviewed studies used widely varying sampling designs across river types, monitoring objectives, and target species, indicating a need for explicit justification of spatial and temporal replication rather than a single standardized design for all freshwater mussel surveys.

5.2 Water volume and filtration

Filtration is the most common method for concentrating on eDNA from water. Typically, glass fiber filters (0.7–1.2 μm) and mixed cellulose ester filters (0.45 μm) are used, but selecting the appropriate pore size involves balancing filtration volume with clogging risk in turbid rivers. Mussel habitats often have high suspended sediment loads, which can cause clogging. In such cases, prefiltering or using large-pore filters may allow for processing more water while still capturing sufficient DNA. Consistent use of the same filter type and pore size across a project is important for reliable detection probabilities (Spens et al., 2017). Table 2 indicates that water volumes greater than 1 L were most commonly used in field surveys, whereas larger pore-size filters were frequently selected in turbid systems to reduce clogging, illustrating the practical balance between DNA capture efficiency and field feasibility.

5.3 Preservation and storage

Preservation methods must stabilize DNA until it is processed in the laboratory. Ethanol and long-term freezing are common, but field-compatible options, such as DNAzol and Longmire’s solution, are increasingly used when samples cannot be kept cold immediately (Renshaw et al., 2015). For unionid mussels, which often contain inhibitors such as humic substances (Schrader et al., 2012), extraction kits may require additional purification steps or inhibitor-removal columns to ensure successful PCR amplification (McKee et al., 2015). Standardization of extraction procedures enhances comparability, and including negative controls in the field helps differentiate contamination from future detections. Cold-chain transport of samples, which enhances DNA integrity (Rees et al., 2014), is likely to be supported by appropriate field-handling practices. Samples should, whenever possible, be kept on ice in the field and until transferred to −20 °C or −80 °C freezers. Long-term storage at room temperature can cause rapid DNA degradation, especially in hot climates (Robinson et al., 2021). The methodological synthesis further indicated that freezing was the most commonly reported preservation strategy, whereas ethanol- and buffer-based methods were more often used in studies where maintaining a cold chain was difficult or impractical, illustrating how field logistics can influence protocol selection.

5.4 DNA extraction methods

Many unionid eDNA investigations have used commercially available silica-based extraction kits (e.g., Qiagen DNeasy, PowerWater, and similar protocols) because of their standardized methods and reproducibility. Nonetheless, extraction efficiency is highly dependent on the filter material, pore size, and preservation technique, and little work has systematically compared extraction methods for unionids. The variation in methods underscored a general need to standardize extraction protocols to improve the utility of mussel eDNA detection (Lopes‐Lima et al., 2017; Stoeckle et al., 2021). Table 2 shows that Qiagen DNeasy-based methods predominated across the reviewed literature; however, PowerWater, NucleoSpin, CTAB, and modified laboratory protocols were also employed, demonstrating that no universally adopted extraction workflow has yet emerged.

5.5 Inhibitor removal strategies

Freshwater systems are often PCR-inhibiting environments due to humic substances, sediment, and organic matter, which reduce amplification efficiency and can lead to false negatives. Many unionid eDNA studies include inhibitor removal using commercial kits or other purification methods, as well as inhibitor-tolerant polymerases, to reduce PCR inhibition; however, the use of these techniques varies across investigations. Little comparative evidence on inhibitor-removal methods in unionid monitoring is available, underscoring the importance of standardized laboratory procedures (Goldberg et al., 2016; Harper et al., 2019). The reviewed studies also showed considerable variation in the implementation of QA/QC procedures, including inhibition testing, negative controls, positive controls, and technical replicates, indicating that quality assurance practices have improved but remain inconsistently reported among freshwater mussel eDNA investigations.

5.6 Molecular markers and analytical platforms

Several mitochondrial markers have been used to detect species in unionid mussel eDNA studies to date (e.g., COI, 16S and ND1) (Coghlan et al., 2021; Klymus et al., 2021). Table 2 shows that COI remained the most frequently used genetic marker for species-specific detection, whereas 16S rRNA and ND1 were widely applied in metabarcoding and unionid-specific assays. More recent studies have increasingly adopted multi-marker strategies and model-assisted analytical frameworks to improve taxonomic resolution and strengthen ecological interpretation (Prié et al., 2023; Marshall et al., 2022).

5.7 Implications for methodological standardization

Table 2 highlights that, although several methodological components are converging toward common practice, considerable variation persists across the freshwater mussel eDNA workflow. Water sampling, glass fiber filtration, freezing-based preservation, column-based DNA extraction, COI-targeted assays, and qPCR have emerged as the most frequently adopted approaches, whereas sequencing platforms, metabarcoding workflows, and quality assurance practices remain more heterogeneous. Together, these standards will enable eDNA monitoring in freshwater mussels to gain further regulatory acceptance and broad practical application by harmonizing of sampling protocols, laboratory procedures, QA/QC frameworks, and reporting standards, thereby improving reproducibility and facilitating cross-study comparisons.

6 Ecological interpretation of unionid mussel eDNA signals

Interpretation of freshwater mussel eDNA signals extends beyond methodological performance and requires consideration of the ecological processes that influence DNA production, transport, persistence, and detection (Figure 5). Table 3 synthesizes the major ecological findings, methodological limitations, conservation applications, and future research priorities identified across the 49 reviewed studies. Collectively, the evidence reflects that eDNA detection is governed by complex biological, environmental, and methodological factors. Consequently, ecological context is essential for interpreting eDNA results and applying them effectively in freshwater mussel conservation and management.

6.1 Ecological factors influencing eDNA detection and interpretation

The reviewed literature indicates that ecological processes strongly influence freshwater mussel eDNA detection and interpretation (Table 3). A few studies reported associations between eDNA concentration and mussel abundance, density, biomass, or catch per unit effort (CPUE) (Stoeckle et al., 2021; Togaki et al., 2020; Wu et al., 2024; Currier et al., 2018), suggesting that eDNA may provide useful estimates of relative abundance under appropriate environmental conditions. However, these relationships were not consistently linear and were influenced by hydrological conditions, sampling strategy, DNA degradation, and analytical methodology. Consequently, current evidence supports the use of eDNA for relative rather than absolute population assessment.

Another major ecological transport factor is hydrological transport. Seven studies showed that the transport of eDNA downstream can affect spatial distribution, particularly in flowing river systems (Table 3). Detection of eDNA might thus occur downstream of locations that support an occurrence, complicating the assessment of species presence and the delineation of occupied reaches. These results highlight the necessity of including definitions of hydrological characteristics in survey design and analysis when monitoring rare or threatened freshwater mussel populations.

Seasonal biological processes also influence detection probability. Eight studies reported that reproductive activity, spawning periods, and seasonal environmental variation significantly affected eDNA detection success (Table 3). Increased DNA release during reproductive periods may enhance detection sensitivity, whereas seasonal changes in flow conditions, temperature, and microbial activity may accelerate DNA degradation or alter transport dynamics. Consequently, sampling seasons should be standardized or explicitly justified when comparing results among studies.

Sampling design further contributed to variation in detection. Eight studies identified sampling location, water depth, filtration volume, and spatial replication as important determinants of eDNA recovery. Appropriate spatial and temporal replication, therefore, remains essential for reducing false-negative detections and improving confidence in freshwater mussel monitoring programs. Taken together, the reviewed evidence indicates that freshwater mussel eDNA signals should be interpreted within their ecological context rather than as direct indicators of local population size or occupancy. Integrating biological knowledge with hydrological and environmental information substantially improves the reliability of eDNA-based conservation assessments.

6.2 Conservation implications and remaining uncertainties

Despite substantial methodological advances, several important limitations remain (Table 3). Twelve studies identified PCR inhibition, turbidity, pH, or poor water quality as major factors that reduced detection success, while nine studies reported false-negative detections in low-density populations or in environmentally challenging systems. These findings emphasize the importance of adequate field replication, inhibitor testing, and robust laboratory quality assurance procedures.

The reviewed studies also highlighted limitations associated with molecular assays. Six studies reported that primer bias, marker selection, or incomplete reference databases reduced taxonomic resolution and influenced species identification, particularly in metabarcoding applications. Continued expansion of regional reference databases and further assay validation are therefore essential for improving detection accuracy.

Although several studies have reported positive relationships between eDNA concentration and mussel abundance, the evidence available to date indicates that eDNA is more reliable for detecting species presence than for estimating population size or demographic structure. Accordingly, eDNA is currently best used as a complementary monitoring tool when demographic information is required; however, continued advances in quantitative assays, hydrological modelling, and occupancy-based approaches may improve its future capacity to support population-level inference. The conservation value of freshwater mussel eDNA monitoring is nevertheless strongly supported by the available evidence. Table 3 reveals that eDNA has been successfully applied for validation against conventional surveys, detection of rare and endangered species, biodiversity assessment, conservation planning, and rediscovery of previously undetected populations. Future research should prioritize protocol standardization, integration of hydrological and occupancy models, expansion of reference databases, and multi-marker approaches to further improve ecological interpretation and support broader regulatory implementation of freshwater mussel eDNA monitoring (Goldberg et al., 2016).

7 Regulatory adoption and global implementation framework for freshwater mussel eDNA

7.1 Current regulatory status and validation requirements

eDNA is increasingly being incorporated into freshwater mussel conservation and biodiversity monitoring as a complementary tool to confirm the presence of rare, threatened, and endangered species. For example, the U.S. Fish and Wildlife Service has incorporated eDNA into conservation genetics and monitoring programs, demonstrating its value for supporting conventional surveys and informing management decisions (Waits et al., 2025). Across the reviewed literature, eDNA consistently demonstrated high detection sensitivity when appropriate sampling strategies, validated molecular assays, and rigorous quality assurance procedures were implemented (Table 3). However, regulatory acceptance still requires broad assay validation, standardized protocols, reproducibility across laboratories and detailed reconciliation of false-positive and false-negative detection rates. Hence, freshwater mussel eDNA is presently viewed as a supplemental line of evidence rather than an absolute substitute for traditional surveys, especially where demographic or habitat data are needed to inform legal or regulatory decisions. Figure 6 summarizes a framework for assay development, validation, standardization and regulatory implementation.

7.2 Operational implementation framework

Successful implementation of freshwater mussel eDNA monitoring requires a coordinated workflow extending from assay development to regulatory adoption (Figure 6). Initial assay development should include verification of taxonomic specificity using reference databases and laboratory validation of detection limits, repeatability, and analytical sensitivity (Valentini et al., 2016; Sepulveda et al., 2020). Subsequent field validation should directly compare eDNA results with conventional mussel surveys under representative environmental conditions (Wilcox et al., 2015). Pilot studies are essential for optimizing sampling strategies, preservation methods, filtration protocols, and bioinformatic workflows before regional implementation. Standardized laboratory procedures, inter-laboratory comparisons, certified reference materials, and comprehensive training programs will further improve reproducibility and facilitate adoption by resource managers and conservation agencies. Early collaboration among researchers, regulatory agencies, and environmental managers is critical to ensuring that scientific advances are efficiently translated into operational monitoring programs (Taberlet et al., 2018; Rodriguez et al., 2025).

7.3 Freshwater mussel-specific regulatory challenges

Although substantial progress has been achieved, several challenges continue to limit the regulatory implementation of freshwater mussel eDNA monitoring. As demonstrated by the evidence synthesis (Table 3), hydrological transport, seasonal variation, PCR inhibition, incomplete reference databases, and imperfect detection probabilities remain important sources of uncertainty. Moreover, while eDNA is highly effective for confirming species presence, it currently provides limited information on demographic parameters such as age structure, recruitment, population size, and population viability, which remain essential for endangered species assessments and environmental permitting. Consequently, freshwater mussel conservation programs will continue to benefit from integrated monitoring strategies in which eDNA is used to prioritize survey locations and complement.

7.4 Future directions for global implementation

Future implementation should prioritize harmonization of international protocols, expansion of regional reference databases, increased data sharing, user-friendly bioinformatics pipelines, and continued development of quantitative molecular approaches (Djaffardjy et al., 2023; Rodriguez et al., 2025). Unionid mussels remain unevenly distributed globally, with major biodiversity hotspots occurring in North America, East Asia, and parts of Africa (Haag and Williams, 2014; Lopes‐Lima et al., 2017). However, most freshwater mussel eDNA studies have been conducted in North America and Europe, leaving substantial geographical knowledge gaps across tropical and subtropical regions. Expanding freshwater mussel eDNA monitoring into underrepresented regions, together with stronger international collaboration and regulatory harmonization, will improve biodiversity assessment, conservation planning, and long-term monitoring of imperiled unionid populations worldwide.

8 Conclusions and future research directions

eDNA has become a valuable tool for freshwater mussel monitoring and conservation. This review synthesized evidence from 49 peer-reviewed studies, providing a comprehensive assessment of research trends (Table 1), methodological developments (Table 2), and ecological evidence, conservation applications, and remaining limitations (Table 3). Relationships between eDNA concentration and mussel abundance are improving, but current evidence remains insufficient for reliably estimating population size or demographic characteristics across most freshwater mussel systems. Consequently, eDNA is currently most effective when integrated with conventional surveys and ecological information. Continued protocol harmonization and standardized reporting will be essential to improve reproducibility, facilitate cross-study comparisons, and support broader regulatory adoption.

Future research should prioritize standardized field and laboratory protocols, improved quantitative relationships between eDNA concentration and abundance, expanded regional reference databases, enhanced bioinformatic pipelines and multi-marker approaches, broader inter-laboratory validation, and the evaluation of emerging technologies, including machine learning-assisted data analysis, automated water sampling systems, portable sequencing platforms, and long-read sequencing technologies, to improve taxonomic resolution, field deploy ability, and routine regulatory implementation. eDNA complements conventional freshwater mussel monitoring. Continued collaboration among researchers, conservation practitioners, and regulatory agencies will be essential to translate methodological advances into standardized monitoring frameworks that strengthen biodiversity assessment and evidence-based freshwater mussel conservation.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcosc.2026.1891198/full#supplementary-material

Inclusion and exclusion criteria used for study selection during the systematic literature review. Government reports and technical documents were consulted only to provide regulatory and management context and were not included in the systematic qualitative synthesis. Reference lists of eligible studies were manually screened to identify additional relevant peer-reviewed publications. Supplementary material at the source

Footnotes

Edited by: Zuogang Peng, Southwest University, China

Reviewed by: Thadickal V Joydas, National Center for Wildlife, Saudi Arabia

Arnelyn D. Doloiras-Laraño, Ehime University, Japan