Frontiers of Environmental DNA in Aquatic Biodiversity Monitoring: From Technical Validation to Ecological Insight

1. Introduction

Globally, aquatic ecosystems are facing unprecedented and multifaceted pressures. Habitat fragmentation, overfishing, dam construction, invasive alien species, and hydrological fluctuations driven by climate change are collectively driving a drastic decline in fish diversity, severely threatening the health and stability of aquatic ecosystems [1,2,3]. Accurate and efficient monitoring of the composition and dynamics of fish communities is the cornerstone for formulating science-based conservation strategies and evaluating management effectiveness. Traditional fish survey methods, such as electrofishing, gillnetting, and trawling, despite contributing indispensable foundational data over recent decades, possess inherent limitations. These include high labor intensity, destructiveness to aquatic organisms, sampling difficulties in complex habitats (e.g., deep waters, rapids), and insufficient capacity to detect rare, cryptic species or those in early life stages [4].

Against this backdrop, the emergence of environmental DNA (eDNA) technology has revolutionized the field of aquatic biomonitoring [5]. This technique enables species identification by extracting and analyzing trace amounts of genetic material shed by organisms into environmental samples (water, sediment, etc.), significantly reducing reliance on direct capture or observation. Specifically, eDNA metabarcoding allows for the simultaneous resolution of multi-species information within an ecosystem, providing an unprecedented tool for rapid, comprehensive, and non-invasive biodiversity assessment [6,7]. Since Ficetola et al. [8] first applied eDNA for invasive species detection in 2008, the technology has rapidly permeated to the frontiers of ecology and environmental science, including target species monitoring, biodiversity inventorying, early warning of invasive species, and ecosystem health assessment [9,10,11].

However, despite its transformative potential, the application of eDNA technology is not without challenges. Issues such as incomplete reference databases and a lack of standardized protocols across studies can introduce uncertainties in data interpretation. Consequently, traditional monitoring methods remain indispensable for validating eDNA findings and ensuring the robustness of biodiversity assessments [12].

This Special Issue—“Mapping and Monitoring Aquatic Biodiversity in Hotspot Habitat Areas Using Environmental DNA (eDNA)”—brings together nine innovative studies utilizing eDNA for aquatic biodiversity research, collectively showcasing the latest advancements and diverse applications in this dynamic field. These investigations span a wide array of aquatic ecosystems, from high-mountain streams to large reservoirs and from urban lakes to international rivers, deep-diving into core issues such as species identification accuracy, diversity assessment efficiency, spatial pattern analysis of communities, and synergistic application with traditional monitoring methods. Based on the logical interconnections of their research foci, these nine articles can be grouped into the following four central themes: (1) Construction of DNA barcode reference libraries and discovery of cryptic diversity; (2) Application of eDNA in fish diversity monitoring and community dynamics analysis; (3) Synergy, comparison, and validation of eDNA with traditional monitoring methods; and (4) Pioneering exploration of eDNA in precise detection of invasive species.

2. Synopsis of the Special Issue

2.1. Construction of DNA Barcode Reference Libraries and Discovery of Cryptic Diversity

Accurate species identification is the logical starting point for the effective application of eDNA, a premise that is highly dependent on comprehensive and reliable DNA barcode reference libraries [13]. Shi et al. [14] conducted a systematic study on fishes of the Yuanjiang River Basin in China, constructing a reference library encompassing 64 morphologically identified species, represented by 764 barcode sequences. This study not only validated the high success rate (>93%) of DNA barcoding for fish identification in this river, but, more importantly, employed barcoding gap analysis and various DNA-based delimitation methods to reveal significant intraspecific genetic divergences exceeding the thresholds in four species. This finding suggests that these morphological species may harbor undescribed cryptic species, providing crucial clues for subsequent taxonomic revision and the delineation of finer-scale conservation units. This work powerfully demonstrates the fundamental role of improving regional DNA barcode libraries in truly revealing and quantifying biodiversity.

2.2. Application of eDNA in Fish Diversity Monitoring and Community Dynamics Analysis

Several papers in this issue showcase the remarkable capability of eDNA in rapidly acquiring baseline data on fish diversity and resolving community spatiotemporal dynamics. In a study on the Qingshui River, Huang et al. [15] detected 91 fish species using eDNA, 20 more than historically recorded, fully embodying the ultra-high sensitivity of this technique for constructing more complete species inventories. Similarly, Yuan et al. [16] confirmed in their study of the Pingzhai Reservoir that eDNA detected significantly more fish species (43) compared to traditional catch surveys (29) and successfully monitored alien species like Micropterus salmoides and Oreochromis niloticus, providing essential baseline information for reservoir ecosystem management.

Regarding the resolution of spatial patterns, Dong et al. [17] focused on the Upper Yangtze River Reserve. Using eDNA metabarcoding, they revealed the spatial distribution characteristics of 93 fish species, discovering that the Chishui River not only possessed the highest species richness but also exhibited a significantly distinct community structure from other dam-impacted rivers, underscoring its unique conservation value as an undisturbed reference system and critical habitat. Liu et al. [18], studying three rivers in the mountainous region of southern Anhui, further elucidated the special ecological function of confluences within river networks. They found that during the dry season, the taxonomic, functional, and phylogenetic diversity at confluences was significantly higher than in the mainstem, providing direct scientific evidence for identifying and protecting biodiversity hotspots.

In exploring spatiotemporal dynamics, the seasonal survey of the Three Gorges Reservoir area by Huang et al. [19], spanning breeding, feeding, and overwintering periods, is particularly noteworthy. The authors found that fish community structures during breeding and overwintering periods were relatively similar, while in the feeding season they differed significantly, with the reservoir’s tail and tributaries identified as preferred fish habitats. Integrating environmental factor analysis, the results highlighted water temperature as the primary environmental driver shaping fish community structure in this reservoir, offering significant insights into understanding the impacts of reservoir operation on fish life history.

2.3. Synergy, Comparison, and Validation of eDNA with Traditional Monitoring Methods

Comparing and validating eDNA data against long-term, systematic traditional monitoring datasets is a critical step in establishing its reliability and facilitating its transition from research to application [20]. Brammell et al. [21] conducted an exemplary comparative study in the high-elevation streams of the Great Smoky Mountains National Park. They systematically compared eDNA metabarcoding results with decades of historical electrofishing data, finding high congruence in species composition. Notably, a primer set targeting a shorter amplicon (97 bp) detected 90% of species, outperforming a primer set targeting a longer fragment (225 bp), suggesting that primer design critically influences detection success in environments where eDNA is prone to degradation [22]. Furthermore, significant positive correlations were found between eDNA read counts and historical biomass data for five common species, providing valuable empirical support for the potential of eDNA technology in quantitative fish stock assessment. This finding echoes the conclusions of Vučić et al. [23] in their study of urban Croatian lakes, where eDNA successfully evaluated the outcome of invasive species removal efforts. By comparing pre- and post-cleanup detection results, they confirmed the absence of most target IAS (e.g., Ameiurus melas, Lepomis gibbosu), thus validating the effectiveness of management actions.

2.4. Pioneering Exploration of eDNA in Precise Detection of Invasive Species

The high sensitivity of eDNA technology confers unique advantages for the early detection of invasive species and precise monitoring of pathogens [24]. In the Three Gorges Reservoir study, Huang et al. [19] successfully monitored two fish species listed in China’s “Key Management Invasive Alien Species Inventory”—Coptodon zillii and Pterygoplichthys pardalis—clearly revealing their spatiotemporal distribution patterns within the reservoir, providing critical intelligence for targeted early intervention and precise prevention and control. Jothinarayanan et al. [25] further extended the application of loop-mediated isothermal amplification (LAMP) technology in fish eDNA detection. Focusing on Lake Borre and the Drammen River in Norway, Esox lucius, Anguilla anguilla, and Salmo salar were selected as target species, encompassing three categories: invasive species, endangered species, and data-deficient species. Two sets of LAMP primers were designed for each species. The study confirmed that LAMP technology can efficiently and specifically identify target fish eDNA, providing a feasible technical pathway for rapid species monitoring and conservation management in aquatic ecosystems.

3. Conclusions

The nine studies in this Special Issue collectively chart the maturation of environmental DNA technology in aquatic biodiversity monitoring. The findings consistently demonstrate that eDNA metabarcoding, with its exceptional sensitivity, broad taxonomic coverage, and non-destructive nature that avoids physical harm to organisms, exhibits significant potential, surpassing or complementing traditional methods in revealing cryptic diversity, constructing comprehensive species inventories, resolving community spatiotemporal dynamics, and providing early warnings for invasive species and pathogens [14,15,16,17,18,19,21,22]. From foundational barcode library construction in the Yuanjiang River to spatiotemporal analyses in the Three Gorges Reservoir and Great Smoky Mountain streams, these works validate eDNA’s reliability and underscore its value as a core tool bridging molecular biology, ecology, and environmental management.

Looking ahead, eDNA technology will inevitably progress beyond “presence/absence detection” toward standardization, quantification, and intelligence [26]. Priorities include constructing high-precision regional DNA barcode databases, optimizing sampling protocols to establish industry standards, and developing algorithms for inferring species biomass from eDNA data [27,28]. Crucially, eDNA must be integrated with long-term traditional monitoring and artificial intelligence to build an integrated multi-source monitoring network [29,30]. Only then can it truly become an indispensable tool for mapping aquatic biodiversity hotspots, providing an irreplaceable scientific foundation for safeguarding aquatic life and achieving sustainable fishery management in a changing world.