# Environmental DNA metabarcoding for catchment-scale detection of aquatic plants, invasive species, and land-use indicators in a large river

Armando Espinosa Prieto, Laurent Hardion, Nicolas Debortoli, Thibaut

Bournonville, Jonathan Marescaux, Karina Anna Elisabeth van der Zon, Jean-Nicolas Beisel

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Armando Espinosa Prieto, Laurent Hardion, Nicolas Debortoli, Thibaut Bournonville, Jonathan Marescaux, et al.. Environmental DNA metabarcoding for catchment-scale detection of aquatic plants, invasive species, and land-use indicators in a large river. Ecological Indicators, 2025, 178, pp.113943. 10.1016/j.ecolind.2025.113943. hal-05413175

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Original Articles

# Environmental DNA metabarcoding for catchment-scale detection of aquatic plants, invasive species, and land-use indicators in a large river

Armando Espinosa Prieto<sup>a,*</sup>,<sup> ID </sup>, Laurent Hardion<sup>a</sup>, Nicolas Debortoli<sup>b,1</sup>, Thibaut Bournonville<sup>b</sup>, Jonathan Marescaux<sup>b</sup>, Karina Anna Elisabeth van der Zon<sup>a</sup>, Jean-Nicolas Beisel<sup>a</sup>

ARTICLE INFO

Keywords:

eDNA

Macrophytes

Invasive species

Biodiversity assessment

Land use

River biomonitoring

A B S T R A C T

Monitoring plant biodiversity in large river systems is essential for ecological assessment but remains logistically challenging due to poor visibility, strong currents, and broad spatial scales. Environmental DNA (eDNA) metabarcoding offers a minimally invasive solution, allowing large-scale biodiversity surveys by analysing aquatic and terrestrial plant diversity from river water samples. Here, we present the first application of multi-marker eDNA metabarcoding for integrated plant biodiversity assessment in a large river system, targeting aquatic vegetation, invasive species, and land-use-associated flora along the Upper Rhine River between Basel (Switzerland) and Strasbourg (France). Our method recovered 24 aquatic plant species (18% of reads), under-scoring the potential of this approach for monitoring macrophytes in large rivers where conventional visual surveys are impractical. Land-use indicators, including crop and ornamental species, comprised 5.5% of reads, and contrasted between the human-modified canal of the Rhine and the natural river channel. We also detected 16 exotic plant species, highlighting the method's potential for monitoring and managing invasive populations. However, the spatial resolution of species detection could benefit from closer sampling along the river and additional samples across its cross-section. This study highlights the utility of multi-marker eDNA metabarcoding as a scalable, non-invasive tool for catchment-wide vegetation monitoring and environmental management in large river systems.

# 1. Introduction

Large rivers are the arteries of human civilisation, supporting large populations, enabling the production of food, industrial products, and power, as well as facilitating the rapid transport of goods and people (Wantzen et al., 2016). Undeveloped large rivers and their floodplains are also among the most diverse ecosystems on Earth (Gore and Douglas Shields 1995), yet anthropogenic stressors have drastically reduced this diversity (Best, 2019). For instance, freshwater animal populations have declined by 83 % on average since 1970—more than in terrestrial or marine ecosystems—highlighting the exceptional vulnerability of freshwater biodiversity to human-induced change (WWF, 2022; Gutiérrez-Rial et al., 2023). Recognising the social and economic importance of healthy rivers, considerable efforts have been made to improve the ecological state of large rivers, but management and restoration are still needed to halt biodiversity decline. Large-scale and

long-term monitoring is of key importance for characterising biodiversity changes, evaluating restoration activities, and identifying risks (Haase et al., 2023). Human activities play a crucial role in shaping plant communities within socio-hydrosystems, either through direct management practices (riverbank maintenance, pruning aquatic plants, etc.) or by altering environmental conditions and landscapes. Ecological studies on urban and human-modified landscapes contribute to understanding the pressures and opportunities shaping plant communities. Surveying the biodiversity of large rivers poses significant challenges, and continuous monitoring of fauna and flora with conventional methods is economically burdensome and time-consuming given the spatial scales (Deiner et al., 2017). Deep and fast-flowing waters can be dangerous to practitioners, and navigating through riparian forests and wetlands is labour-intensive. However, the significance of ecological surveys cannot be overstated, given the precarity of these ecosystems in Europe (Haase et al., 2023). Furthermore, in socio-hydrosystems with a

<footnote><sup>a</sup> University of Strasbourg, CNRS, ENGEES, LIVE UMR 7362, 67000 Strasbourg, France</footnote>

<footnote><sup>b</sup> E-BIOM, SA, Les Isnes, Belgium</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: armando.espinosa@live-cnrs.unistra.fr (A. Espinosa Prieto).</footnote>

<footnote n="1">¹ Present address: Namur Molecular Tech, CHU UCL Namur, Yvoir, Belgium.</footnote>
strong anthropogenic influence, the introduction and colonisation of exotic and potentially invasive species are particularly high, calling for early detection.

Environmental DNA (eDNA) metabarcoding, the simultaneous detection of multiple taxa based on their DNA extracted from an environmental sample (Taberlet et al., 2018), is significantly broadening the methodological toolkit for biodiversity surveys, especially in aquatic environments (Altermatt et al., 2023). It offers potential solutions to the difficulties in monitoring large rivers. First, it standardises the sampling process, as the water collection from the surface is rapid and does not require heavy equipment; it also enhances safety by eliminating the need for potentially hazardous activities such as diving. Additionally, eDNA enables the detection of cryptic and benthic species, given the high degree of sensitivity to minute DNA traces in the environment. The potential of standardising the sampling campaign along the river facilitates repeatable and comparable results in space and time, allowing for large-scale studies. Finally, eDNA enables plant species identification year-round, whereas traditional surveys often depend on seasonal traits like flowering, which restrict the survey window.

The importance of aquatic plants as biological quality elements in the assessment of river health is well established under the European Water Framework Directive (WFD). The first European effort to include eDNA surveys in large rivers dates to 2019, the Joint Danube Survey (JDS), an international research project focused on the Danube River and its tributaries. The JDS aims to comprehensively assess the ecological status and water quality of the Danube River Basin. The 2019 survey utilised the eDNA metabarcoding approach to survey different aquatic and terrestrial fauna in parallel to conventional survey methods. However, eDNA was not used to survey aquatic vegetation during the 2019 survey as few studies had applied the method to aquatic plants in large rivers (Cannon et al., 2016; Ji et al., 2021). In a recent study on the Vosges rivers (Espinosa Prieto et al., 2024), we found that terrestrial species could also be detected through eDNA, highlighting the potential for a multi-marker approach to survey both aquatic and terrestrial vegetation simultaneously. This finding is particularly relevant in the context of large, anthropogenically altered rivers such as the Rhine, where surrounding land use and human activities may influence the eDNA signal. In fact, the contribution of the surrounding landscape to eDNA, particularly in relation to plant species, has received limited attention in the literature (Evrard et al., 2019; Foucher et al., 2024; Reji Chacko et al., 2023). Our study aims to fill this gap by applying a multi-marker approach to explore the detection of aquatic, amphibious, and terrestrial species, while also considering the broader socio-ecological context of the river system.

We present a proof-of-concept for three applications of multi-marker eDNA metabarcoding within a large river socio-hydrosystem. First, we verify the validity of eDNA metabarcoding for detecting aquatic and amphibious plant communities in large rivers. Second, we investigate whether this approach can be used in a socio-ecological context to retrieve the eDNA fingerprint of land use and human activities on the Rhine. Third, we explore the unique opportunity to quickly assess the presence of exotic invasive species with what could be a routine universal metabarcoding approach as part of biomonitoring programmes.

## 2. Methods

### 2.1. Study area

Like many large rivers worldwide, the Upper Rhine River has undergone significant anthropogenic modifications, particularly for navigation, agriculture, and hydroelectric production (Brauns et al., 2022). Major engineering works began in the 19th century, with successive phases of rectification, regularisation, and canalisation (Cioc, 2002; Schmitt et al., 2019), which drastically altered the river's hydrology and morphology. To support navigation and energy production, the Grand Canal d'Alsace (GCA) was constructed between 1928 and 1958 as a

concrete bypass channel parallel to the more natural Old Rhine. While these measures reduced flood risks and enabled economic development, they also caused substantial ecological degradation, including the loss of longitudinal and lateral connectivity and the disappearance of over 100,000 ha of functional floodplains between Basel and Bingen (Vanderpoorten and Klein, 1999; Arnaud et al., 2019; Wantzen et al., 2022).

Urban expansion towards the river further intensified pressure on ecosystems (Beisel et al., 2021), while the drop in the water table desiccated many aquatic habitats and significantly reduced the regeneration of alluvial hardwood forests (Deiller et al., 2001; Schnitzler, 1997). Physical barriers, like channelised banks and altered riverbeds, limited habitat availability for amphibious and aquatic plants. Thousands of natural islands and their characteristic vegetation were removed. However, artificial levees created secondary habitats for drought-tolerant plant communities previously found on river islands (Carbiener & Schnitzler, 1990). Today, the Upper Rhine Valley hosts a mosaic of protected areas under Natura 2000, ZNIEFF, and Ramsar designations, making it one of Europe's richest regions in terms of biodiversity, despite ongoing challenges (Schnitzler, 1994).

### 2.2. Sampling and data processing

The approximately 126 km long segment of the Upper Rhine River between Strasbourg (France) and Basel (Switzerland) was surveyed in October 2020. Starting at the French border immediately downstream of Basel, five water samples were collected from the Grand Canal d'Alsace (GCA) at 11 km intervals and two samples from the former Old Rhine River (hereafter the Rhine) at the beginning and end of the Rhine Island (Fig. 1). This island has three layers of regulatory protection and harbours protected and rare species. It is designated as a Ramsar area, a Natura 2000 site, and two ZNIEEF areas of 1269 ha and 781 ha cover the entire Rhine Island. An additional sample was taken downstream of Strasbourg, about 74 km from the closest upstream sampling site (Fig. 1). The wet lab and bioinformatic workflows were identical to those described by Espinosa Prieto et al. (2024) (available at https://zenodo.org/records/12571296) adapting the pipeline when necessary (see Appendix A for summary of methods). In this study, we used

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Location of the eight sampling sites. Purple and green dots correspond to the samples within the Grand Canal d'Alsace and the Old Rhine, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
relative read abundance (RRA) to filter out unreliable detections and was calculated as the number of reads assigned to a species divided by the total number of assigned reads in a sample (plant species and genus level assignments). To minimise false positives, we applied a relative read abundance (RRA) threshold of 0.005 %, based on an empirical evaluation of total RRA values for each taxon across the full dataset. We examined how taxa were progressively excluded at increasing thresholds and selected the cut-off that removed likely artefacts (implausible taxa) while retaining species known to occur along the river. This threshold was consistently applied across samples and aligns with Elbrecht & Steinke (2019), who recommend adapting minimum abundance thresholds to sample type and study goals. A read abundance distribution curve (Fig. A.1) and a stepwise table of excluded taxa (Table B.1) illustrate the effect of thresholding on taxon retention. We used RRA to visualise detections because, despite some taxon-specific biases, it can offer a more accurate representation of relative composition across samples than occurrence data, which tend to overemphasise rare taxa as shown by Deagle et al. (2019).

To assess whether eDNA captured land use and human activity along the river, we retrieved a list of cultivated crops within a 3 km radius of each sampling site. This was based on the Registre Parcellaire Graphique (RPG) 2020 dataset of declared agricultural parcels, obtained from the French National Institute of Geographic and Forest Information (IGN) from https://geoservices.ign.fr/rpg. Crop types were identified through visual inspection of parcel attributes in GIS through the visualisation portal (https://www.geoportail.gouv.fr/carte). Urban and industrial areas were also noted where visible.

The species were classified into terrestrial, amphibious, and aquatic categories based on their soil moisture index (SMI) values from Julve's Baseflor (Baseflor version 2021, available from https://www.tela-bota.nica.org/projets/phytosociologie/porte-documents/), with terrestrial species ranging from drought-tolerant to tolerating inundation periods (SMI 1–8), amphibious species (helophytes) rooted in permanently wet soils (SMI 9–10), and aquatic species requiring continuous immersion (SMI 11–12). We also adapted the native status of the flora from the vascular plant catalogue of Île-de-France (Conservatoire Botanique National Bassin Parisien, MNHN, https://cbnbp.mnhn.fr/cbnbp) to our study region, which classifies plants into six categories: indigenous, eurynaturalised, stennaturalised, spontaneous, accidental and cultivated and planted plants (Table B.2). Furthermore, we retrieved species lists from nearby ZNIEFF and Ramsar protected areas to evaluate eDNA detections of species of ecological or conservation interest.

Although the term *exotic* refers to all species intentionally or unintentionally introduced into a territory outside their natural range, *invasive* only applies to those exotic species that proliferate in their new environment (Pyšek and Richardson, 2010). The risk analysis of (Weber and Gut, 2004) classifies plant species based on their invasiveness, which can help identify management priorities. We used a modified invasiveness classification system, from established invasive to exotic non-invasive, of the list of exotic species from the Grand-Est region in France (Table B.3).

# 3. Results

## 3.1. eDNA metabarcoding

After bioinformatic analyses, we recovered from the eight eDNA samples a total of 205 plant species belonging to 162 genera and 69 families, of which 3 species were mosses (Bryophytes), 2 ferns (Pteridophytes), 3 conifers (Gymnosperms) and the remaining 197 flowering vascular plants (Angiosperms). Additionally, 36 taxa were only recovered at the genus level, representing 5 % of all assigned reads. In total, 37 species represented 90 % of the assigned reads, eight of which were aquatic plants, three amphibious, two cultivated, two exotic species, and the remaining 22 were indigenous terrestrial species (Table B.2). The aquatic and amphibious plant species-level assignments corresponded to

18 % of all assigned reads (n = 25 species) (Fig. 2), of which 95 % were submerged aquatic plants (n = 19 species, Table B.2). Meanwhile, indigenous terrestrial and naturalized plants represented 75 % of the reads (n = 129 species), while cultivated plants (crops, ornamental, etc.) 5.5 % (n = 40 species) (Fig. 2).

Terrestrial vegetation dominated the read abundances and corresponded to the expected dominant and common indigenous species of the riparian forests (Table B.2). For example, hygrophilous species, those tolerating inundation periods of weeks to months, were the most abundant in terms of read count and in terms of biomass, as these were mainly tree species. When examining combined taxonomic assignments at both species and genus levels, a notable 10.5 % of the total read count belonged to willows, the genus *Salix* which encompasses hygrophilous trees typical of riverbanks and wetlands (Table B.2). Other common species of the Rhine riparian forest recovered predominantly at the genus level were *Rubus* (6 %), *Acer* (5.4 %), *Alnus* (3.6 %), *Quercus* (2.3 %), *Populus* (2.3 %), and *Ulmus* (2.5 %). Interestingly, we observed a diversity of terrestrial species that span a broad spectrum of edaphic moisture conditions that closely reflect the different habitats around the GCA and the Rhine. Four xerophilous species characteristically growing on the levees of the GCA were recovered sporadically in some samples, including *Sedum acre* L., *Arrhenatherum elatius* (L.) P.Beauv. ex J.Presl C. Presl, *Scabiosa columbaria* L., *Galium verum* L. The detection of *Bituminaria bituminosa* is noteworthy since its distribution is predominantly Mediterranean. However, the high relative abundance (2.5 % of all reads, Table B.2) suggests that the species has established local populations, presumably profiting from the dry riverbanks of the GCA. Other species of the interstices and paths included the very common stinging nettle (*Urtica dioica* L.) and the ribwort plantain (*Plantago lanceolata* L.) that were recovered in all samples and represented 9.5 % and 3.4 % of the assigned reads, respectively, which is on par with other high-biomass species such as trees and bushes.

## 3.2. Aquatic and amphibious plants

Indigenous macrophytes (n = 17) dominated in terms of RRA the two branches of the river (the GCA and the Old Rhine). Eight species were commonly found at least six of the eight sites (Fig. 3). Most were characteristic of meso- to eutrophic waters as expected in the upper Rhine River, where human pressure is important. Eight macrophytes represented close to 90 % of all reads for this group of plants, of which 64 % of the reads belonged to three species that were recovered in all samples. Specifically, 30 % belonged to the Eurasian watermilfoil (*Myriophyllum spicatum* L.), followed by 18 % for sago pondweed (*Stuckenia pectinata* L.) and 16 % for horned pondweed (*Zannichellia palustris* L) (Fig. 3,

<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Proportion of reads at the species level for the four plant groups relative to assigned reads at the species level. The number of species attributed to each group appears in parentheses. A total of 205 species were detected, but five species appear in both cultivated and exotic groups. An additional 46% of the reads were assigned at the genus level.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Relative read abundance (RRA) of aquatic and amphibious plants in the GCA (G1 to G6) and the Old Rhine (R1, R2), ordered from upstream (left) to downstream (right) for each river branch. Bubble sizes are scaled using a square root transformation to enhance the visual representation of smaller values. Values < 0.01 are represented as 'b'.</figcaption></figure>

Table B.2). Similarly, three species of amphibious plants represented 62 % of the reads, split almost equally between pale persicaria (*Persicaria lapathifolia* (L.) Delarbre), marsh-marigold (*Caltha palustris* L.) and common reed (*Phragmites australis* (Cav.) Trin. Ex Steud.), at 22 %, 21 % and 19 %, respectively (Fig. 3).

We only recovered one macrophyte (*S. pectinata*) from the seven ecologically determinant macrophytes identified on the ZNIEFF and Ramsar lists and potentially occurring on the Rhine Island and in the drainage canal parallel to the GCA (Table B.5). Similarly, only one amphibious species (*Butomus umbellatus* L.) was recovered at the species level, while seven other taxa were recovered at the genus level, including *Carex*, *Juncus*, *Rumex*, *Salix* and *Sparganium* (Table B.2). The genetic resolution of the markers influenced the detection of some taxa, as barcodes of closely related species may be indistinguishable. Although *Carex pendula* Huds. was recovered at species level at site G2, only genus-level assignments were possible at the other sites, ranging from 0.02 % to 0.3 % RRA per site (Fig. 3) (Table B.2). Similarly, for *Salix* and *Potamogeton*, several species were detected (Fig. 3) but 99.9 % and 96 % of the *Salix* and *Potamogeton* reads, respectively, were assigned at rank genus (Table B.4).

### 3.3. Cultivated, ornamental, and spontaneous plants

A total of 40 species of cultivated plants were recovered from eDNA water samples, either for the agro-food industry (12 species) or for ornamental purposes (28 species). In GCA samples, two to five crop species were detected, while on the Rhine, only one crop plant was found at site R1 and none at R2 (Fig. 4). The dominant crops in terms of surface area along both banks of the river are corn (*Zea mays* L.) and common wheat (*Triticum aestivum* L.). This is reflected in their higher detection rates in more samples compared to other crops, which are less widely cultivated (Fig. 4). However, the RRA for corn varied considerably between sites, from 0.01 % (80 reads) for G3 to 12 % (61382 reads) for G5 (Fig. 4). From two sites located within a few hundred metres of silos for grain storage, G5 and G2 (Fig. 5), different proportions of corn and wheat were recorded (Fig. 4), while at site G1, located 3 km downstream of a silo, only wheat was detected with an RRA of 0.14 % (792 reads) (Fig. 4). Site G4 had the second highest RRA for corn, 0.98 % (>12000 reads) and the closest corn field was 200 m behind a forest strip.

The remaining cultivated plants, including orchard species such as plums, apricots, apples, and pears, were recorded sporadically at
<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Relative read abundance (RRA) of aquatic plants in the GCA (G1 to G6) and the Old Rhine (R1 and R2), ordered from upstream (left) to downstream (right) for each river branch. Bubble sizes are scaled using a square root transformation to enhance the visual representation of smaller values. Values < 0.01 are represented as ‘b’.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 5.** Location of G1, G2, and G5 sites on the Grand Canal d'Alsace and the location of the grain silos (stars). The black arrows indicate the flow of water and, for site G2, the small arrow indicates the inlet of the drainage canal into the GCA.</figcaption></figure>
different sites. In particular, hemp (*Cannabis sativa* L.) was detected at sites G4 and G5, corresponding to an RRA of 0.058 % (720 reads) and 0.1 % (529 reads), respectively (Fig. 4). However, we did not retrieve land parcels of industrial hemp in public referenced land parcels from 2020 near or upstream of the sites.

Of the 32 ornamental plants, the yellow-paint maple (*Acer pictum* Thunb.) was recorded in all samples and represented 71 % of all the reads from ornamental plants. In contrast to crops, we detected six and five species at sites R1 and R2 on the Rhine, respectively (Fig. 4), perhaps originating from the urban environment surrounding the two sites on the Rhine Island. At sites G4 and G6, we found the most ornamental plants, possibly due to their proximity to the urban landscape (Fig. 4).

### 3.4. Naturalised exotic and invasive species

A total of 16 exotic species, both invasive and non-invasive, were identified, accounting for 1.5 % of all assigned reads (Fig. 2). Among these, the black locust (*Robinia pseudoacacia* L.) was detected in all samples, with its RRA ranging from 0.007 % (34 reads) at site G5 to 1 % (12,827 reads) at site G4 (Fig. 6). This was the most abundant species in the data, representing 52 % of all reads from invasive species. Two additional terrestrial invasive species frequently found in alluvial environments are the Japanese knotweed (*Reynoutria japonica* Houtt.) and the Giant knotweed (*Reynoutria sachalinensis* (F.Schmidt) Nakai). The former was detected in five sites, including at site R1 on the Rhine, and the later was co-occurring only at sites G2 and G4 (Fig. 6).

Only two naturalised invasive macrophytes were detected at varying read counts and sites. The Canadian waterweed (*Elodea canadensis* Michx.) was only recovered at low read abundance at site R1 in the Rhine (0.02 % RRA, 180 reads) (Fig. 6). The variable-leafed watermilfoil (*Myriophyllum heterophyllum* Michx.) was only recovered at G6 downstream of Strasbourg with a high read abundance (1.2 % RRA, 7151 reads) (Fig. 6). This correlates with the current known distribution of this species, for which the only known occurrence in the studied area is a large and permanent population in Strasbourg. The detection of additional reads of *Elodea* and *Myriophyllum* at the genus level across all sites hints at additional species of these genera, particularly to *E. nutalli* and *M. verticillatum*, which are expected in the studied reach (Fig. 6, Table B.2).

## 4. Discussion

### 4.1. Aquatic vegetation

The environmental DNA recovered from the Rhine and the GCA reflected the composition of aquatic vegetation within the river and in the surrounding landscape, with signals largely dominated by the most abundant macrophyte species. Compared to macrophytes, amphibious plants were only found sporadically in our eDNA samples, hinting at a localised eDNA contribution of these plants or to fewer eDNA in the water samples overall. On the contrary, the contribution of riparian plants to eDNA in river water in other systems is significant (Espinosa Prieto et al., 2024; Reji Chacko et al., 2023). However, there is no generalisable relation between the number of reads and the abundance, cover, or population size of species for aquatic plant eDNA (Doi et al., 2021; Kodama et al., 2021). However, the total relative read abundance on the reach scale revealed patterns of dominance and rareness of species in our study, which is consistent with previous observations on macrophytes (Doi et al., 2021). To further utilise relative read abundance in plant metabarcoding, extensive research is needed on the release rates of plant eDNA and other abiotic factors and how they may relate to population indices in natural environments.

Despite using five markers, several common amphibious plants in the Rhine River could only be detected at the rank genus (*Carex*, *Salix*, and *Juncus* among others). Studies interested in these taxa could use group-specific markers and primers capable of discriminating between closely related species to improve taxonomic resolution (De Barba et al., 2014). Alternatively, if species lists exist for the study area, reference databases restricted to those species could be used over a general database (Espinosa Prieto et al., 2023). Additionally, for species with difficult taxonomic identification, errors in public reference databases could originate from erroneous species assignments. A questionable detection in our samples was *Potamogeton praelongus* Wulfen, an endangered macrophyte that grows in oligotrophic waters in mountain lakes or slow-moving rivers. While the majority of *Potamogeton* sequences were correctly assigned to genus level, a small number of sequences were attributed to *P. praelongus*, which raised concerns about over-classification. Upon manual inspection of the local BLAST output, these turned out to be ITS1 sequences, and the assignments likely reflect reference database ambiguities and the taxonomic complexity of the group, rather than true species-level detections. High sequence

<figure><img src="image_7.png" /><figcaption>**Fig. 6.** Relative read abundance (RRA) of exotic plants in the GCA (G1 to G6) and the Old Rhine (R1, R2), ordered from upstream (left) to downstream (right) for each river branch. Bubble sizes are scaled using a square root transformation to enhance the visual representation of smaller values. Values < 0.01 are represented as ‘b’.</figcaption></figure>
similarity among congeners and hybridization within Potamogetonaceae reduce the reliability of species-level calls. Its detection was most likely due to the similarity between the barcodes of this species and *P. natans* L. (Kuzmina et al., 2018), which is more likely present in the study area (observed very locally in the floodplain in 1990 based on old botanical surveys). Similar challenges have been reported for other taxonomically complex or hybridogenic groups such as *Rumex*, *Carex*, *Salix*, and *Taraxacum* in eDNA studies (Espinosa Prieto et al., 2024b).

Only two Ramsar and ZNIEFF determinant species known to occur within the Rhine Island were recovered in the eDNA survey. Consequently, rare ecological determinant species that naturally occur in small numbers in the surrounding environment and are not directly present in the Rhine were not recovered in our Rhine water samples, nor in the GCA. Several factors can explain this observation, including a dilution effect, sampling design, and PCR bias. The dilution of DNA from low-abundance species in the water column could be significant compared to more abundant taxa resulting in a more stochastic sampling of rare DNA, even when sampling 24 L of water. In this case, sampling closer to the confluence or increasing the water volume could yield the expected results. Even if rare eDNA was captured, PCR bias additionally reduces the chances of detecting the species given the strong competition for primers and polymerase, where the most abundant and best-matching sequences will amplify best (Moinard et al., 2023).

## 4.2. Cultivated and ornamental plants

In this study, eDNA was used to capture the socio-hydrological imprint of the Upper Rhine region, reflecting not only land use but also agricultural practices and industrial activities. Land use and industrial port activities along the GCA appear to have contributed significantly to the eDNA detected in the river, as evidenced by the presence of cereal crops and orchards. We do not discard that some crops consumed by human and cattle may come through waste waters but more sampling is needed to confirm the origin of eDNA. Beyond land use detection, our data may also reflect specific agricultural practices, such as intercropping. In particular, hemp (*Cannabis sativa*), a crop in the Cannabaceae family that is grown for fibre, seed oil, and food, was detected at two sites with RRA values comparable to those of co-occurring aquatic and terrestrial plants. Hemp is known for its rapid growth cycle and is commonly used as a secondary crop after the main harvest. The nearest declared fields were located over 60 km inland from the Rhine, making their detection unlikely based on known patterns of airborne eDNA dispersion (Tournayre et al., 2025), though still possible given the long-distance dispersal capabilities of Cannabis pollen (Nimmala et al., 2024). However, if this were the case, we would expect detections at other sites as well. A more plausible explanation is the local, undeclared use of Cannabis for intercropping in corn fields adjacent to the sites where it was detected. Its detection in October also aligns with typical post-harvest planting practices. While direct evidence of intercropping is not available, the detection suggests small-scale or undeclared cultivation, and demonstrates the potential of eDNA to reveal subtle or unregistered agricultural activity.

The recovery of cereal crops at certain sites may be attributed to storage silos, where corn is exposed to the environment during open air loading onto boats, contributing to eDNA in the river. Other sources of eDNA cannot be ruled out, as these crops have also been found in sites far from silos but adjacent to fields. Environmental DNA from neighbouring fields can be transported by wind through suspended dust particles or pollen (Johnson et al., 2021a, 2021b; Johnson et al., 2019) and could be deposited on the river, contributing to its eDNA fingerprint. Xiong et al. (2024) demonstrated that sewage water contributes to the detection of commercial fish in freshwater rivers. In the study by Reji Chacko et al. (2023), although the authors do not study the human fingerprint of the landscape, they identified that eDNA recovers the local land cover signal in river catchments through plant and animal compositions characteristic of different land uses, particularly forests and

high grasslands.

The detection of ornamental plants in our study also underscores the complexity of eDNA sources in anthropogenic systems. We suggest that proximity to sampling sites strongly influences the recovery of ornamental species, but additional factors, such as plant form (e.g., tree, shrub, or grass), reproductive mechanisms (e.g., wind-pollinated versus self-pollinated), and usage patterns (e.g., public or private spaces), may also affect retrieval rates and read abundance. The differences between the GCA and the Old Rhine in crop species likely reflect the influence of landscape features on the dispersal of eDNA. Forest strips, 250 m to 1 km wide, surrounding the Rhine sampling sites drive the diversity and composition of eDNA detected in water samples and could act as both physical barriers to the air transport of eDNA from distant crop fields and technical barriers, diluting crop-derived eDNA within the wider diversity of forest eDNA as suggested by differences in relative read abundances. Sites G1 and G6, located immediately downstream of Basel and Strasbourg respectively, showed qualitative differences in the richness of ornamental plant eDNA, with G6 registering 11 species compared to only 6 at G1. While further sampling is needed to confirm the influence of urban proximity, our observations suggest a potential urban effect on ornamental plant detection, further supported by the high number of ornamentals detected at site G4 near a village.

While this study provides compelling evidence for the detection of local land use and human activities, further investigation is needed to distinguish between the potential sources of this eDNA. Clarifying these contributions would enhance our understanding of the dynamics of eDNA in anthropogenically influenced river systems and help resolve ambiguities in detecting agricultural and industrial impacts.

## 4.3. Invasive species

Previous studies have demonstrated the utility of eDNA barcoding for detecting invasive aquatic plants, but these efforts primarily focused on species-specific assays targeting individual organisms (see references in Espinosa Prieto et al., 2023). Our study extends this approach by employing “universal” plant primers and eDNA metabarcoding to detect aquatic and terrestrial invasive plants in a large river environment, unprecedented until this study.

The detection of *R. japonica* and *R. sachalinensis* in our study underscores the presence of disturbed ecosystems, often associated with an excess of phosphates and nitrates (Schnitzler-Lenoble and Muller, 1998). These invasive plants contribute significantly to the displacement of native species (Staentzel et al., 2020), altering the structure and function of ecosystems. The co-occurrence of *R. japonica* and *R. sachalinensis* at two sites may suggest the presence of their hybrid Reynoutria × bohemica. Distinguishing this hybrid morphologically is challenging due to its strong resemblance to *R. japonica*. However, eDNA metabarcoding could enable its detection, especially when using appropriate non-recombinant markers as in this study, offering a valuable complement to traditional identification methods. Although our results are not conclusive on hybrid presence, we only highlight the potential of eDNA for detecting such hybridization events as demonstrated in other studies (Stewart and Taylor, 2020).

The presence of *Elodea canadensis*, a naturalised invasive species assessed as high risk by Weber and Gut (2004), illustrates the long-standing impacts of invasions in the region. First recorded in 1875 (Issler et al., 1982), *E. canadensis* competes with indigenous flora and contributes to eutrophication and sedimentation in river ecosystems (Thiebaut et al., 1997). Although it is less invasive than *Elodea nuttallii* (Planch.) H.St.John, both species present considerable ecological challenges. In our study, *E. nuttallii* and *E. canadensis* were distinguishable using barcoding markers, but species-level identification of *E. nuttallii* was hindered by mislabelled sequences in reference databases. However, the high relative read abundance at the genus level provided a robust proxy for the detection of *E. nuttalli*. This case highlights the need for continuous curation and validation of barcode databases to enhance
taxonomic resolution. It also shows that knowledge of the local distri-
bution and ecology of species is crucial to properly interpreting eDNA
results. In this case, knowing that *E. nuttallii* was the most abundant
species and that *E. canadensis* was the only other likely candidate helped
us interpret genus-level assignments and avoid false negatives.

Although eDNA metabarcoding could distinguish closely related
species, it may lack the power to discern ecologically significant sub-
species. This limitation is exemplified by the detection of *Lamium
galeobdolon* L., where uncertainty remains between the native *L. g. subsp.
galeobdolon* L. and the emerging invasive ornamental *L. g. subsp. argen-
tatum* (Smejkal) J. Duvign. Both subspecies exhibit identical barcode
sequences for the ribosomal and plastid markers used in this study. In
contrast, visual surveys could rapidly differentiate them, as the orna-
mental subspecies features distinctive silver patches on its leaves. Given
its documented spread near Basel (Rusterholz et al., 2023), including at
the G1 sampling site, it is plausible that the invasive subspecies was
detected by eDNA; however, field confirmation remains necessary.

Our findings underscore the potential of eDNA metabarcoding as an
early warning tool for the detection of new invasions and the identifi-
cation of invasive species hotspots, allowing for timely and efficient
management responses. Although metabarcoding excels in providing a
broad overview of species diversity, it is limited in its ability to provide
precise abundance data (Deiner et al., 2017, but see Moinard et al.,
2023), which could instead be obtained with species-specific barcoding
methods (Kodama et al., 2021, Matsuhashi et al., 2019). The relative
read abundance (RRA) approach used in this study, while subject to
amplification biases and not directly reflective of organism abundance,
can nonetheless convey ecologically meaningful patterns. For example,
the elevated read proportions of the invasive tree Robinia pseudoacacia
may correspond to its dominance in the landscape and high biomass,
which likely contribute to increased eDNA release.

With eDNA metabarcoding, invasive species detection could be in-
tegrated into large-scale biodiversity surveys. Sampling at distant in-
tervals provides an initial indication that can guide targeted field
surveys and conventional biomonitoring to confirm species presence
and quantify population extent. To achieve a finer spatial resolution,
sampling intervals shorter than the 11 km used here could be imple-
mented. The detection of a species at a site confirms its presence at that
location or upstream; however, the absence of detection, whether
through eDNA or visual surveys, should be interpreted with caution, as it
may not accurately reflect the species’ distribution downstream due to
environmental factors (eDNA precipitation, degradation, etc.), or limi-
tations in detection methods.

## 4.4. Spatial design and sampling considerations

Despite its promise, eDNA surveys in large rivers may come with
their own set of challenges. Despite its promise, eDNA surveys in large
rivers may come with their own set of challenges. Our aim was to
demonstrate the feasibility of using a simple and accessible eDNA pro-
tocol to survey riverine and riparian vegetation, native, invasive and
cultivated plants, in a large and logistically complex hydrosystem. The
sampling in our study was carried out during the senescence period,
considered the time of year with the most abundant plant eDNA load
(Anglès d'Auriac et al., 2019; Kodama et al., 2021; Matsuhashi et al.,
2019; Miyazono et al., 2021). This fits the aim of the study to demon-
strate a "one-shot" vegetation survey approach since more eDNA of
different species should be captured in this way. Sampling at other times
of year may be performed depending on target organisms and research
hypothesis. Our sampling strategy involved eight sites spaced c. 11 km
apart, including two sites downstream of two major cities, and was
limited to single riverbank filtration of 24 L of water. This design reflects
practical constraints encountered in many real-world settings, where
mid-channel sampling (requiring boats) or dense spatial grids may not
be feasible due to limited access, permits, or resources. For example,
many large rivers constitute country borders, and one may not have

access to both riverbanks. While our approach may not capture cross-
sectional heterogeneity of eDNA in large rivers, it offers a pragmatic
baseline. Uneven distribution of eDNA in the water column across the
river section potentially affects the accurate detection of vegetation
(Altermatt et al., 2023) and future studies could benefit from a sampling
design across the river's width. However, field studies report mixed
outcomes regarding cross-sectional sampling (Kodama et al., 2021)
which could depend on the width and other hydromorphological pa-
rameters of the river. For instance, the river studied by Kodama et al.
(2021) is a fourth of the width of the GCA and the Rhine which measure
c. 250 m. In our study, sites G3 and G4, located on opposite riverbanks of
the GCA, exhibited differences in plant community composition and
relative read abundances, even for species detected at both sites.
Although anecdotical, this observation aligns with cross-sectional het-
erogeneity of eDNA. Future applications could also benefit from denser
spatial sampling especially if interested in recovering protected species
that may have a very localised or patchy distribution. Meanwhile,
filtering large volumes of water, as done in this study, may help
compensate for sparse spatial coverage by enhancing eDNA retrieval and
reducing the influence of PCR inhibitors (Schabacker et al., 2020). The
potential for eDNA to travel long distances in large, fast-flowing rivers,
such as the GCA with a discharge of ~1,000 m³/s, may increase spatial
coverage and influence detection patterns. However, we found no evi-
dence of downstream accumulation of eDNA over the 11 km between
sites, as indicated by differences in taxon lists, suggesting that denser
sampling could still improve spatial resolution.

4.5. Challenges for eDNA-based macrophyte assessment under the
European WFD

Macrophytes are among the Biological Quality Elements (BQEs) used routinely to assess the ecological status of water bodies under the European WFD. Macrophyte-based indicators rely on field surveys involving morphological species-level identification and estimation of plant cover or frequency through semi-quantitative descriptor scales or percentage classes, which are categorical data rather than precise biomass measurements. Hering et al. (2018) evaluated the readiness of eDNA-based identification for routine ecological assessments under the current WFD based on eleven criteria. They argued that eDNA “methods are least suited for BQEs that are surveyed rather than sampled”, which applies to macrophytes. At the time, research on macrophyte eDNA was limited (Espinosa Prieto et al., 2023), but the conclusions still largely hold today, as progress remains slow compared to that for the animal kingdom. Using Hering et al. (2018) criteria and expert assessment as a reference, we discuss how our results contribute to addressing two of the most critical limitations for integrating eDNA into existing macrophyte indicators, abundance estimation and species-level identification.

Environmental DNA metabarcoding currently cannot provide abun-
dance estimates, as the relation between read abundance and plant
coverage remains unvalidated. Moinard et al. (2023) highlighted exist-
ing biases underlying this limitation, notably primer amplification bia-
ses, and proposed a modelling approach to address them, though its
applicability is restricted by the need for a mock community containing
all expected species to calibrate the model. The universal primers used in
this study produced disproportionately high read counts for a few
dominant, high-biomass species, with 90 % of reads assigned to just 37
taxa. This has important implications for the WFD, as such dominant
signals from non-target taxa may obscure ecologically relevant groups.
Our findings provide a clear rationale for using group-specific primers in
future applications aimed at optimising detection of indicator taxa for
WFD monitoring. However, despite the conceptual appeal, it remains
impractical and unlikely to design a metabarcoding assay using primers
that exclusively amplify a selected group of macrophytes across multiple
families for common markers (rbcL, trnL, ITS1 and ITS2) without also
amplifying non-target plants, due to the conservation of primer binding
sites across taxa (Espinosa Prieto et al. 2024b). Nonetheless, our results
show that the relative read abundances obtained from five different
markers convey ecologically meaningful patterns (section 4.4 and 4.3)
and serve as a starting point for developing new metrics tailored to the
specificities of eDNA data. Hering et al. (2018) also recommended
exploring alternatives to abundance estimation, including relative
abundances and quantitative eDNA approaches such as barcoding.

Frequencies of occurrence across multiple eDNA samples or markers could align well with the categorical structure of current macrophyte indicators, as demonstrated for amphibians (Pilliod et al. 2013). Additionally, presence-absence data, as generated in this study, provide valuable information for tracking species distributions and entire plant communities across river networks in a systematic and reproduceable manner across space and time. Such data are particularly relevant for total biodiversity assessments and for the detection of sensitive taxa, supporting complementary monitoring objectives within the WFD framework. As also noted by Hering et al., (2018), future frameworks for eDNA-based assessments may require alternative metrics tailored to the strengths of molecular data.

Environmental DNA barcoding targets individual species using
species-specific assays and can provide abundance estimates based on
DNA copy numbers measured by qPCR or dPCR (Kodama et al. (2021);
Matsuhashi et al. (2019), to cite a few). These studies show that the
correlation between DNA copy number and plant coverage appears to be
species-specific and system-dependent. While similar works also relied
on qPCR to estimate DNA copy numbers, results may be improved by
using dPCR, which enables absolute quantification without relying on
standard curves. The main limitation of species-specific assays is the
requirement of multiple assays to cover all ecologically determinant
species; to date, such assays have only been developed for a few exotic
invasive taxa (Espinosa Prieto et al. 2023).

Low taxonomic resolution, whether intrinsic to the marker or due to
poor database curation, can limit the precision of WFD macrophyte as-
sessments, which rely on species-level identification to derive ecological
status metrics. According to the European Standard EN 14184:2014,
surveyors are expected to identify macrophytes to species level, as
specific taxa often serve as indicators of environmental quality. Genus-
level detections in eDNA data can obscure differences in species'
ecological preferences, reducing the sensitivity of metrics based on
species richness, indicator presence, and abundance. However, this
limitation is not unique to eDNA, as conventional surveys also report
genus-level identifications when species-level identification is not
feasible. In our study, the inability to resolve certain genera to species
level reflects known taxonomic challenges, particularly for groups that
are hybridogenic or poorly differentiated morphologically and geneti-
cally. The European standard recommends sampling specimens for in-
lab verification, expert confirmation, and rigorous training to achieve
species-level identification. For eDNA analysis, group-specific primers
and markers could improve their taxonomic resolution and enhance
their detectability in the overall data (De Barba et al., 2014, Coghlan et
al 2020). But as previously discussed, this may be impractical and
cumbersome in the eDNA workflow. In the long term, integrating locally
vouchered reference sequences of family-specific markers may provide a
sustainable improvement in species-level resolution for problematic
groups. While lower taxonomic resolution may limit the direct appli-
cation of current eDNA methods to certain aspects of WFD classification,
it does not preclude the use of eDNA data in broader ecological assess-
ments, as demonstrated in this study.

5. Conclusions

Our study demonstrates that eDNA metabarcoding can simulta-
neously detect aquatic and terrestrial plant species from river water
samples, providing a broader picture of biodiversity in large river sys-
tems such as the upper Rhine. The identification of 16 exotic species,
including both invasive and non-invasive taxa, highlights the method's
potential for early detection and spatial delineation of species of

concern. Although promising, this approach still requires improvements
in spatial resolution and sampling design to reliably locate populations
and interpret species' distributions. As such, eDNA metabarcoding
should be viewed as a complementary tool to conventional monitoring
methods, particularly suited to enhance early warning and catchment-
scale ecological assessments in complex riverine landscapes.

6. Data statement

Scripts and data associated with the analyses are archived in Zenodo
(https://doi.org/10.5281/zenodo.12571296). Raw sequence reads and
related metadata are deposited in the SRA (BioProject PRJNA1290332).

7. Funding statement

This work was supported by the scientific counsel of the Ecole
Nationale du Génie de l'Eau et de l'Environnement de Strasbourg, the
Observatoire Hommes-Milieux Fessenheim, the University of Stras-
bourg, and the French State as part of the Projet de territoire de
Fessenheim.

Declaration of Generative AI and AI-assisted technologies in the
writing process

During the preparation of this work, the authors used Writefull to
improve the language and readability of the manuscript. After using this
service, the authors reviewed and edited the content as needed and take
full responsibility for the content of the publication.

CRediT authorship contribution statement

**Armando Espinosa Prieto:** Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Laurent Hardion:** Writing – review & editing, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization. **Nicolas Debortoli:** Writing – review & editing, Supervision, Software, Resources, Formal analysis. **Thibaut Bournonville:** Writing – review & editing, Investigation. **Jonathan Marescaux:** Writing – review & editing, Supervision, Project administration. **Karina Anna Elisabeth van der Zon:** Writing – review & editing. **Jean-Nicolas Beisel:** Writing – review & editing, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare the following financial interests/personal re-
lationships which may be considered as potential competing interests:
Armando Espinosa Prieto reports article publishing charges was pro-
vided by University of Strasbourg. Armando Espinosa Prieto reports
financial support was provided by French State as part of the Projet de
territoire de Fessenheim. Armando Espinosa Prieto reports financial
support was provided by National School for Water and Environmental
Engineering of Strasbourg. If there are other authors, they declare that
they have no known competing financial interests or personal re-
lationships that could have appeared to influence the work reported in
this paper.

Acknowledgments

This work was supported by the scientific counsel of the Ecole
Nationale du Génie de l'Eau et de l'Environnement de Strasbourg, the
Observatoire Hommes-Milieux Fessenheim, the University of Stras-
bourg, and the French State as part of the Projet de territoire de Fes-
senheim. We also gratefully acknowledge the support from the Interreg
project EcoServ, the Agence de l'eau Rhin-Meuse, and the Parc Naturel
Régional des Vosges du Nord. The authors would like to thank the HighPerformance Computing Center of the University of Strasbourg for providing scientific support and access to computing resources. Additionally, part of the computing resources used in this work were funded by the Equipex Equip@Meso project (Programme Investissements d'Avenir) and the CPER Alsac calcul/Big Data.

Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ecolind.2025.113943.

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