Ecology, Plant Science, Ecological Modeling, Management, Monitoring, Policy and Law
7
Scopus Publications
Scopus Publications
A comparative analysis of hybridisation capture and PCR-based eDNA metabarcoding for monitoring bryophytes in riparian ecosystems Thomas Reinhart, Armando Espinosa Prieto, Thomas Begoc, Hugues Tinguy, Francis Bick, Etienne Chanez, Jean-Nicolas Beisel, Laurent Hardion Metabarcoding and Metagenomics, 2025 Despite their ecological importance, mosses remain under-represented in ecological studies due to their challenging detection in field surveys and morphological identification, exacerbated by the lack of expert botanists. In this study, we optimise an environmental DNA method for the detection of bryophytes from river water samples, with the aim of facilitating their inclusion in biodiversity assessments. We compared three different methods in terms of species detection and community dissimilarity at seven sites along a river. The methods include (i) visual transect surveys conducted by bryologists based on macro- and micro-morphology, (ii) multi-marker PCR metabarcoding of the rbcL and the ITS2 markers with newly designed primers targeting bryophytes, and (iii) hybridisation capture (HC) for the same markers. We found that PCR metabarcoding recovered more than 50% (n = 37) of the species observed in the field, while hybridization capture detected only 16% (n = 11). PCR metabarcoding identified the most species, 101 species compared to 68 observed in the field and 27 with HC. Both the PCR and HC metabarcoding approaches identified bryophyte species not recorded in field surveys but expected in the catchment. Molecular methods, particularly PCR metabarcoding, recovered rare and elusive species difficult to observe in the field and occurring outside our transect. The two markers used in the molecular approaches contributed uniquely to species detection, making a multi-marker approach necessary to study this group. Environmental DNA and field surveys represent integrative methods that collectively enhance detection of inconspicuous species and yield the most comprehensive species inventory.
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, et al. Ecological Indicators, 2025 • eDNA enables efficient monitoring of plant diversity in large, complex river systems. • Simultaneously detect aquatic, riparian, and land-use associated plant taxa. • Identified 16 invasive species, potential for risk assessment in large rivers. • Cultivated and ornamental plants are more prevalent in the canal than in the Rhine. • Adapting the sampling design to large rivers is needed to improve species detection. 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), underscoring 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.
A comparative analysis of eDNA metabarcoding and field surveys: Exploring freshwater plant communities in rivers Armando Espinosa Prieto, Laurent Hardion, Nicolas Debortoli, Thibaut Bournonville, Thibaut Mathot, Jonathan Marescaux, Etienne Chanez, Cybill Staentzel, Jean-Nicolas Beisel Science of the Total Environment, 2024 While environmental DNA (eDNA) metabarcoding holds promise as a holistic approach to assess vegetation changes and community composition across diverse spatial and temporal scales, systematic investigations of its efficacy compared to conventional field surveys remain scarce in the literature. The present study explores the differences in plant diversity recovered from field surveys and captured with a multi-marker eDNA metabarcoding approach (two nrDNA ITS1 and ITS2, and two cpDNA rbc L and trn L) from river water samples. The eDNA metabarcoding approach retrieved 46 aquatic plants (hydrophytes and helophytes) and 245 terrestrial plants, compared to 24 and 127 species identified from field surveys. On average, eDNA samples collected immediately downstream of the survey sites recovered 43 % and 39 % of the aquatic and terrestrial species observed, respectively. Discrepancies were explained by differences in taxonomic resolution, the stochasticity of the retrieval of rare and elusive species, and the presence of reference sequences. We found a significant positive correlation between spatial and community distances at scales ranging from 2 to 9 km and identified turnover as the driving force of these differences. Metabarcoding demonstrated sensitivity to community changes and both approaches converge on a similar community structure. Interestingly, eDNA samples collected immediately upstream of the survey sites exhibited significant species overlap with the downstream samples (c. 100 m apart). Overall, our results demonstrate that within-site species mismatches between the methods are nonnegligible, and they question the use of eDNA for generating complete species lists at scales comparable to our field surveys (< 100-m transects). However, with adequate sampling and a multi-marker metabarcoding approach, eDNA has the potential to approximate catchment gamma diversity with less sampling effort than conventional surveys. • Metabarcoding detects twice as many riparian and riverine plants as field surveys. • A multi-marker approach improves species recovery across the four main plant phyla. • eDNA is not cumulative along rivers and detects turnover in community composition. • Methodological biases are strongest at the site-scale, where species lists differ. • eDNA sampling design should not be neglected for accurate biodiversity assessment.
Finding the perfect pairs: A matchmaking of plant markers and primers for multi-marker eDNA metabarcoding Armando Espinosa Prieto, Laurent Hardion, Nicolas Debortoli, Jean‐Nicolas Beisel Molecular Ecology Resources, 2024 As the scope of plant eDNA metabarcoding diversifies, so do the primers, markers and methods. A wealth of primers exists today, but their comparative evaluation is lacking behind. Similarly, multi‐marker approaches are recommended but debates persist regarding barcode complementarity and optimal combinations. After a literature compilation of used primers, we compared in silico 102 primer pairs based on amplicon size, coverage and specificity, followed by an experimental evaluation of 15 primer pairs on a mock community sample covering 268 plant species and genera, and about 100 families. The analysis was done for the four most common plant metabarcoding markers, rbcL, trnL, ITS1 and ITS2 and their complementarity was assessed based on retrieved species. By focusing on existing primers, we identify common designs, promote alternatives and enhance prior‐supported primers for immediate applications. The ITS2 was the best‐performing marker for flowering vascular plants and was congruent to ITS1. However, the combined taxonomic breadth of ITS2 and rbcL surpassed any other combination, highlighting their high complementarity across Streptophyta. Overall, our study underscores the significance of comprehensive primer and barcode evaluations tailored to metabarcoding applications.
An integrative taxonomic revision of the Chaerophyllum hirsutum complex (Apiaceae) using morphological and molecular markers Thomas Reinhart, Lucile Guillon, Thomas Begoc, Pauline Chapotin, Jean-Pierre Reduron, Armando Espinosa Prieto, Laurent Hardion Plant Ecology and Evolution, 2024 Background and aims – Chaerophyllum hirsutum represents a complex of taxa with varying treatments and ranks across floras. Using both morphometric and molecular markers, we assessed the robustness of C. hirsutum, C. elegans, C. villarsii, and C. villarsii var. cicutariiforme. Material and methods – Ten morphometric variables and two ratios were calculated. Based on the sequencing of six plastomes, the rps16 intron was selected as the more variable region and sequenced on a broader sampling. Additionally, we also sequenced the nrDNA internal transcribed spacer 2 (ITS2) using Illumina technology to obtain intra-individual allelic diversity. Key results – Morphologically, the most easily differentiated taxon was C. elegans, especially using the number of subterminal umbels. The distinction between C. hirsutum and C. villarsii was rather clinal, but is mainly based on the degree of carpophore division. Finally, C. villarsii var. cicutariiforme was less easily distinguishable from the three others, but partly using the carpophore length and the total length of basal leaf blade. The cpDNArps16 clearly distinguished C. elegans from the three other taxa of the complex, which rather showed a geographical pattern of cpDNA diversity. The nrDNA ITS2 partially distinguished C. villarsii from the other taxa, without distinction of C. elegans. Conclusions – The present study supports the species differentiation of C. elegans based on both morphology and chloroplast genome. Furthermore, C. villarsii var. villarsii and C. villarsii var. cicutariiforme could potentially be recognized as distinct varieties within C. hirsutum. This will need to be confirmed by future studies using a larger sampling size and more comprehensive markers, covering a broader portion of the nuclear genome.
Toward freshwater plant diversity surveys with eDNA barcoding and metabarcoding Armando Espinosa Prieto, Jean‐Nicolas Beisel, Pieternel Verschuren, Laurent Hardion Environmental DNA, 2023 Providing reliable, cost‐effective data on species distribution is critical to ensuring biodiversity conservation. However, many species may go unrecorded by conventional surveys, especially in aquatic environments. Environmental DNA (eDNA) barcoding and metabarcoding are alternative approaches that could complete biodiversity estimates based on species observations. While eDNA surveys are being standardized for some animal groups (e.g., fish and amphibians), research on eDNA approaches for freshwater plant communities is just starting to bear fruit. Here, we synthesized the 22 studies that used eDNA barcoding and metabarcoding to survey plant biodiversity in freshwater systems. We present evidence that contemporary aquatic plants (macrophytes) and terrestrial plants can be detected in water and surficial sediment eDNA from lakes and rivers. The phenology (e.g., senescence) of the target taxa strongly influences species detection. The main application of eDNA barcoding targets the monitoring of invasive macrophytes, and barcoding assays are available for 14 taxa. The metabarcoding approach shows a range of applications: the detection of rare macrophytes, catchment‐scale floristic inventories, and sediment fingerprinting. Barcodes on the plastid genome (cpDNA) are preferred for both approaches: matK and trnH‐psbA for barcoding, trnL, and rbcL for metabarcoding. The intergenic transcribed spacer 1 (ITS1) from the nuclear ribosomal DNA (nrDNA) was used for designing eDNA barcoding assays on five invasive macrophytes. In contrast, only three metabarcoding studies used the ITS1 or IST2 with newly designed primers. However, metabarcoding applications should routinely use a multi‐locus approach, combining cpDNA and nrDNA barcodes. Barcode combinations and existing primers need further testing on eDNA samples. We recommend using local barcode reference databases (BRDs), ideally self‐made, to circumvent taxonomic gaps and heterogeneous sequences in public BRDs. Finally, new technologies and developments like droplet digital PCR and hybridisation capture offer new perspectives for eDNA‐based biodiversity monitoring approaches.
