Mapping the northernmost transnational non-native population of Xenopus laevis using pooled eDNA sampling

227 Mapping the northernmost transnational non-native population of Xenopus laevis using pooled eDNA sampling Teun Everts1,2* , Loïc van Doorn3* , Tim Adriaens4, Sabrina Neyrinck1, Kevin Kerckhofs1, Jeroen Speybroeck3, Rein Brys1 1 Research Institute for Nature and Forest (INBO), Genetic Diversity, Geraardsbergen, Belgium 2 KU Leuven, Department of Biology, Plant Conservation and Population Biology, Heverlee, Belgium 3 Research Institute for Nature and Forest (INBO), Monitoring and Restoration of Aquatic Fauna, Linkebeek, Belgium 4 Research Institute for Nature and Forest (INBO), Wildlife Management and Invasive Species, Brussels, Belgium Corresponding author: Teun Everts ([email protected]) Copyright: © Teun Everts et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract Early-detection-rapid response (EDRR) programs are invaluable for managing incipient invasions, but require monitoring strategies that maximize search coverage and minimize costs. While environmental DNA (eDNA)-based monitoring techniques are now widely used for monitoring aquatic invasions, the large monitoring efforts in EDRR programs demand further improvements in sampling strategies. This need is exemplified by the recently discovered African clawed frog Xenopus laevis (Daudin, 1802) invasion along the French-Belgian border region – its northernmost introduced population – where it was first detected in France in 2018 near the Belgian border. In response to the risk of its permanent establishment and cross-boundary spread, we employed quantitative eDNA barcoding analyses in the context of an EDRR program. We (i) developed and validated a novel cost-efficient sampling strategy that involves pooling water from multiple sites allowing for broader area coverage, (ii) delineated the spatial extent of the invasion, and (iii) assessed whether lotic systems serve as cross-boundary dispersal corridors. In 2020, 2022, and 2023, we determined the presence and eDNA concentrations of X. laevis from a total of 426 sites by pooling water from 366 sites in 83 samples, and sampling 59 additional sites individually. We found that this pooled approach can accurately determine X laevis presence and approximate its average eDNA concentration across pooled sites. We detected X. laevis in 26 samples, revealing an interconnected population spanning an area of 103 km2, contradicting prior assumptions of an early invasion stage. We provide evidence that the river Lys and the Douvebeek facilitate cross-boundary movement. The pooled sampling technique presented here is a cost-effective method for providing timely, actionable data to inform management decisions; however, further validation is needed to confirm its reliability and broader applicability. The X. laevis invasion along the French-Belgian border underscores the complexities of managing biological invasions across national and administrative boundaries, providing valuable insights for other transboundary invasions. Key words: African clawed frog, biological invasions, dispersal corridor, droplet digital PCR (ddPCR), early-detection-rapid-response (EDRR), environmental DNA (eDNA), non-native invasive species, rapidly expanding distribution Academic editor: Filipe Ribeiro Received: 14 February 2025 Accepted: 12 August 2025 Published: 7 October 2025 Citation: Everts T, van Doorn L, Adriaens T, Neyrinck S, Kerckhofs K, Speybroeck J, Brys R (2025) Mapping the northernmost transnational non-native population of Xenopus laevis using pooled eDNA sampling. In: Anastácio P, Ribeiro F, Chainho P (Eds) Invasions in Aquatic Systems. NeoBiota 102: 227–248. https://doi. org/10.3897/neobiota.102.150311 NeoBiota 102: 227–248 (2025) DOI: 10.3897/neobiota.102.150311 * Contributed equally as the first author. Advancing research on alien species and biological invasions A peer-reviewed open-access journal NeoBiota 228 NeoBiota 102: 227–248 (2025), DOI: 10.3897/neobiota.102.150311 Teun Everts et al.: Pooled eDNA sampling to map the northenmost Xenopus laevis invasion Introduction Freshwater ecosystems harbour ten percent of all species and one-third of all vertebrate species while covering less than one percent of the Earth’s surface (Dudgeon et al. 2006). However, freshwater biodiversity is declining at an alarming rate (Sayer et al. 2025), with the ongoing establishment and spread of non-native invasive species (NIS) identified as a significant contributor. These biological invasions can result in species extinctions, biotic homogenization, and disruptions to ecosystem functioning (Katsanevakis et al. 2014; Blackburn et al. 2019; van Rees et al. 2021; Andrés et al. 2023). Despite ongoing management efforts, the influx of NIS is expected to continue (Seebens et al. 2017). To counter newly incoming NIS, establishing early-detection-rapid-response (EDRR) systems is essential (Hulme 2006; Vander Zanden and Olden 2008). To be effective, EDRR systems must employ cost-efficient monitoring methods capable of detecting species at low densities and having the potential to operate effectively across extensive spatial scales (Myers et al. 2000; Mehta et al. 2007; Greenhalgh et al. 2022). In recent years, a growing body of evidence has demonstrated the effectiveness of environmental DNA (eDNA)-based analyses for the early detection of aquatic NIS, underscoring their value in EDRR systems (Trebitz et al. 2017; Everts et al. 2023). Typically stemming from skin cells, mucus, gametes, and other biological materials, eDNA represents the entirety of DNA directly extractable from environmental samples, including water, soil, and air, eliminating the necessity to observe or capture the organism itself (Ficetola et al. 2008). eDNA-based detection methods are generally more cost-efficient and sensitive compared to conventional monitoring methods – such as netting, acoustic monitoring, or visual surveys – particularly when surveying inaccessible habitats or targeting cryptic species (Fediajevaite et al. 2021; Keck et al. 2022; Moss et al. 2022). Beyond species detection, eDNA-based analyses can, in certain circumstances, also provide approximate estimates of species densities (Yates et al. 2019; Rourke et al. 2021; Carvalho et al. 2022; Everts et al. 2022). However, the accuracy of eDNA-based analyses can vary significantly depending on factors such as the specific ecosystem and target species (Fediajevaite et al. 2021; Keck et al. 2022). Interpreting eDNA data in open systems, such as marine or riverine environments, can be more complex than in closed environments such as ponds or small lakes due to intricate effects of admixture, diffusion, and mixing on the distribution of eDNA particles (Rourke et al. 2021; Hinz et al. 2022). Even within a given system, the performance of eDNA-based monitoring can be affected by species-specific factors, including eDNA shedding rate, species density, phenology, and mobility (Adams et al. 2019; Everts et al. 2021; Thalinger et al. 2021). Overall, eDNA-based methods are expected to provide the greatest value in detecting early invasion stages of fish and amphibian species in ponds or small streams (Secondi et al. 2016; Fediajevaite et al. 2021; Everts et al. 2022; Keck et al. 2022). The African clawed frog, Xenopus laevis (Daudin, 1802), serves as a pertinent example of a NIS for which an eDNA-based EDRR system may prove highly effective (Measey et al. 2012; Secondi et al. 2016; Vimercati et al. 2019). Native to Sub-Saharan Africa, X. laevis has been used as a model organism in biological research and pregnancy testing for decades (Gurdon and Hopwood 2000; Wallingford et al. 2010). It has also become a lucrative commodity in the pet trade (Measey 2016), leading to its global exportation in the 20th century and to the successful establishment of populations in various regions across North-America, 229 NeoBiota 102: 227–248 (2025), DOI: 10.3897/neobiota.102.150311 Teun Everts et al.: Pooled eDNA sampling to map the northenmost Xenopus laevis invasion South-America, Asia, and Europe. Given its aquatic lifestyle, effective camouflage, and underwater mating calls (Tinsley and Kobel 1996), introductions of X. laevis often remain unnoticed for extended periods (Measey et al. 2012). The species primarily breeds in ponds and disperses via streams and rivers (Moreira et al. 2017), with periodic overland movement that typically occurs during wet periods and heavy rainfall (Measey et al. 2012; Measey 2016). Xenopus laevis has a largely aquatic ecology, can adapt to a wide range of environments, is highly fertile, and can disperse up to 5.4 kilometres per year (Pagano et al. 2024). It can exert a profound impact on native aquatic fauna due to predation, competition, and disease transmission (Weldon et al. 2004; Lillo et al. 2011; Courant et al. 2018). The cryptic and invasive nature of X. laevis makes it a highly suitable target for eDNA-based monitoring systems, which offer a sensitive and cost-effective approach for generating critical information to support the management of biological invasions (Secondi et al. 2016; Brys et al. 2021; Everts et al. 2022, 2023, 2024). In Europe, established X. laevis populations can be found in Portugal, Italy, and France (Measey et al. 2012). In France, X. laevis was first recorded in the western department Deux-Sèvres in the 1980s (Fouquet and Measey 2006). Since then, the species has spread at an estimated rate of 1.2 km per year, reaching an invaded area of 8,400 km2 by 2024 (Pagano et al. 2024). Further north, a new population was discovered in 2018 in the Armentières canton (Nord department, France), just a few kilometres from the Belgian border (van Doorn et al. 2022). By 2021, the presence of X. laevis had been confirmed in two adjacent administrative regions of Belgium – Flanders and Wallonia (Adriaens et al. 2023; Pauwels et al. 2023). However, the spatial extent of this new invasion in Belgium, as well as the pathways facilitating cross-boundary expansion, remained largely unknown. To address this knowledge gap, we sampled 426 water bodies in the French-Belgian border region between 2020 and 2023 for quantitative eDNA-based barcoding analyses to determine the presence and associated eDNA concentrations of X. laevis throughout the area. We developed and preliminarily tested the effectiveness of a novel sampling strategy that pools water from multiple sites (Fig. 1), enabling extensive area coverage in a short timeframe with limited resources, and discuss its strengths and limitations. We mapped the current distribution of X. laevis in the border region as a part of an EDRR program, and assessed whether two waterways – the river Lys and its tributary, the Douvebeek – serve as potential dispersal corridors (Pauwels et al. 2023). Given that X. laevis is currently regarded as being in an early invasion stage where complete eradication might still be feasible (Reniers et al. 2023), the insights gained from this study could support effective transboundary management strategies. We further address the challenges posed by the expansion of NIS across national and administrative borders and propose potential solutions to mitigate these issues. Material and methods Study area The French-Belgian border region is characterized by a temperate oceanic climate with warm summers, but is substantially colder compared to other European areas containing established non-native X. laevis populations. Here, vast agricultural fields are interspersed with small towns (Fig. 2), similar to other regions in Europe that are invaded by X. laevis (Measey et al. 2012). The study area contains numer- 230 NeoBiota 102: 227–248 (2025), DOI: 10.3897/neobiota.102.150311 Teun Everts et al.: Pooled eDNA sampling to map the northenmost Xenopus laevis invasion Figure 1. Schematic overview of pooling eDNA samples across locations under three different scenarios. In this hypothetical example, three ponds were sampled and filtered through a single filter. a. When X. laevis is absent in all three ponds, no X. laevis eDNA is detected (i.e, no signal). This could also occur when X. laevis is present in very low densities (i.e., false-negative); b. When X. laevis is present in a small proportion of the sampled ponds, a low amount of X. laevis eDNA is expected to be amplified (i.e., weak signal), unless X. laevis density and/or the ratio target-positive to target-negative ponds are high; c. When X. laevis is present in all three sampled ponds, in either low or high densities, a large amount of eDNA is expected (i.e., strong signal). ous ponds, typically used for cattle or irrigation, which are known to be suitable habitats for X. laevis (Lobos and Jaksic 2005; Faraone et al. 2008; Rebelo et al. 2010). Given the aquatic nature of X. laevis, the river Lys and the Douvebeek are assumed to serve as dispersal corridors between France and Belgium (Adriaens et al. 2023; Pauwels et al. 2023). The Lys is a partially canalized river, extending 202 kilometres in total length. Within the study area, the river ranges from 30 to 70 metres in width and reaches a maximum depth of 4.5 metres (Fig. 2). It originates in Pas-de-Calais, France, and flows northeast along the French-Belgian border before draining in the river Scheldt in Belgium. The Douvebeek is a small tributary of the river Lys that is 231 NeoBiota 102: 227–248 (2025), DOI: 10.3897/neobiota.102.150311 Teun Everts et al.: Pooled eDNA sampling to map the northenmost Xenopus laevis invasion Figure 2. Map of the sampled area along the French-Belgian border. France is labelled as “FR”, while the two Belgian Regions, Wallonia and Flanders, are labelled “BE(W)” and “BE(F)”, respectively. Coloured circles indicate the sampled locations per sampled year. The river Lys and the Douvebeek are depicted as thick blue lines, with other waterways represented by thinner blue lines. approximately 21 kilometres long, originating along the hillside of the Mont Noir in France. This stream continues southeast into Flanders, where it flows along the Flemish-Walloon border, draining into the river Lys in the Walloon municipality of Comines-Warneton. The Douvebeek can be characterized as a turbid creek with discharge levels that strongly fluctuate in response to precipitation events. The source area is considered ecologically valuable with several adjacent natural areas harbouring high biodiversity including large pondscapes with important relict populations of native amphibians, such as four local newt species, including the protected great crested newt Triturus cristatus (Laurenti, 1768), which are vulnerable to X. laevis invasion (Lillo et al. 2011; Courant et al. 2018). The middle and lower reaches of the stream, however, have poor water quality due to erosion, wastewater discharge, and nutrient inflows. Sampling Both lentic (i.e., ponds) and lotic (i.e., ditches, streams, canals, and rivers) systems were sampled for eDNA in April 2020 (86 sites), August 2022 (211 sites), and August 2023 (129 sites). These sampling periods coincide with the reproductive season and peak activity of X. laevis, which spans from March to October, thereby increasing the likelihood of detection (Courant et al. 2018; Everts et al. 2021). Sampling locations covered the French-Belgian border region (i.e., in Flanders, Wallonia, and France), mainly focusing on the river Lys, the Douvebeek, and the 232 NeoBiota 102: 227–248 (2025), DOI: 10.3897/neobiota.102.150311 Teun Everts et al.: Pooled eDNA sampling to map the northenmost Xenopus laevis invasion surrounding areas (Fig. 2). In 2020, sampling focused on the western part of the region, based on the assumption of overland cross-border dispersal. Results from this initial campaign informed the 2022 sampling strategy, which shifted to the eastern part of the study area and considered both overland and lotic dispersal pathways. In 2023, the sampling design was further refined based on insights from previous years, with a focus on evaluating the role of the Douvebeek and river Lys as potential corridors for transboundary dispersal (Fig. 2). In total, 303 lentic and 123 lotic sites were sampled. To minimize the number of required eDNA analyses while maintaining spatial coverage, we implemented a novel sampling strategy. This strategy involved pooling water from multiple sampling locations (mean: 4.4, range: 2–8), and filtering this pooled water through a single eDNA filter (i.e., pooling across locations). Adjacent ponds (mean distance to centroid: 309 m, range 7–1,333 m), as well as segments and tributaries of the Douvebeek and river Lys (mean distance to centroid: 632 m, range 52–2,692 m), were therefore grouped into sampling clusters. Within each cluster, water samples were merged into one integrated water sample (Fig. 1). This sampling strategy was used in areas with uncertain prior knowledge of X. laevis presence, and allowed us to assess its occurrence in 366 sites using only 83 eDNA filters. An additional 59 sites, located in regions where X. laevis presence was considered more likely (e.g., the downstream portion of the Douvebeek, proximal to waterways) were individually sampled, yielding a grand total of 142 eDNA filters. To evaluate the performance of the pooled sampling approach, we selected two clusters, each comprising four ponds, of which the initial pooled sample indicated the presence of X. laevis DNA. These ponds were then sampled both individually and again via pooled sampling, generating an additional ten eDNA filters for analysis. Upper water layers were sampled for eDNA from the shore, using a sterile sampling pole with a 0.5 L sterile bag attached at the end. As eDNA particles are heterogeneously distributed in lentic systems, detection sensitivity was increased by implementing a merged sampling (i.e., pooling of water within a location), as described by Brys et al. (2021). As such, water was collected from all sites attributed to a single sampling cluster, and immediately filtered in the field over a single disc filter containing a 0.8 µM PES membrane combined with a 5 µm glass fibre prefilter (NatureMetrics, Surrey, England). Water was filtered until clogging, and the total volume of filtered water was recorded. Filters were then sealed off at both ends and immediately placed in a freezer box. At the end of the day, the filters were transferred to storage at -21 °C, where they remained until molecular analyses were conducted. All reusable sampling material (i.e., sampling pole and bucket) was disinfected with a 2% Virkon S solution (Antec DuPont, Suffolk, UK) to minimize the risk of DNA cross-contamination. To test for potential contamination during field sampling, five negative controls were included by filtering 2 L of mineral water in the field (i.e., field blanks): one at the end of the 2020 sampling campaign and one at the end of each of the two sampling rounds in both the 2022 and 2023 sampling campaigns. Molecular analyses All eDNA samples were stored and processed in a PCR-free building dedicated to low copy number template extractions. This facility hosts DNA-free high-efficiency particulate air (HEPA)-filtered compartments with positive pressure to prevent eDNA sample contamination. To test for PCR inhibition and to evaluate the ex- 233 NeoBiota 102: 227–248 (2025), DOI: 10.3897/neobiota.102.150311 Teun Everts et al.: Pooled eDNA sampling to map the northenmost Xenopus laevis invasion traction efficiency of each filter, an internal positive control (IPC) was added in the first step of the extraction together with the lysis buffer (for details on the IPC see Everts et al. 2021). DNA extraction was performed using Qiagen’s DNeasy Blood & Tissue Kit and purified with the DNeasy PowerClean Cleanup Kit (Qiagen) according to the guidelines provided by the manufacturer. To determine X. laevis presence and estimate its abundance, an 83-bp barcode located on the 12S region of the obtained DNA extracts was analysed and quantified via droplet digital PCR (ddPCR) using the primer/probe assay developed by Secondi et al. (2016). Even though this assay cannot discriminate between 12 (sub)species of Xenopus, it is expected that only X. laevis currently occurs in the sampled area (Pauwels et al. 2023), and thus a positive detection indicates the presence of X. laevis. For each eDNA sample, an average of 3 (range: 1–6) replicate ddPCR reactions were conducted. The unequal number of replicates across samples reflects the adaptive nature of the EDRR framework in which this study was conducted, where resource allocation was adjusted in subsequent years based on field conditions and preliminary findings. Specifically, a higher number of replicates was generally analysed for samples collected in areas with limited prior knowledge of species presence (e.g., locations increasingly distant from the known French population in Armentières), in more inland sites given the expectation that X. laevis primarily disperses via aquatic systems, and in larger water bodies where detecting a few individuals would require increased analytical effort (e.g., the river Lys compared to small ponds). To test for potential PCR failure or contamination, each ddPCR plate included two positive controls containing 10 pg/µL of DNA extracted from X. laevis tissue, along with no-template controls (NTCs) that served as PCR blanks by omitting DNA from the reaction. The advantages of ddPCR compared to conventional quantitative PCR (qPCR) are fourfold: (i) it eliminates the need for calibration curves through absolute quantification, (ii) its high level of sample partitioning enables highly precise measurements, (iii) it reliably detects low concentrations of target DNA even in the presence of abundant non-target DNA, and (iv) it exhibits greater resilience to PCR inhibitors, making it particularly suitable for complex environmental samples (Guri et al. 2024). Data analyses Xenopus laevis was considered present if successful PCR amplification occurred in at least one ddPCR replicate. Target eDNA concentrations (in copies/µL per L filtered water), used as a proxy for species density, were calculated following Everts et al. (2022), and averaged across replicates. The results of pooled samples (i.e., X. laevis presence/absence and target eDNA concentration) were plotted as the centroid of all the sites attributed to a single sampling cluster, representing a conservative approach to visualizing pooled eDNA data. We tested the effectiveness of pooled eDNA sampling for two clusters each containing four ponds. Specifically, we compared (i) the presence/absence of X. laevis in each individually sampled water body with its detection in the pooled sample, and (ii) whether the average target eDNA concentrations from individually sampled ponds corresponded to the target eDNA concentration in the pooled sample, functioning as measures of similarity across sampling methods. To quantify the spatial distribution of X. laevis in the sampled area, we calculated a buffer-based approximation of the potentially occupied range. This 234 NeoBiota 102: 227–248 (2025), DOI: 10.3897/neobiota.102.150311 Teun Everts et al.: Pooled eDNA sampling to map the northenmost Xenopus laevis invasion approach incorporates the locations of positive detections and the known dispersal capacity of the species to delineate areas where X. laevis may plausibly be present. Although the dispersal capacity of X. laevis varies widely (Pagano et al. 2024), we adopted a reasonable estimate of 2 km per year. This is considerably lower than the maximum predicted dispersal speed of 5.4 km in Chile, yet slightly higher than the 1.2 km per year reported in Southern France (Pagano et al. 2024), ensuring that the potential invaded area is not underestimated. Buffers with a 2 km radius were constructed around each target-positive eDNA detection (i.e., either the centroid for pooled samples or the exact locations for individual samples). Overlapping buffers were merged and the total surface area was calculated. The number of resulting polygons can be informative on the historical trajectory of the invasion, including the occurrence and number of introductions (Everts et al. 2023). In order to facilitate the visualization of the merged eDNA sampling, we generated heatmaps using the kernel density estimation tool in QGIS v3.34.7. The obtained ddPCR data from each of the ponds and streams were analysed collectively to obtain an overall understanding of the invasion in the studied area. Given the highly localized nature of eDNA signals in small streams (Van Driessche et al. 2022), we expect the results from lotic systems to primarily reflect local conditions, rather than an accumulation of upstream signals (cf. Deiner et al. 2016). Weight was given to either the presence (0 when X. laevis was not detected or 1 when X. laevis was detected) or the log-transformed target eDNA concentrations in two separate heatmaps. Both heatmaps were generated using a 2 km linear decay, meaning that the influence of any given point on the heatmap value diminishes linearly to zero at a distance of 2 km. Other settings were left at default. Whereas this approach does not account for uneven sampling density, weighting by species presence or target eDNA concentration allows the generated heatmaps to provide an approximate qualitative (interpolation of X. laevis presence) and quantitative (i.e., interpolation of X. laevis eDNA concentrations) overview of the invaded area. However, in cases where sampling sites are excessively spatially clustered, we recommend employing more advanced interpolation methods that account for the uneven density of sampling locations, such as kriging or spatially explicit generalized linear mixed models (GLMMs). Results The generated ddPCR data exhibited high quality, with no DNA detected in field or PCR blanks, consistent recovery of expected target DNA concentrations from positive controls, and successful IPC amplification confirming the absence of PCR inhibition. Consequently, the likelihood of false positive detections due to laboratory or field contamination, as well as false negative detections resulting from extraction and PCR failure was considered unlikely. Individual sampling of each of the four ponds within the two pond clusters that were selected for the pooled sampling validation and tested positive on X. laevis presence, revealed that X. laevis was present in only one pond per cluster (Fig. 3). Despite a substantially lower target eDNA concentration of that pond in one cluster compared to the other (0.18 vs 4.20 copies/µL per L filtered water), X. laevis eDNA could clearly be detected in the pooled sample of both clusters. Tar- 235 NeoBiota 102: 227–248 (2025), DOI: 10.3897/neobiota.102.150311 Teun Everts et al.: Pooled eDNA sampling to map the northenmost Xenopus laevis invasion Figure 3. The X. laevis invasion along the French-Belgian border. Samples of streams and ponds are represented by triangles and squares, respectively. For pooled samples, the centroid of the sampled locations is mapped. Xenopus laevis detection is indicated in red, while its non-detection is shown in grey. The grey shading represents the buffer-based occupied area and was constructed with a buffer of 2 km around the target-positive locations. Insets display two clusters where the pooled sampling methodology was empirically tested. White and red dots represent ponds where the individual sample was negative and positive, respectively. The red-coloured filter represents a target-positive pooled sample. The table shows X. laevis eDNA concentrations (expressed in copies/µL), the average concentration across individually sampled ponds, the concentration of the pooled sample, and the similarity between the latter two. get eDNA concentration of the pooled sample in the higher-concentration cluster (1.02 copies/µL) closely matched the average concentration of the individual samples (1.05 copies/µL; 97.5% similarity). This similarity was less pronounced in the lower-concentration cluster (pooled sample: 0.07 copies/µL; average of individual samples: 0.045 copies/µL; 64.3% similarity). Xenopus laevis DNA was detected in 26 of the 142 collected samples (12 lotic and 14 lentic; 11 individual and 15 pooled samples; 0 in 2020, 3 in 2022, and 23 in 2023), resulting in a total occupied area of 103 km2 within the study area (Fig. 3; Suppl. material 1). The spatial proximity of positive X. laevis detections resulted in a single, continuous polygon, suggesting the presence of one interconnected population. All positive detections occurred within one kilometre of either the river Lys or the Douvebeek. The distribution of X. laevis extends from the headwaters of the Douvebeek to its confluence with the river Lys, and upstream along the river Lys and a small tributary (Courant du Pont Bertin) near Armentières, encompassing both France and the Walloon and Flemish Regions of Belgium. The frequency of positive detections and X. laevis eDNA concentrations were higher in the Douvebeek than in the river Lys (Fig. 4). Positive detections were relatively evenly distributed within the Douvebeek, whereas the majority of samples exhibiting high target eDNA concentrations were located more downstream near its confluence with the river Lys. Nine sites (6 lentic, 3 lotic; 3 sampled in 2022, 6 sampled in 2023) exhibited target eDNA concentrations similar to or exceeding those at the three known breeding sites reported in Adriaens et al. (2023); eight were near the Lys-Douvebeek confluence, and one lentic site was near the source of the Douvebeek (Suppl. material 1). 242 NeoBiota 102: 227–248 (2025), DOI: 10.3897/neobiota.102.150311 Teun Everts et al.: Pooled eDNA sampling to map the northenmost Xenopus laevis invasion Use of AI No use of AI was reported. Funding Publication of this study was partly supported by Research Foundation Flanders (1S01822N) and the European Union’s Horizon Europe HORIZON-CL6-2024-BIODIV-01 project “GuardIAS – Guarding European Waters from IAS”, under grant agreement no. 101181413 (Katsanevakis et al. 2024). Author contributions Conceptualization: RB, TE. Data curation: KK, TE. Formal analysis: KK, TE. Funding acquisition: TE, LD, RB, TA. Investigation: LD, TE. Methodology: TE, RB, SN. Project administration: RB, TA. Supervision: RB. Validation: SN, RB, KK, LD, TE. Visualization: TE, KK. Writing – original draft: TA, TE, LD. Writing – review and editing: SN, RB, JS. Author ORCIDs Teun Everts https://orcid.org/0000-0001-7862-4209 Loïc van Doorn https://orcid.org/0000-0003-3497-9168 Tim Adriaens https://orcid.org/0000-0001-7268-4200 Sabrina Neyrinck https://orcid.org/0000-0002-7379-9269 Jeroen Speybroeck https://orcid.org/0000-0002-7241-7804 Rein Brys https://orcid.org/0000-0002-0688-3268 Data availability All of the data that support the findings of this study are available in the main text or Supplementary Information. References Adams CIM, Hoekstra LA, Muell MR, Janzen FJ (2019) A brief review of non-avian reptile environmental DNA (eDNA), with a case study of painted turtle (Chrysemys picta) eDNA under field conditions. 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The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/neobiota.102.150311.suppl1