The study site concerned a section of the lowland brook Tungelroyse beek (51°14.38'N, 005°52.086'E – 51°14.26'N, 005°47.77'E) near the village Tungelroy in the Province of Limburg, the Netherlands (Fig. 1). This brook of 35 km length has several tributaries and discharges into the Meuse River. The sampling site was selected because it harbors the largest known M. bipartitus population in the Netherlands (Binnendijk et al. 2017). Over most of its course, the brook was meandering and harbored well-developed riparian and hydrophyte vegetation. During sampling the mean water temperature was 16.0 °C, conductivity 664 µS/cm, pH 7.3, water velocity 0.2 m/s, depth 70 cm, and Secchi depth 60 cm. The brook width ranged between 5–8 m and its bed substrate predominantly consisted of sand.

Figure 1. 

A The location of the study site in the Netherlands is framed in red. B Denotation of the sampled stretch of the brook Tungelroyse beek.

Samples of fish, macroinvertebrates, macrophytes, dead organic material, and bottom soil substrate were collected in October 2019 in the stretch of the brook that is denoted in Fig. 1. Fish were caught using handheld electrofishing equipment (Bretschneider EFGI 650). After catching, the fish were euthanized using a neutralized benzocaine solution of 100 mg l^-1 (Close et al. 1997; Neiffer and Stamper 2009; Department of Primary Industries 2015). Macroinvertebrates were collected using 70×55 cm dip nets with a mesh size of 1 mm. Helophytes, floating-leaved, and submerged aquatic macrophytes were collected by hand. Soil samples were collected by means of a hollow soil sampling tube with a diameter of 5 cm. Fish were identified using Kottelat and Freyhof (2007). Several keys were used for the identification of macroinvertebrates: amphipods (Zettler and Zettler 2017), crayfish (Koese and Soes 2011), dragonflies (Franke 1979), water beetles (Friday 1988), water bugs (Tempelman and Van Haaren 2009), and freshwater mollusks (Gittenberger et al. 2004). A Nikon SMZ800 stereo microscope with a 10–63 magnification was used for the identification of small macroinvertebrates. For the identification of macrophytes, we used the flora of Duistermaat (2020).

All samples were transported to the laboratory and stored separately at -18 °C until preparation. To obtain muscle samples a piece of 0.5–1 cm of the dorsal tissue of each fish was dissected (Pinnegar and Polunin 1999). Muscle tissue samples were also dissected from crayfish and unionid mussels. For crayfish, the muscle tissue of the abdomen was used after the removal of the intestine. Other macroinvertebrates were stored alive for two days at 5 °C to empty their intestinal contents. Subsequently, these invertebrates were rinsed with tap water and after that with demineralized water before processing. From unionid mussels, muscle tissue of a similar size was dissected. Of small mollusks, whole soft bodies were used. For small mollusks and other macroinvertebrates, individuals of the same species were pooled to obtain enough material (0.22–0.26 mg) for analyses. The stems, leaves, and roots of individual plants were pooled.

After preparation, all samples were stored at -80 °C until freeze-drying. Freeze-drying was carried out at -90 °C for 24–48 hours for fish and macroinvertebrate samples (Paalvast and Van der Velde 2013; Verstijnen et al. 2019). Plant, dead organic material, and soil samples were freeze-dried for at least 48 hours. After freeze-drying, the samples were grounded with aluminum balls, for 2 min at 30 rpm, using a Retsch MM 400 (Verstijnen et al. 2019). Subsequently, the grounded samples were weighted in tin cups (Elemental Microanalysis 8 × 5 mm) and prepared for isotope analyses. For the fish and invertebrate samples, 0.22–0.26 mg dry weight was weighted. For plants and soil, separate samples were weighted for carbon (10 mg) and nitrogen (40 mg) analyses.

Carbon and nitrogen stable isotopes were measured using a Thermo Scientific FLASH 2000 HT Elemental Analyzer with a Thermo Scientific DELTA V Advantage Next Generation Isotope Ratio mass spectrometer. Reference gasses were calibrated with the IAEA standards (IAEA-N-2 and IAEA-CH-6), with a maximum deviation of 0.15‰. As an internal standard control, caffeine was used and the ¹³C/¹²C and ¹⁵N/¹⁴N of every sample were determined (in ‰). The isotope ratios (R) δ¹³C and δ¹⁵N are relative to Vienna PDB and atmospheric N₂ and were calculated by:

δ¹³C or δ¹⁵N = (R_sample/R_standard − 1) * 1000

Quantification of energy sources and trophic structure of communities and organisms was conducted by comparing relationships of naturally occurring stable isotope ratio distributions of nitrogen ¹⁵N:¹⁴N and carbon ¹³C:¹²C. Stable isotope ratios of nitrogen (δ¹⁵N) are enriched between prey and predator tissues, thus providing a measure for the trophic position of a consumer in a food web. The trophic position of each fish species in the food web was calculated following Vander Zanden and Rasmussen (1999). Stable isotope ratios of carbon (δ¹³C) show little or no trophic level enrichment and are useful for identifying the production sources for consumers (Vander Zanden et al. 1999). For calculating the primary consumer δ¹⁵N-δ¹³C baseline, the δ¹⁵N (‰) measured values of the mollusks were used (Table 1).

Convex hull areas were used to display isotopic overlap between fish, mollusks, other macroinvertebrates, and plants. Isotopic niche widths of benthic species were compared using Bayesian standard ellipse areas (SEA) (Jackson et al. 2011; Syväranta et al. 2013). SEA are less sensitive to increasing sample sizes, whereas Convex hull areas tend to increase as the sample size increases for comparing isotopic niche widths. SEA with a 95% confidence interval of benthic fish species and overlap between SEA were computed using the Stable Isotope Bayesian Ellipses in R (SIBER) package (Jackson et al. 2011). This approach calculates Bayesian SEA parameters by running 100,000 Markov-Chain Monte Carlo (MCMC) simulations. Subsequently, isotopic niche areas (‰²) and proportion overlap (%) were estimated on the posterior distribution of the covariance matrix for each benthic fish species and compared. The estimates were corrected for sample size (SEAc) by two standard deviations and plotted (Jackson et al. 2011; Ryan et al. 2013). The relationships between benthic fish length and their δ¹⁵N and δ¹³C signatures (‰) were analyzed using Pearson correlation tests.

Modeling benthic fish species diet source proportion was done with a Bayesian mixing model that generates robust probability estimates using the Stable Isotope Analysis in R (SIAR) package (Parnell et al. 2010; Parnell and Jackson 2013). The initial diet sources concerned all sampled macroinvertebrates with the exemption of spiny-cheeked crayfish (Faxonius limosus) > 40 mm since they are too large to be a prey of the benthic fish species of concern. Thus, the model was first run with a wide range of 28 possible diet sources per consumer. Via a backward stepwise selection procedure, a dietary item that contributed the least was then excluded. Eventually, stable isotope signatures of the five most important dietary items were included in a final model. It is never clear what the accurate fractional contribution of an isotopic source is to a consumer’s tissue (Martínez del Rio et al. 2009). Therefore, we modeled proportions of diet sources for the consumers on the basis of four different stable isotope fractionation factors (ΔN and ΔC) and associated standard deviations, in accordance with Verstijnen et al. (2019). These are: A) ΔN 3.4 ± 1.0 and ΔC 0.4 ± 1.3 (Post 2002: established mean trophic fractionation based on a literature review), B) ΔN 2.9 ± 1.2 and ΔC 1.3 ± 1.3 (McCutchan et al. 2003: fractionation in muscle tissue), C) ΔN 2.3 ± 1.6 and ΔC 0.4 ± 1.2 (McCutchan et al. 2003: aquatic consumers) and D) ΔN 3.1 ± 1.6 and ΔC 2.0 ± 0.8 (Caut et al. 2009: based on a selection of data of fractionation in muscle tissue of fish). Computing all analyses and statistical tests was done with the program R version 3.6.2 (R Core Team 2021).

In total, eight fish species were particularly abundant: Barbatula barbatula, Cobitis taenia, Gobio gobio, Gasterosteus aculeatus, M. bipartitus, P. semilunaris, Rhodeus amarus, and Rutilus rutilus (Table 1). Barbatula barbatula, C. taenia, G. gobio, and P. semilunaris were caught at the bottom of the brook, mostly on bare (unvegetated) bed substrate. Rhodeus amarus and R. rutilus were mostly caught in vegetated zones of the brook. Misgurnus bipartitus was most frequently caught in dense vegetation zones close to the banks. Gasterosteus aculeatus was caught in several habitats. The isotopic signatures of all sampled fish ranged between -39.29 (R. amarus) and -25.64 (P. parva) for δ¹³C, for δ¹⁵N this ranged between 8.63 (P. parva) and 20.27 (R. rutilus).

The δ¹⁵N signature of fish demonstrates that Silurus glanis is most likely the top predator in the brook Tungelroyse beek (Fig. 3). It also showed the highest mean trophic position of the fish community (Fig. 4). Individual trophic positions varied between 1.24 (G. gobio individual) up to 3.90 (G. aculeatus individual). Of the 14 fish species included in the cluster analysis, M. bipartitus ranked 8^th with a mean trophic position of 2.9, respectively preceded and followed by the benthic G. gobio (3.0) and P. semilunaris (2.9). The two other benthic species B. barbatula and C. taenia showed a lower mean trophic level of 2.6. Cluster analysis based on mean δ¹³C and δ¹⁵N values shows that four groups can be distinguished and two species occupy a distinct position (Fig. 5). The latter two species concern a relatively small planktivorous species (P. parva) and a small predominantly herbivorous cyprinid (R. amarus) that forages mainly on diatoms. Group 1 consists of an omnivore (R. rutilus) and three benthivores (B. barbatula, C. taenia, P. semilunaris). Group 2 consists of two benthivorous species (G. gobio, M. bipartitus) and an omnivore (Cyprinus carpio). Group 3 consists of two piscivorous species (Esox lucius, Perca fluviatilis) and zooplankton feeding juvenile cyprinids that were too small to identify at the species level. Group 4 consists of two insectivorous and piscivorous species (G. aculeatus, S. glanis).

Figure 3. 

Figure of trophic signatures of fish species and taxa depicting mean δ¹⁵N and δ¹³C values (‰). The lines denote the standard deviations. The number of used individuals per species or taxa, and associated standard deviations and ranges are presented in Table 1. Benthic fish species are depicted with a * and alien species are indicated in bold.

Figure 4. 

Figure of the mean trophic position of fish species and taxa for which 4 or more individuals were collected. Bars represent standard deviations. Benthic fish species are depicted with a ^* and alien species are indicated in bold.

Figure 5. 

Dendrogram obtained from complete linkage cluster analysis (Euclidean distance) of mean δ¹³C and δ¹⁵N for fish species or taxa of which 4 or more individuals were encountered. Benthic fish species are depicted with a ^* and alien species are indicated in bold.

Pearson correlation analyses of all three native benthic fish species revealed that body size correlated negatively in a significant way with their δ¹⁵N signatures: B. barbatula (r = -0.60, t(12) = -2.56, p < 0.05), C. taenia (r = -0.54, t(17) = -2.67, p < 0.05), and G. gobio (r = -0.70, t(13) = -3.56, p < 0.01) (Fig. 6). In addition, significantly positive correlations between body size and δ¹³C signatures were observed for C. taenia (r = 0.58, t(17) = 2.92, p < 0.01) and G. gobio (r = 0.68, t(13) = 3.32, p < 0.01).

Figure 6. 

Correlations between total length (mm) and stable isotope ratios (δ¹⁵N and δ¹³C in ‰) of five benthic fish species in the brook Tungelroyse beek. Significant Pearson correlations are indicated with a p-value and a trend line.

The niche space of five benthic species: B. barbatula, C. taenia, G. gobio, M. bipartitus, and P. semilunaris was determined using the 95% confidence interval Bayesian standard ellipses of δ¹³C and δ¹⁵N (‰) (Fig. 7). Juvenile and adult individuals were included in this analysis. Isotopic niche area overlap was observed among all benthic species. The largest isotopic niche area was observed for B. barbatula and the smallest for P. semilunaris and M. bipartitus, respectively (Fig. 8). Misgurnus bipartitus displayed a significant niche overlap with G. gobio (91.8%), B. barbatula (37.1%), P. semilunaris (8.9%), and C. taenia (1.1%) (Table 2). Proterorhinus semilunaris displayed significant isotopic overlap with B. barbatula (91.2%), C. taenia (66.1%), G. gobio (37.4%), and M. bipartitus (8.9%).

Figure 7. 

The 95% confidence interval corrected Bayesian standard ellipse areas (SEAc) of δ¹³C and δ¹⁵N values (‰), demonstrating significant isotopic niche partitioning among five benthic fish species in the brook Tungelroyse beek.

Figure 8. 

Measures of uncertainty and central tendency of corrected Bayesian standard ellipse areas (‰²) based on 100,000 posterior draws of parameters showing 95, 75, and 50% credibility intervals from light to dark grey respectively. Black dots denote the mean.

Using Bayesian mixing models, the diet source proportion of the five benthic fish species with four different fractionation factors was modeled. The estimation of five out of 28 possible dietary items that mostly contributed to the diet elucidated notable differences in the diet of alien and native fish species (Table 3). Between different fractionation factors, the model generally produced a similar output. For B. barbatula, the most important dietary items were the snails Lymnaea stagnalis and Bithynia tentaculata, the water boatman Sigara falleni, larvae of non-biting midges (Chironomidae), and the small water boatman Micronecta scholtzi made up 18–30%, 11–24%, 14–23%, 16–22%, and 14–21% of the diet, respectively. The most important dietary items of C. taenia were B. tentaculata (6–34%), L. stagnalis (16–31%), the snail Radix labiata (13–27%), S. falleni (11–21%), and M. scholtzi (11–19%). For G. gobio, these were chironomid larvae (15–34%), larvae of the damselfly Calopteryx splendens (14–31%), the snail Planorbarius corneus (9–23%), the water stick insect Ranatra linearis (9–23%), and B. tentaculata (17–21%). The most important dietary items of M. bipartitus were chironomid larvae (15–75%), dragonfly (Anisoptera) larvae (4–32%), B. tentaculata (13–29%), R. linearis (3–29%), and Gammarus roeselii (4–21%). Due to the small size of their mouth, B. barbatula, C. taenia, and M. bipartitus probably only eat small individuals of snails. For P. semilunaris, the most important dietary items were Corbicula leana (15–50%), S. falleni (11–40%), B. tentaculata (10–27%), G. roeselii (5–25%), and R. labiata (5–22%).

The mixing models showed that the native species B. barbatula and C. taenia had a similar diet since they had four out of five most important dietary items in common (Table 3). These were two snails and two boatman species. The native G. gobio had three dietary items in common with the alien M. bipartitus. Chironomid larvae were the most important dietary item for both species. The alien P. semilunaris had three dietary items in common with C. taenia: two snails and a boatman. The dietary items that appear to be the most important for C. taenia were less important for P. semilunaris (Table 3).

Analyses of stable isotope ratios (δ¹⁵N and δ¹³C) have provided insight into the food web of a Western European lowland brook. Two alien benthic fish species have been established in the brook Tungelroyse beek in the last two decades (NDFF 2023a, b; Van Kessel et al. 2013). Both invaders do not dominate the benthic fish community and are considered moderately abundant, similar to the native B. barbatula (Binnendijk et al. 2017). Cobitis taenia and G. gobio are considered highly abundant. The mean trophic levels of the five benthic fish species ranged between 2.6 and 3.0, indicating that these benthic species are all secondary consumers (i.e., carnivores that mostly eat herbivores). The analyses demonstrated that M. bipartitus predominantly consumed native and alien macroinvertebrate species. The larvae of non-biting midges (Chironomidae) and dragonflies (Anisoptera), two mollusk species and a gammarid were the most important dietary items. This is in line with the results of a diet study on the closely related Oriental weatherfish Misgurnus anguillicaudatus (Urquhart and Koetsier 2014). No such studies have yet been conducted for M. bipartitus. Based on stomach analyses, it was determined that chironomid larvae, dragonfly or damselfly larvae (Odonata), mollusks, and detritus were the most frequently consumed prey of M. anguillicaudatus. Another stomach analysis study of M. anguillicaudatus also demonstrated that chironomid larvae and mollusks were important dietary items (Keller and Lake 2007). However, both studies found that worms (Oligochaeta) and water fleas (Cladocera) were also relatively important dietary items of M. anguillicaudatus (Keller and Lake 2007; Urquhart and Koetsier 2014). During our sampling, we did not catch Oligochaeta or Cladocera. No species belonging to these taxonomic groups were also found during previous macrofauna sampling occasions (Basten et al. 2010). The fauna sampling by dipnets with a 1 mm mesh size was probably less effective for some taxonomic groups of benthic invertebrates and zooplankton. For instance, more taxa of benthic Oligochaeta and Chironomidae can be expected in a nutrient-rich lowland stream such as the Tungelroyse beek (Redeke 1948; Tolkamp 1980). Therefore, in addition to the use of a fine-meshed dip net, a core sampler and plankton net are also recommended in future studies. Nevertheless, the information on the trophic levels of the benthic fish species shows that the examined species predominantly prey on macroinvertebrates. Combining results of multiple sampling techniques with novel approaches for dietary analyses (e.g., mixing models, eDNA from intestines, food experiments) will reduce uncertainty in the results of future studies on the diet of benthic fish species in the Tungelroyse beek. Proterorhinus semilunaris consumes native and alien macroinvertebrates. A water boatman, a gammarid species, and three mollusk species were identified as the most important dietary items. Stomach analyses demonstrated that mollusks and gammarids can be important dietary items of P. semilunaris (Vašek et al. 2014; Všetičková et al. 2014), but also revealed that chironomid larvae were the main food sources of this species (Adámek et al. 2010; Vašek et al. 2014; Všetičková et al. 2014; Endrizalová et al. 2020). The latter is not the case in our study, despite the presence of chironomid larvae in the brook. This may indicate niche differentiation between various populations of P. semilunaris (see section Niche differentiation).

A comparison of the Bayesian standard ellipses shows a significant isotopic niche space overlap between the alien and native fish species. Based on their isotopic niche overlap, potential competitive interactions can be expected between M. bipartitus and G. gobio (91.9% overlap), and to a lesser extent with B. barbatula (37.1% overlap) and C. taenia (1.1% overlap). Results of Bayesian mixing models also demonstrate that partial food overlap between M. bipartitus and G. gobio is likely. Out of 28 modeled dietary items, chironomid larvae were most important for both fish species. Furthermore, they feed on larvae of Odonata (dragonflies and damselflies), the faucet snail Bithynia tentaculata, and the water stick insect Ranatra linearis. These macroinvertebrates with a relatively large body size are associated with nutrient-rich lowland brooks and mainly occur in submerged aquatic vegetation (Redeke 1948; Verdonschot et al. 1992). Potential competitive interactions with respect to food can also be expected between the alien P. semilunaris and native B. barbatula (91.2% overlap), C. taenia (66.1% overlap), and to a lesser extent G. gobio (37.4% overlap). Results of Bayesian mixing models also indicate food overlap between P. semilunaris, B. barbatula, and C. taenia. Out of 28 modeled dietary items, two dietary items were identified as the main communal dietary items between the latter three species: B. tentaculata and the water boatman Sigara falleni. The aquatic snail Radix labiata was an important dietary item for P. semilunaris as well as for C. taenia.

Niche differentiation may allow species to co-exist by avoiding competition or resource partitioning. Proterorhinus semilunaris displays an opportunistic and plastic feeding pattern outside its native range (Pettitt-Wade et al. 2015; Nagelkerke et al. 2018; Tarkan et al. 2018, 2019). For M. bipartitus, this has not yet been studied. However, diet studies on the related M. anguillicaudatus and European weatherfish (Misgurnus fossilis) demonstrated that these species are opportunistic feeders (Keller and Lake 2007; Urquhart and Koetsier 2014; Norris 2015; Pyrzanowski et al. 2021). Therefore, we assume that both benthic invaders of the present study are food generalists. The isotopic niche spaces of both alien fish species were smaller than those of native benthic species. This indicates niche differentiation, promoting coexistence due to the food specialization of the invaders (Jackson and Britton 2014; Jackson et al. 2014; Tran et al. 2015; Top-Karakuş et al. 2021). The feeding areas they frequently visit may be smaller and more restricted than for the native species because of competition for space or territory (Baltz and Moyle 1993). In fact, the fish could select diet items and feeding areas avoiding natives. The degree of isotopic niche overlap between M. bipartitus and P. semilunaris was low (8.9% overlap), indicating little resource competition between the invaders. In contrast to M. bipartitus, P. semilunaris did not have chironomid larvae as an important dietary item, whereas these larvae appeared to be the main food source in several other studies (Adámek et al. 2010; Vašek et al. 2014; Všetičková et al. 2014; Endrizalová et al. 2020). In its native range, the isotopic niche space area of P. semilunaris was significantly larger than co-occurring fish species (Tarkan et al. 2018). The isotopic niche overlap between P. semilunaris, B. barbatula, and G. gobio is in line with the results of a study based on the functional feeding traits of these species (Nagelkerke et al. 2018). They concluded that P. semilunaris is a generalist feeder and its trophic profile overlaps with B. barbatula and G. gobio. Cobitis taenia and M. bipartitus were not assessed. The available body of knowledge on the feeding strategies of M. bipartitus, P. semilunaris supports the coexistence of these alien species with native species is likely due to niche differentiation by a large plasticity of their feeding behavior. We cannot demonstrate a shift in resource partitioning of native species because data on the reference situation before the arrival of the alien species are not available. Moreover, we lack dietary information in time and space.

The body size of native fish species correlated negatively in a significant way with their δ¹⁵N (‰) signature (Fig. 6). This indicates that different life stages of these species prey on other food items (Smyly 1955; Copp and Vilizzi 2004). We found that aging individuals of native species were less N enriched. Larger individuals prey on larger organisms and larger species with a lower δ¹⁵N (‰) signature. The N enrichment of both alien fish species did not differ for individuals with different body sizes, probably because these species are more opportunistic and plastic in their way of feeding. A shelter competition experiment demonstrated that the presence of P. semilunaris did not cause shifts in the habitat choice of B. barbatula (Van Kessel et al. 2011). The lack of competitive behavior between these species most likely resulted in their isotopic niche overlap. Moreover, it may also explain the high degree of niche overlap (99.2%) between the native B. barbatula and C. taenia. According to the Bayesian mixing models, these species also have a considerable overlap in dietary items. This is notable because previous research has shown that several life stages of both species have different habitat preferences (Copp and Vilizzi 2004).

Isotopic niche spaces were identified by determining Bayesian standard ellipses, a relatively new and proven method that has already been applied in several studies of on various aquatic invasions (e.g., Jackson et al. 2012; Mumby et al. 2018; Balzani et al. 2020; Cerveira et al. 2021). The imperfections in the sampling of benthic macrofauna and zooplankton do not affect our results on niche widths, since these 95% confidence interval standard ellipse areas are determined based on the isotopic signatures of the benthic fish species (Jackson et al. 2011). The sample sizes used in the present study for comparing the benthic species ranged from n = 14 to n = 34. It is recommended to use the corrected standard ellipse area (SEAc) value if sample sizes are small (n < 30) or unequal (Jackson et al. 2011). SEAc results in a slightly large ellipse but the shape is similar to that of the regular SEA and is evenly reliable. Simulations of the Bayesian model with smaller sample sizes than those used in this study (e.g., n = 3, Ryan et al. 2013) can also result in robust SEAc values (Jackson et al. 2011). The use of Bayesian mixing models is a valuable addition to comparing Bayesian standard ellipses since they yield robust estimates of the diet (Parnell et al. 2010; Jackson et al. 2012). It has been demonstrated that mixing models even yield better estimates of prey identification and quantification than regular stomach analysis (Koester et al. 2022). However, this study only focused on isotopic niches of benthic fishes. Hutchinson (1957) defined a niche as the set of biotic and abiotic conditions in which a species is able to persist and maintain stable populations over time. A niche is not fixed but can change over time due to changes in the environment or interactions with other species. This denotes a limitation of the present study as it did not examine prey abundance or foraging strategies and habitat use of different benthic fish species during the day and night, and over seasons. Diet preferences of benthic fish can be seasonal (e.g., Smyly 1955; Copp and Vilizzi 2004; Adámek et al. 2010). This could mean that there is a seasonal effect on the trophic niche overlap of co-occurring fish species. The isotopic signature measured from fish sampled in October is that of a period of high food resource richness. The trophic niches overlap may be different during periods with lower resource richness. Therefore, follow-up research should focus on unraveling seasonal variability of food niche overlap between species. Moreover, analyses of stable isotopes in this study concern a snapshot in the time of year, but also in the colonization process. To better understand the colonization process of M. bipartitus and P. semilunaris, it is also recommended that analyses be repeated at a later time, with this study serving as a baseline.

The use of stable isotope analyses can be a reliable approach to quickly assess the potential impacts of invaders on native species and their environment (Balzani and Haubrock 2022). Here, we use this technique to predict the impact of two benthic invaders on three native species in a lowland brook. Lowland rivers and brooks in western Europe are usually characterized by high nutrient contents (Hering et al. 2010; De Klein and Koelmans 2011), which is also the case for the brook investigated in this study (Basten et al. 2010). Due to the high nutrient load in this brook, it is likely that food resources are not limiting the abundance of benthic fish species (Giller 1984). Therefore, the coexistence of sympatric native and alien benthic fish species is a plausible scenario.

There is evidence that both alien species are expanding their geographical range in Western Europe (Cammaerts et al. 2012; Manné et al. 2013; Verreycken 2021). In 2012, the first M. bipartitus population in the Netherlands was found in the brook Tungelroyse beek (Van Kessel et al. 2013). In subsequent years, the population continued to expand in this brook (Binnendijk et al. 2017). In 2020, a specimen was found in a 12 km downstream tributary of the Meuse River (Binnendijk 2020; NDFF 2023a). It is likely that the M. bipartitus population will further expand and will utilize the Meuse River as a dispersal corridor to colonize other tributaries, as P. semilunaris did (Cammaerts et al. 2012; Manné et al. 2013). This makes it likely that habitats of the native M. fossilis will be invaded in the long run. In addition to risks of hybridization and disease transmission (Wanzenböck et al. 2021), resource competition may pose a significant threat to M. fossilis populations. Proterorhinus semilunaris, first found in the Netherlands in 2002, has become a widespread species (NDFF 2023b). Given the range expansion of both alien species, it is possible that they will eventually colonize systems with fewer food resources where competition may eventually lead to the displacement of native benthic fish species.