All the three studied species, monkey goby, racer goby and perch, were sampled from three sites (Fig. 1) located in the Bug River (the Vistula River system’s largest eastern tributary, Baltic Basin, Poland) in 11–14 August 2007. The Bug River maintained its natural character of a lowland, meandering river within a wide valley. It is 772 km long and the watershed covers 39,420 km². Its sources are in Ukraine, but after 185 km, a stretch of 363 km comprises the border between Poland, Ukraine and Belarus. Later, it turns to the west until it joins the Narew River, shortly before its confluence with the Vistula River. Sampling sites Z (52°23.57333'N, 22°42.25833'E), R (52°42.08667'N, 22°09.73333'E) and site B (52°37.41000'N, 21°35.03000'E) were situated in the lower Bug River, which flows entirely through the territory of Poland. The river in this section is more than 100 m wide and relatively shallow, but with a heterogeneous depth profile (pools and riffles), mainly with a sandy bottom, though with some contribution of gravel and stones (Table 1) and scattered submerged vegetation, such as Elodea canadensis, Potamogeton perfoliatus, Myriophyllum sp. The riverbed is naturally meandering with a sequence of eroded and deposited banks with some emerging macrophytes (Typha sp., Scirpus sp., Juncus sp., Sparganium sp., Glyceria maxima). Only in site B, there was a short section of the bank that had a limestone embankment and a paved area at a small bay and a platform created as a recreational area in the village. The racer goby was very abundant there, as it used such artificial structures as hiding places. The surrounding landscape mainly comprised pastures and other agricultural lands (Table 1).

Figure 1.

Location of sampling sites Z, R, B in the Western Bug River and places of first record of racer goby (red dot) and monkey goby (green dot) in Poland.

Fish were sampled at depths from 0.5 m to 1.7 m, along the riverbank by electrofishing with a battery-powered unit, 350 V, 20–100 Hz, wading ca.100 m upstream along the bank and from the boat drifting 500 m downstream.

Fish were anaesthetised (MS-222) and preserved in 4% formaldehyde. In the laboratory, the fish were measured for total length (L_T; to the nearest 1 mm) and weighed (with 0.01 g accuracy). Their alimentary tracts were dissected. The gut contents (in each fish, the same section of alimentary tract, i.e. stomach and first half of intestine) were weighed (to 0.0001 g accuracy) and prey items were identified under a stereomicroscope. Chironomidae larval stages can be identified to genera or groups of closely-related species, but only rarely to the species level. Their remains from the fish gut lack many features that are necessary for precise identification and, thus, following the main key used in this study (Brooks et al. 2007), they were identified to the morphotype cf. level.

Animal prey remains were identified to the lowest readily recognisable taxon, counted and the proportional weight was estimated. The percentage contribution by weight of each food category to the biomass of total stomach content was estimated visually (Hyslop 1980) and then recalculated into real weights, based on the weight of total gut content. The frequency of occurrence (defined as percentage of fish guts containing given prey category in relation to the total number of fish with guts containing any food: %F), percentage of biomass (weight of given food category in relation to total weight of gut content: %B) and relative abundance (number of given prey category in relation to total number of prey: %N) were quantified for each food category at each sampling site.

The Amundsen et al. (1996) modification of the Costello (1990) graphical method was applied to describe feeding strategy and to identify dominant prey items for the fish species, as well as feeding phenotypic plasticity.

Dietary overlap between each pair of fish species was calculated using Schoener’s index (Wallace 1981): α = 1–0.5 [Ʃ_{n = 1} (p_ij – p_ik)], where p_ij is the proportion of the ith resource used by species j and p_ik is the proportion of the ith resources used by species k; overlap values exceeding 0.6 were regarded as high or biologically significant (Wallace 1981). As the proportion in Schoener’s index calculation, we used %N proportion: numbers of given prey type to the total number of prey found in fish gut. To show how the accuracy of prey identification influences the evaluation of dietary overlap, we estimated it based on protocol 1 – considering main food categories, i.e. prey pooled into taxonomic groups usually applied in fish diet studies, for example, Amphipoda, Chironomidae (called later Schoener’s index 1) and protocol 2 – considering detailed food categories, i.e. prey identified to the lowest possible taxonomic level (called later Schoener’s index 2).

To compare the taxonomic composition of the diet between fish species overall (all sites pooled) and at each sampling site, one-way permutation analysis of similarity (ANOSIM, Bray-Curtis similarity coefficient) was used, based on prey. ANOSIM is analogous to an ANOVA procedure, with non-parametric permutation applied to rank similarity matrix of samples. The similarity percentage procedure (SIMPER) was used to identify which prey taxa were most likely responsible for the patterns detected by ANOSIM. SIMPER provided the average dissimilarities between the species and identified which prey taxa made the greatest contribution to any dissimilarities between analysed categories (Clarke and Warwick 1994). All multivariate analyses were performed using PAST software (ver. 3.15; Hammer et al. (2001)).

Dietary niche width was calculated as a Simpson diversity index: 1 – D = 1 – Σpi ² and Shannon diversity index: H = – Ʃpi log₂ pi, where pi is the proportion of different prey in the diet (Ghent 1991).

Both goby species and European perch fed on the macrozoobenthos. The perch is known to shift toward piscivory with its ontogenetic development (Hjelm et al. 2000; Rezsu and Specziár 2006). In our study, only a few individuals of perch, i.e. > 120 mm predate on juveniles of fish. Bleak Alburnus alburnus, bitterling Rhodeus amarus and unidentified fry of other cyprinids were recorded in its diet in the Bug River. In the case of the studied gobies, we recorded piscivory only in the racer goby, but identification of the prey species was impossible due to the advanced stage of the digestion process. Piscivory was already reported, both for racer goby and monkey goby, but such a food category was not considered important and generally occurred only in the largest individuals (Grabowska and Grabowski 2005; Grabowska et al. 2009; Grabowska et al. 2023).

In general, the diets were more similar between the goby species than between either of the gobies and perch. However, the diet of perch was more similar to that of the racer goby than to that of the monkey goby. Both the Eurasian perch and the racer goby fed on prey that indicated their association with macrophytes. Macrophyte patches are refuges for small fish, as well as hiding places or substrate for several macrozoobenthic groups, such as amphipods, insect larvae, for example, Diptera and Zygoptera larvae or gastropods, that are attractive food for many fish species (Gulati et al. 1990; Van den Berg et al. 1997; Dukowska et al. 2012; Dukowska and Grzybkowska 2014; Grzybkowska et al. 2020). In fact, they were common prey for perch and racer goby in our study.

Amphipods were especially important food items for perch and racer goby in the Bug River. Depending on the site, these fish mainly ate Pontogammarus robustoides or Dikerogammarus villosus and less D. haemobaphes. Field observations have shown that all three species are rather eurytopic (Bącela and Konopacka 2005; Grabowski et al. 2007; Żytkowicz and Kobak 2008). Nevertheless, they show some species-specific habitat preferences. For example, D. villosus and D. haemobaphes were reported to prefer stony substrates (Boets et al. 2010; Clinton et al. 2018). On the other hand, in comparison to adult individuals, juveniles of P. robustoides are known to prefer various macrophytes as their main habitat (Czarnecka et al. 2010). The high contribution of P. robustoides to the diet of the racer goby and the co-occurring European perch has also been reported in our earlier studies in the Włocławski Reservoir (Grabowska and Grabowski 2005). Amphipoda are known to be eaten by racer goby in the main channel of the Vistula River and in the large dam-reservoir built on it (Kakareko et al. 2005), as well as in its native range, i.e. in the middle Dnieper River (Pinchuk et al. 2003). Considering chironomid larvae, both perch and racer goby predated relatively large species, such as Glyptotendipens cf. pallens. This morphotype of Glyptotendipes spp. is common in various freshwater habitats (Moller Pillot 2009). Often, it is associated with macrophytes and coarse organic matter (Kornijów 1997; Moller Pillot 2009; Čerba et al. 2022). Glyptotendipes pallens is a plant tissues miner and scraper (Koperski 1998; Beiger 2004), but it also inhabits other types of substrates, for example, plant detritus, wood debris and mud (Moller Pillot 2009; Čerba et al. 2022). Macrophytes are traps for organic matter in running waters and create ideal microhabitats for bottom dwelling chironomids, thus, many Glyptodendipes spp. are common on macrophytes, as well as in mud gathered around them (Grzybkowska et al. 2020). Glyptotendipes sp. were also one of the most important Chironomidae taxa in the diet of racer goby in lowland rivers in the Dnieper River system (Didenko et al. 2021a) where, in line with our findings, the diet of racer goby also indicated its association with plants, as has been reported from the Vistula River (Kakareko et al. 2005). Epiphytic chironomids were found to be the main prey of perch in pondweed (Potamogeton spp.) patches, while typically benthic species were preferred by ruff (Gymnocephalus cernuus) in the lowland Warta River (Dukowska and Grzybkowska 2014). We did not record Chironomus riparius in the diet of perch at any site, while it was quite common in gut content of both goby species, however, with different contributions to the overall species diet. This sediment burrowing chironomid is probably more difficult to obtain by perch, contrary to both goby species, as they have a habit of hiding in sediments (Kakareko 2011), which may give them more opportunities to find C. cf. riparius larvae in mud. However, the European perch, considered to be an epi-benthic predator, was found to penetrate bottom sediments to some depth searching for food in lake littoral, where it fed on large individuals of Chironomus plumosus larvae (Kornijów 1997). Despite that, amongst the same lentic sedimentary benthos communities associated with littoral macrophytes, predation by perch was most intensive on motile invertebrates, such as isopods and amphipods, while chironomids contributed less to perch diet (Kornijów et al. 2016). This is consistent with our results, suggesting that amphipods may be the most important prey for the European perch.

Both goby species and European perch feed on small gastropods, such as Bithynia sp., Valvata sp. and Potammopyrgus antipodarum, which are also associated with submerged macrophytes (Van den Berg et al. 1997). These gastropods were frequently recorded in the diet of the racer goby in the Włocławski Reservoir (Kakareko et al. 2005). Locally, they were even the dominant food category for that fish species (Kostrzewa and Grabowski 2003). In our study, in comparison with racer and monkey gobies, European perch rarely ate gastropods. Such prey was scarcely reported in previous studies on the diet of perch, even if they were abundant in the macroinvertebrate assemblages (Rezsu and Specziár 2006; Kornijów et al. 2016). Macroinvertebrates of such low mobility are not attractive prey for sight-dependent diurnal predators like perch (Craig 2008; Kornijów et al. 2016). Another food item that differentiated gobies and perch in terms of diet were the Sphaeridae bivalves. That typical benthic group of molluscs was found quite frequently in the diet of both racer and monkey gobies, while none was recorded from perch. Sphaeridae were also an important prey of both goby species in the Vistula River and in the Włocławski Reservoir (Kakareko et al. 2005). Coleopteran larvae were occasional prey of gobies and perch in the Bug River. In the gut content of racer goby, we even found an adult of Gyrinidae. The presence of pleuston organisms, such as whirligig beetles, suggests that racer goby utilised a wider range of microhabitats when searching for food, from the surface of the water to the riverbed. In fact, the diversity of the gobies’ diets, especially in the case of racer gobies, was higher than in the case of perch.

In summary, alien gobies, in particular the racer goby, and European perch possibly used similar habitats for foraging, i.e. macrophyte patches in areas of more stagnant water and muddy bottom. However, perch with a body length similar to that of co-occurring gobies, was more piscivorous. The dietary overlap between perch and gobies usually comprised prey items that are very common in the riverine environment, such as amphipods and large chironomid larvae (Dukowska et al. 2012; Dukowska and Grzybkowska 2014). The two fish display different foraging strategies. Perch searches actively for prey, is a sight-dependent diurnal predator (Craig 2008) and prefers rather motile prey that are easier to detect (Kornijów et al. 2016). Activity of the prey seems to be less important for a nocturnal predator, such as the racer goby (Grabowska and Grabowski 2005; Kakareko et al. 2013). In experimental conditions, the racer goby fed equally effectively on immobilised and mobile amphipods, choosing prey species rather according to their quality than their mobility (Błońska et al. 2015), which suggests that, to detect food, the racer goby uses not only sight, but also other senses. Furthermore, perch is morphologically and anatomically better adjusted for active hunting and pursuing escaping prey than gobies that do not possess a swim bladder and have a less streamlined body shape.

Racer goby and monkey goby had similar diets. They fed mainly on Chironomidae larvae, on the basis of the relative abundance and frequency of this prey in the fish gut content. The detailed identification of taxa within this food category showed that, in fact, the gobies foraged in different microhabitats, even at the same sites and their mode of foraging was also slightly different. Our study shows that several taxa of chironomids contributed to 60% dissimilarity between the diet of the studied goby species. Chironomids are a prevalent group in the freshwater macrozoobenthos, often standing out in their abundance and species and functional diversity, which makes them key elements of freshwater food webs (Armitage et al. 2012). Their ecological characteristics allow them to fill many niches and serve as a varied functional groups in aquatic ecosystems. Different groups of chironomid larvae are associated with different types of substrate: mud, sand, gravel, stones, plants (Moller Pillot 2009). They inhabit periphytic communities that develop on various hard surfaces or exploit the substrate by drilling into plant or animal tissue, mining wood, burrowing into the sediment surface or attaching to the bodies of other invertebrates (Moller Pillot 2009; Grzybkowska et al. 2016; Antczak-Orlewska et al. 2021). Chironomid larvae, being such a diverse group of macroinvertebrates and important food for many aquatic organisms, can be used as an additional indicator of habitat preferences, based on their contribution to the predator’s diet.

Glyptotendipes cf. pallens dominated amongst chironomid larvae in the diet of racer goby. In summer, this taxon can be found in silty tubes built on macrophytes, mining in their decaying parts, but also on other firm surfaces, such as decaying wood or stones. In large rivers, in particular, the larvae of this species are more numerous on stones than on plants. This taxon avoids fast running waters and prefers more stagnant parts of the river channel (Moller Pillot 2009). Similarly, racer goby is more abundant in lentic areas, where it prefers habitats with a muddy bottom and moderate macrophyte cover, but also stones, for example, rip-raps along the river banks (Kakareko 2011; Płąchocki et al. 2020) or single stones scattered on the bottom (Kakareko et al. 2016). Thus, the high abundance of G. cf. pallens in the racer goby diet derives from similarity in habitats occupied by the prey and its predator. In addition to Glyptotendipes cf. pallens, the other chironomids associated with macrophytes (Dicrotendipes nervosus and Polypedilum sordens) were found in the gut content of the racer goby more frequently and in higher abundance than in the gut of the monkey goby. Similar chironomid taxa also dominated the racer goby diet in the Dnieper River system (Didenko et al. 2021a, b). Another indicator of racer goby habitat preferences are chironomids that use stones as one of the possible substrates, such as Rheocricotopus chalybeatus, which also frequently settles on plants and uses stones if plants are unavailable (Moller Pillot 2013). The R. cf. chalybeatus was recorded in the gut of racer goby more often than in monkey goby at two out of the three sites. Compared to racer goby and perch, the monkey goby ate many large larvae of the Chironomus cf. riparius. This pelophilous species is very common in chironomid communities associated with mud and sand, but sometimes also with submerged aquatic plants, burrowing in soft sediment trapped by the roots (Dukowska and Grzybkowska 2014; Grzybkowska et al. 2020; Leszczyńska et al. 2021). The species can be very numerous also on stones or concrete bottoms covered by a thin layer of mud (Moller Pillot 2009). Chironomus riparius is often considered to be a characteristic inhabitant of flowing waters, also fast-flowing sections of brooks and streams and even rapids, providing that the organic silt is on the bottom, as it feeds on organic particles. It was also recorded as the dominant chironomid taxon in the diet of monkey goby in the Vistula River and less numerous in the gut content of the co-occurring racer goby, which eats mainly epiphytic species (Kakareko et al. 2005). The monkey goby is usually associated with sandy or gravelly bottoms in lotic parts of rivers, while it is less abundant at sites with moderate vegetation cover (Kakareko 2011; Płąchocki et al. 2020). It also prefers higher water velocity in comparison to the racer goby, as has been shown experimentally (Kakareko 2011) and can be found more often in the main flow of the river, where there are spots with slower water velocity caused by varied obstacles, such as macrophytes or stones. The latter form refuges for several organisms that are prey for fish, for example, for monkey goby. Such patches of macrophytes and stones covered by periphyton and accumulating sediment rich in organic matter, are very productive (Grzybkowska et al. 2020). Besides C. cf. riparius, other chironomid bottom-dwellers are more frequent in monkey goby gut content than in racer goby, for example, Cladopelma gr. viridulum, Cryptochironomus, Lipiniella moderata, Microtendipes cf. pedellus, Stictochironomus cf. rosenschoeldi (Brooks et al. 2007; Moller Pillot 2009). Some chironomid taxa recorded in the diet of monkey goby are typically associated with a fast water current and stony gravel substrate (e.g. R. cf. chalybeatus, Rheotanytarsus sp.) or with sand like, for example, Lipiniella moderata (Moller Pillot 2009; Klukowska et al. 2011). Moreover, the considerable contribution of caseless Trichoptera larvae, for example, Hydropsyche sp., which use water current to catch suspended organic matter, is another indicator that monkey goby occurs in lotic habitats (Stuijfzand et al. 1999). This supports the hypothesis of niche separation between the monkey goby and the racer goby and concurs with previous findings that monkey goby consumed mainly sand- and mud-dwelling, burrowing chironomids, while the racer goby has a more diverse diet, including both bottom-dwelling burrowing and phytophilous morphotypes of Chironomidae, as well as other macrophyte-associated macroinvertebrates (Kakareko et al. 2005; Didenko et al. 2022b). Shift in diel feeding activity can be another way to avoid food competition between co-occurring alien gobies. The racer goby is predominantly a nocturnal feeder (Grabowska and Grabowski 2005; Kakareko et al. 2013), while the monkey goby is more active during the day (Didenko et al. 2017) or shows no difference between day and night (Grabowska et al. 2009). Similarly, the co-existence of the other invasive gobies, i.e. round goby (Neogobius melanostomus) and big head goby (Ponticola kessleri) in the middle Danube River was suggested to be possible by resource partitioning and slightly different feeding strategy (Števove and Kováč 2013). The previously published revisions of ecological interactions of five alien Ponto-Caspian gobies in their non-native range (Kornis et al. 2012; Grabowska et al. 2023) emphasised that they are a diverse group considering their ecological demands and functional ecology, including types of prey (e.g. Didenko et al. (2022b)) and diet shift with ontogeny (e.g. Števove and Kováč (2016)); thus, their invasions in European inland waters impact native biota in diverse ways.

To conclude, we show that detailed prey identification to the lowest possible taxon is crucial to properly justify the diet overlap between co-occurring fish species and to verify the suggested impact of alien invaders on native species through interspecific competition. Resource partitioning considering prey types and foraging habitats is one of the ways of allowing the co-existence of closely-related alien gobies with similar food preferences in the invaded waters and their co-occurrence with local fish species. Together with an opportunistic feeding strategy, it is likely to be a major factor behind their invasion success observed in European waters in the last decades. We therefore recommend that, in order to gain more detailed insights into the foraging strategy of fish, in future studies, researchers should not limit their dietary analysis only to the identification of higher taxa, but should identify prey down to the lowest possible level, especially in taxonomic groups consisting of species that differ in the microhabitats they occupy.

The authors have declared that no competing interests exist.

No ethical statement was reported.

No funding was reported.

Conceptualization: JG. Formal analysis: JG, MP. Investigation: MG, MP, JG. Resources: JG. Visualization: JG. Writing – original draft: MG, JG. Writing – review and editing: MP.

Joanna Grabowska https://orcid.org/0000-0001-9924-0650

Mateusz Płóciennik https://orcid.org/0000-0003-1487-6698

Michał Grabowski https://orcid.org/0000-0002-4551-3454

All of the data that support the findings of this study are available in the main text or Supplementary Information.