Discussion

Diet profile

The DNA metabarcoding method provided a clear taxonomic resolution of the partially digested stomach content and potentially detected highly diverse food taxa. This high taxonomic resolution is in line with those of previous dietary studies based on DNA-based approaches (e.g., Harms-Tuohy et al. 2016; Pan et al. 2021; Rees et al. 2020; Sakaguchi et al. 2017). The results of this study indicate that the diet ofCampylomormyrus and Gnathonemus species is composed mainly of three types of prey items, i.e., benthic invertebrates, allochthonous invertebrates, and macrophyte material. The most dominant prey taxa found in the gut contents of these species belong to benthic invertebrates, especially aquatic insects. In particular, dipterans (Chironomidae, Simuliidae, Drosophilidae, and Tephritidae), coleopterans (Zopheridae, Carabidae, Histeridae, and Scarabaeidae), trichopterans (Hydropsychidae, Lycaenidae, and Elateridae), ephemeropterans (Leptophlebiidae, Baetidae, and Ephemerellidae), and odonatans (Coenagrionidae, Chlorocyphidae, and Chlorogomphidae) are important diet constituents of all the species. The larvae of these insects live usually in holes and interstitial spaces of the riverbed. Although the DNA approach used here cannot tell the stage of the aquatic insects found in the diet, it is reasonable to assume that the aquatic larvae, rather than the terrestrial imagines, were targeted byCampylomormyrus and Gnathonemus .
Beside aquatic insects, annelid worms (such as Glossoscolecidae, Naididae, and Megascolecidae) were also found in the diet of allCampylomormyrus and Gnathonemus species in this study. Similar to the insects’ larvae, the annelid worms hide in mud and among aquatic vegetation in the substrate of the riverbed. Other benthic invertebrates found were freshwater snails (Gastropoda, orders Pachychilidae and Stylommatophora), and crustaceans (Malacostraca, orders Decapoda, Copepoda, Cladocera, and Amphipoda).
The second group of food items found in the diet ofCampylomormyrus and Gnathonemus species is allochthonous invertebrates. The most abundant prey taxa from this group are Hymenoptera (including Formicidae, Mymaridae, and Braconidae) and Lepidoptera (including Nymphalidae, Lycaenidae, and Hepialidae). Additionally, Araneae (Arachnida) were frequently found in the diet.
The third group of food items is plants, including grasses, such as Poaceae of the Poales order, and flowering plants, such as Fabaceae and Asterales.
It must be noted that we cannot exclude some of these taxa having derived from the diet of the primary prey (secondary predation; Sheppard et al. 2005) or comprise small organisms and plant debris unintentionally ingested during grasp suction. However, the stomach contents of Campylomormyrus and Gnathonemus species found in this study using a DNA metabarcoding approach are compatible withCampylomormyrus mainly (about 90 %) feeding on aquatic insects (Nwani et al., 2008; Roberts & Stewart, 1976). A previous study, based on morphological observation, reported that stomach contents of someCampylomormyrus species contain larvae of chironomids, Povilla, trichopteran, ephemeropterans and odonates, dead plant debris, and decomposing animal debris (Roberts & Stewart, 1976). This study reported also that the stomach content of a specimen of C. rhynchophorus had Chironomidae, Simuliidae, and trichopterans, and a few small ephemeropterans. Another study on the stomach contents ofC. tamandua using morphological observation reported similar food taxa (Nwani et al., 2008). The few available dietary studies on other fish species inhabiting the Congo River such as Schilbe intermedius (Dirat et al. 2019) and Distichodus antonii ,D. affinis and D. lusosso (Zebe et al. 2010) showed similar prey spectra.

Diet comparison among species

The dietary compositions of the Campylomormyrus andGnathonemus species suggest that their feeding behavior is opportunistic, hence depending on food availability and accessibility. Based on our results, all species exploit diverse food niches in their habitats. For instance, the prevalence of benthic invertebrates, such as larvae of dipterans and coleopterans and annelid worms, in the diet of all species suggests that these species exploit the bottom of the riverbed, while the occurrence of allochthonous invertebrates, such as Formicidae, Nymphalidae and Arachnida spiders, may indicate a certain degree of surface feeding. Further, the diets also include food items from the water column, such as copepods. Accordingly, these species may exhibit high trophic flexibility and diverse feeding behaviors.
However, the RRA results revealed significant differences among the dietary compositions among the species, potentially associated with EOD and snout length. For example, the diet of some species, such asC. compressirostris , C. curvirostris , and G.petersii , contains more dipterans, while the diet of C. tshokwe contains more coleopterans. The diet of C. numenius was exceptional, as it contained large amount of grass. Unfortunately, this latter finding remains anecdotal, as we had only one sample of this species available.
Species with long EOD had preferentially fed on other taxonomic groups (i.e., coleopterans, ephemeropterans, spiders, annelids), compared to the species with short EOD, where dipterans dominated in the diet (Fig. S6.1 in the Supplementary File S6). Similar differences were found according to snout length (Fig. S6.2 in the Supplementary File S6). Note, however, that species with long EOD often also exhibit a long snout (Fig. 4; cf. Lamanna et al. 2016).

Dietary analysis and the radiation scenario

Our study was motivated by potentially providing further support for the hypothesis that radiation of Campylomormyrus is caused by an adaptation to different food sources, associated with diversification of EOD. The results proved that all the species tested in the current study are able to exploit diverse niches, especially the bottom fauna, regardless of their snout shape and EOD.
The current study still provides some evidence that different snout morphologies and the associated divergence in the EOD translate into different prey spectra. As a different morphology of the feeding apparatus among the species may constitute a functional adaptation to exploiting different substrates (Amen et al. 2020), the different prey spectra could simply reflect differential availability of different prey in the respective microhabitats. Unfortunately, no data on substrate-specific benthofauna are available for the Congo River. However, it has been argued that the duration of the EOD plays a crucial role during food detection by determining the prey items that can be detected best (Harlan Meyer 1982). In fact, prey detection via electrolocation is a frequency-based process. Our results cannot establish a causal link between EOD characteristics and prey spectra, yet they are compatible with the hypothesis that the divergence in EOD could be of adaptive value during feeding as well, beside its proven function as a prezygotic isolation mechanism. In this case, EOD would be a ‘magic trait’ triggering both adaptation and reproductive isolation (Feulner et al. 2009). One approach to further investigate a potential dual function of the EOD divergence (feeding specialization and reproductive isolation) would be to expand on the choice experiments performed by Amen et al. (2020) by presenting a variety of food items. This will though still be confounded by the association of long EOD with long snout. Ultimately, one would like to know prey-specific detection probabilities, relative to the physical properties of the different discharges. To achieve such knowledge remains a challenge.
In general, the snout morphology of Campylomormyrus may enhance their grasp suction mode of feeding (Marrero & Winemiller 1993). This enhanced mode enables them to obtain the aquatic insects that burrow into the different substrate structures. This functional foraging specialization allow an efficient exploitation of the rich bottom fauna of benthic invertebrates, especially aquatic insect larvae, which other fishes may not reach. Such trophic specializations may have triggered the observed radiation of Campylomormyrus in the Congo river.