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). Our results indicate that the diet of Campylomormyrusand 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 by Campylomormyrus and Gnathonemus . Indeed,
larvae of aquatic insects were reported as food items found in the
stomach of mormyrid fish (Blake, 1977; Hyslop, 1986).
Beside aquatic insects, annelid worms (such as Glossoscolecidae,
Naididae, and Megascolecidae) were also found in the diet of allCampylomormyrus and Gnathonemus species. 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). Previous studies on the diet of
mormyrids using morphological observations did not report annelid worms.
The absence of this food taxon may be due to the progressed digestion
before the morphological examination.
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.
These food items are usually located in the habitat at the surface of
the water.
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 our 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 (Roberts & Stewart, 1976), reported that
stomach contents of some Campylomormyrus species contain larvae
of chironomids, Povilla , trichopteran, ephemeropterans and
odonates, dead plant debris, and decomposing animal debris. Roberts &
Stewart (1976) reported also that the stomach content of a specimen ofC. rhynchophorus had Chironomidae, Simuliidae, and trichopterans,
and a few small ephemeropterans. Another study on the stomach contents
of C. 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 Campylomormyrusphenotypes
The dietary compositions among Campylomormyrus andGnathonemus suggest that their feeding behavior is related to
phenotypical traits (EOD duration and snout morphology). Based on our
results, , different prey spectra were found in the stomach of the
different groups of Campylomormyrus species, which may relate to
species exploiting 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 they 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 C. compressirostris contains more dipterans,
while the diet of C. tshokwe contains more coleopterans.
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.
Similar differences were found according to snout length. 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
the 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 also reflect differential availability of different prey
in the respective microhabitats. 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 also be
of adaptive value during feeding, 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.
The presence of distinct prey spectra among Campylomormyrusspecies with different snout morphologies implies a potential
association between snout morphology and feeding behavior. Specifically,
the species-specific snout morphology in Campylomormyrus may be
related to variations in their grasp suction mode of feeding, as
previously described by Marrero and Winemiller (1993). Enhanced grasp
suction enables them to obtain the aquatic insects that burrow into the
different substrate structures. This functional foraging specialization
allows for an efficient exploitation of the rich bottom fauna of benthic
invertebrates, especially aquatic insect larvae, which other fish may
not reach. Such trophic specializations may have triggered the observed
radiation of Campylomormyrus in the Congo River.
Potential limitations of our dietary
analysis
Sample sizes and quality of DNA. Our study was limited by the
availability of stomach content samples we collected from the field,
i.e., the Congo River. Despite of a four weeks effort during our 2012
expedition, we did not yield equally high sample sizes for all target
species, such that the small number of individuals per species did not
allow for diet comparisons among the individual Campylomormyrusspecies, except for C.
compressirostris and C. tshokwe . Moreover, all species examined
here are nocturnal, so the stomach contents are expected to be highly
digested, due to the time between the last meal and the collection
during day time. Our samples had been stored for relatively long time (7
years) prior to DNA extraction. We followed established protocols to
store the samples (Seutin et al. 1991) which had been successfully
applied for degraded samples (Epp et al. 2010; Stoof-Leichsenring et al.
2012). Note also that Campylomormyrus tissue samples taken and
stored under the same conditions (i.e., during Congo expeditions in
2004, 2006, and 2012) yielded amplifiable DNA (entire cytochrome b gene,
>1000bp) when analyzed up to 10 years later (Lamanna et al.
2016). While we cannot exclude that storage may have contributed to DNA
degradation, our positive control (a fresh sample taken from a C.
compressirostris raised in our lab and fed with defined food) yielded a
similar read number (419,872) as the stomach samples taken in Congo and
stored for several years prior to analysis (\(M\text{ean}=335,020\);\(SD=234,220\)). We hence conclude that progressed digestion may be
the major cause of the DNA degradation encountered in our study.
Relative Read Abundance (RRA) and biomass correlation. The
number of reads belonging to a particular food taxon is utilized as a
proxy for the biomass of this taxon to obtain semi-quantitative measures
on the relative amounts of different food taxa consumed byCampylomormyrus and Gnathonemus species. This approach is
used in most of the dietary studies, and it implicitly assumes a
correlation between the number of sequences and the biomass. The logic
behind this assumption is that a large biomass of a particular food item
in the diet would translate into a large amount of DNA in the dietary
sample and, consequently, more recovered DNA sequences (reads). However,
potential biases may arise from technical (such as PCR efficiency,
primer choice, DNA extraction, etc.) and biological issues (such as
among-prey species variation in tissue cell density, survival of DNA
during digestion, and the state of digestion), obscuring the
quantitative signal. Despite of these caveats, RRA has been considered
valid as a proxy for abundance in eDNA (e.g., Kiemel et al. 2022) and
sedaDNA (e.g., Krueger et al. 2021) studies. We hence argue that
utilizing RRA as a semi-quantitative measure of prey biomass – with all
caution – provides relevant additional information relative to merely
reporting the presence of prey taxa, which implicitely overstates the
importance of rare food items. Moreover, technical biases may affect all
samples in a similar manner and hence not compromise our comparison of
relative diet composition among different EOD/snout phenotypes.
Taxonomic resolution of dietary items.The dietary differences among the
species and phenotypes are more visible in the low taxonomic levels,
i.e., at genus and species levels. However, the relatively low
percentages of reads assigned to the family, genus, or species level
(see Sec. 3.4) may compromise the confidence in the inferred prey
spectra at these taxonomic levels. Therefore, we limited the statistical
analysis of diet overlap and assessment to the order level (see
Sec. 3.4) which we argue could be more reliably inferred (as atested by
a lower percentage of unassigned reads). Notwithstanding this
limitation, the study confirmed significant differences in the diet
among EOD and snout phenotypes. It is plausible to expect that these
differences may be even more pronounced at the species/genus levels.