Results

Sequencing results

The sequencing for all samples generated 6.9Gb raw sequencing data of FASTQ formatted reads. The raw data have been deposited to the open repository Zenodo (Amen et al. 2022). The total number of raw sequences from the 27 stomach samples is 19531074, ranging from 19226 to 867093 per individual sample. Out of them, a total of 18357160 sequences were obtained after quality filtering, ranging from 16703 to 828735 per individual.

Taxonomic classification

The taxonomic classification of the filtered sequences against the custom marker database using Kraken2 classified all the reads into seven phylogenetic levels (domain, kingdom, phylum, class, order, family, genus, and species) or unclassified reads. The percentage of the unclassified reads reached 33.22 % of the pooled sequences of all samples. On the individual level, the percentage of unclassified reads ranged from 8.38 % to 71.67 %. The high percentage of unclassified reads is attributed to sequences outside the range of our custom marker database (eukaryotic COI and rbcL only). When the entire nucleotide database (www.ncbi.nlm.nih.gov/nucleotide/) is used instead of our custom database, the percentage of unclassified reads dropped to 10.11 % of the pooled sequences of all samples. We limited the analysis to the taxonomic classifications using the eukaryote COI/rbcL custom database, however, to keep the focus on the diet analysis (instead of prokaryotes in the gut microbiomes).
The classified reads pooled for each species are visualized using the metagenomics data explorer Pavian and depicted in the supplement (Supplementary File S3). Comparing the distributions of the reads for the food taxa classified at each phylogenetic level reveals that most of the reads are assigned to few food taxa, while many of the classified taxa are represented by only a few reads. We excluded records from primates and birds, which were presumably contamination. Taxa represented by less than 0.01 % of reads were further excluded, as they may constitute contamination/background noise (Alberdi et al. 2018). The remaining identified prey items were assigned to 146 taxa of different taxonomic level: 20 classes, 34 orders, 97 families, 105 genera, and 90 species (Tables S4.1-S4.6 Supplementary File S4).

Relative abundance

Campylomormyrus had a broad spectrum of prey items. On the class level, Insecta dominated by far. They were found in all samples, representing more than 90% of the total reads (Figure 1). Other classes were also found in all samples but with less percentage of the total reads, such as Clitellata, Arachnida, Malacostraca, and Hexanauplia. At the order level, the most abundant prey items were Diptera, Coleoptera, and Hymenoptera (all Insecta) class, all of which were in all samples (Figure 1). Also, the Haplotaxida (Clitellata) and Araneae (Arachnida) were found in all samples. Further insect orders such as Lepidoptera, Trichoptera, Ephemeroptera, and Hemiptera, as well as Decapoda (Crustacea) were found in more than 83 % of the samples.
The Relative Read Abundances (RRA) of the primary food taxa for the studied species are depicted in Figure 2. Most species share the same food taxa, albeit in different proportions. The most dominant prey taxon at class level is Insecta, followed by Clitellata. At the order level, the most abundant orders are all insects, i.e., Diptera, Coleoptera, Hymenoptera, and Lepidoptera.
The RRA data were used to assess the dietary niche width by calculating the Shannon diversity index (Figure 3 and Table S5.1 in the Supplementary File S5). The Shannon diversity index differed considerably amongthe studied species, pointing towards different dietary niche width. As an example, the lowest Shannon index values are found for C. compressirostris samples (1.62±0.02 SE), while the highest are found for C. tshokwe (2.21±0.03 SE).

Diet overlap

The diets of most of species significantly overlap at class and order phylogenetic levels (Table 1): At these levels, Pianka index values showed statistically significant niche overlap based on comparison with 1,000 null models (see the Supplementary File S8 for more details) and Schöner index scored more than 0.6 for all species comparisons except when compared to C. numenius . At lower taxonomic level (family, genus, species), there was much less overlap in the diet among species. The degree of diet overlap is further confirmed by the Bray–Curtis dissimilarity index (0: similar; 1: dissimilar), as shown in Table S5.2 in the Supplementary File S5.
As many reads could not be assigned to family, genus, or species level, we performed further statistical analyses on read assignments at the order level. A perMANOVA on the Bray–Curtis dissimilarity index data derived from the RRA values at order level indicates significant dietary differences among species (F =3.596,r2 =0.275, p ≤0.001), excluding the species with small sample size (C. alces , C. curvirostris , and C. numenius ). We performed a post hoc pairwise perMANOVA using a Bonferroni correction of the p -values to find out which species drive these results. C. compressirostris versus C. tshokwe and G. petersii versus C. tshokwe were statistically significantly different in their diet (p <0.05), while C. compressirostris and G. petersii did not show a significant difference.
A perMANOVA on the EOD groups (long vs. short EOD) indicated significant dietary differences between the two groups (F =8.5,r2 =0.254, p ≤0.001, Bray–Curtis dissimilarity index data derived from the RRA values at order level). Similarly, for snout morphology (short vs. medium vs.long), a perMANOVA on the Bray–Curtis dissimilarity index data (derived from the RRA values at order level) showed significant dietary differences among the three groups (F =2.676,r2 =0.182, p ≤0.001). The post hoc pairwise perMANOVA using a Bonferroni correction of the p -values indicated that only the long snout versus the medium snout is statistically significant (p<0.002).
The patterns of dietary difference among samples are visualized in Figure 4 by ordinating the Bray–Curtis dissimilarity index values in two dimensions using NMDS. The stress level for the NMDS was 0.133, which indicates a good representation (Clarke 1993). The NMDS plot shows the segregation of samples based on EOD and the degree of diet overlap/dissimilarity based on species and snout length.