Most water quality parameters showed no statistically significant differences between the two sampling sites. Water temperature (which ranged from 11°C to 18°C throughout the study) was the only parameter that was significantly different (p = 0.0052) between the sampling sites, being higher in Veleiros (Table 1).
Periphytic bacterial community
The bacterial diversity (Shannon; p = 0.52), richness (Chao1; p = 0.11), and evenness (Pielou; p = 0.95) indices were not significantly different between the two sites (Figure 2A). The same was observed when the indices were used to assess the diversity of the community across the time points, except for evenness. After the initial formation of a microbial biofilm (T1), the periphytic community changed, and a significant difference was observed in evenness between T1 and T2 and remained significantly different until T4 (Pielou; p = 0.027).
A MDS analysis was performed for both sampling sites (Figure 2B). The distance between groups was assessed for the variable time (Permanova; R2 = 0.19; p = 8.9x10^-4), and for the variable site (Permanova; R2 = 0.12; p = 9.9x10^-5). For Jangadeiros, the bacterial periphytic community presented a more similar composition in T1, T2, and T3, differing from T4. Surprisingly, in Veleiros, T1, T2, and T4 clustered together and T3 was more dissimilar to the other time points. This implies that, in Veleiros, a disturbance of periphytic succession occurred between T2 and T3, and then the bacterial composition was re-established at T4. These observations are also discerned in the Venn diagrams. The periphytic succession in Jangadeiros includes an increase in the number of taxa commonly present in T1 to T3 (T1-T2, n = 72 and T1-T3, n = 89), but this is strongly reduced between T1 and T4 (n = 53) (Figure 2C). In Veleiros, the number of common bacterial taxa at T1 and T2 (n = 56) was reduced at T3 (n = 36), but at T4 (n = 50) it returned to a similar level relative to the initial stage of the bacterial succession (Figure 2D).
A total of 28 bacterial phyla were identified during the study at the two sites. Of these, 11 presented a relative abundance higher than 1% at one or more sampling times. Proteobacteria dominated both sampling sites, with abundances ranging from 62.1% to 77.3% of the total community (Table S2). Bacteroidota was the second most abundant at both sites, followed by Nitrospira and Cyanobacteria. Acidobacteriota, Actinobacteriota, and Planctomycetota were also present at abundances higher than 1%, with Planctomycetota presenting an abundance higher than 6% at T4 in Veleiros.
Among the most abundant ASVs in Jangadeiros, Citrobacter, a gram-negative coliform bacteria from the family Enterobacteriaceae, was the most abundant genus at T1 (up to 43% of the total sequences), disappearing at the other time points (Figure 3A; Table S3). In Veleiros, Serratia, a gram-negative coliform (Figure 3B; Table S3), was highly abundant at T1 (up to 44% of the total sequences), along with Pseudomonas, which appeared mainly at the first time point. Nitrosomonas, Candidatus Nitrotoga, Nitrosospira, and Flavobacterium were constantly present at the four time points.
Periphytic eukaryotic communities
Among eukaryotes, the most abundant group found at both sites was Alveolata, followed by “Excavates”, Diatomea, and other Stramenopila (Figure 4A). Chlorophyta showed peaks of high abundance in Veleiros, but the same pattern was not observed in Jangadeiros. Ciliophora was the most abundant group within the Alveolata at both sites, representing more than 75% of the sequences, followed by Dinoflagellata, with up to 25% relative abundance (Figure 4B). A Venn diagram of the distribution of Ciliophora taxa at different time points in Jangadeiros revealed that T1 harbored three unique taxa, while T2 and T3 presented five, and T4 had four unique taxa (Figure 4C; Table S4). In Veleiros, T1 harbored only two unique taxa, while T2, T3, and T4 harbor six, one, and four, respectively (Figure 4D; Table S4).
Among ciliates, the periphytic community at both sites was primarily composed of peritrichs, with the colonial genus Epistylis being the most abundant taxon (Figure 4E). Its highest relative abundance was observed at T2 in Jangadeiros and at T1, T3, and T4 in Veleiros. The genera Vorticella and Zoothamnium also exhibited high abundance. Vorticella reached a peak in Jangadeiros at T3, while Zoothamnium had its highest abundance in Veleiros at T2. Other peritrich genera were also present in the community, but at low abundance at both sites.
With regard to other ciliate genera, the heterotrich Stentor had a high relative abundance at T4 in Jangadeiros, but in Veleiros it was present at very low abundance along the microbial succession. Among predators, Amphileptus had an abundance of up to 12% at T1 in Jangadeiros. In Veleiros, the same genus showed a slightly lower abundance at T3 and T4 (Figure 4E).
Correlation between bacterial and ciliate communities
Spearman’s correlation between Ciliophora and Bacteria taxa performed at genus level (or corresponding annotation) demonstrated potential ecological relationships among these groups along the periphytic microbial succession. A larger number of significant correlations was found in Jangadeiros compared to Veleiros (Figure 5). In Jangadeiros, genera associated with methanotrophy and methylotrophy, along with the nitrite-oxidase Nitrospira, presented significant correlations with ciliates. Considering the Ciliophora community, five genera showed positive correlations with a broad range of bacterial taxa (Figure 5A). In Veleiros, the methane oxidizer Crenothrix presented a positive correlation with three ciliate genera, and no negative correlations were observed at this site (Figure 5B).
Ciliate morphology-based analyses
The peritrich ciliate assemblage was composed of seven genera and nine morphologically identified species. All genera and species were recorded at both sites, except for Mioschiston duplicatum and Vaginicola tincta, recorded only in Jangadeiros (Figure 6). Epistylis was the most abundant peritrich genus in Jangadeiros at T1, with E. portoalegrensis reaching a peak of abundance, followed by E. smalli and E. plicatilis (Figure 6A). In Veleiros at T1, the most abundant genus was Vorticella, with V. convalaria reaching a high peak of abundance (Figure 6B).
At T2 in Jangadeiros, different species of Epistylis were still abundant, but Vorticella spp. showed an increase in abundance, reaching more than 1.000 inds/mm2 (Table S5). A similar situation was observed at T2 in Veleiros, where Vorticella represented more than 45% of the peritrichs present in the samples (Figure 6A). At T3, both sites presented a dominance of genus Vorticella. Jangadeiros had a low abundance of peritrichs at T3, with Vorticella reaching an abundance of 150 inds/mm2. In Veleiros, the highest abundance of peritrichs during the study was observed at T3, with Vorticella reaching more than 90% of the peritrich records observed in the samples.
The lowest abundance of peritrichs in Jangadeiros was observed at T4. Species of Vorticella accounted for more than 80% of the peritrichs observed in the samples. In Veleiros at T4, a similar result was observed for Vorticella, with other genera presenting very low abundances (Figure 6A).