3. Results
All primer sets produced successful sequencing results for most samples. However, one sample (SF08) for COI did not produce any amplification, even after further DNA purification, quantification, and a new PCR adjustment. Nevertheless, SF08 was successfully amplified and sequenced using the 12S markers Mifish and Neofish. A low number of reads were observed for SF04 and SF08, resulting in only one and three reads, respectively, despite the latter not presenting any problems in the amplification process, resulting in 93.33% (two failed samples out of 30) sequencing success. Notably, both 12S markers resulted in 100% amplification and sequencing success, with at least 38,697 reads in a sample (SF27) for MiFish, and 37,228 reads (SF10) for NeoFish.
The sequencing effort resulted in 4,505,309 reads for all markers after quality filtering. The number of reads showed considerable differences among markers and samples. COI produced 584,309 reads, averaging 19,477 reads per sample, ranging from one (SF04) to 29,485 (SF11). MiFish presented 1,919,545 total reads, averaging 63,985, with a minimum of 38,697 (SF27) and maximum of 83,734 (SF23). Sequencing with NeoFish resulted in 2,001,455 reads, with an average number of 66,715 per sample, varying from 37,228 (SF10) to 96,285 (SF23). After BLASTn searches, 1,699 COI reads remained without taxonomic assignment, and 69 were assigned to Bacteria. On the other hand, all MiFish and NeoFish reads were assigned to fish taxa.
ASVs were assigned to 26 fish taxa, from which 22 were identified at the species level, two at the genus level and three at the subfamily level. The 12S marker NeoFish was able to detect the highest number of orders, families, genera, and the same number of species as MiFish. In contrast, COI detected fewer species, genera, and families than the other markers and the same number of orders as MiFish (Table S1, Figs. 1, S1).
The combined use of the three markers increased the genera detection rates by 25% to 87.5% when considering an initial analysis with only NeoFish or COI, respectively (Fig. 2). The improvement in species recovery rates with the use of all three markers combined ranged from 31.25% to 61.54% when considering an initial analysis with either 12S gene markers or COI, respectively (Fig. 2).
The COI marker detected 16 taxa belonging to 13 species, eight genera, six families, three orders and one class (Fig. S1). Besides the 13 taxa identified at the species level, one was identified at the genus level, one at the subfamily level and another at the family level (Table S1). Among the 13 species, three were detected exclusively by the COI gene (Bergiaria westermanni , Leporinus friderici andProchilodus lineatus ). The species B . westermanniwas present in 12 samples, with an average RRA of 2.29%, ranging from 0.13% to 6.16%. The anostomid L . friderici was detected in eight samples, with the RRA ranging from 0.13% to 32.64%, and an average of 7.81%. Lastly, P . lineatus was present in three samples and had an average RRA of 3.60%, ranging between 1.20% to 7.12% (Fig. 3).
With the 12S gene markers, 21 taxa were detected, including 18 species, 14 genera, 11 families, four orders and one class (Table S1). One of these taxa was identified at the genus level (Characidium sp.) and two at the subfamily level (Doradinae and Stevardiinae). Of the 18 identifications at species level, eight were exclusively detected with the 12S gene, all indigenous to the São Francisco Basin:Cetopsorhamdia iheringi , Megaleporinus elongatus ,M . reinhardti , Pachyurus squamipennis ,Planaltina myersi , Pseudoplatystoma corruscans ,Steindachnerina elegans and Sternopygus macrurus (Fig. 1, Table S1).
Four of the eight species detected exclusively with the 12S markers were detected by both markers, but with some variation in samples and abundance. For example, the species C . iheringi was detected by MiFish and NeoFish in sample SF15, with 0.07% and 0.02% RRA, respectively (Fig. 3).
Overall, RRA and taxon detection was not consistent between each marker (Fig. 3). For instance, the most abundant taxon P . pohlihad a total of 1.348.589 (70.79% of the total) for MiFish and 424.499 (73.02%) reads recovered for Neofish. Notably, the highest RRA detected for NeoFish was M . elongatus , with 236.332 (20.90% of the total) reads. Additionally, in some samples (e.g., SF05, SF13, SF14 and SF15) where both MiFish and COI detected multiple taxa for Pimelodidae, NeoFish was not able to identify any taxa for this family (Fig. 3).
The PERMANOVA evidenced significant differentiation of fish communities using distinct molecular markers (Table 1). The influence of primer choice on taxa recovered in each sample was significant for both presence-absence (Jaccard) and RRA (Bray-Curtis) analyses (Table 1). While there is considerable overlap between both 12S markers considering only presence-absence of each taxon in each sample, as can be seen in the PcoA plot (Fig. 4a), the analysis taking taxa abundance into account revealed a slight overlap between MiFish and COI (Fig. 4b).