Multi-marker Metabarcoding Assessment of Biodiversity within Stream Biofilm Communities along an Acid Mine Drainage Recovery Gradient

In southeastern Ohio, active remediation of streams affected by Acid Mine Drainage (AMD) has proven to be successful for some streams, while others have not recovered based on macroinvertebrate assessment. In this study, biofilms were collected from three Moderately Impaired, three Recovered, and two Unimpaired streams. The biodiversity was characterized by metabarcoding using two universal barcode markers (16S & 18S) along with two algal specific markers (UPA & rbcL) and high-throughput amplicon sequencing. For each marker, the ordination of Bray-Curtis Index calculated from the total operational taxonomic units (OTUs) present in each stream showed the Unimpaired and Recovered streams clustered, while Moderately Impaired streams were more distant. Focusing on the algal OTUs only, the Shannon index for the rbcL and UPA markers showed significantly lower alpha diversity in Moderately Impaired streams compared to Unimpaired streams, but the Recovered streams were not significantly different from the other two stream types. Further examination of the UPA marker revealed a decrease in relative abundance of diatoms in Moderately Impaired streams compared to Recovered and Unimpaired streams. The same 10 algal phyla were identified with the 18S and UPA marker, but the UPA included Cyanobacteria as well. A total of 21 diatom genera were identified from the rbcL marker data and these were investigated for taxa that have been reported to be indicators of high water quality. Overall, results from this study corroborate previous findings using individual groups of organisms to assess stream impairment from AMD.