Stream Algal Biofilm Community Diversity Along An Acid Mine Drainage Recovery Gradient Using Multimarker Metabarcoding

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 and 18S) along with two algal specific markers (UPA and rbcL) and high-throughput amplicon sequencing. For each marker, the ordination of Bray-Curtis Index calculated from the total Amplicon Sequence Variants (ASVs) present in each stream showed the Unimpaired and Recovered streams clustered, while Moderately Impaired streams were more distant. Focusing on the algal ASVs, 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 categories. The two universal markers together captured all algal phyla providing an outline of the diversity, but the two algal specific markers produced a greater number of ASVs and taxonomic depth for algal taxa. Further examination of the UPA marker revealed a drastic decrease in relative abundance of diatoms in Moderately Impaired streams compared to Recovered and Unimpaired streams. Likewise, diatom genera identified in the rbcL data and indicative of stream water quality showed marked differences in relative abundance among stream categories. Although all markers were useful, the algal-specific UPA and rbcL contributed more insights into algal community differences among stream categories.