Mats from wadable sites were collected using a suction device and placed in sterile Whirlpak® bags, then put on ice for transport to the Annis Water Resources Institute (AWRI, Muskegon, MI, USA). Three replicate mat samples were collected from each habitat type at each site during each sampling event. Mats from MIS were collected by NOAA divers using a coring device, and transported to AWRI as cores in plastic tubes on ice. Plankton tow samples were also collected at GSS and ECB to determine taxa that may be considered part of the surrounding planktonic community, rather than active members of the microbial mat community. Each mat sample collected was subsampled, with one subsample used for generating unialgal cultures and the other for metabarcoding.

To isolate cyanobacterial taxa, mat samples were spread onto solid Z-8 medium (Rippka et al. 1988) and nitrogen-free Z-8 medium to isolate a wider range of cyanobacteria, and grown under ambient conditions (23 °C, ∼16:8 h light:dark photoperiod). Colonies were individually picked and plated until unialgal cultures were achieved. Morphology of the strains was analyzed via light microscopy (Nikon Eclipse Ni with DIC), and taxonomic identification was assessed using Wehr et al. (2015) and Komárek and Anagnostidis (2005). Images were taken with a high-resolution camera (Nikon digital sight DS-U3). Direct PCR was performed as follows: cells were placed into -20 °C for 30 mins, centrifuged, and the supernatant containing DNA collected. The partial 16S rRNA and the whole 16S–23S ITS region (Gaylarde et al. 2004) was amplified using primers CYA8F and CYAB23R (Neilan et al. 1997). The 50 µL PCR reaction contained: 27 µL DNA containing supernatant, 0.5 µL of each primer (0.01 mM concentration), and 22 µL PCR Master Mix (Promega, Madison, WI, USA). PCR amplification proceeded as detailed in Casamatta et al. (2005), and products were frozen and sent to Eurofins Scientific (Louisville, Kentucky) for Sanger sequencing.