Each site was visited in the spring (April-May), summer (June-July), and fall (September) periods. Exceptions include MIS and OAK, which were only sampled during the summer period. During each visit, a YSI multiprobe (Yellow Springs Instruments, Inc., Yellow Springs, OH, USA) was used to measure temperature, specific conductance, and percent dissolved oxygen. Due to multiprobe malfunction, data from a summer 2021 YSI deployment was used to characterize MIS water parameters. In addition to YSI parameters, 250 mL acid-washed Nalgene bottles were used to collect water samples for nutrient analyses at each sampling point. Each water sample was subsampled into two vials, of which one was refrigerated and one was frozen within 24 h of collection. The refrigerated subsample was used to determine orthophosphate (SRP) concentrations using USEPA method 365.1 (O’Dell 1996). The frozen subsample was used to determine dissolved silica concentrations using USEPA method 370.1 (USEPA) and chloride, sulfate, and nitrate using USEPA method 300.0 (Pfaff 1993).

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.

Individual diatom cells were isolated from each culturing subsample via micropipette serial dilution to establish unialgal cultures. Monocultures were maintained in WC+Si liquid medium (Guillard & Lorenzen 1972) at 10 °C and a 12:12 light cycle. For morphological identification of cultures, live material was boiled in HNO3 for one hour, repeatedly washed and settled with ddH2O, dried on coverslips, and mounted on slides using Naphrax®. Each culture was identified to species under 1000× using a Nikon Eclipse Ni-U light microscope with DIC and Krammer and Lange-Bertalot (1986, 1988, 1991a,b). When monocultures had grown to a sufficient density for DNA extraction, cells were harvested by centrifugation and a Chelex extraction was performed following Richlen & Barber (2005). The rbcL region of each culture was amplified using primers rbcL66+ (Alverson et al. 2007) and DPrbcL7- (Jones et al. 2005) using Cytiva PuReTaq ™ Ready-To-Go ™ PCR beads (Cytiva, Marlborough, MA, USA) and a thermocycler protocol of 94°C for 3 mins 30 s, then 36 cycles of 94°C for 50 s, 52°C for 50 s, 72°C for 80 s, with a final extension at 72°C for 15 mins (Stepanek et al. 2015). The PCR products were frozen and sent to Eurofins Scientific (Louisville, Kentucky) for Sanger sequencing using the PCR primers as well as internal primers CfD+ (Hamsher et al. 2011) and rbcL1255- (Alverson et al. 2007).