"This study quantified chlorophyll-a (as a proxy for biomass of microbial photoautotrophs) on rock surfaces (µg/cm2) and in surface waters (µg/L) collected from non-perennial stream sites (descriptions below). These samples were collected as part of the NSF-funded project AIMS (Aquatic Intermittency Effects on Microbiomes in Streams) Approach 2 ('seasonal sampling') and Approach 3 (spatially-intense synoptic sampling) for three non-perennial stream systems in Alabama, starting in January 2022 and ending in February 2024.
For rocks, rock surfaces were scraped with a sterile wire brush over a known area, and the loosened epilithic biofilms were rinsed into a sterile container with a known volume of sterile deionized water. Then 10 mL of the resulting biofilm slurry was filtered through a 0.7 µm GFF filter. For water samples, a known volume of stream water was filtered through a 0.7 µm GFF filter. These Chl-a samples (GFF flters) were stored at -20ºC.
For quantification, chlorophyll-a was extracted from collected GFF filters in 90% ethanol at 80ºC for 5 minutes, steeped overnight (4ºC in darkness) and immediately quantified using high performance liquid chromatography (HPLC) via fluorescence detection (Halvorson et al., 2019; Meyns, Illi, & Ribi, 1994) with a Shimadzu 10ADvp series equipped with a Shimadzu RF10Axl fluorescence detector (excitation 430nm, emission 670 nm). Chlorophyll-a content was calculated as µg per cm2 of rock surface area sampled, and µg per L of surface water sampled.

Talladega Site Description:
Talladega (stream code=TL) research watershed (outlet location: 33.76219799, -85.59550775) in the Talladega National Forest (Cleburne County, AL, USA). The watershed drains a non-perennial unnamed tributary of Pendergrass Creek, and contains 0.92 km^2 of mixed coniferous and deciduous forest in the Piedmont Upland physiographic section. Located near Anniston, AL, the watershed spans an elevation range from 345 to 456 m above sea level and is a tributary to the Coosa River (within the larger Mobile-Tombigbee basin). The region has a humid subtropical climate, with mean daily January and July air temperatures of 5.3°C and 25.3°C respectively, and mean annual precipitation of 1,400 mm/yr.

Paint Rock Site Description:
Paint Rock (stream code=PR) research watershed (outlet location: 34.96861724, -86.16501705) on privately owned property in Jackson County (AL, USA). The watershed drains a non-perennial unnamed tributary to Burks Creek, and contains 2.97 km^2 of deciduous forest in the Cumberland Plateau physiographic section. Located near Estillfork, AL, the watershed spans an elevation range from 211 to 550 m above sea level, and is a tributary to the Paint Rock River (within the larger Tennessee basin). The region has a humid subtropical climate, with mean daily January and July air temperatures of 4.4°C and 25.4°C respectively, and mean annual precipitation of 1,390 mm/yr.

Shambley Creek Site Description:
Shambley Creek (stream code=WH) research watershed (outlet location: 32.98410915, -88.01334337) was on privately owned property in Greene County (AL, USA). The watershed drains a non-perennial unnamed tributary to Shambley Creek, and contains 0.70 km^2 of coniferous forest managed for silviculture in the East Gulf Coastal Plain physiographic section. Located near Eutaw, AL, the watershed spans an elevation range from 63 to 94 m above sea level, and is a tributary to the Sipsey River (within the larger Mobile-Tombigbee basin). The region has a humid subtropical climate, with mean daily January and July air temperatures of 7.3°C and 27.4°C respectively, and mean annual precipitation of 1,350 mm/yr.