We sampled macroinvertebrates along a 100 m reach every 20 m alternating channel location from left, to center, to right for a total of six samples per reach. We generally used a surber sampler, though if water was too deep, a D-net was used to scrub a similar area (0.09 m2) or when channels became too constrained a mini surber was employed (0.02 m2). We compiled all six samples into a composite sample, removed debris and leaf matter via manual inspection of material and elutriation, and sieved the remaining sample through a 500 µm sieve. We then preserved the sample in 95% ethanol and returned it to the lab. In the lab, ethanol was refreshed before we froze the sample in a -20 ºC freezer. We shipped samples to a commercial lab (Jonah Ventures, Boulder, CO) for metabarcoding analysis of the invertebrate community. Briefly, Jonah Ventures homogenized community samples with a hand immersion blender and extracted DNA with a DNeasy Powersoil Kit following the manufacturer’s protocol. They then amplified samples using two primers from the CO1 gene (CO1 F230 fragment Hajibabaei et al. 2012; CO1 BE fragment Folmer et al. 1994, Gibson et al. 2015). They conducted PCRs with initial denaturation at 95 ºC for 5 min, followed by 40 cycles of 40s at 95 ºC, 1 min at 46 ºC, 30s at 72 ºC and a final elongation at 72 ºC for 10 min. They then cleaned samples using Exo1/SAP, and pooled, normalized and indexed. Samples were sent for sequencing at the CU Boulder BioFrontiers Sequencing Center where the Center used the v2 500-cycle kit with appropriate quality control measures. Jonah Ventures then demuliplexed sequenced samples using phigs v2.1.0 followed by removal of gene primers and merging read pairs. Read pairs were then clustered using unoise3 denoising algorithm in vsearch and with sequences with less than 8 reads discarded. Taxonomy was assigned using a custom best-hits algorithm with reference to NCBI Gen Bank to each Exact Sequence Variant (ESV) (See linked Jonah Venture documents for full methods). Additional habitat data can be found on HydroShare.

TAL- This study was conducted in the Talladega 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.

WHR-This study was conducted in the Shambley Creek research watershed (outlet location: 32.98410915, -88.01334337) 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. 

PRF-This study was conducted in the Paint Rock 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.