design: All samples were processed using standardized protocols based on the Earth Microbiome Project [22]. Upon receipt, fecal material was isolated from the preservation buffer by pelleting (centrifugation at 10,000 g for 5 min, pouring off supernatant), and genomic DNA was extracted using the 100-prep Qiagen DNeasy PowerSoil DNA Isolation Kit (Germantown, MD, USA). Briefly, samples were placed in bead tubes containing C1 solution and incubated at 65 C for 10 min, and this was followed by 2 min of bead beating. After, the manufacturers protocol was followed as written. Amplicon libraries of the V4 region of the 16S rRNA gene (primers 505F/816R) were generated using a dual-indexing one-step PCR with complete fusion primers (Ultramers, Integrated DNA Technologies, Coralville, IA, USA) with multiple barcodes (indices) [23]. PCR reactions contained 0.330 ng template DNA, 0.1 L Phusion High-Fidelity DNA Polymerase (Thermo Fisher, Waltham, MA, USA), 1X HF PCR Buffer, 0.2 mM each dNTP, and 10 M of the forward and reverse fusion primers. The PCR conditions were as follows: initial denaturing at 98 C for 30 s, 30 cycles of 10 s at 98 C, 30 s at 55 C, and 30 s at 72 C, 30, an incubation at 72 C for 4 min 30 s for a final extension, and a hold at 6 C.