Soil samples were collected in various field seasons between 1993-1994 and 2018-2019 according to the protocols established in the McMurdo Dry Valleys in 1993 (Freckman and Virginia 1993). Sequencing and analyses were performed in April 2021.

DNA extraction was on each sample using the provided standard protocol with DNeasy PowerSoil Kit (Qiagen). For each sample 0.25 grams of homogenized whole soil was used for each extraction, replicated 6-fold per sample. A total of 5 negative controls were also performed on pure PCR-grade water and one reagent-only control. Sequencing libraries were prepared from each sample following established protocols (Kozich et al. 2013). 4 ul of extracted DNA was added to a 96 well plate including sample-specific primers with 17 ul of AccuPrime Pfx Supermix, and 2 ul of each paired set of duel-indexed primers for the V4 region of the 16S rRNA gene supplied by the Earth Microbiome Project. To prevent evaporation, a drop of mineral oil was added to each well. The plate was amplified in individual 20 ul reactions on a thermocycler at 95 °C for 2 minutes followed by 30 cycles of 95 °C for 20 seconds, 55 °C for 15 seconds, and 72 °C for 5 minutes, then 72 °C for 10 minutes and held at 4 °C. Amplified DNA was run on gel electrophoresis using 4 ul of amplified DNA and 4 ul of loading dye in a 1% agarose gel at 100v for 60 minutes alongside a ladder. PCR amplicons were normalized and pooled using the Charm Biotech’s Just-a-PlateTM Normalization kit to 20ng/ul of total DNA per sample. These amplicons were submitted for sequencing on a MiSeq, 2x250v2 chemistry at the Arizona State University genomics core sequencing center. All post-extraction steps included a reagent only negative control to remove contaminant sequences in data analysis.

Environmental analyses were performed by the BYU Environmental Analytics Lab. In short, pH was measured using a Thermo Orion Model 410A+ by Thermo Electron, Waltham, MA. Organic matter content was detected using chromic acid titration (Walkley and Black, 1934) and nitrate content measured following the methods described in Keeney and Welson (1982) on a Fialyzer 2000, FIALAb, Seattle, WA. Phosphorous and Potassium content were extracted using 0.5M sodium bicarbonate following the Olsen et al. (1954) and Schoenau and Karamos (1993) protocols respectively.

References cited:

• Freckman, D. W., and R. A. Virginia. 1993. Extraction of nematodes from Dry Valley Antarctic soils. Polar Biology 13:483–487.
• Keeney, D.R., and D.W. Nelson. Nitrogen - Inorganic forms. pp. 643-698. In: A.L. Page (ed.), Methods of Soil Analysis Part 2. 1982. American Society of Agronomy, Inc. Madison, WI.
• Kozich, J. J., S. L. Westcott, N. T. Baxter, S. K. Highlander, and P. D. Schloss. 2013. Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform. Applied and Environmental Microbiology 79:5112–5120.
• Olsen, S.R., C.V. Cole, F.S. Watanabe, and L.A. Dean. 1954. Estimation of available phosphorus in soil by extraction with sodium bicarbonate. USDA Cir. No. 939.
• Schoenau, J.J., and R.E. Karamonos. Sodium Bicarbonate Extractable P, K, and N. pp. 51-58. In: M.R. Carter (ed.), Soil Sampling and Methods of Analysis. 1993. Canadian Society of Soil Science. Ottawa, Ontario, Canada.
• Walkley, A., and I.A. Black. 1934. An examination of the Degtjareff method for determining soil organic matter and a proposed modification of the chromic acid titration method. Soil Science 37:29-38.