A study was conducted to examine soil microbial communities and associated geochemical parameters at potential glacial refugia and glaciated control sites throughout the McMurdo Dry Valleys region of Antarctica. Soil samples were collected as part of ongoing long-term monitoring efforts by the McMurdo Dry Valleys Long Term Ecological Research program (MCM LTER). The oldest samples used in this study were collected during the 1993-1994 austral summer, and the newest from the 2018-2019 austral summer. "Refugia" sites were selected based on geographical positions and elevations indicative of potential glacial refugia status. Each refugia site was paired with a lower elevation "glaciated" site in the same dry valley that was not likely to have functioned as a refugium. Six replicate soils per sampling site were sequenced with 16S primers following Earth Microbiome Project protocols, filtered using the DADA2 pipeline, and clustered to amplicon sequence variant using the SILVA reference database to generate the microbial classification table included herein. Soil samples were also analyzed for various geochemical parameters as part of this study, which include P, K, NO3-, gravimetric water content, percent organic matter, pH, and electroconductivity.
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.
Funding for this work was provided by the National Science Foundation for Long Term Ecological Research, most recently grant #OPP-1637708.