Mitochondrial COXI sequences and associated metazoan abundances in soils collected from the McMurdo Dry Valleys, Antarctica from 1995 to 2022

About 500 g of the top 10 cm of soil were collected with clean plastic scoops and sterile Whirlpak bags from sites in the MDVs during the Austral summer between 1996 to 2022. Soils were transported to the Crary Laboratory in McMurdo Station where they were stored at 4 °C until soil invertebrates and geochemistry could be performed within 48 hours. A sugar centrifugation extraction (Freckman and Virginia, 1993) was performed with a subsample of 100 g to calculate metazoan abundance. Soil invertebrates were counted and identified to species, as living or dead, life stage (juvenile or adults), and sex. Soil moisture, pH, and electrical conductivity were measured following standard protocols of the MCM LTER project (e.g., http://mcm.lternet.edu/content/soil-elevational-transect-experiment). Subsequent subsamples of 100 g were extracted in the same way at Brigham Young University to collect individual Scottnema lindsayae. Following Powers and Harris (1993) nematode extraction methods, individual S. lindsayae (Timm, 1971) were hand-picked with an eyelash tool onto 10 μl of elution buffer on a cover slip. Using a clear, sterilized 10 μl micropipette tip, the nematode was ruptured by gentle pressure and lysate confirmed in the sterile water. Lysate was moved to a 0.2 ml microcentrifuge tube. The cover slip was further cleaned with 5.2 μl of sterile water, which was added to the microcentrifuge tube. Nematode lysate was combined with 2.4 μl each of 20 μM diluted forward primer (COI-Scott-F1: 5'-GTTACAACTTTTTTGCTTATTCTCTCAC-3’) and reverse primer (COI-Scott-R2: 5’-CTGTAAAATAAGCTCGACTRTCWG-3’). Additional extractions were performed with an alternative reverse primer with higher fidelity, but shorter sequence reads (COI-Scott-R4 5’-GCRTCRATACCTGTTACRTATATRTG-3’). Additionally, 15 μl of JumpStart™ REDTaq® ReadyMix™ Reaction Mix (Sigma-Aldrich, DE) was added to the mixture and vortexed for 30 seconds to mix. The mixture was placed in a DNA thermal cycler (Mastercycler X50s, Eppendorf, Germany) on the following settings: initial denaturation at 94°C for 5 minute, 40 cycles of denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds, extension of 0.5°C/second ramp to 72 for 90 seconds. Followed by a final extension at 72°C for 5 minutes and hold at 4°C. Following PCR amplification, the products were screened on a 1% TAE agarose gel stained with ethidium bromide and visualized with a UV image capture (GelDoc Go, Bio-Rad Labratories, USA). Successful DNA amplifications were cleaned with a magnetic bead cleaning protocol. Mag-Bind® TotalPure NGS (Omega Bio-tek, GA) was added in a 1.2x concentration to the remaining PCR product, vortexed for 10 minutes at 2,000 rpm, centrifuged for 5 seconds, placed on a magnetic rack until bead solution was cleared. The supernatant was removed and thrown away, while the beads were washed with 200 μl 80% ethanol for 30 seconds twice. After removing all ethanol, residual ethanol was allowed to evaporate for 10 minutes. Tubes were removed from the magnetic rack as 30 ml of elution buffer (Omega Bio-tek, GA) was added and vortexed to mix. Tubes were returned to the magnetic rack to separate the supernatant. The supernatant was removed and added to a clean 0.2 ml tube and beads discarded. DNA concentration was quantified by Quibit 4 Flurorometer (Invitrogen, USA). DNA was diluted or concentrated to 10 ng for Sanger dideoxy sequencing at the Brigham Young University Sequencing Center (Provo, UT). Analysis was conducted with a combination of ARLEQUIN v3.5.2.2 (Excoffier and Lischer., 2010), R Studio (R Core Team, 2002) utilizing pegas (Paradis, 2010), haplotypes (Aktas, 2020), poppr 2.9.3 (Kamvar, Tabima and Grunwald, 2014; Kamvar, Brooks, Grunwald, 2015), and adegenet (Jombart, 2008; Jombar and Ahmed 2011) packages. 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