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. Phylogeographic reconstructions were recreated in IQ-tree with the built-in model finder and ultrafast bootstrap values (Kalyaanamoorthy et al., 2017; Hoang et al., 2018; Minh et al., 2020) and FigTree v1.4.4 (available at http://tree.bio.ed.ac.uk/software/figtree/). Bayesian Skyline plots were computed with BEAST v2.6.7 (Bouckaert et al., 2019) and tracer.

References Cited:

• Aktas, C. (2020) “haplotypes: Manipulating DNA Sequences and Estimating Unambiguous Haplotype Network with Statistical Parsimony.”
• Bouckaert, R. et al. (2019) “BEAST 2.5: An advanced software platform for Bayesian evolutionary analysis.” PLoS computational biology. doi: e1006650.
• Excoffier, L. and Lischer, H. E. L. (2010) “Arlequin suite ver 3.5: A new series of programs to perform population genetics analyses under Linux and Windows.” Molecular Ecology Resources, pp. 10:564-567.
• Freckman, D. W. and Virginia, R. A. (1993) “Extraction of nematodes from Dry Valley Antarctic soils,” Polar Biology, 13(483–487). doi: https://doi.org/10.1007/BF00233139.
• Hoang, D. T. et al. (2018) “UFBoot2: Improving the ultrafast bootstrap approximation.” Molecular Biology and Evolution, pp. 35:518-522.
• Jombart, T. (2008) “adegenet: a R package for the multivariate analysis of genetic markers.” Bioinformatics, pp. 24:1403-1405. doi: 10.1093/bioinformatics/btn129.
• Jombart, T. and Ahmed, I. (2011) “adegenet 1.3-1: new tools for the analysis of genome-wide SNP data.” Bioinformatics. doi: 10.1093/bioinformatics/btr521.
• Kalyaanamoorthy, S. et al. (2017) “ModelFinder: Fast model selection for accurate phylogenetic estimates.” Nat. Methods, pp. 14:587-589. Available at: https://doi.org/10.1038/nmeth.4285.
• Kamvar, Z. N., Brooks, J. C. and Grunwald, N. J. (2015) “Novel R tools for analysis of genome-wide population genetic data with emphasis on clonality.” Frontiers in Genetics, p. 6:208. doi: 10.3389/fgene.2015.00208.
• Kamvar, Z. N., Tabima, J. . and Grunwald, N. J. (2014) “Poppr: an R package for genetic analysis of populations with clonal, partially clonal, and/or sexual reproduction.” PeerJ. doi: 10.7717/peerj.281.
• Minh, B. Q. et al. (2020) “IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era.” Molecular Biology and Evolution, pp. 37: 1530-1534.
• Paradis, E. (2010) “pegas: an R package for population genetics with an integrated-modular approach.” Bioinformatics, pp. 26: 419-420.
• Powers, T. O. and Harris, T. S. (1993) “A polymerase chain reaction method for identification of five major meloidogyne species.,” Journal of nematology, 25(1), pp. 1–6. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19279734%0Ahttp://www.pubmedcentral.n....
• R Core Team (2002) “R: A language and environment for statistical computing. R Foundation for Statistical Computing.” Vienna, Austria. Available at: https://www.r-project.org/.
• Timm, R. W. (1971) “Antarctic soil and freshwater nematodes from the McMurdo Sound Region,” Proceedings Helminth Soc Washington, 38(1), pp. 42–52.