Field Methods
During January 2019, we collected hyporheic sediments nine transects across pools, meanders, and riffles along Von Guerard Stream in Taylor Valley, Antarctica. Each transect was composed of five sampling sites at different lateral positions across the stream channel: two sampling sites were located at the margins of the visible wetted zone, one sampling site was directly under the thalweg, and two sampling sites were located halfway between the wetted margin and the thalweg. Transects ranged from 6.6 m to 13.6 m in total length across the stream channel. At each sampling site, we removed the surface sediment and mat material in order to focus our sampling on the underlying hyporheic sediments. We collected bulk sediment samples down to 10 cm; these samples were used for all laboratory analyses. Samples were frozen immediately after sample collection and remained frozen until laboratory analyses.

Laboratory Analyses
To quantify chlorophyll and its degraded phaeopigments, we used an acetone extraction followed by spectroscopy. Approximately 30 g of hyporheic sediment was extracted in 90% buffered acetone for 24 hours. The suspended material was then centrifuged for 10 minutes at 1500 RPM and the extracted supernatant was decanted into glass scintillation vials. The remaining sediment portion was dried at 55ºC and weighed. We measured the chlorophyll fluorescence of the extracted pigment spectroscopically using a FluoroMax F3 fluorometer (HORIBA Jobin Yvon, Paris, France) following well-established methods (Holm-Hansen et al., 1965; Wetzel & Likens, 2013). We measured phaeophytin concentrations on the same samples used for chlorophyll by taking the difference in fluorescence after acidification (100 µl 0.1M hydrochloric acid). Fluorescence values were standardized into concentrations of chlorophyll a and phaeophytin by dry weight.

To quantify the amount of organic matter in the hyporheic sediments, we dried and combusted sample subsets. Approximately 20 g of wet, homogenized sediment was subsampled for each site, weighed and then dried at 105ºC for 24 hours. Samples were reweighed to determine dry weight and gravimetric moisture content. Dried samples were then combusted in a muffle furnace at 450ºC for 4 hours and reweighed to calculate loss on ignition (LOI) relative to dry weight.

We measured inorganic N adsorbed to the sediments using a 2M KCl extraction. Approximately 20 g of wet, homogenized sediment from each site was weighed into sterile HDPE sample cups. After 80 ml of 2M KCl was added, the cups were inverted three times and placed on an elliptical shaker table at 400 rpm for 1 hour. Samples were removed from the shaker table, allowed to settle for 1 hour, and filtered through Whatman Qualitative Grade 1 filter paper. Filtered extracts were stored frozen until the time of analysis. Both extractable nitrogen as ammonium (N-NH₄⁺) and as combined nitrate and nitrite (N-NO₂^- + NO₃^-, referred to as N-NO₃^- hereafter) were measured using a Lachat Quickchem Flow Injection Analysis System (Hach, USA) according to standard protocols 4500-NH₃ (phenolate FIA) and 4500-NO₃ (cadmium reduction FIA). Concentrations for each N species were normalized to sample dry weights, which were estimated using gravimetric moisture content determined during the LOI protocol. The minimum detection limits were 0.005 mg N-NH₄⁺ L^-1 and 0.004 mg N-NO₃^- L^-1.

The KCl extraction used to measure inorganic N is also expected to desorb organic matter from the sediment (Gabor et al., 2015). To evaluate the potential relationship between inorganic N and soluble organic matter, we also determined the UV absorbance spectra of the KCl extracts using an Agilent 8453 spectrophotometer. The humic fraction of the soluble organic matter is expected to have an absorbance peak at 254 nm associated with the absorbance by aromatic ring structures (Gabor et al., 2015).

We measured the percent biogenic silica of the hyporheic sediments using a hot NaOH extraction, following the standard operating procedure from the National Lacustrine Core Facility (LacCore, University of Minnesota). To account for the low levels of biogenic silica in our samples, we used 300 mg of oven-dried sediment for the extractions (Spaulding et al., 1997). To digest the samples, we added 38 mL of 0.5 M NaOH to the samples and immediately placed them in an 85˚C water bath. We subsampled aliquots at 60, 90, 120, and 200 minutes to account for the slow dissolution of clay minerals (DeMaster, 1981). Aliquots were dyed molybdate blue and measured spectroscopically using a FluoroMax F3 fluorometer (HORIBA Jobin Yvon, Paris, France). To arrive at the percent biogenic silica for each sample, we calculated the linear equation for the 90, 120, and 200-minute aliquots for each sample and assumed the intercept to be the percent biogenic silica with no additional silica from clay dissolution. We disregarded the first time point (60 minutes) because it consistently did not follow the linear relationship of the subsequent three time points. We believe this was due to an incomplete dissolution of the biogenic silica at the first time point, potentially due to the presence of intact and viable diatom frustules in our samples.

References

• DeMaster, D. J. (1981). The supply and accumulation of silica in the marine environment. Geochimica et Cosmochimica Acta, 45(10), 1715–1732. https://doi.org/10.1016/0016-7037(81)90006-5
• Gabor, R. S., Burns, M. A., Lee, R. H., Elg, J. B., Kemper, C. J., Barnard, H. R., & McKnight, D. M. (2015). Influence of Leaching Solution and Catchment Location on the Fluorescence of Water-Soluble Organic Matter. Environmental Science & Technology, 49(7), 4425–4432. https://doi.org/10.1021/es504881t
• Holm-Hansen, O., Lorenzen, C. J., Holmes, R. W., & Strickland, J. D. H. (1965). Fluorometric Determination of Chlorophyll. ICES Journal of Marine Science, 30(1), 3–15. https://doi.org/10.1093/icesjms/30.1.3
• Spaulding, S. A., McKnight, D. M., Stoermer, E. F., & Doran, P. T. (1997). Diatoms in sediments of perennially ice-covered Lake Hoare, and implications for interpreting lake history in the McMurdo Dry Valleys of Antarctica, 18.
• Wetzel, R. G., & Likens, G. E. (2013). Limnological Analyses. Springer Science & Business Media.