Biotic interactions are an unexpected yet critical control on the complexity of an abiotically driven polar ecosystem

Abiotic and biotic factors control ecosystem biodiversity, but their relative contributions remain unclear. The ultraoligotrophic ecosystem of the Antarctic Dry Valleys, a simple yet highly heterogeneous ecosystem, is a natural laboratory well-suited for resolving the abiotic and biotic controls of community structure. We undertook a multidisciplinary investigation to capture ecologically relevant biotic and abiotic attributes of more than 500 sites in the Dry Valleys, encompassing observed landscape heterogeneities across more than 200 km2. Using richness of autotrophic and heterotrophic taxa as a proxy for functional complexity, we linked measured variables in a parsimonious yet comprehensive structural equation model that explained significant variations in biological complexity and identified landscape-scale and fine-scale abiotic factors as the primary drivers of diversity. However, the inclusion of linkages among functional groups was essential for constructing the best-fitting model. Our findings support the notion that biotic interactions make crucial contributions even in an extremely simple ecosystem.


Analysis of Soil Samples (Field).
In the field laboratory, bulk soil samples were further aliquoted and processed for additional analyses: 1.
DNA-based analyses (~80 g soil): aliquoted from homogenized bulk soil sample into a sterile 50 mL centrifuge tube. Analyses were carried out at the University of Waikato.

2.
Soil total ATP analysis (100 mg soil x 2): aliquoted from homogenized bulk soil sample into sterile ATP assay tubes.

3.
Soil geochemistry (~80 g soil): aliquoted from homogenized bulk soil sample through a 2 mm sieve into a 4 oz. Whirl-Pak. Analyses were carried out at Virginia Tech, Blacksburg, VA, USA.

4.
Soil pH and conductivity (2 mL volume): aliquoted from homogenized bulk soil sample through a 2 mm sieve into a 15 mL centrifuge tube.

5.
Soil water activity (Aw) (~15 g): aliquoted from homogenized bulk soil sample through a 2 mm sieve into an Aw measurement cup.
Total soil ATP was measured in duplicates (triplicates where the duplicates did not match) using a 3M Clean-Trace Beverage Test Kit (Acorn Scientific, Auckland, NZ) with a modified protocol. In short, 100 µL of Extractant Buffer was added to 100 mg of soil and allowed to incubate for 60 seconds. 75 µL of ATP Assay Solution was then added to the sample, which was immediately read using a 3M Clean-Trace NG Luminometer (Acorn Scientific). Total soil ATP levels were recorded as relative fluorescence units, and pure ATP solutions were used to check for signs of inhibition in samples with low readings. Soil pH and conductivity were measured using the slurry method (Lee et al. 2012). In brief, 10 mL of deionised water was added to a soil aliquot (2 mL) and mixed thoroughly. The pH and conductivity of the resulting slurry was  (Barrett et al. 2004). Microinvertebrates (i.e., nematodes, tardigrades and rotifers) were extracted from soils using a modified sugar-centrifugation technique (Freckman and Virginia 1997) and identified and enumerated using bright-field microscopy (Olympus CK40 Inverted Microscope, Olympus America Inc., Center Valley, PA). Population abundances were recorded as numbers of individuals per kg soil, corrected to oven-dry weight equivalent. Demographic information for nematode populations (i.e., gender, juvenile/adult, alive/dead) was also recorded but not used for the construction of the SEM. Observed protozoan (i.e., flagellates, amoebae, and ciliates) abundances were recorded, but the data were not included in the SEM since reliable characterizations of protozoan abundance and diversity greatly exceeded our logistical capability (Bamforth et al. 2005).
Soil samples for DNA-based analyses were transported to Scott Base within 72 hours of collection, where they were stored at -20°C. They were then shipped to University of Waikato under refrigerated conditions and stored at -80°C until analyzed. DNA was extracted from soils and analyzed as described below. A subsample of soil was air-dried and ground in a ball mill to a fine homogenous powder for geochemical analyses. Organic carbon and total nitrogen (inorganic For samples yielding DNA concentrations less than 1.8 ng/µL, extractions were repeated manually, without processing on the X-tractor Gene, to increase yields. The lysis steps were Supplementary Information (Lee et al) completed as outlined above, and the method after the chloroform step was modified as follows.
The final ammonium acetate concentration of the lysate was brought to 2.5 M, 300 µL chloroform:isoamyl alcohol (24:1) was added, samples were vortexed, and centrifuged at 16,000 g for 5 minutes. The entire aqueous phase was transferred to a new tube and the chloroform step repeated with an equal volume of chloroform:isoamyl alcohol (24:1). The aqueous phase was transferred to a new tube and DNA was precipitated with addition of 0.54 volumes of isopropanol followed by centrifugation at 16,000 g for 20 min. Pellets were washed by adding 1 mL 70 % ethanol, centrifuged at 16,000 g for 5 min, and the supernatant discarded. Dried pellets were resuspended in 30 µL TE pH 8.5.