Site Selection:
In the fall of 2005, three sites were selected that varied primarily in deciduous shrub abundance, hereafter referred to as low, medium and high shrub sites. Sites were chosen to have similar state factors (climate, relief, parent material, and time) but varied in the abundance of deciduous shrubs (Jenny, 1994). The sites represent a natural gradient of increasing shrub abundance because the same species of deciduous shrubs (Betula nana, and Salix pulchra) are found at all three sites (except S. richardsonii, which is found only at the medium shrub site); however, their percent cover increases from 15 to 94 %. Our sites are within 1 km of each other, and have similar parent material, and time since last glaciation (Itkillik I, deglaciated ca. 60 000 yr), and regional climate, although microclimates vary across sites due to differences in slope and aspect. The low shrub site is located on top of gently rolling hills, while the medium and high shrub sites are located in depressions along water tracks of ephemeral streams fed by spring snowmelt.
Our low shrub abundance site is located in moist acidic tussock tundra where the vegetation consists of approximately equal biomass of graminoids (Eriophorum vaginatum and Carex bigelowii), dwarf deciduous shrubs (B. nana, Vaccinium uliginosum, and S. pulchra), evergreen shrubs (Ledum palustre ssp. decumbens and V. vitis-idea), and mosses (Hylocomium splendens, Aulacomnium turgidum, Dicranum spp., and Sphagnum spp.) (Shaver and Chapin, 1991). In our medium shrub abundance site, vegetation consists of graminoids (primarily C. bigelowii), deciduous shrubs (B. nana, V. uliginosum, S. pulchra and S. richardsonii), and mosses (H. splendens and Dicranum spp.). Our high shrub abundance site has predominantly deciduous shrubs (B. nana, S. pulchra, and some Potentilla fruticosa) with some evergreen or wintergreen shrubs (V. vitis-idaea and Linnaea borealis), forbs (Polygonum bistorta, Petasites frigidus, Stellaria longipes, Valeriana capitata, and Artemisia alaskana), graminoids (Poa arctica, C. bigelowii, and Calamagrostis canadensis), and mosses (Sphagnum spp. and H. splendens).
Snow Manipulation:
To manipulate snow depth, snow fences (1.5 m high and 62 m long) were set up in the fall of 2005 at the low and medium sites to manipulate snow depth. For the high site, the patchy nature of the shrub stands made it necessary to set up two separate snow fences (1.5 m high and 32 m long) in patches with similar shrub composition and density. Our purpose for adding snow was to simulate the amount of snow that might be trapped by deciduous shrubs; therefore, the height of the snow fences was selected to match the maximum shrub height within the region. Fences were oriented E-W, and snow drifts accumulated on the northern side of the fences. Two treatments (control=ambient snow and drift=manipulated snow) were set up at each site. The drift plots were set up 4 m from the fence on the northern side of the fence, because this was the zone of maximum snow accumulation. At the low and medium sites, the control plots were set up on the southern (non-drift) side of the fence, 10 m from the fence at the low site, and 7 m from the fence at the medium site. These buffer zones were left to prevent the control from being exposed to snow trapped by the fence. At the high shrub sites, control plots were located in line with one of the fences, beginning 5 m from its end, and in 3 discontinuous blocks of tall shrubs to the south (control side) of the fence, beginning approximately 15 m from the fence. This arrangement was chosen because the cover of tall shrubs was discontinuous on the southern (control) side of the fences. For all sites, plots on the drift side of the fences were located in the zone of maximum snow accumulation, which was relatively uniform. Within each treatment, 18 2 by 10 m plots, with 1 m buffer strips between, were established. For this study, six plots per treatment (n=6) were randomly assigned to measure N mineralization and nitrification. Remaining plots were used for additional experiments.
Soil temperature:
Soil temperature, at 5 cm within the organic layer, was measured continuously (1-3 h intervals) from June 2006-June 2007 in each study plot (n= 3-4) by using Ibutton temperature data loggers (IButtonLink, LLC, East Troy, WI). Weekly mean soil temperatures were calculated from mid-June 2006 through mid-May 2007 for all treatments and sites. Annual soil temperatures were calculated for each site and treatment and included 336 days of measurements.
Net N-mineralization:
We used the in situ soil incubation method (Di Stefano and Gholz 1986, Hart and Firestone 1989) to assess (1) site differences in net N mineralization and nitrification, (2) snow effects on net N mineralization and nitrification, and (3) SOM versus microclimate effects on net N mineralization and nitrification. To determine N availability across our sites, we measured net N mineralization in the unmanipulated control plots (n=6) at each site (N=18). To test the effects of snow on N availability, soil cores were removed from the control plots at each site and incubated in the snow addition plots (n=6) in each site (N=18). To assess the relative importance of SOM quality and site microclimate on net N mineralization, soil cores were removed from the control plots (n=6) at each site (N=18) and either replaced for in situ incubation or reciprocally transplanted to the control side at each of the other sites. In each replicate of incubation experiment, four soil cores were removed from the top 10 cm in either June or September of 2006 for each layer sampled. Organic soils were sampled with a 5 cm diameter metal corer; new frost boils and Eriophorum vaginatum tussocks were avoided. One core (initial) was removed from the ground, chilled, and processed (within 48 h of sampling) for pools of inorganic N (N-NH4+ and N-NO3-), dissolved organic N (DON), chloroform-fumigated microbial biomass N (MB-N), bulk soil percent C and N, and soil moisture. The other three cores (final) were placed in a 12 cm long and 5 cm diameter PVC tube and capped at the top with one resin bag, and at the bottom with two resin bags. Resin bags were made of nylon that was soaked in 1.2M HCl for 2 h before filling with ion exchange resins. Bags contained 17 g fresh weight (49.4% moisture, 8.28 g oven-dry equivalent) of mixed-bed ion exchange resins (IONAC® NM-60 H+/OH- form, type I beads 16-50 mesh; J. T. Baker, Phillipsburg, New Jersey, USA). One core was then returned to its original hole in the control plot, one core was transplanted to a randomly assigned snow addition plot within the same site for incubation, while the other two remaining cores were each transplanted to a randomly assigned control plot from one of the other two sites for incubation. By doing this, we held the substrate constant but altered the environment of the incubation. All final cores were incubated for 74 days in the summer (mid June 2006-Sept 2006) and 280 days in the winter (Sept 2006-mid June 2007) to look at seasonal affects on N availability. At the end of the incubation periods, soil cores were removed and the soil was processed (within 48 hours of sampling) for pools of N-NH4+ and N-NO3-, DON, MB-N, bulk soil percent C and N, and soil moisture in exactly the same way as the initial core. Resin bags were removed from cores and frozen until processing (see below). Net N mineralization was calculated as the difference between the DIN (NH4+ and NO3-) in the initial soil core and the DIN in the final soil core plus the DIN accumulated on the middle resin bag. Net N nitrification was calculated as the difference between the nitrate in the initial soil core and the nitrate in the final soil core plus the nitrate accumulated on the middle resin bag. The percent of mineralized N that was nitrified was calculated by dividing the N nitrified by the amount of N that was mineralized and multiplying by 100. Nutrient pool sizes and annual net N mineralization were calculated using the soil bulk density obtained from soil harvest conducted in 2007.
Prior to analysis, soils were homogenized by hand and the >2 mm diameter fraction (e.g., roots, rhizomes, course woody debris, and rocks) was removed. Soil water content was calculated by subtracting the dry weight of the soil (60 °C for 48 h) from the wet weight of the soil and then dividing by the dry weight of the soil. To determine bulk soil percent C and N, a subsample of <2mm soil fraction was dried at 60º C for 48 h, ground to a fine powder on a Wiley-mill with a #40 mesh screen, and then analyzed using an ECS 4010 elemental analyzer (Costech Analytical, Valencia, California, USA).
Pools of dissolved inorganic N (N-NH4+ and N-NO3-) were measured by extracting 10 g of fresh soil with 50 ml of 0.5 M K2SO4. The soil slurry was agitated on a shaker table for 2 h, allowed to sit overnight in a cooler, and then vacuum filtered through a Whatman GF/A filter. Filtrate was frozen until analyzed colorimetrically, on segmented flow autoanalyzer (Astoria analyzer, Astoria-Pacific, Inc, Clackamas, Oregon, USA).
Dissolved organic N (DON) was measured on a subsample of the K2SO4 extract that was digested with a persulfate oxidation digestion procedure (Sollins et al., 1999) prior to colorimetric analysis. Because this digestion procedure converts all forms of N to NO3-, DON was calculated by subtracting the DIN measured previously from the total N that was determined in the digestion procedure.
Microbial biomass N was determined using the chloroform fumigation method. Ten grams of fresh soil was incubated with 100 ml of pentene stabilized chloroform in a glass dessicator for 24 h. Post incubation, soils were extracted with 0.5 M K2SO4 exactly the same way as for DIN. Fumigated extracts were digested using the same persulfate oxidation procedure used for DON analysis. Nitrate was then measured colorimetrically. Chloroform labile-N was calculated by subtracting the DON and DIN concentrations from the initial, un-fumigated sample from the total N that was extracted from the post-fumigated sample.
After incubation, resin bags were rinsed with deionized water to remove soil and then extracted with 50 ml of 2N KCl. Resins and KCl were agitated for 1 h and then filtered through a pre-leached Whatman #1 filter. Filtrate was immediately frozen. At time of analysis, extracts were thawed and measured for N-NH4+ and N-NO3- colorimetrically.

Data has been published in DeMarco et al. 2014. Effects of arctic shrub expansion on biophysical versus biogeochemical drivers of litter decomposition. Ecology In press.
See Publication for further explanation of methods and references.