These methods, instrumentation and/or protocols apply to all data in this dataset:| Methods and protocols used in the collection of this data package |
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| Description: | This study was conducted at the Niwot Ridge Long-Term Ecological Research site (40° 03ʹ N, 105° 35ʹ W, elevation 3520 m), located in the Front Range and specifically in the Roosevelt National Forest, Boulder County, Colorado. We worked on the West Knoll, the site of Dearing’s previous studies on pikas and alpine avens (Dearing 1996a; 1996b; 1997a; 1997b). At this site, the west-facing slopes are generally wind-blown and support dry-meadow and fellfield vegetation. In contrast, snow accumulates on the east-facing slopes, where scattered snow beds remain until mid-summer (Suding et al. 2015).
Recent samples – In late July/early August 2010, 2013, 2014, 2017, and 2018, we collected 1-2 leaves (0.3 - 0.5 g) from 10-15 individual alpine avens plants from foraging areas in active pika territories on the West Knoll. Sampled plants were at least 50 m apart, and we targeted leaves that were green and fully intact. Leaves were cut just above the ground and kept on ice in the field, then transferred to a -80°C freezer until analysis. A subset of leaves was weighed fresh and then dried to measure water content, as described in detail below. In 2018, we also collected leaves of alpine avens from five additional locations, including one additional site in the Front Range, and 1-3 locations in each of four other mountain ranges in the southern Rocky Mountains (San Juan, Cimarron, La Sal, and West Elk). At each of these five locations, we collected 1-2 leaves from at least 3 individual plants at each of at least 3 sampling locations (separated by > 500 m). We did not have historical samples from any of these five locations, but this sampling allowed us to investigate spatial variation in plant chemistry within a year.
Historical samples – We also reanalyzed “historical samples” of alpine avens that were collected by Dearing on the West Knoll of Niwot Ridge in 1992. These samples (frozen leaves) had been stored at - 80°C until 2014, when they were tested as described below. We analyzed these frozen leaves collected in 1992 as an assay control and compared our results to the values reported in Dearing (1996a; 1997a; 1997b).
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| | Description: | Plant chemistry & nutrition assays
Total phenolic activity of alpine avens was measured using the Folin–Ciocalteu method (Waterman and Mole 1994) for both recent and historical alpine avens samples. Although this method is less accurate in measuring absolute phenol content compared to more sophisticated analytical approaches (e.g., LC-MS), it provides a consistent estimate of biological activity in ecological studies when activity depends on phenolic oxidation, such as plant–herbivore interactions (Appel et al. 2001; Moreira et al. 2020). Furthermore, we wanted to compare our results with those of previous studies that used the same assay (Dearing 1996a; 1997a; 1997b). Phenolic content of ~ 0.2 g of frozen plant material was extracted into a solution of 95% methanol by grinding with a Polytron PT3100 Mixer (Kinematica, Lucerne, Switzerland) at 12,000 rpm for 30 s. After 48 hours in the dark at room temperature, samples were centrifuged for 5 min at 3,300 rpm, and the supernatant was used in the Folin–Ciocalteu reaction, following the protocol of Ainsworth and Gillespie (2007). As in Dearing’s previous studies, tannic acid was used as the standard for the standard curve. We used a subsample of each plant sample to calculate water content by weighing fresh samples in the field (fresh weight; FW) and re-weighing after drying for 24 hours at 40°C (dry weight; DW). All phenolic activity values were converted to phenolic activity per g dry weight (DW). Total phenolic activity is expressed as mg tannic acid equivalent (TAE) per g DW of plant material.
Total fiber content (neutral detergent fiber, NDF) and cellulose/lignin content (acid detergent fiber, ADF) of recent and historical alpine avens samples were measured in a fiber analyzer according to the manufacturer’s instructions (ANKOM 200 Fiber Analyzer; ANKOM Technology, Macedon, New York). Fiber analyses required 0.5 g of dried plant material. Nitrogen content was measured by combusting 5 mg of dried, ground plant material in an Elemental Combustion System (Costech Analytical Technologies, Valencia, California) coupled to a Delta Plus Advantage mass spectrometer (Thermo Finnigan, San Jose, California) operating in the continuous-flow mode. Nitrogen analyses were conducted for both historical samples and for recent samples collected in 2013 and 2017; however, additional replicates of avens collected in 2017 failed in the instrument, leaving only one replicate of nitrogen content for that year.
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| | Description: | Overwinter preservation experiment
We also tested whether changes in plant chemistry or microhabitat characteristics (e.g., duration of snowpack or insolation) would affect overwinter preservation of pika haypiles (i.e., the amount of biomass remaining in early summer after snowmelt). To do so, we repeated an experiment originally conducted in 1992 – 1993 by Dearing (1997b), in which samples of a high phenolic plant species (alpine avens) and a low phenolic plant species were exposed to winter haypile conditions. Specifically, we collected alpine avens and mixed graminoid samples (Carex spp & Deschampsia spp) in August 2017 from West Knoll. We then created two types of “artificial haypiles”, each consisting of 15-25 g FW of either avens or graminoids (n = 5 artificial haypiles of each sample type, as in Dearing (1997b)). Each artificial haypile was contained in a bag constructed of fiberglass window-screening mesh, which was then placed in a wire suet-feeder cage, allowing sample exposure to free air while preventing damage to or theft of samples by rodents or pikas. These cages were placed in the talus to mimic the placement of pika haypiles (i.e., each was positioned under a large rock where it was exposed to air but not to direct sunlight). These cages were left in place from August 2017 until July 2018, when their contents were dried at 40°C for 48 hours and re-weighed. The % biomass remaining was calculated as: (DW remaining, g) / (starting FW, g × dry matter, %). We recognize that many other factors could affect preservation of actual pika haypiles, including the size, structure, and composition of the haypile (Jakopak, Hall, and Chalfoun 2017) or pika activities such as deposition of feces/urine or reorganization, but by controlling these extraneous factors, this experiment provides important insight into the basic preservation ability of the plants that comprise the pika haypile.
We placed these artificial haypiles (n = 10 total) at varying aspects around the West Knoll so that potential effects of microhabitat (slope aspect and slope angle, which affect insolation, microclimate, and snowpack duration) on overwinter preservation could also be investigated. Although we did not directly measure microclimate or snowpack duration at each location, we calculated an insolation index to capture the effects of incident sunlight on each artificial haypile. Following Jeffress et al. (2013), we calculated insolation as sin(aspect) × cos(slope angle). Thus, insolation values range from -1 (indicating a steep, south-facing slope with high sun exposure) to 0 (indicating a flat slope) to +1 (indicating a steep, north-facing slope with very little sun exposure).
Although Dearing’s original experiment used clover (Trifolium parryi) as the low phenolic plant sample, we chose to include graminoids instead in this experiment because graminoids are now much more common than T. parryi in haypiles and in the environment, and they make up a significant part of the pika’s summer diet at this site (Bhattacharyya and Ray 2015). Dearing (1997b) included the low phenolic plant in her experiment to test whether a) high-phenolic plant species preserved better than low-phenolic plant species, and b) whether high-phenolic plant species could enhance preservation of low-phenolic plant species in a mixed sample. We did not attempt to determine whether avens affected graminoid preservation in a mixed sample, nor did we compare current graminoid preservation to past clover preservation. Thus, while it is possible that pikas have shifted to using graminoids more than clover because nutritional shifts have made clover a less palatable food source (e.g., through increased phenolics), this substitution should not affect the conclusions of our experiment, which tested for changes in preservation of avens and for how microhabitat affects overwinter preservation of haypiles.
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