This study was carried out at three elevational sites spanning the krummholz transition zone between subalpine forests and alpine tundra communities on Pennsylvania Mountain (Park County, CO, USA; 3682, 3626, and 3588 m a.s.l.). At this location, a mosaic of open meadow and willow understorey microsites dominates the krummholz region. Alpine willows (Salix glauca and Salix brachycarpa) play an important role in these ecosystems, insulating herbaceous plants during the winter and competing for light, water and nutrients during the growing season (Totland & Esaete 2002; Dona & Galen 2006, 2007). As a result, willows can greatly impact the surrounding plant community and potentially the community of organisms that associate with alpine perennials. This study was conducted across the willow-meadow ecotone in nutrient-limited dry alpine meadows where mycorrhizal associations are predicted to be particularly important (Theodose & Bowman 1997).
In krummholz regions of the central Rocky Mountains, S. glauca is the primary ectomycorrhizal (EM) host, although stunted conifer trees (Abies lasiocarpa, Picea engelmannii and Pinus aristata) also occur. In other systems, willows are colonized by both EMF and AMF (Wang & Qiu 2006); however, using staining techniques we have not seen evidence of AMF colonization of willows at this location in 4 years of study (K. Becklin, pers. obs.). A survey of EMF colonization across the willow-meadow ecotone indicates that, as expected, EMF are more abundant in the willow understorey where the density of willow roots is high (Becklin & Galen 2009). On average, EMF colonize 33% (range = 0–64%) of root tips in samples collected from the willow understorey compared to 2% (range = 0–33%) in samples collected from the open meadow.
Dry alpine meadows contain a diverse mixture of mycorrhizal and nonmycorrhizal grasses, sedges and forbs. Many of these herbaceous plant species occur in both open meadow and willow understorey habitats; however, plant density and diversity are generally higher in the open meadow (Becklin 2010). Arbuscular mycorrhizal (AM) hosts are common within the herbaceous community although other types of mycorrhizal fungi, including EMF, likely colonize some herbaceous alpine plants (Cripps & Eddington 2005; Wang & Qiu 2006). Surveys of four common herbaceous forbs, Taraxacum ceratophorum, Taraxacum officinale, Polemonium viscosum and Polemonium delicatum, indicate that AMF composition is similar across the willow-meadow ecotone (Becklin 2010), whereas AMF colonization is greater in the open meadow (Becklin & Galen 2009).
Effects of Above- and Below-Ground Interactions
To test whether above- or below-ground interactions drive spatial patterns in AMF colonization across the willow-meadow ecotone, we manipulated three aspects of the willow habitat (canopy cover, willow presence and EMF abundance) then monitored AMF and EMF colonization over time. This experiment was replicated in 2007–08 and 2008–09.
To isolate the below-ground effects of willows and EMF, we transplanted S. glauca cuttings into open meadow microsites then manipulated subsequent colonization by EMF. Willow cuttings were collected from source populations on Pennsylvania Mountain in 2002 (Dudley & Galen 2007), rooted in a sterile 1 : 3 mixture of sand and peat moss, and grown for 5–6 years in the greenhouse (University of Missouri, Columbia, MO, USA). In early June of 2007 and 2008, we transplanted the willow cuttings back into the open meadow. At the time of transplanting, willows were 40–93 cm (average = 63 cm) tall with 3–6 (average = 3) branches, and 0–21% (average = 6%) of root tips were colonized by EMF. To control for differences in initial size, willows were transplanted in pairs of approximately equal size at least 3 m from each other (Fig. 1). Willow transplants were placed in holes twice the size of the rootball, and the extra space was filled with sterile peat moss. For one randomly selected willow within each pair, we lined the transplant hole with landscaping cloth (DeWitt Pro-5 Weed Barrier; DeWitt Company, Sikeston, MO, USA). This cloth served as a barrier minimizing contact with EMF present in the field soil, resulting in willow plots with a low abundance of EMF [nonmycorrhizal willow (NW); Fig. 3a]. The other willow within each pair was left open to colonization by naturally occurring fungi over the course of the experiment, resulting in plots with a high abundance of EMF [ectomycorrhizal willow (MW); Fig. 3a]. Compared to naturally occurring willows, the transplants had very small canopies ( >150 vs. <50 cm in diameter); thus, potential canopy effects were likely minimal in NW and MW treatments. After 2 years, the willow transplants were excavated. The root mass of NW and MW willow transplants was visually similar in size, indicating that the landscaping cloth did not restrict root growth of NW transplants. At that time, we also assessed final EMF colonization of each willow transplant to verify treatment efficacy and mycorrhizal status (see below for method of determining EMF colonization). To isolate shading effects independent of the presence of willows or EMF, we placed 80% shade cloth (Gempler’s, Janesville, WI, USA) over open meadow plots (S). We also established unmanipulated control plots in the open meadow (OC) and understorey of naturally occurring willows (WC). Individual plots were c. 0.25 m2 and there were 10 replicates per site for a total of 30 replicates per treatment. All treatments within a replicate were at least 3 m from each other and from the nearest naturally occurring willow (Fig. 1). Due to elk damage and transplant mortality, the total sample sizes for NW and MW treatments were reduced to 18 and 21, respectively.
Figure 1. To test the effects of above- and below-ground interactions on arbuscular mycorrhizal fungal colonization (experiment 1), we manipulated canopy cover, willow presence and ectomycorrhizal fungal (EMF) colonization in five experimental treatments. Within each replicate experimental plots (0.25 m2) were at least 3 m from each other and from the nearest naturally occurring willow. Landscaping cloth was used to minimize EMF colonization of nonmycorrhizal willow transplants (NW). Ectomycorrhizal willow transplants (MW) were open to colonization by naturally occurring fungi. Shade cloth was erected in the open meadow to mimic the effects of a willow canopy (S). Unmanipulated plots in the willow understorey (WC) and open meadow (OC) served as controls. Four herbaceous forbs (Polemonium delicatum, Polemonium viscosum, Taraxacum ceratophorum and Taraxacum officinale) were transplanted in each plot to minimize variation in plant community composition.
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To assess treatments effects on mycorrhizal fungal colonization, we collected soil cores (6 × 10 cm) in July of each year. Soil cores were collected from as close to the centre of each plot as possible and stored at 4 °C until processed. We isolated and washed the roots from each core; we then examined 300 root tips for EMF colonization using a dissecting microscope at 10× magnification. EMF root tips were identified based on shape, colour, branching pattern and the presence of a mantle. EMF colonization was calculated as the proportion of EMF root tips. After examining the roots for EMF, we subsampled the herbaceous roots from the soil core, avoiding woody roots that were likely from willow hosts. The herbaceous roots were cleared in 10% KOH at room temperature for 48 h, briefly acidified in 0.1 N HCl, stained with 0.05% trypan blue for 24 h and destained in lactoglycerol for 24 h (Phillips & Hayman 1970). Using a compound microscope at 400× magnification, we examined a total of 100 points along ten haphazardly selected root fragments for AMF colonization (McGonigle et al. 1990). AMF colonization was calculated as the proportion of herbaceous root length containing AMF structures (i.e. arbuscules, vesicles and coils).
As mycorrhizal fungal colonization depends on the presence of appropriate hosts, variation in plant community composition among plots could influence the results of this experiment. We used two approaches to account for variation in the herbaceous community among the five treatments. First, for each plot, we estimated the percentage cover by graminoid and forb host species in both years of the experiment. Second, we transplanted adults of four dominant alpine forbs at our study site (P. viscosum, P. delicatum, T. ceratophorum and T. officinale) into each plot, including the OC and WC. These species were examined in a previous survey of mycorrhizal associations on Pennsylvania Mountain, and results from that study indicate that AMF readily colonize all four plant species (Becklin & Galen 2009). The herbaceous transplants were collected from resident populations on Pennsylvania Mountain in early June of each year. Although these transplants only comprised a small portion of the total plot vegetation (average = 30%), transplanting these species into all five treatments decreased variation in plant community composition that could obscure treatment effects. By the end of the experiment, AMF colonized all herbaceous transplants (see Table S1 in the Supporting Information).
Abiotic conditions and resource availability can also impact mycorrhizal colonization. To evaluate whether abiotic conditions varied among treatments, we monitored air temperature, relative humidity and photosynthetic photon flux density (PPFD) in a subset of the plots at each site (N = 6 per treatment). Air temperature and relative humidity were measured using hobo H8 environmental data loggers (Onset Computer Corporation, Bourne, MA, USA). PPFD was measured using quantum light sensors connected to a CR-10 environmental data logger (Campbell Scientific, Inc, Logan, UT, USA). Data were collected between 09:00 and 15:00 on 13–18 July 2007. We also collected soil cores (2 × 10 cm) from a subset of plots in mid-July of each year after at least 3 days without rain to determine soil pH, percentage organic matter, extractable Bray I phosphorus concentration, extractable nitrate concentration, extractable ammonium concentration, exchangeable calcium concentration, exchangeable magnesium concentration, exchangeable potassium concentration and soil water content (N = 6 per treatment; Soil and Plant Testing Laboratory, University of Missouri, Columbia, MO, USA). Methods for the soil analyses are listed in Appendix S1 (see Supporting Information).
Variation in environmental conditions in the first experiment (soil pH, percentage organic matter, soil nutrient concentrations and soil water content) was analysed using multivariate analysis of variance (manova) with site (3682, 3626 and 3588 m a.s.l.), treatment and their interaction as fixed factors. A separate analysis was conducted for above-ground conditions (air temperature, relative humidity and PPFD) as these variables were only measured in S, OC and WC treatments. Differences in percentage plant cover over time were analysed using repeated measures analysis of variance (rm-anova) with site, treatment and their interaction as fixed factors. Treatment effects on colonization by EMF (proportion of colonized root tips) and AMF (proportion of colonized root length) over time were also assessed using rm-anova with site, treatment and their interaction as fixed factors. As arbuscules are indicative of functional AM associations, we conducted this analysis for arbuscular colonization as well as total AMF colonization. Planned contrasts were used to evaluate whether the presence of a canopy (S vs. OC), willow (NW vs. OC) or EMF (MW vs. NW) affected AMF colonization. Planned contrasts were also used to evaluate whether the effect of EMF on AMF colonization was similar in the experimental and control plots (MW vs. WC). The quantitative relationship between EMF and AMF colonization was evaluated using analysis of covariance (ancova) with treatment as a fixed factor. To evaluate the effect of soil conditions on EMF and AMF colonization, we first conducted a principle components analysis to transform the soil components (soil pH, percentage organic matter, soil nutrient concentrations and soil water content) into predictor variables. We then analysed the relationship between mycorrhizal colonization and each principle component (eigenvalue > 1) using ancova with treatment as a fixed factor.
In the second experiment, we evaluated the effect of leaf litter on EMF and AMF colonization using anova with year, site, habitat, leaf litter treatment and their interactions as fixed factors and replicate as a random factor. The quantitative relationship between EMF and AMF colonization in plots with and without leaf litter was evaluated using ancova with site, habitat, litter treatment and their interactions as fixed factors. The effect of leaf litter biomass on EMF and AMF colonization was also evaluated using ancova with site, habitat and their interaction as fixed factors.
All statistical analyses were conducted with the statistical program jmp 8.0 (SAS Institute Inc., Cary, NC, USA). Normality and homoscedasticity were improved by arcsine-square root transforming AMF and EMF colonization, percentage plant cover and soil water content. Soil pH, phosphorus concentration and nitrate concentration were log transformed. All other variables met the assumptions of normality and equal variances.