Geographic locations and tree species
Study sites were located within the ATE of the Snowy Range in south-east Wyoming (41°20′30″ N 106°14′30″ W) and the Beartooth Mountains in north-central Wyoming (45°01′59″ N 109°24′21″ W). The climate for both mountain ranges is characterized by cold winters from September to June with minimum annual air temperatures from −25 to −40°C. Summers typically have daily maximum air temperatures below 25°C with frequent clear skies and nocturnal frost. Much of the annual precipitation comes during winter as snow, and snowbanks frequently persist into August. Two codominant conifer species, Picea engelmannii Parry and Abies lasiocarpa (Hook.) Nutt., occur in islands interspersed among alpine tundra within the ATE.
The ATE consists of three zones: forest, timberline, and treeline. At timberline, trees of mature stature form a patchy mosaic of tree islands. Treeline is the upper limit of tree growth, where trees are also in tree islands but are severely reduced in growth with krummholz morphology. No conifer seedlings or juveniles were found at treeline, and we therefore limited our field study to conifers found in the forest and at timberline.
Seedling occurrence and percent colonization
The frequency of C. geophilum colonization was measured for all naturally occurring P. engelmannii and A. lasiocarpa seedlings and juveniles found at our study sites. Seedlings were distinguished from juveniles based on the presence of cotyledons and primary needles, and wood formation in stem. In 2002, measurements were made on 101 juveniles found along transects of 2 m width and 30 m length, which were positioned in representative areas of forest and timberline zones of each mountain range (n = 3 per life zone). Additionally, the distance to the nearest adult tree was measured for each juvenile found at timberline. No first-year seedlings were detected in 2002, however, 83 conifer seedlings were found in 2003 along two transects that traversed the forest and timberline life zones in the ATE of the Snowy Range (N = 2 transects per life zone).
Root systems of young conifers were carefully excavated from the soil, gently washed with water to remove soil debris, and stored in 70% ethanol. All root lengths were later examined for the presence of fungal colonization under 40× magnification. Cross sections of colonized root tips were examined for the presence of hartig nets, and colonization levels were calculated for individual conifers as the percent of root tips colonized. All fungi that formed mantles and hartig nets on roots of our subject plants had the distinctive morphological features of C. geophilum (e.g. Goodman et al., 1996; Hagerman et al., 1999; Agerer, 2002). Roots of older conifers near our younger subject plants appeared to have a much higher diversity of black and ectomycorrhizal fungi, as revealed by morphological and molecular assessments (Hasselquist et al., unpublished data). Our study focused on C. geophilum because all other fungal species we observed on roots were not ectomycorrhizal. Any other ectomycorrhizal species that may have been present on roots of subject plants, but were not detected, would have had very small abundances.
Seeds of P. engelmannii were sterilized in 30% H2O2 for 10 min, rinsed three times with deionized water, and then placed into 20 cm × 20 cm × 1 cm Plexiglass microcosms. These microcosms were filled with a commercial organic potting mixture (FertiLome, Voluntary Purchasing Groups Inc., Bonham, TX, USA) that was pasteurized at 80°C for 4 h in a soil sterilizer (model SST-60R, Pro-grow Supply Corp., Brookfield WI, USA). Soil in the microcosms was shaded from light to inhibit algal growth. Following germination, approx. 1 cm3 of agar that either contained C. geophilum or was sterile (control group) was placed within 1 cm of seedling roots in each microcosm (n = 14 per treatment) and grown for two months to ensure fungal colonization, which was verified visually.
All 28 seedlings were later transplanted individually into 400 ml pots containing a sterilized sandy loam with a pH of 7.2 and a bulk density of 1.4 g cm−3. Volumetric soil water content was monitored gravimetrically and maintained near 0.20 m3 H2O m−3 soil with daily additions of distilled water. Approx. 72 mg of nitrogen as aqueous ammonium nitrate was initially added to each pot. Transplanted seedlings were placed under a 16 h: 8 h (day: night) photoperiod generated by natural sunlight and two 400-watt metal halide lamps (G3400-1G+, Rudd Lighting Inc., Racine, WI, USA), which generated maximum photon flux densities near 1000 µmol m−2 s−1 (400–700 nm). Air temperatures ranged diurnally from 10 to 25°C. After 6 wk of growth, daily watering stopped and photosynthesis measurements began for four consecutive days, during which volumetric soil water content decreased from 0.20 to 0.068 m3 H2O m−3 soil. Destructive measurements of water status, biomass, and nutrient concentrations were performed on the fourth day following cessation of watering. Volumetric soil water content was determined gravimetrically each day as the soils were allowed to dry (n = 28).
Photosynthesis was measured on P. engelmannii seedlings for each of the 4 d soils were allowed to dry (n = 7 for colonized seedlings and n = 14 for noncolonized seedlings). Instantaneous photosynthetic carbon assimilation rates (A), transpiration (E), and water use efficiency (WUE = A/E) were measured with a closed-flow gas exchange system (LI-6400, Li-COR Biosciences, Inc., Lincoln, NE, USA) equipped with a light source. Readings were taken at ambient CO2 levels (approx. 370 ppm), at a light level of 1000 µmoles m−2 s−1, and at temperatures near 27°C. Measurements were made on entire seedlings, and reported on a silhouette leaf area basis, which is the amount of leaf area perpendicular to, or illuminated by the artificial light source in the chamber of the LiCOR 6400. Leaf areas were determined by photographing seedlings from the angle in which they were illuminated during measurements, with objects of known size in the field of view (model Coolpix 990, Nikon, USA). Leaf areas in the resulting digital image were then determined using image-processing software (Image J, Scion Image, Frederick MD, USA).
Xylem water potential were measured for all seedlings using a Scholander-type pressure chamber (model 1000, Plant Moisture Stress, Corvallis, OR, USA). Root systems were carefully harvested and thoroughly rinsed, and examined under 40× magnification to determine the percent of root tips colonized by C. geophilum. Biomass of roots and shoots were measured to the nearest tenth of a milligram at the end of the experiment, following 24 h of drying at 80°C. Phosphorus concentrations were determined by first digesting each entire seedling in a 10 : 1 nitric acid : sulfuric acid mixture using the method of Hall (1995), and then measuring absorbance at 880 nm using an auto-analyzer after reacting the resulting orthophosphate with molybdenum in the presence of antimony in sulfuric acid, using the manufacturer's methodological specifications (for Standard Methods of US Environmental Protection Agency; Alpken FS 3000, OI Analytical, College Station, TX, USA) at the Center for Ecological Research and Education at Idaho State University.
Chi-square analysis was performed to determine if there was a significant difference in the relative abundance of conifer species between the forest and timberline life zones. Because of the low degrees of freedom associated with the data sets (d.f. = 1), we used the Yates correction for continuity while performing the chi-square analysis (Zar, 1999). One-way analyses of variance (anova), followed by Tukey tests (α < 0.05) for mean separation (Zar, 1999, JMP Version 3.2.2., SAS Institute, Cary, NC, USA), were employed to detect significant differences in C. geophilum colonization between conifers species (P. engelmannii and A. lasiocarpa) and between each life zone (forest and timberline). Least-squares regression analysis was used to investigate the correlation between C. geophilum colonization levels at timberline and the distance to nearest adult tree. One-way anova was also used to detected differences in physiological measurements between colonized and noncolonized seedlings. Regression analysis was used to determine correlations between the percent of root tips colonized and measurements of host conifer physiology.