Catena 147 (2016) 216–224

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Soil microbial community profiles and functional diversity in limestone
cedar glades
Jennifer Cartwright a,⁎, E. Kudjo Dzantor b, Bahram Momen c
U.S. Geological Survey, Lower Mississippi-Gulf Water Science Center, 640 Grassmere Park, Suite 100, Nashville, TN 37211, USA
Tennessee State University, 3500 John A Merritt Blvd., Nashville, TN, 37209, USA
University of Maryland, College Park, 1425 Animal Sciences/Agricultural Engineering Building, College Park, MD 20742, USA

a r t i c l e i n f o a b s t r a c t

Article history: Rock outcrop ecosystems, such as limestone cedar glades (LCGs), are known for their rare and endemic plant spe-
Received 11 November 2015 cies adapted to high levels of abiotic stress. Soils in LCGs are thin (b25 cm), soil-moisture conditions fluctuate sea-
Received in revised form 5 May 2016 sonally between xeric and saturated, and summer soil temperatures commonly exceed 48 °C. The effects of these
Accepted 6 July 2016
stressors on soil microbial communities (SMC) remain largely unstudied, despite the importance of SMC-plant
Available online 16 July 2016
interactions in regulating the structure and function of terrestrial ecosystems. SMC profiles and functional diver-
sity were characterized in LCGs using community level physiological profiling (CLPP) and plate-dilution frequen-
Limestone cedar glades cy assays (PDFA). Most-probable number (MPN) estimates and microbial substrate-utilization diversity (H) were
Rock outcrop ecosystem positively related to soil thickness, soil organic matter (OM), soil water content, and vegetation density, and were
Environmental microbiology diminished in alkaline soil relative to circumneutral soil. Soil nitrate showed no relationship to SMCs, suggesting
Soil microbial community lack of N-limitation. Canonical correlation analysis indicated strong correlations between microbial CLPP patterns
Community level physiological profiles and several physical and chemical properties of soil, primarily temperature at the ground surface and at 4-cm
depth, and secondarily soil-water content, enabling differentiation by season. Thus, it was demonstrated that sev-
eral well-described abiotic determinants of plant community structure in this ecosystem are also reflected in SMC
Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license

1. Introduction extreme temperatures at the soil surface combined with widely-fluctu-
ating hydrologic conditions. Soil surface conditions are characteristically
Rock-outcrop ecosystems in the USA contribute to regional and hot and xeric in summer and experience prolonged saturation in winter
global plant biodiversity at levels vastly disproportionate to their small (Baskin and Baskin, 1989). These stress factors in LCGs effectively ex-
geographic footprints (Quarterman et al., 1993). For example, limestone clude most vegetation of the surrounding mesophytic forest and thus
cedar glades (LCGs) of the southeastern USA support unique assem- help maintain habitat for endemic plants (Baskin and Baskin, 2003,
blages of plant species including rare endemics listed under the U.S. En- 1989, 1999). LCGs function as edaphic climax ecosystems where soils
dangered Species Act (Baskin and Baskin, 1999, 1989). Despite their are too thin to support the growth of trees. Instead, the vegetation is
botanical importance, many LCGs face ongoing degradation (Baskin et dominated by C3 forbs and C4 summer annual grasses, accompanied
al., 1995; Quarterman et al., 1993). Indeed, roughly 50% of total LCG by mosses, lichens, and cyanobacteria in microhabitats of extremely
area and 90% of ecologically intact LCGs have been lost from Middle Ten- thin soil or exposed limestone bedrock (Baskin and Baskin, 1999;
nessee, rendering LCGs an “endangered ecosystem” (Noss et al., 1995). Baskin et al., 2007).
Within their geographic range, LCGs provide a unique physical envi- Plant communities in LCGs have been systematically described (e.g.
ronment, characterized by very thin soils that experience seasonally- Baskin and Baskin, 2003; Somers et al., 1986; Taylor and Estes, 2012)
but their associated soil microbial communities (SMCs) are largely un-
studied. Indeed, SMCs have received little scientific attention in rock
Abbreviations: CCA, Canonical correlation analysis; CF, Canonical function; CLPP, outcrop ecosystems throughout eastern North America, despite the rec-
Community level physiological profiling; H, Shannon-Weaver Index representing ognized importance of these ecosystems to global plant biodiversity
microbial substrate-utilization diversity; LCG, Limestone cedar glade; LOI, Loss on (Quarterman et al., 1993). This is a key knowledge gap, because plant-
ignition; MPN, Most probable number; OM, Organic matter; PDFA, Plate-dilution SMC interactions influence a host of ecological processes such as bio-
frequency assay; SMC, Soil microbial community.
⁎ Corresponding author.
geochemical cycling, plant-pathogen dynamics, succession, and soil dis-
E-mail addresses: (J. Cartwright), turbance (de Vries et al., 2012; Reynolds et al., 2003; van der Heijden et
(E.K. Dzantor), (B. Momen). al., 2008).
0341-8162/Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (

Molano-Flores and Bell. PDFA was performed on USA). 1950.. going from the least Twelve LCGs were selected to study spatial and temporal patterns of to the highest concentration in a dilution series. NJ. and forbs and the remainder within a 3-m buffer of pre. analyzed for organic matter (OM). Noss et al. culturable microbes in soil. CO. pH.3. Materials and methods At each sampling event. supplementary material)..2. within the park are Middle Ordovician limestones (Thornberry. Benton. USA). The vegetation cover at each quadrat was characterized based on vi- able management practices for fragile ecosystems such as LCGs sual Within LCGs at this site. 2. ples per quadrat) and ground-surface temperature were measured system to support scientifically-sound management of LCGs. Oak Brook. 1 Data used in the analysis for this study are available from http://dx. checked for contamination after 48 h. 2012). the objectives of this study were (1) to char. and sparsely-distribut. Van der Putten. CA). and nitrate concentration. Soils were then plain in the Nashville Basin section of the Interior Low Plateaus physio. USA). J. or shrub. a thin. tion category. 1996. Inc. forb. cm-diameter metal probe into the soil until it contacted bedrock (4 sub- vation challenges. respectively. following Cofer et al. such as projected land-use and climate changes. assessed using community-level physiological profiling (CLPP). Frank- cedar forest (Cofer et al.5. such as from climate change or exotic each quadrat at each sampling event (see Section 2. Loveland. Two soil associations are present: the Gladeville-Rock pH Meter (Spectrum Technologies. with each quadrat sampled 4 times at approximately 4-month intervals. levels were estimated using a Hach Platinum Series combination nitrate Department of Agriculture Soil Conservation Service. lichens.. Dickinson. USA). zones of exposed bedrock surrounded by thin soil support mosses. and 10−7) were inoculated soil in bedrock cracks and to ecotones with the surrounding oak-red onto plate count agar (Difco™. Improved understanding of SMCs and their relationships to centage of each category was estimated for each quadrat and catego- abiotic stressors is thus vitally important to the long-term conservation rized as “none. 86°25′58″W). CA. OM graphic province (Fenneman and Johnson. a portion of each soil USA (35°52′35″N.4. 2008. The per- al. OH. 16 months (February 2012 to May 2013). using a Taylor 9842N digital thermometer and a Taylor 1523 digital thermometer/hygrometer. Plainfield.5066/ 1991. Study area glass jars and transported on ice to the laboratory for analysis.1. SMC metabolic profiles were Barfield soils. 2. A number of in- vestigators have indicated an urgent need for development of sustain. Quadrats were sampled according to a rotational schedule over soil samples collected from May 2012 to May 2013. to estimate MPN of culturable microbes per mL of soil suspension at dominantly Juniperus virginiana forest surrounding glades. respectively (Taylor Inc. with the dark at 25 °C for 7 days. 2010). 2004). Gravi- metric soil water content (3 subsamples per soil sample) was measured Soil microbial investigations1 were performed in LCGs at Stones using a Mettler Toledo HB43 halogen moisture analyzer (Mettler Tole- River National Battlefield (Fig.5. do.3 (Esri. Nordman. the total number of marked circles with pos- approximately 80% (28 quadrats) located in areas dominated by moss. Quadrat po. 2004. lin Lakes. Harris and Sommers. In areas with PDFA (Harris and Sommers. Soil nitrate outcrop-Talbott and the Talbott-Barfield-Rock outcrop complex (U. 10− 3. dozen LCGs are present within the park. Hayward.” “30% to 70%. in petri dishes. Preston-Mafham et al. 2012). IL.1. ranging from b70 m2 to ap- proximately 2800 m2. both of which have surface layers of silty clay loam. These glades form a roughly ring-shaped config. Plate-dilution frequency assay of microbial populations ed herbaceous plants (Fig. along with a 95% confidence interval on MPN sition was randomly assigned using ArcGIS v. Vascular plants were broadly categorized as (Baskin et al. S1. 2008. Vegetation characterization these processes (Harris. USA). The petri dishes were prepared from dehydrated agar. which is Service. 10− 5. acterize spatial and temporal patterns of soil microbial numbers and functional diversity in LCGs.S.5.. Several electrode (Hach Company. 10− 6. Thornberry-Ehrlich. 10− 4. (2008). For chemical analysis. species invasions (Cofer et al. 2012). 1968) was used to estimate an MPN of greater soil thickness. vine.2. 1973).. 1977).. 1).” “b 30%. / Catena 147 (2016) 216–224 217 Ongoing efforts to restore glade environments and to understand multiple subsamples were averaged to obtain a single measurement for and predict ecological changes. USA).. could be strengthened by studies of SMC dynamics that interact with 2. 1) in Rutherford County. Microbial community level physiological profiling (CLPP) CLPP based on sole-carbon substrate utilization (Garland and Mills. and Company. C4 summer annual grasses and C3 forbs are pre. 2. 1968). Soil microbiological characterizations uration surrounding a topographic high underlain by a structural basin (Cofer et al. This park is within a karst sinkhole sample was air-dried and sieved using a 2-mm screen. IL. Soil samples were sealed in autoclaved 2.5-m quadrats were established. Soil temperature at 4-cm soil depth (3 subsam- study also contributes to a holistic ecological understanding of this eco. Soil pHH2O (1:1) was measured using a calibrated FieldScout SoilStik Ehrlich. and pre-marked 2. 2009. For certain soil properties. 1946)..S.5-m × 0.4). Thirty six 0. 9. 2002) was performed F7NV9G9C using 96-well Biolog® EcoPlates (Biolog Inc. This study provides baseline information useful for Depth to bedrock (soil thickness) was measured by inserting a 1- monitoring abiotic and biotic changes in LCG soils in the face of conser. 1995. This samples per quadrat). Areas between glades are predominantly Talbott and a most probable number (MPN) method. culated for each quadrat as the sum of individual scores for each vegeta- In service of these goals. 1977). Quarterman et graminoid. (Cochran. 2007. cyanobacteria. and (2) to relate these patterns to key soil 2. assigned from 0 to 3. Bedrock formations was estimated using the loss-on-ignition (LOI) method (Davies.” or “N70% covered” with scores of plant biodiversity in LCGs and related ecosystems under changing en. 2008. Following incubation in SMCs (Fig. Nordman. with woody species generally confined to pockets of deep sample (10− 2. silty ator (4 °C) within 6 h of leaving the field and was processed for micro- clay loam with limestone flags throughout the soil and forming a layer bial characterization within 24 h. soil samples were collected at a depth of 3– 4 cm using sterile techniques. Sampling design with eight circles per dilution level. An overall vegetation score was cal- vironmental conditions. Tennessee.. the 10−2 dilution level. Soil microbial populations were at the soil surface (U. Garland. 1993). Redlands. itive growth (colony development) was referenced to statistical tables graminoids. For each . Ten microliters (10 μL) from each di- lution were inoculated into the center of each circle. LCGs consist of outcrops A separate portion of each soil sample was transferred to a refriger- of thinly-bedded limestone surrounded by Gladeville soil. Department of Agriculture Soil Conservation characterized using a plate-dilution frequency assay (PDFA)..doi. Columbus. Six levels of serial dilutions from each soil dominant. Soil physical and chemical analysis physical and chemical properties that define the abiotic stress regime in this ecosystem. Cartwright et al. 2.

. / Catena 147 (2016) 216–224 Fig. Physiographic divisions are from USDA (2015). Map of sampling locations in 12 selected limestone cedar glades (LCGs) at Stones River National Battlefield. 1. Cartwright et al.218 J. Polygons representing LCGs are courtesy of the National Park Service.

.. T ¼ ðR−C Þ=AWCD ð1Þ higher water content.46 22. CLPP patterns for soil microbial communities taken per quadrat or soil sample were averaged prior to statistical anal. all other properties were measured 4 times per quadrat over 16 area under the incubation curve) to the sum of the microbial activities months. and high-MPN among the constructed functions (Lattin et al.53 38.26 Gravimetric soil water 144 3 3. For certain properties. higher soil OM. NC. To determine the effects of various soil physical and chemical properties The soils of the LCGs in this study were generally thin (all b 23 cm on MPN. ferent (P b 0.07 29. 150 μL of the 10−2 dilution level suspension from the PDFA Because plots of H against soil physical and chemical properties indi- procedure (Section 2. March 2013. SAS Institute. To correct for inoculum-density effects. chemical properties. Immediately fol.6. 2002). This approach has advantages over the use of quantile regression at only one or a few quantile levels because limitations on both the maxi- 2. Hayward. USA) at an absor. contained 31 carbon substrates replicated 3 times on each plate. on all substrates for a given EcoPlate. Soil and ground-surface tempera- tures were often very hot in summer.1 × 106. EcoPlates were read at factor (Cade and Noon. multiple subsamples 2. 94 out of 99 soil samples were categorized as follows: low: 2. Gravimetric soil water content ranged from xeric (PROC ANOVA and PROC GLM. Cary. bance of 590 nm to monitor changes in respiration over time. Results organisms per mL of suspension of the 10−2 dilution level.40 5. where T represents transformed substrate-level response values. 2). one-way ANOVA and Tukey's post-hoc tests were performed depth to bedrock). each measurement represents an average of R subsamples.3 × 105 to 7. 96.12 0. neutral to slightly alkaline.1) were inoculated into an EcoPlate. EcoPlates were covered and incubated in the dark Heteroscedasticity implies that multiple rates of change (slopes) may at 25 °C.5.32 To assess community-level microbial substrate-utilization diversity Vegetation score 36 – 1 8 2.40 22.05). of carbon substrates (31 for EcoPlates). the area under the incubation curve—from 48 h to 120 h of Soil organic matter 144 – 0. such that between-group MPN estimates were significantly dif.3 × 106 to 13. USA). Cartwright et al. CCA was performed on CLPP data from soils (PROC CANCORR. Soil physical and chemical properties in relation to MPN MPN groups so these samples were not retained for further analysis. 2012).43 strates. LOI) Mafham et al.. and more dense vegetation (Fig. Higher MPN was significantly associated with thicker soils.1.63 8. 2011).61 11. quadrat (36 quadrats). exceeding 38 °C and 48 °C 2.6.40 22. as can portions of the response variable distribution most All statistical analyses were performed using SAS 9. soil properties and H (PROC QUANTREG. Neither soil nitrate nor either of the temperature variables (ground-surface temper- hXn i AWCD ¼ ðR−C Þ =n ð2Þ ature and soil temperature at 4-cm depth) were significantly related to i¼1 MPN.50 content (percent) 2. To investigate relationships between CLPP patterns of SMCs ysis to avoid pseudoreplication. At each sampling event. J. Soil sample categorization based on MPN of linear combinations of the original variables within each set Based on PDFA analysis. 2003). plate-level transformations MPN estimates of culturable microbes in 94 soil samples ranged for carbon substrate data followed Garland and Mills (1991): between 2.2. Table 1 strate).05 incubation—was calculated and used in subsequent analysis (Preston. . EcoPlates cated highly heteroscedastic.3 (SAS Institute. and canonical corre- lation analysis (CCA). limited by the limiting factor(s) under consideration (Cade and Noon. measured and unmeasured limiting factors (Schmidt et al. and MPN and CLPP were analyzed using analyses of variance (ANOVA). describe the relationships between the response variable and a limiting ride..7 × 106 3.50 48.04 8. SAS Institute.62 8.. the Shannon-Weaver index (H) was calculated as: Ground-surface 144 – −1. Relationships between vegetation.3. Microbial substrate-utilization diversity Soil nitrate (μg g−1 NO3-N) 144 – 1. SAS Institute. tetrazolium chlo.92 temperature (°C) Xn Soil temperature at 144 3 0.28 7. CLPP Quantile process plots show the slope parameter and associated 95% was performed on soil samples collected from February 2012 to confidence interval at all quantile levels for the quantile regressions. 2010). R is the mean absorbance of the response wells (3 wells per carbon sub. Inc. medium: 1.59 4.67 from CLPP data.2). Quantile regression is well-suited to such intervals of 24. see Section 2.27 0. and 120 h after inoculation using a Biolog distributions when ecological responses are constrained by multiple MicroStation plate reader (Biolog.4. depth to bedrock and vegetation were measured once per where pi is the ratio of the microbial activity on each substrate (T values. wedge-shaped distributions (Section 3. 2011). 2003. Three quantile regression was used to investigate relationships between these wells contained sterilized water to serve as controls. Schmidt et al. Ye and Wright.67 16. provided a measure of substrate utilization. high: 4. with variable levels of using a general linear model with the three MPN groups as categories OM (Table 1). quantile regression analysis. Soil pH 144 – 7. 2011). Color development of a respiration indicator.3 × 105 and 13.8 × 105. groups.7 × 106. CFs) were calculated to maximize the correlation were used to assign soil samples to low-. Transformations for CLPP data respectively. Statistical analyses mum and minimum values of the biological response variable can be identified. 72. (below 5%) to saturated (above 45%). and various soil physical and chemical properties. medium-. soil physical and 2003.4.94 H ¼ − i¼1 pi ð ln pi Þ ð3Þ 4-cm depth (°C) N = number of measurements.46 49. Pairs 2.6. lower (more neutral) pH. lowing inoculation. and n is the number pled from February 2012 to May 2013. T). C is the mean absorbance value of the 3 control wells. CA. but within-group MPN estimates were not.6.78 1. 48. In this way. To integrate time-series data N R Min Max Mean Standard deviation from multiple EcoPlate readings (for AWCD and for individual sub- Depth to bedrock (cm) 36 4 2.1. AWCD is Soil physical and chemical properties across 36 quadrats in 12 limestone cedar glades sam- average well color development for the EcoPlate.04 0. (fraction. Estimates for 5 samples were significantly lower (1) or higher (4) than any of the 3. 95% confidence intervals on MPN estimates (Canonical Functions.6. 2012). / Catena 147 (2016) 216–224 219 soil sample.84 5.20 8.0 × 106 to 3.80 0.

For positive for 0. 2. For quantile ranges. C. P N 0. . of the soil property on H is considered positive (P b 0. Conversely. (92%) above 3. soil nitrate. 3A). 3). soil temperature at 4-cm depth. D. quantile process plots based on quantile regression across all quantile The association between soil thickness (depth to bedrock) and H was levels. E.05). depth to bedrock. τ.05). Distribution by most probable number (MPN) category of A. the effect values of H were not different across MPN groups (F = 2. ground-surface temperature and H. the slope parameter estimate of the regression function can be plotted (vertical axes in Fig. τ% of H values are ≤ the τ quantile regression func. at any given circumneutral soils and decreased with increasing alkalinity (Fig.75 (Fig. Cartwright et al. soil organic matter. highly heteroscedastic distributions (Fig. 2003). / Catena 147 (2016) 216–224 Fig. with 115 samples out of 125 the soil property under consideration affects H.2. indicating that H was higher in tion of the given soil property (Cade and Noon. Several soil properties showed significant associations with H across Consequently.38.21. G. P N 0. Differing letters above each boxplot represent significant differences (P b 0. supplementary material). gravimetric soil water content. 3. τ (Fig.05) and where the entire shaded area is above zero on the vertical axis.74.220 J.05). B. 3).0.15 b τ b 0. confidence interval (shaded area) can be compared to zero to test if lization diversity ranged from 2. soil property effect on H is negative.00 to 3. with inferences following Schmidt et al. H was uncorrelated with MPN (r = 0. 3) and its 95% Values of the Shannon-Weaver Index (H) for microbial substrate-uti. 3C).25 b τ.05). τ indicating that microbial substrate-utilization diversity was not merely values for which the entire shaded area is below zero indicate that the a function of the numbers of culturable microbes in soils. and H was negative for 0. Values of τ for which the shaded Plots of H versus soil physical and chemical properties indicated area includes zero indicate no effect of the soil property on H (P N 0. S2. (2012). The association between soil pH any quantile level τ. Microbial substrate-utilization diversity τ value (horizontal axes in Fig. vegetation score. soil pH. these relationships were investigated by means of most of the intermediate (not extremely high or low) quantile ranges. Thus. F.

G. 3G and H.15 b τ b 0. ground-surface temparature and H. Cartwright et al. ground-surface temperature and soil temperature at 4-cm depth had Other soil properties showed no significant associations with significant negative associations with H across limited quantile ranges: H. Gravimetric soil water content had a positive association with H for level (Fig. E. soil temperature at 4-cm depth. ranges.60 and 0.50 and 0. soil pH. Quantile process plots showing slope parameter estimates and 95% confidence intervals (shaded areas) for the Shannon-Weaver Index (H) of microbial substrate utilization for A. D. soil organic matter. B.30 b τ b 0.80 (Fig. with H only for 0.65. gravimetric soil water content. J. soil nitrate. Soil nitrate was not associated with H at any quantile respectively). 3D). 3F).30 b τ b 0. The association with vegetation score was pos. Both itive for 0. / Catena 147 (2016) 216–224 221 Fig. depth to bedrock.25 b τ b 0. respectively (Fig. 3E). C.85 (Fig. 3. All horizontal axes are quantile levels (τ). . whereas soil OM had a significant positive association 0.10 b τ b 0. or their associations were limited to relatively narrow quantile 0.65 (Fig. 3B). vegetation score.55 b τ b 0. F. meaning that τ% of H values are ≤ the quantile regression function of the given soil property.

48⁎ 0. / Catena 147 (2016) 216–224 Table 2 culturable microbes. and could in fact be qualitatively different from. significantly positive only for limited ranges of τ based on quantile regression.93⁎ 0. 2003). given that all Soil organic matter (LOI) −0. soil pH ranged from b4. The absence of a relationship between soil nitrate and any of the SMC indicators in this study may indicate that N was not a limiting nu- trient in soil at this study site. SMCs also help deter- 1994). (Table S1.27⁎ 0. soil chemistry is influenced by lime- ⁎ indicates significant correlation at P b 0. mine plant community composition (Wardle et al. P b 0. Nordman. The carbon substrates that were correlated with CFsubs1 and CFsubs2 from litter and root exudates (Thomson et al. 4. 2009).. thin and relatively alkaline soils were associated both Ground-surface temperature (°C) 0. 1993.. the full suite of carbon sources available for mi- crobial metabolism in LCG soils is likely to be vastly more complex than. suggesting that 4. may account in part for previous findings of reduced soil respiration in solute values of the correlations between original variables and the ca. Soil temperature at 4-cm depth (°C) 0. Influence of soil physical and chemical properties on soil microbial tial variability in soil OM was reflected in CLPP data.37⁎ 0. rel- atively alkaline zones support lower numbers of culturable microbes Of the CFs constructed.. The second function (CFsoil2) primarily reflected soil OM con.. 2003. Plant community composition influences SMCs ground-surface temperature.26⁎ 0.69⁎ 0. the first function (CFsoil1) Not surprisingly.44⁎ −0. that represented by the 31 substrates present on the EcoPlates in this study (Konopka et al. 4).. 2010. and (2) all six of the EcoPlate substrates that are components of Findings from this study indicate that microbial substrate-utilization root exudates were significantly correlated with either the first or sec- diversity in LCG soils is not simply a reflection of relative numbers of ond CF. carboxylic acids. plant community composition that directly affect soil microbial re- amino acids. Our findings suggest that these thin-soil.29⁎ al. All carbon substrates on the EcoPlates that are known compo.75⁎ −0.30⁎ fects of soil thickness and pH were so clearly discernible.30⁎ 0. respectively. soil water content was positively related both to MPN and to H.12 In particular.25⁎ soils in this study were very thin and soil pH values were within a rela- Depth to bedrock (cm) −0. Thin- soil areas support distinct vegetation communities (Quarterman et al. sponses (Cong et al. physical and chemical properties. and polymers (Zak et al. 3. N-fixing cyanobacteria (e. This pattern for 57% of the total variability of the multivariate data. certain soil properties were prominently related both to microbial abundance and to substrate utilization diversity. supplementary material) were spread fairly evenly across all Differences in quantity and quality of these inputs are reflections of six of the substrate guilds present on the EcoPlates: carbohydrates. Findings from this study highlight the importance of seasonally-var- iable soil conditions to SMCs in LCGs. in LCGs result from intense evaporation during summer and early . and secondarily to soil water content through microenvironmental effects (e. and that soil physical and chemical properties ap- Correlation coefficients between the first two sets of canonical functions (CFs) and soil pear to influence these microbial indicators in different ways. Quarterman. Based on the ab. circumneutral soil. 1983. for example nents of root exudates (Campbell et al.49⁎ Soil nitrate (ppm NO3-N) −0. If the primary mechanism by which vegetation cover influences ples were plotted along with cool-season (November through February) MPN and H is through organic inputs and food-web interactions. and that SMCs in these zones display reduced substrate-utilization di- cantly (r = 0. 2004). 2004). CFsubs1 and CFsubs2 are the first two canonical functions were suppressed by seasonally dry soil conditions.222 J. 2014). Nostoc spe- cies) are generally prominent constituents of LCG ecosystems (Baskin and Baskin. 1950a). 2002).11 −0. in that: (1) soil communities OM was the most strongly-correlated soil property with the second CF.39⁎ 0.3. suggesting their eco- Variable CFsoil1 CFsoil2 CFsubs1 CFsubs2 logical relevance to SMCs. CFsoil1 and CFsoil2 were constructed from soil properties. via detrital food webs in which different SMCs generate different nutri- ther CFsub1 or CFsub2. Future investigations to quantify expression levels of genes involved in specific microbial metabolic pathways could help elu- cidate the role played by various forms of OM in soil food webs of LCGs. thin-soil areas of LCGs (Cartwright and Hui. Zak et al. It is noteworthy that the ef- Gravimetric soil water content (percent) −0.22⁎ 0.36⁎ 0.45⁎ −0.05.40⁎ −0.44⁎ −0.18 0. suggesting that numbers Fig.21⁎ 0. temperature effects from (Table 2)... Wardle et al.72⁎ 0.1. CLPP patterns for soil microbial communities 1993.11 −0. profiles were apparent when warm-season (May through August) sam.98⁎ 0. vegetation score was positively associated with was most strongly related to soil temperature at 4-cm depth and to both MPN and H. In particular. On the one hand.5 to N8.35⁎ across terrestrial ecosystems in North and South America.g.28⁎ tively narrow range (by comparison. shading. the first and second sets correlated signifi... 2004). In thin-soil areas of LCGs. esters.18 0. 2015..35⁎ 0. On the other hand. 1998). CFsubs1 and CFsubs2 were constructed from CLPP carbon substrate data. Indeed.29⁎ 0. 1997) were correlated with ei. amines. might expect that soil OM would also be positively associated with these SMC indicators. nonical functions related to soil properties. Ordination plot of soil samples collected from May through August versus from November through February based on canonical correlation analysis of microbial of culturable microbes and microbial substrate-utilization diversity substrate utilization profiles. soil OM was strongly related to MPN. Seasonally dry soils constructed from CLPP carbon substrate data.01) and together accounted versity compared to zones of thicker. one samples in an ordination plot of CFsubs1 and CFsubs2 (Fig. This is perhaps not surprising given the complexity of factors regulating microbial metabolism in soil environments (Torsvik and Øvreås..g. includ- ing the glades at this study site (Dubois. We found mixed support for this conclusion.. Cartwright et al. Discussion higher rates of carbon additions to soil in more densely-vegetated zones of LCGs supported greater numbers of culturable microbes. the association of OM with H was relatively weak. However.5 Vegetation score −0. Somers et al. i.72. stone parent material and by relative scarcity of leaf-litter inputs.e. soil moisture effects from transpiration) and nutrient inputs tent.83 and 0. Spa- 4. 2003). Martin and Sharp. Seasonal differences in substrate utilization ent pools for plant use (Reynolds et al. Lauber et Soil pH 0.17 with reduced MPN and with lower H values. 1986).

2010. 2012.. prominent variable based on canonical correlation analysis of commu- In contrast to soil-moisture effects. 206–219. Tennessee Acad.. E. basis (Fig. Quarterman et al... Khanafer. the U. 1998. Most-probable number estimates along with mi- soil moisture always ≥37%). biodiversity-rich ecosystem. C. 369. D. Club 122.. 2009. or product names is for descriptive purposes lipid fatty acid (PLFA) profiles and fatty acid methyl esters (FAME) only and does not imply endorsement by the U. Table 1) with field logistics.. pH. J. The authors thank William Wolfe (U. 2002). thickness. However. discerned between SMCs and soil properties in LCGs using culture-de. dle Tennessee cedar glades. This research was supported by the National Park Service. Supplementary data 4. 2. this study found mixed results nity-level physiological profiles. / Catena 147 (2016) 216–224 223 autumn due to soil thinness combined with full insolation from lack of organisms capable of rapid metabolism of a limited number of carbon canopy cover (Baskin and Baskin. 1999. Quarterman. In future studies. 64. Baskin. 2003. Conclusions Talmon et al.. B. Soc. Rastogi and Sani. further research is warranted circumneutral soil than in alkaline soil. Separation of soil samples in or. Somers et al. B. Botanists sources under laboratory (not field) conditions. PI). Zogg et al. and vegetation score. 1995). it nonetheless indicated have long noted the prevalence of drought-tolerance adaptations clear differences in SMCs between soil samples (Fakruddin and among LCG vegetation. Sutton. 2013. and a review of their aut- volve multiple.. 101–118.g. clear and statistically significant patterns were Baskin. analysis. 1989. can circumvent many of the limitations associated with culture depen.. Torrey Bot. phospho. 2011. 1. J.S. 2013. 2011). a combination of cul- not represented in the laboratory protocols for this study. In future studies.. Flow diagrams for plant succession in the Mid- versity than either approach alone (Al-Awadhi et al. ship with SMC indicators. and Rock Outcrop Plant Communities Because culture-dependent and culture-independent approaches in. Government. Savannas. which includes succulents. Radwan. 1993. i.doi. Preston-Mafham et al. 1131–1140. Baskin. allowing proliferation of culturable microbes that in this endangered and biodiversity-rich ecosystem... In a variety of arid and semi-arid ecosys. Cedar glade endemics in Tennessee. Cade. 1997).g.R. ture-dependent methods with molecular approaches could enable a inoculated agar plates for PDFA analysis and EcoPlates for CLPP were in..07. Baskin. M.. N. Cedar Glades of the Southeastern United States. and moisture availability) known to be of primary impor- 1995. Appendix A.. pp.. Wolfe. 2003. two approaches may allow better representation of true microbial di. New York. Ali.. 1993).S. 412–420. this weakly related to H. Le et al. Environ. Although CLPP necessarily restricted our analysis to Front. Quarterman et al. S. 4).. R. it is noteworthy that temperatures at the soil surface Acknowledgements and at a depth of 4 cm were both strongly correlated to the first pair of CFs based on CCA analysis of CLPP data. Malik et al. J. Geological Survey) and Gib fects on SMCs in this ecosystem by employing a range of seasonally-rel. This study of SMCs in LCG soils employed culture-dependent These data include point locations representing the sampling quadrats methods.. This study indicates that patterns of soil water availability in LCGs 1999. 5. Al-Mailem. C. soil nuals that survive dry summer conditions using dormancy (Baskin et al.. In: Anderson. these differ- crassulacean acid metabolism photosynthetic pathway. Noon. ences were associated with a number of environmental factors (e. 63–74. Sci. the limitations of which are thoroughly documented described in this article. Barrens.2. 1993). 2007. N. more comprehensive assessment of microbial diversity and function cubated at ~ 25 °C. have demonstrated temperature-related shifts in soil microbial community structure (Frey et al. (Eds. suggesting possible functional differ- for temperature effects on SMCs. Bull. ranging from 0 °C to 40 °C. Future studies could seek to clarify soil-temperature ef. Baskin. J... Dzantor. lems in Culture-Independent Analysis of Environmental Bacterial Communities: A 2010). References ful information for characterizing SMCs in a variety of environmental Al-Awadhi.. Baskin. J. A floristic plant ecology study of the limestone glades of northern Alabama. J. 1997. 2011). crobial diversity at a particular site (Dahllöf. Specifically. e. Inst. at doi:http://dx. soil water content. 2003. Fralish. of North America. Bot.. no single molecular technique can comprehensively describe mi. D.e. Temperatures at the soil surface and ences in microbial communities based on season. bial community structure across seasons.S. The vascular flora of cedar glades of the southeastern United States and its phytogeographical relationships.. 2008. and winter-an.. tems. Geological Survey) and anonymous reviewers. 2002. C. non-overlapping sets of biases.. Springerplus. 2013). Cambridge University Press. Baskin. dency. This manuscript was improved based on reviews or by applying culture-independent methods to characterize soil micro. Because seasonal moisture limitation is so crobial substrate-utilization diversity were positively related to soil ecologically important to plant communities in LCGs (Quarterman. However. Cartwright et al. J. Soil temperature. p.2016. J. Baskin. 2003). pendent methods.S. Baskin. Baskin.. 2008). Geological Survey. Garland et al. culture-dependent methods continue to generate use. tance to plant community structure in LCGs (Baskin and Baskin. H. Soil nitrate showed no relation- concerning its effects on microbial community structure and function. Ecol. Furthermore. Martin and Sharp (1983) found no clear relation- ship between soil moisture availability and species richness for protozoa Several soil properties that are key components of abiotic stress re- in LCGs. Quarterman. Bias Prob- contexts (Frąc et al. concurrent use of the ecology.g. may have been dormant under extremely cold or hot temperatures in the field. C..1016/j. Backlund and Troy Morris (National Park Service) for their assistance evant incubation temperatures (e. significantly negative only for limited ranges study provides useful information to begin investigating plant-SMC re- of τ. by Thomas Byl (U. 1986).. J. 1995. are also relevant to soil microbial ecology. PI). firm. Although a variety of was a in the southeastern United States (Mearns et al.. molecular methods Representative Study on Hydrocarbonoclastic Bacteria. In this study. species using the Mannan. A likely explanation is that temperature regimes in the field were lationships in these ecosystems. 1950b).S. soil moisture availability is a strong control on microbe-mediated processes such as heterotrophic soil respiration (Munson et al. . Texas 1.. Any use of trade. suggesting that N may have been non-limiting especially in light of predicted increases in summer drought severity in these soils. Konopka et al. A gentle introduction to quantile regression for ecologists. Quarterman et al.. 226–242. 1999. and were higher in 1950a.. J. and Tennessee functionally-different SMCs were present in LCG soils on a seasonal State University (USDA/NIFA award 2010-38821-21594. Baskin. J. thickness. Webb.. (Degens and Harris. Because soil at a depth of 4 cm were both unrelated to MPN estimates and were microbial analysis has been lacking for rock outcrop ecosystems.). 130...catena. at the surface and at 4-cm depth. C. Torrey Bot. Such methods. (Study# dination space based on substrate-utilization profiles suggests that STRI-00025. Soil moisture limitation can hybrid approaches could be employed—coupling the methods de- inhibit microbial function by constraining rates of substrate diffusion scribed here with culture-independent procedures—to further elucidate and by cytoplasmic dehydration that reduces enzymatic efficiency the role of environmental factors in shaping SMCs in this threatened and (Stark and Firestone. Dashti. Res. Methodological considerations Supplementary data associated with this article can be found in on- line version. possibly because of relatively limited sampling (5 sites each gimes in limestone cedar glades showed clear relationships with soil sampled twice) that did not include xeric conditions (average available microbial indicators...010.

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