Shannon diversity index data based on Community Level Physiological Profiling (CLPP) of soil samples from 120 point locations within limestone cedar glades at Stones River National Battlefield near Murfreesboro, Tennessee

This dataset contains data collected within limestone cedar glades at Stones River National Battlefield (STRI) near Murfreesboro, Tennessee. This dataset contains information on soil microbial metabolic diversity for soil samples obtained from certain quadrat locations (points) within 12 selected cedar glades. This information derives from substrate utilization profiles based on Biolog EcoPlates (Biolog, Inc., Hayward, CA, USA) which were inoculated with soil slurries containing the entire microbial community present in each soil sample. EcoPlates contain 31 sole-carbon substrates (present in triplicate on each plate) and one blank (control) well. Once the microbial community from a soil sample is inoculated onto the plates, the plates are incubated and absorbance readings are taken at intervals.For each quadrat location (point), one soil sample was obtained under sterile conditions, using a trowel wiped with methanol and rinsed with distilled water, and was placed into an autoclaved jar with a tight-fitting lid and placed on ice. Soil samples were transported to lab facilities on ice and immediately refrigerated. Within 24 hours after being removed from the field, soil samples were processed for community level physiological profiling (CLPP) using Biolog EcoPlates. First, for each soil sample three measurements were taken of gravimetric soil water content using a Mettler Toledo HB43 halogen moisture analyzer (Mettler Toledo, Columbus, OH, USA) and the mean of these three SWC measurements was used to calculate the 10-gram dry weight equivalent (DWE) for each soil sample. For each soil sample, a 10-gram DWE of fresh soil was added to 90 milliliters of sterile buffer solution in a 125-milliliter plastic bottle to make the first dilution. Bottles were agitated on a wrist-action shaker for 20 minutes, and a 10-milliliter aliquot was taken from each sample using sterilized pipette tips and added to 90 milliliters of sterile buffer solution to make the second dilution. The bottle containing the second dilution for each sample was agitated for 10 seconds by hand, poured into a sterile tray, and the second dilution was inoculated directly onto Biolog EcoPlates using a sterilized pipette set to deliver 150 microliters into each well. Each plate was immediately covered, placed in a covered box and incubated in the dark at 25 degrees Celcius. Catabolism of each carbon substrate produced a proportional color change response (from the color of the inoculant to dark purple) due to the activity of the redox dye tetrazolium violot (present in all wells including blanks). Plates were read at intervals of 24 hours, 48 hours, 72 hours, 96 hours and 120 hours after inoculation using a Biolog MicroStation plate reader (Biolog, Inc., Hayward, CA, USA) reading absorbance at 590 nanometers.For each soil sample and at each incubation time point, raw absorbance values were transformed according to the equations:T = (C-R) / AWCD; and AWCD = [Σ (C – R)] / nwhere T represents transformed substrate-level response values, C is the absorbance value of control wells (mean of 3 controls), R is the mean absorbance of the response wells (3 wells per carbon substrate), AWCD is average well color development for the plate, and n is the number of carbon substrates (31 for EcoPlates). To integrate time-series data from multiple EcoPlate readings (both for AWCD and also for individual substrates, T), the area under the incubation curve, from 48 hours to 120 hours of incubation time, was calculated.To assess community-level microbial diversity, the Shannon-Weaver index (H) was calculated as follows:H = - ∑ p(ln p)where p is the ratio of the activity of each substrate (T values, area under the incubation curve) to the sum of the activities of all substrates for a given EcoPlate. Thus, the numeric values contained in the fields of this dataset represent H values (Shannon-Weaver index of diversity) based on substrate-utilization diversity of the entire microbial community of each soil sample. Higher values indicate that the entire microbial community metabolized a greater diversity of substrates present on the EcoPlates during the incubation period under consideration. Detailed descriptions of experimental design, field data collection procedures, laboratory procedures, and data analysis are presented in Cartwright (2014).References:Cartwright, J. (2014). Soil ecology of a rock outcrop ecosystem: abiotic stresses, soil respiration, and microbial community profiles in limestone cedar glades. Ph.D. dissertation, Tennessee State UniversityCofer, M., Walck, J., and Hidayati, S. (2008). Species richness and exotic species invasion in Middle Tennessee cedar glades in relation to abiotic and biotic factors. The Journal of the Torrey Botanical Society, 135(4), 540–553.Garland, J., & Mills, A. (1991). Classification and characterization of heterotrophic microbial communities on the basis of patterns of community-level sole-carbon-source utilization. Applied and environmental microbiology, 57(8), 2351–2359.Garland, J. (1997). Analysis and interpretation of community‐level physiological profiles in microbial ecology. FEMS Microbiology Ecology, 24, 289–300.Hackett, C. A., & Griffiths, B. S. (1997). Statistical analysis of the time-course of Biolog substrate utilization. Journal of Microbiological Methods, 30(1), 63–69.Insam, H. (1997). A new set of substrates proposed for community characterization in environmental samples. In H. Insam & A. Rangger (Eds.), Microbial Communities: Functional versus Structural Approaches(pp. 259–260). New York: Springer.Preston-Mafham, J., Boddy, L., & Randerson, P. F. (2002). Analysis of microbial community functional diversity using sole-carbon-source utilisation profiles - a critique. FEMS microbiology ecology, 42(1), 1–14. doi:10.1111/j.1574-6941.2002.tb00990.x

本数据集采集自田纳西州默弗里斯伯勒附近的斯通河国家战场（Stones River National Battlefield, STRI）内的石灰岩雪松裸地。本数据集包含从12处选定的雪松裸地内的样方点位采集的土壤样品的土壤微生物代谢多样性信息。该信息基于Biolog EcoPlates（美国加利福尼亚州海沃德市Biolog公司）的底物利用谱获得，每块平板均接种含有对应土壤样品中全部微生物群落的土壤悬液。EcoPlates平板包含31种单一碳底物（每块平板上每种底物设置3个重复孔）以及1个空白对照孔。将土壤样品的微生物群落接种至平板后，对平板进行培养并定期读取吸光度值。

针对每个样方点位，使用经甲醇擦拭、蒸馏水冲洗过的抹刀在无菌条件下采集一份土壤样品，将其放入带密封盖的高压灭菌罐中并置于冰上。土壤样品全程冰藏运送至实验室后立即冷藏。野外采样后24小时内，使用Biolog EcoPlates对土壤样品进行群落水平生理剖面（community level physiological profiling, CLPP）分析。

首先，使用梅特勒托利多HB43卤素水分分析仪（美国俄亥俄州哥伦布市梅特勒托利多公司）对每份土壤样品的重量法土壤含水量（soil water content, SWC）进行3次重复测定，以3次SWC测定的平均值计算每份样品的10克干重当量（dry weight equivalent, DWE）。每份土壤样品取10克DWE的新鲜土壤，加入盛有90毫升无菌缓冲液的125毫升塑料瓶中，制备一级稀释液。将塑料瓶置于手腕式摇床振荡20分钟，使用灭菌吸头从每份样品中吸取10毫升悬浮液，加入90毫升无菌缓冲液中制备二级稀释液。将每份样品的二级稀释液塑料瓶手动振荡10秒后倒入无菌托盘，使用设定为150微升的灭菌移液器将二级稀释液直接接种至Biolog EcoPlates的每个孔中。每块平板立即加盖，放入带盖的盒中，于25摄氏度黑暗条件下培养。由于所有孔（包括空白孔）中均添加了氧化还原染料氯化三苯基四氮唑（tetrazolium violet，原文拼写为violot，疑似笔误）的活性，每种碳底物的分解代谢会产生成比例的颜色变化响应（从接种液的底色变为深紫色）。接种后分别于24小时、48小时、72小时、96小时及120小时，使用Biolog MicroStation酶标仪（美国加利福尼亚州海沃德市Biolog公司）在590纳米波长下读取平板吸光度值。

针对每份土壤样品及每个培养时间点，原始吸光度值按照下述公式进行转换：
T = (C-R) / AWCD; 且 AWCD = [Σ (C – R)] / n
其中，T为经转换的底物水平响应值，C为对照孔的吸光度值（3个对照孔的平均值），R为反应孔的平均吸光度值（每种碳底物对应3个孔的平均值），AWCD为平板的平均孔颜色发展值，n为碳底物的总数（EcoPlates平板为31种）。为整合多次EcoPlates读取的时间序列数据（包括AWCD值及各底物的T值），计算了培养时间48小时至120小时区间内的培养曲线下面积。

为评估群落水平的微生物多样性，采用香农-威纳指数（Shannon-Weaver index, H）进行计算，公式如下：
H = - ∑ p(ln p)
其中，p为单个底物的活性（即T值，培养曲线下面积）与对应EcoPlates平板所有底物活性总和的比值。因此，本数据集各字段中的数值即为基于每份土壤样品全部微生物群落底物利用多样性计算得到的香农-威纳多样性指数（H值）。数值越高，代表在对应培养周期内，该土壤样品的全部微生物群落能够代谢EcoPlates平板上更多种类的碳底物。

实验设计、野外数据采集流程、实验室操作流程及数据分析的详细说明参见Cartwright（2014）的研究。

参考文献：
1. Cartwright, J.（2014）. 岩石裸露生态系统的土壤生态学：石灰岩雪松裸地中的非生物胁迫、土壤呼吸及微生物群落剖面. 田纳西州立大学博士学位论文
2. Cofer, M., Walck, J. & Hidayati, S.（2008）. 田纳西州中部雪松裸地的物种丰富度与外来物种入侵及其与非生物和生物因子的关联. 《Torrey植物学会杂志》, 135(4), 540–553
3. Garland, J. & Mills, A.（1991）. 基于群落水平单一碳源利用模式对异养微生物群落进行分类与表征. 《应用与环境微生物学》, 57(8), 2351–2359
4. Garland, J.（1997）. 微生物生态学中群落水平生理剖面的分析与解读. 《FEMS微生物生态学》, 24, 289–300
5. Hackett, C. A. & Griffiths, B. S.（1997）. Biolog底物利用时间进程的统计分析. 《微生物方法学杂志》, 30(1), 63–69
6. Insam, H.（1997）. 一套用于环境样品群落表征的新型底物. 收录于：Insam, H. & Rangger, A. 编. 《微生物群落：功能与结构方法》(pp. 259–260). 纽约：Springer
7. Preston-Mafham, J., Boddy, L. & Randerson, P. F.（2002）. 基于单一碳源利用谱分析微生物群落功能多样性：一项评述. 《FEMS微生物生态学》, 42(1), 1–14. doi:10.1111/j.1574-6941.2002.tb00990.x