Luquillo LTER

The elevation gradient is located in eastern Puerto Rico and is composed of five forest types each with characteristic vegetation. Soil samples were collected every three months from March 2003 thru December 2004. Soil fungal and bacterial communities were analyzed using fatty acid methyl esters (FAME) and TRFLP profiles.

In each forest type, five plots of 5 x 5 m were established and soil samples were collected every three months starting in March 2003 thru December 2004. A polyvinyl chloride plastic tube 5 cm in diameter and 10 cm in length was used to take a random core sample from each plot after removal of surface litter. A total of 150 soil cores were analyzed but only 79 of these yielded usable DNA extractions. Samples were stored in sterile plastic bags at -20°C for fatty acid or DNA extraction. Each sample was subdivided by depth into 0-5 and >5-10 cm strata. Fatty acids were extracted directly from 3 g of soil using the method for ester linked fatty acid methyl ester (EL-FAME). Briefly, the method used a mild alkaline methylation followed by pH neutralization with 1M acetic acid and FAME extraction using hexane. Extracts were cleaned using a NH₂ column to remove humic substances. Samples were stored at -20°C until analyzed. Samples were analyzed in a GC-MS (Hewlett Packard 6890). DNA was extracted from 0.3 g of sample using MoBio Ultra Clean Soil DNA Isolation Kit. The 16S bacterial rDNA was amplified using primers 27F-FAM and 1525R, and digested with MnlI. The fungal ITS region was amplified using primers ITS1-FAM and ITS4, and digested with HaeIII. Samples were analyzed in an ABI 3130 using GenScan 500 Liz Size Standard.