title: diazotrophic community (nitrogen fixing microorganisms) response to change in the land use of tropical forests
design: A spatially explicit sampling design was established for three plots within each of the primary forest, adjacent pasture (38 years old), and secondary forest (12–17 years old) areas. The nested sampling design established 0.01, 0.1, 1.0, 10 m2 quadrats nested within a 100 m2 quadrat, and 12 samples were collected within each 100 m2 plot. Overall, three 100 m2 quadrats were established at 0, 1, and 10 km apart within both primary forest and pasture sites (36 samples per land use system). No secondary forest site was available at exactly 1 km distance, so the second 100 m2 sampling plot for secondary forest was at about 3 km (0, 3, and 10 km). Soil cores (5 cm diameter) collected at 10 cm depth were used for DNA extraction and geochemical analysis. The nifH gene was amplified using poly F & R primers (Poly et al., 2001) and products were sequenced using the 454 GS FLX Sequencer (Michigan State University Genomics Facility).