This protocol is adapted from Russo et al. 2023 methods for high-molecular weight DNA extraction. I modified the protocol to use reagents and incubation conditions used by the Ferris Lab at Tulane University to extract DNA from Mimulus spp. (monkeyflower) tissue (leaf and buds), see protocol. The original CTAB:Chloroform protocol used by the Ferris lab traces back to the Willis Lab at Duke University. The objective of these DNA extractions are to collect fungal DNA present in leaf tissue. I have incorporated sample preparation procedures used by the Arnold Lab at The University of Arizona to eliminate contamination of samples and optimize DNA extractions from preserved leaf tissue for future fungal ITS Illumina MiSeq sequencing. Solid phase reversible immobilization beads (SPRI) utilized are adapted from Rohland and Reich 2012 and Liu et al 2023. All extractions were performed in a sterile biosafety cabinet to prevent sample contamination. Prior to being placed in CTAB, photosynthetic tissue from plants was surface sterilized with sequential washes in 95% EtOH (10 s) , 0.5% sodium hypochlorite (1 min), and 70% EtOH (1min) and air dried under sterile conditions.Due to the small size of monkeyflower plants, the maximum amount of leaf tissue produced per host were placed in 750- 1000 µL of CTAB buffer and kept at RT until DNA extraction process. *Several aspects of this protocol, mainly those with reagent amounts and molarities, have changed compared to the Russo et al 2023 protocol and the Willis Lab standard CTAB extraction protocol from ca. 2010.

This protocol is adapted from Russo et al. 2023 methods for high-molecular weight DNA extraction. I modified the protocol to use reagents and incubation conditions used by the Ferris Lab at Tulane University to extract DNA from Mimulus spp. (monkeyflower) tissue (leaf and buds), see protocol. The original CTAB:Chloroform protocol used by the Ferris lab traces back to the Willis Lab at Duke University. The objective of these DNA extractions are to collect fungal DNA present in leaf tissue. I have incorporated sample preparation procedures used by the Arnold Lab at The University of Arizona to eliminate contamination of samples and optimize DNA extractions from preserved leaf tissue for future fungal ITS Illumina MiSeq sequencing. Solid phase reversible immobilization beads (SPRI) utilized are adapted from Rohland and Reich 2012 and Liu et al 2023. All extractions were performed un a sterile biosafety cabinet to prevent sample contamination. Prior to being placed in CTAB, photosynthetic tissues from plants were surface sterilized with sequential washes in 95% EtOH (10 s) , 0.5% sodium hypochlorite (1 min), and 70% EtOH (1min) and air dried under sterile conditions.Due to the small size of monkeyflower plants, the maximum amount of leaf tissue produced per host were placed in 750- 1000 µL of CTAB buffer and kept at RT until DNA extraction process. *Several aspects of this protocol, mainly those with reagent amounts and molarities, have changed compared to the Russo et al 2023 protocol and the Willis Lab standard CTAB extraction protocol from ca. 2010.

Shade trees provide important ecological services that support productivity in coffee agroforestry systems. Processes such as biological nitrogen fixation play a key role in this. Less is known, however, about potential indirect mechanisms by which nitrogen fixation supports coffee productivity. One potential route for this to occur is by providing ecological benefits to other above-and belowground organisms that enrich the overall function of agroecosystems. A useful lens with which to evaluate the ecological benefits to these communities under shade trees is to assess how ground-dwelling ant communities respond to the quality of leaf litter from established nitrogen (N)-fixing tree species. Here, we use two trees commonly planted in coffee agroecosystems: Inga micheliana, an N-fixing species, and Alchornea latifolia, a non-N-fixing species. In this study, we set out to answer the following questions: (1) How does the leaf litter environment differ between I. micheliana and A. latifolia? (2) Do differences in environmental factors between I. micheliana and A. latifolia correlate with differences in ant abundance and species richness? and (3) Do differences in environmental factors between I. micheliana and A. latifolia correlate with differences in ant community composition? Twenty-eight randomly selected sites (14 I. micheliana and 14 A. latifolia) were established within a 45-ha plot in a shaded organic coffee farm in Chiapas, Mexico. Three 1-m 2 quadrats within a 5-m radius from the base of the selected trees were established, and the leaf litter within the quadrats was removed and sieved.Ant specimens were extracted from leaf litter collected from quadrats using the mini-Winkler method and identified to genus and species, or morphospecies, level.Results indicate that I. micheliana, the N-fixing species, has a lower C:N ratio than A. latifolia. Differences in C:N ratios are correlated with ant abundance but not with ant species richness. Distance to edge (in meters) has significant effects on leaf litter ant abundance, richness, and species composition. Results suggest that there may be unaccounted feedbacks from N-and non-N-fixing vegetation to brown food webs enabling them to sustain similar ground-dwelling ant communities.