Geology and climate influence rhizobiome composition of the phenotypically diverse tropical tree Tabebuia heterophylla

and from there, further digging was done—in a depth from 1–1.5 foot to 5 feet deep- until we could collect secondary roots branching out from the main root. For each tree, four secondary roots (each root per tree represents a subsample) extending between 46–91 cm were collected, cut and stored in a 50ml falcon tube prior to being transported in ice to the laboratory. Addi- tionally bulk soil close to every tree was also collected. At each of four points ~ 30.5 cm from each selected tree, soils were sampled down to 15 cm with a soil auger. The bottom 10 cm of the soil samples were stored in sterile 50 ml Falcon tubes for transport from the field to the lab- oratory. Sample collection followed protocols from a previous study [20], and was performed under permit number DRNA 2011-IC-018, issued by the Puerto Rico Department of Natural Resources, to the Biology Department of the University of Puerto Rico-Rı´o Piedras Campus. In the lab, soil samples were immediately frozen at -80˚C until gDNA extraction, while roots were washed with 25ml PBS/Tween20 and shaken at 240 rpm horizontally for 1 hour. After roots were bare, we removed them from the PBS solution and froze them in new Falcon tubes at -80˚C. The PBS/Tween20 solution with the rhizosphere was centrifuged at 9,000g for 30 min at 4˚C. The resulting pellet with the rhizosphere was frozen at -80 C until DNA extraction. Genomic DNA extractions and 16S rRNA amplifications Rhizosphere and bulk soil samples were processed for metagenomic DNA extraction and

amplification of 16S rRNA genes. The genomic DNA was extracted from the rhizosphere and bulk soil subsamples (4 subsamples per individual for a total of 24 subsamples per site, or 144 subsamples in total), using the PowerSoil DNA isolation kit (MO BIO, Carlsbad, CA, USA) following the manufacturer’s instructions. The genomic DNA samples were sent to the Joint Genome Institute of the Department of Energy for amplification of the V4 hypervariable region (~291bp length) of the 16S ribosomal RNA (rRNA) and sequencing was performed using the Illumina MiSeq platform following an in-house protocol. Itag data can be downloaded from the JGI repository (https://genome.jgi.doe.gov/portal/ pages/dynamicOrganismDownload.jsf?organism=Tabitaplate2_FD). Community profiling analyses The raw reads were subjected to pre-processing, using JGI’s pipeline iTagger, which performed read QC (filter contaminants such as PhiX control, sequencing library adapter dimers, human Table 1. Physical properties of the sites used in this study and their respective number of OTUs and sequences corresponding to sum of all samples (replicates and trees) per site. Analyses henceforth were done with a rarefaction level of 30,170 sequences per sample, corresponding to the sample with the lowest read count. Site Location (GPS) Geology Elevation (m) Precipitation (mm) Temp. (˚C) pH Read Count OTU Count Rhizo Soil Rhizo Soil S1 Cayey (18˚ 06’ 28.9’’ N 66˚ 04’ 20.7’’ W) Volcanic 647High 2077.7a 21.5d 5.4 1311511 139907 23478 8480

S2 Guayama (18˚ 01’ 24.4’’ N 66˚ 07’ 44’’ W) Volcanic 314Low 1661.4b 23.9c 6 1041329 694638 21825 21013 S3 Maricao (18˚ 09’ 33.4’’ N 66˚ 59’ 55.5’’ W) Serpentine 711High 2392.9a 23.1c 7.1 781076 402700 31430 21925 S4 Cabo Rojo (18˚ 07’ 51.1" N 67˚ 09’ 34.4" W) Serpentine 70Low 1352.0c 25.3b 6.5 219426 565129 16444 22466 S5 Guanica (17˚58018@N 66˚54029@W) Karst 10Low 783.7d 26.3a 8.7 2323394 1172332 30242 25240 S6 Arecibo (18˚ 22’ 49.1’’ N 66˚ 41’ 09.6’’ W) Karst 250High 1864.9b 24.5b 7.3 3326940 1832653 39446 32406 a Very high b High c Medium d Low https://doi.org/10.1371/journal.pone.0231083.t001 PLOS ONE Rhizobiota of the tropical tree Tabebuia heterophylla PLOS ONE | https://doi.org/10.1371/journal.pone.0231083 April 7, 2020 4 / 16