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

Results and discussion We collected rhizosphere and bulk soil samples from 18 Tabebuia trees that grew in soils of different geologies (i.e. volcanic, serpentine and karst). Per each geology, we chose two sites that differed in elevation, and we selected three T. heterophylla trees per site to collect our sam- ples (Table 1). Trees were sampled in four geographically and climatically varying sites (Fig 1A). Sites 1 and 2 had more acidic pH and corresponded to volcanic sites of high and low alti- tude, respectively (Table 1). Sites 3 and 4 had a neutral-like pH and corresponded to serpentine sites of high and low altitude and Sites 5 and 6 corresponded to more alkaline pH of karst ori- gin of low and high altitude respectively (Table 1). The 18 trees used to characterize T. heterophylla’s rhizosphere microbiome yielded ~13.8 million non-chimeric 16S rRNA sequence reads with an average of 383,640 sequences per sample. These reads represented a total of 36,601 OTUs belonging to 69 different phyla. Rarefi- cation of the samples to a uniform depth of 30,170 sequences reduced the total OTUs observed to 26,075, with an average of 3,591 OTUs observed per sample and reduced the number of phyla to 62. Table 1 summarizes the number of sequences and OTUs per site before and after the rarefaction. Analyses considering only OTUs present in at least 40% of the samples resulted in 26 Phyla. Species richness (Chao 1) of rhizosphere and bulk soil was greatest in serpentine (with neu- tral pH)- in both high and low elevation, followed by karst and volcanic sites (Fig 2A and 2B).

Serpentine sites were significantly richer compared to volcanic (p-value <0.05), but not signif- icantly higher than Karst, except for high altitude, humid sites, where richness was significantly higher in serpentine sites, compared to karst (S1 Table). The same differences were found regarding Shannon diversity with decreased diversity in volcanic sites (p-value <0.05) (S1 Fig). Overall, rhizosphere samples possessed significantly higher species richness than bulk soil samples (p-value <0.05), though per sample site this was not the case (S2 Fig, S1 Table). The individual trees at each site didn’t have a defined variability pattern (S2 Fig); sites where rhizo- sphere had low variability could have either low or high bulk soil species richness, although in most cases the rhizosphere was more diverse than the bulk soil at each of the individual trees (S2 Fig). Within geology, we found less species richness in low elevation sites, where temperature was higher and precipitation was lower (Table 1), especially for bulk soil compared to rhizo- sphere (Fig 2A, S2 Fig). Indeed, trees in the humid serpentine site had the highest richness of all samples (Fig 2C) and significantly different from all other geological sites at both altitudes. Additionally, volcanic bulk soil samples had significantly different diversity between low and high elevation sites (p-value = 0.00), while there were no differences in the rhizosphere com- munities of these same sites (p-value = 1.000) (S2 Fig, S1 Table). In general, the rhizosphere and bulk soil bacterial communities within each site clustered

separately both in nMDS and in Principal Coordinates Analysis (Fig 3A and 3B respectively). Primary clustering was by samples of sites under same geology (ANOSIM statistic R: 0.569, p- value = 0.001). In fact, karst samples (regardless of altitude and microhabitat) clustered together and separated from the rest of the parent materials (NMDS1). Clustering also occurred in samples from similar environmental conditions (elevation) (ANOSIM statistic R: 0.3888, p-value = 0.001) (Fig 3A). Rhizosphere and bulk soil samples of the low elevation karstic site (S5) were clearly separated from other sites, while the high elevation volcanic site (S1 Fig), was in the opposite extreme of the nMDS ordination. This is potentially caused by the temperature and moisture gradient from the hot and dry low elevation karst site, to the colder and wetter high elevation volcanic site (Fig 3A; Table 1). Combined replicates per tree(n = 36), PLOS ONE Rhizobiota of the tropical tree Tabebuia heterophylla PLOS ONE | https://doi.org/10.1371/journal.pone.0231083 April 7, 2020 6 / 16