Microbiomes of Velloziaceae from phosphorus-impoverished soils of the campos rupestres, a biodiversity hotspot

Abstract

The rocky, seasonally-dry and nutrient-impoverished soils of the Brazilian campos rupestres impose severe growth-limiting conditions on plants. Species of a dominant plant family, Velloziaceae, are highly specialized to low-nutrient conditions and seasonal water availability of this environment, where phosphorus (P) is the key limiting nutrient. Despite plant-microbe associations playing critical roles in stressful ecosystems, the contribution of these interactions in the campos rupestres remains poorly studied. Here we present the first microbiome data of Velloziaceae spp. thriving in contrasting substrates of campos rupestres. We assessed the microbiomes of Vellozia epidendroides, which occupies shallow patches of soil, and Barbacenia macrantha, growing on exposed rocks. The prokaryotic and fungal profiles were assessed by rRNA barcode sequencing of epiphytic and endophytic compartments of roots, stems, leaves and surrounding soil/rocks. We also generated root and substrate (rock/soil)-associated metagenomes of each plant species. We foresee that these data will contribute to decipher how the microbiome contributes to plant functioning in the campos rupestres, and to unravel new strategies for improved crop productivity in stressful environments.

Fig. 1

The Brazilian campos rupestres are rocky seasonally-dry environments with some of the world’s most phosphorus (P)-impoverished soils. (a) The study was conducted in a campo rupestre site in the Brazilian state of Minas Gerais, as shown on the map (left). Campo rupestre areas are shown in dark gray. The sites where plants of each Velloziaceae species were collected are indicated in the aerial image of the study area (right). (b) Barbacenia macrantha was found in a rocky area (left), where it grows over exposed rocks (right). (c) Vellozia epidendroides specimens were collected in an area (left) where they grow in patches of shallow soil (left).

Fig. 2

Overview of the workflows used to obtain and process the data. (a) Six individuals of both Vellozia epidendroides and Barbacenia macrantha were collected from their natural habitats and individually processed to assess the microbiomes from seven different environments through extraction of microbial DNA. The DNA extracted from three samples of four distinct communities (B. macrantha substrate, B. macrantha rhizosphere, V. epidendroides substrate and V. epidendroides rhizosphere), totaling 12 samples, was sequenced on an Illumina HiSeq platform to generate data for the metagenomic assembly. DNA from all six samples of all the assessed communities, totaling 84 samples, was used to generate 16S V4 and ITS2 amplicons, which were sequenced on an Illumina MiSeq platform. BS = bulk soil, ER = exposed rock, RX = rhizosphere, RN = endophytic root, SX = exophytic stem, SN = endophytic stem, LX = epiphytic leaf, LN = endophytic leaf. (b) The microbial community analysis started with the removal of primer sequences from the sequenced amplicons. Next, reads were denoised using the DADA2 pipeline, and the identified ASVs were assigned to bacterial and fungal taxa though comparison with the SILVA and UNITE databases, respectively. After filtering out ASVs from mitochondria and chloroplasts and low-prevalence amplicons, the phyloseq and vegan packages were used to analyze community composition. (c) The metagenomes were assembled using SPAdes software and then annotated using the standard DOE-JGI MGAP v.4 annotation pipeline. In the structural annotation step, the metagenomes were surveyed to identify CRISPRs, tRNA genes, rRNA genes, other classes of ncRNA genes and protein-coding genes. Next, the protein-coding sequences were functionally annotated and assigned to ortholog groups, metabolic pathways, chemical reactions and protein families.

Fig. 3

Alpha diversity of the Vellozia epidendroides and Barbacenia macrantha microbiomes. Alpha diversity, quantified using Shannon’s equitability index, of the (a) 16S V4 and (b) ITS2 loci retrieved from several microbial communities associated with V. epidendroides and B. macrantha. BS = bulk soil, ER = exposed rock, RX = rhizosphere, RN = endophytic root, SX = exophytic stem, SN = endophytic stem, LX = epiphytic leaf, LN = endophytic leaf.

Fig. 4

Community composition of the Vellozia epidendroides and Barbacenia macrantha microbiomes at the phylum level. Relative abundance of (a) prokaryotic and (b) fungal phyla retrieved from 16S V4 and ITS2 amplicon sequencing, respectively. Each column represents a single sample and samples were grouped according to the environment from which the communities were accessed. BS = bulk soil, ER = exposed rock, RX = rhizosphere, RN = endophytic root, SX = exophytic stem, SN = endophytic stem, LX = epiphytic leaf, LN = endophytic leaf.