Amplicon Sequencing of Rock-Inhabiting Microbial Communities from Joshua Tree National Park, USA

ABSTRACT Endolithic microorganisms have been reported to date in hot and cold drylands worldwide, where they represent the prevailing life forms ensuring ecosystem functionality, playing a paramount role in global biogeochemical processes. We report here an amplicon sequencing characterization of rocks collected from Joshua Tree National Park (JTNP), USA.

U nderstanding the microbial community structure and diversity in dryland regions is of paramount importance in both microbial ecology and evolution. Under these conditions, where most life-forms cannot survive, rocks represent the main refuge for life. Indeed, the endolithic habitat provides thermal buffering, physical stability, and protection against UV and solar radiation, ensuring the ecosystem functionality and creating positive feedback for further colonization (1). Although endoliths are ubiquitous in drylands worldwide, including the hyperarid drylands, from Atacama Desert to the Antarctic Dry Valleys (for examples, see references 2 to 8), a comprehensive assessment of worldwide rock diversity remains insufficiently studied, and comparisons between geographic locations and different climatic conditions are still missing (9). The present study, to our knowledge, represents one of the few available amplicon sequencing reports within the hot desert of Joshua Tree National Park (JTNP) in California (for an example, see reference 10).
Ten rock samples were collected from three different sites in the JTNP (GPS, 34.10, 2115.45; the elevation for all sites is between 1,040 and 1,340 m); the presence of endolithic colonization was assessed by direct observation in situ. Rocks were collected under sterile conditions using a geologic hammer and a chisel, placed in sterile bags, transported in a cooler with ice, and stored at 280°C at the University of California, Riverside (CA), until downstream analysis. DNA extraction was performed using 0.5 g of powdered rock with a PowerSoil DNA extraction kit (MoBio Laboratories, Carlsbad, CA, USA).
The fungal internal transcribed sequence 1 (ITS1) region was amplified using the primers ITS1F and ITS2 (11), while the bacterial 16S rRNA V4 region was amplified using 515F and 806R (12), according to the Earth Microbiome Project protocols (https:// earthmicrobiome.org/protocols-and-standards/). Two replicates were amplified for each sample. PCRs were carried out with a total volume of 25ml, containing 1 ml of each primer, 12.5 ml of Taq DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA), 9.5 ml of nuclease-free water (Sigma-Aldrich, St. Louis, MO, USA), and 5 ng of DNA template using an automated thermal cycler (Bio-Rad, Hercules, CA, USA). The ITS1 locus was amplified following an initial denaturation at 94°C for 1 min and 35 cycles at 94°C for 30 s, annealing at 52°C for 30 s, and extension at 68°C for 90 s, followed by a final extension at 68°C for 7 min. The PCR for the V4 region followed a protocol of an initial denaturation at 94°C for 3 min, 35 cycles at 94°C for 45 s, annealing at 50°C for 1 min, and extension at 72°C for 90 s, followed by a final extension at 72°C for 10 min. The amplicons were quantified using the Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) assay kit (Life Technologies, USA), purified using the Qiagen PCR cleanup kit (Macherey-Nagel, Hoerdt, France), and then pooled to produce an equimolar mixture. Sequencing was performed at the Institute for Integrative Genome Biology (IIGB; https://iigb.ucr.edu/), University of California, Riverside (USA), with the MiSeq reagent kit v3 on the Illumina MiSeq platform (2 Â 300-bp paired-end format).
The fungal ITS resulted in 960,527 quality-filtered reads clustered in 869 valid ASVs, while a total of 1,059,591 valid output reads were obtained for 16S rRNA, resulting in 3,221 ASVs. The majority of the identified ITS sequences recovered from all samples belonged to the phylum Ascomycota, followed by Basidiomycota, while Actinobacteria and Proteobacteria predominated in the 16S rRNA data set.
Data availability. The bacterial 16S rRNA and fungal ITS1 data sets generated and analyzed in the current study are available in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA706555 and in the Zenodo repository (https://doi.org/10.5281/zenodo.3960774).

ACKNOWLEDGMENTS
The samples were collected under Joshua Tree National Park permit number JOTR-2017-SCI-0010. We thank the National Park Service for permission to conduct research. The fungal ITS and 16S rRNA primer sequences and arrayed barcodes were provided by the Alfred P. Sloan Foundation Indoor Microbiome Project. C.C. was supported by an Italian National Program for Antarctic Research (PNRA) postdoctoral fellowship. N.P. was supported by a Royal Thai government fellowship. J.E.S. is a CIFAR fellow in the Fungal Kingdom: Threats and Opportunities program. Sequencing was supported by funds through the U.S. Department of Agriculture National Institute of Food and Agriculture Hatch project CA-R-PPA-5062-H to J.E.S. Data analyses were performed on the High-Performance Computing Center at the University of California, Riverside, in the Institute of Integrative Genome Biology, supported by NSF DBI-1429826 and NIH S10-OD016290. We declare that we have no competing interests.
We thank Mike Allen, Derreck Carter-House, and Sawyer Masonjones for assistance with the rock sampling and transportation.
The rock samples were collected by C.C. and N.P. at Joshua Tree National Park (May 2015); C.C. conceived and designed the experiment, C.C. and N.P. performed the experiments, C.C. analyzed the raw data, and C.C. wrote the first draft. All authors have read and agreed to the published version of the manuscript.