DOI: 10.5061/dryad.kh18932c4
title: Convergent evolution of desiccation tolerance in grasses

description: Desiccation tolerance has evolved repeatedly in plants as an adaptation to
 survive extreme environments. Plants use similar biophysical and cellular
 mechanisms to survive life without water, but convergence at the
 molecular, gene, and regulatory levels remains to be tested. Here, we
 explore the evolutionary mechanisms underlying the recurrent evolution of
 desiccation tolerance across grasses. We present genomes of three
 resurrection grasses native Sub-Saharan Africa. We leveraged comparative
 genomic and transcriptomic approaches to identify patterns of convergence
 and divergence across these species. We observed substantial overlap in
 gene duplication and expression associated with desiccation, and syntenic
 genes of shared origin are activated across species, indicative of
 parallel evolution. In other cases, similar metabolic pathways are
 induced, but using different gene sets, pointing towards phenotypic
 convergence. Species-specific mechanisms supplement these shared core
 mechanisms, underlining the complexity and diversity of evolutionary
 adaptations. Our findings provide insight into the evolutionary processes
 driving desiccation tolerance and highlight the roles of parallel mutation
 and complementary pathway adaptation in response to environmental
 challenges.
description: These data were used to identify core mechanisms of desiccation
 tolerance shared across grasses. We subjected replicated sets of the
 resurrection grasses <em>Oropetiuum</em>
 <em>capense</em>, <em>Tripogon</em>
 <em>minimus</em>, and <em>Microchloa</em>
 <em>caffra</em> to a controlled dehydration-rehydration
 timecourse. Tissues were collected at six comparable timepoints (four
 during drying and two during recovery) for each of the three species.
 Plants were sampled at targeted hydration states including from
 well-watered, partially dehydrated, fully desiccated, and 24 and 48 hours
 post rehydration. The hydration status of tissues was validated by
 measuring relative water content. Gene expression data (RNAseq) was
 generated by extracting total RNA using Spectrum Plant Total RNA kit
 according to the manufacturer's instructions. Total RNA was then
 cleaned to remove impurities and contaminants using Zymo Clean &
 Concentrator kit. RNAseq libraries were constructed by Novagene following
 a standard polyA+ enrichment strategy and sequenced on an Illumina HiSeq
 4000 for 150 bp paired end reads. We leveraged comparative genomic and
 transcriptomic approaches to identify patterns of convergence and
 divergence across these species.
description: # Convergent evolution of desiccation tolerance in grasses
 Here, we provide
 physiological metadata, annotation information, and other useful
 intermediate files from our analyses. ## Description of the data and file
 structure These data are organized in directories. In the parent
 directory, we provide physiological data in the file
 *3_grasses_timecourse_metadata.csv*. Plants were sampled at targeted
 hydration states during the process of dehydration, including well watered
 (WW), partially dehydrated (D1, D2, and D3), fully desiccated (D4), and
 rehydrated (R1 and R2). We validated the physiological status of tissues
 by measuring relative water content (RWC) and photosynthetic efficiency
 (Fv/Fm), and these data are summarized in the
 *3_grasses_timecourse_metadata.csv* file. The parent directory also
 contains a matrix of all the syntenic orthologs for each study species in
 the file *Syntenic_orthogroups.tsv*. We used the MCScan toolkit (v1.1)
 implemented in python
 []
 using the chromosome-scale Oropetium thomeaum genome as an anchor.
 Syntenic blocks were identified using gene models aligned using LAST with
 a minimum of five overlapping syntenic genes. A set of 18,428 conserved
 syntenic orthologs (syntelogs) across five desiccation tolerant grasses
 was created and used for downstream comparative genomic and cross-species
 transcriptomic analyses. The sub directory *Gene_expression* contains
 expression matrices for three species (*Microchloa caffra, Oropetium
 capense, and* Tripogon minimus) as well as the combined expression matrix
 for the syntelog expression across all three species. Trimmed reads were
 sudo-aligned to reference genomes using Salmon (v 1.9.0), and the
 resulting quantification files were processed with tximport (v 3.18) and
 DEseq2 R package (v 1.42.0) to generate normalized expression matrices. We
 computed the expression of syntenic orthologs for each species by summing
 the expression of all genes assigned to that syntelog. The sub-directory
 *DEGs* within *Gene_expression* contains lists of differentially expressed
 genes (DEGs) for each of the three species under both dehydration and
 rehydration conditions. DEGs were identified independently for each
 species with DEseq2 using RWC as a covariate. These analyses produced
 species-specific lists of DEGs during dehydration and rehydration with
 significant (FDR adjusted P-value &lt;0.05) associations with RWC. DEG
 files include the gene name, the reference expression level (basemean) the
 log2FC, standard errors, p-value and FDR adjusted p-values. Syntelog
 (synt) IDs are also included. Empty cells indicate that no syntelog was
 identified for that gene. The subdirectory *Co-expression_modules*
 contains sub-directories for each of the three species with lists of the
 genes and syntelogs in each co-expression module. We generated
 co-expression networks using Weighted Gene Co-expression Network Analysis
 (WGCNA) R package (v1.7). The sub-directory *Gene_ontology* contains the
 GO annotations for each of the three species as well as lists of enriched
 GO terms in each co-expression module and DEG set. GO terms were assigned
 through homology with the well annotated genome of sister species
 *Oropetium thomaeum*. This was done through a BLASTP (v 2.14.0) search of
 all *O. thomaeum* protein sequences against the protein sequences of each
 study species. Parameters were set to return the single best match for
 each peptide and an e-value cutoff of 1e-10. We assigned the GO terms from
 *O. thomaeum* to the homologous genes in our target species. The resulting
 files list the GO terms assigned to each gene. We then used TopGO R
 package (v 2.54.0) to identify significantly enriched GO terms
 (P-value&lt;0.05) within co-expression modules and sets of DEGs for up-
 and down-regulated genes during dehydration and rehydration in each target
 species. GO enrichment files show the p-values for each gene in the
 different conditions. The sub directory *KEGG* contains the KEGG
 annotations for each of the three species. KEGG annotations were generated
 for each species using BLASTKoala
 () on the complete set of annotated
 peptide sequences. ## Sharing/Access information Other data associated
 with this study, including genome assemblies of three resurrection grasses
 native to Sub-Saharan Africa and comprehensive gene expression datasets
 are deposited in NCBI BioProject PRJNA1044305.