DNA and RNA isolation and sequencing

DNA was extracted from leaf tissue for 69 of the 74 taxa using a DNeasy Plant Kit (Qiagen, Germantown, Maryland, USA). To increase yield, slight modifications to the manufacturer’s protocol included increasing lysis buffer incubation time to 1 h and using 25 µL of buffer to elute the final sample. TruSeq library preparation (Illumina, San Diego, California, USA) and genome survey sequencing (GSS, also known as skim sequencing) on a NextSeq instrument were carried out at the University of Missouri, resulting in 2 × 150 bp reads.

RNA from leaf tissue was collected and immediately flash frozen using liquid nitrogen. For 38 samples, RNA was isolated using an Invitrogen PureLink RNA Mini Kit (Thermo Fisher Scientific, Carlsbad, California) followed by TruSeq library preparation and sequencing on the NextSeq, resulting in 2 × 75 bp reads (Appendix S3). For 16 samples, RNA was isolated using an Invitrogen PureLink RNA Mini Kit then sequenced on an Illumina HiSeq instrument, resulting in 2 × 100 bp reads (Appendix S3). For 17 samples, RNA was sequenced on a HiSeq for 2 × 100 bp reads but using a Qiagen RNeasy Plant Kit for RNA isolation (Appendix S3). Two samples were isolated using a ThermoFisher Invitrogen PureLink RNA Mini Kit) and sequenced on a HiSeq for 2 × 250 bp reads (Appendix S3). All sequencing and library preparation for the above samples was performed by the University of Missouri DNA Core Facility.

At the University of Alberta, the sample Cleomella serrulata had tissue pooled from leaves, apical meristematic tissue, and floral tissue of different developmental stages including small, medium, and large buds and open flowers from two plants. Total RNA was extracted using a Qiagen RNeasy Plant Mini Kit following the manufacturer’s protocol, then treated with DNAse I (New England Biolabs, Ipswich, Massachusetts, USA) for 30 min at 37°C to remove residual DNA from the total RNA. Sequencing was conducted by Plateforme d’analyses génomique (l’Université Laval, Quebec City, Quebec, Canada) with Illumina TruSeq RNASeq for library preparation and Illumina for sequencing for paired‐end 2 × 100 bp reads.