Chloroplast gene sequencing
To look for differences in maternal subgenome between P. distichum and P. vaginatum and to confirm that the coarse-textured ecotypes are part of the Disticha clade (see Results), we sequenced the intron of the rpl16 chloroplast ribosomal genes of 16 accessions (3 P. distichum , 13 P. vaginatum ) using the F71 and R1661 primers described in Giussani et al. (2009). TheP. vaginatum samples included 3 fine-textured and 10 coarse-textured ecotypes. Direct Sanger sequencing of purified PCR products was performed at the University of Wisconsin Biotechnology Center using an ABI 3730xl DNA Analyzer. Sequences were aligned against the P. vaginatum rpl16 sequence (accession KF853027; Scataglini, Zuloaga, Giussani, Denham, & Morrone, 2014) and compared using BioEdit (Hall, 1999).