DNA extraction, PCR amplification, cloning, and sequencing

Total genomic DNA was extracted from silica gel-dried leaves or herbarium material using the Plant Genomic DNA Kit (Tiangen Biotech, Beijing, China) following the manufacturer’s instructions.

For each individual, five cpDNA regions (matK, rbcL, psbA–trnH and rps4 + rps4–trnS) and the nuclear gene region (LEAFY) were separately amplified with standard PCR. The matK region was amplified using primers and PCR protocols introduced by the CBoL Plant Barcoding Working Group (http://www.barcodinglife.org/index.php/Public_Primer_PrimerSearch). The rbcL region was amplified using primers 1F⁹⁸ and 1351R⁹⁹, following the PCR protocol described by Hasebe et al.¹⁰⁰. The psbA–trnH region was amplified using primers psbAF and trnHR according to the protocol outlined by Chen et al.¹⁰¹. Amplification primers and protocols to amplify rps4 + rps4–trnS were those described by Nadot et al. Smith and Cranfill^102,103. LEAFY was amplified using primers 1dF and 3dR, which were designed by Zhao⁴⁸.

The PCR products were purified using PEG 8000 or the TIANgel Midi Purification Kit (Tiangen Biotech, Beijing, China) following the manufacturer’s protocol. Then they were directly sequenced in both directions using the amplification primers with an ABI Prism^TM BigDye Terminator Cycle Sequencing Ready Reaction kit (Perkin Elmer, Norwalk, CT, USA). Sequences were analysed using at the ABI 3730XL automated sequencer (Applied Biosystems, Foster City, CA, USA). Cloning of samples with allelic variation in LEAFY was conducted with a pEASY-T3 Cloning Kit according to the manufactures’ protocols (Transgen Biotech), and 6 to 12 clones were sequenced for each sample. GenBank accession numbers are listed in Supplementary Table ^S2.