*2.3. PCR Amplification and Sequencing*

Four standard plant DNA barcode loci (Table 1) were amplified, including the two core barcoding regions for land plants, *rbcL* and *matK* [20] and two additional loci that have been proposed as additional plant DNA barcoding loci, ITS2 and *psbA-trnH* [21–23]. PCR was performed using Bioline Taq polymerase (New England Biolabs, Ipswich, MA, USA) and a standard thermal cycling profile including an initial denaturation for 5 min at 95 ◦C, 35 cycles each including 95 ◦C denaturation for 30 s, a locus-specific annealing temperature (see Table 1) for 30 s, and an extension cycle at 72 ◦C for 40 s, followed by a final extension of 72 ◦C for 10 min. Amplified PCR products were cleaned using ExoSAP-IT (ThermoFisher Scientific, Waltham, MA, USA) following the manufacturer's protocols. Cycle sequencing was performed in 96-well plates using the same PCR primers, the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems®, Norwalk, CT, USA), and

sequenced on the Automated ABI3730 Sequencer (Life Technologies, Carlsbad, CA, USA). Raw chromatograms were edited in Geneious Prime (2021, https://www.geneious.com, accessed on 15 January 2022) and annotated before uploading to BOLD (https://www. boldsystems.org, accessed on 30 January 2022) and GenBank (See Supplementary Table S1 for accession numbers).

**Table 1.** Locus information for each plant DNA barcoding marker used in this study, including forward and reverse primer names, primer sequences, annealing temperature, and citation.

