*4.2. DNA Extraction, Sequencing and Assembly*

All the methods in this article were based on the methods of Zhou et al. [40]. Total genomic DNA was extracted from samples using the DNeasy Plant Mini Kit with a standard protocol (Qiagen Co., Hilden, Germany). The DNA was sequenced according to the manufacturer's manual for the Illumina Hiseq X. Approximately 6.2 Gb of raw data from *M. cochinchinensis*, 6.5 Gb of raw data from *M. tricolor*, and 6.3 Gb of raw data from *M. bibracteolatus* were generated with 150 bp paired-end read lengths. The software Trimmomatic (version 0.39, Institute for Biology, Aachen, German) [41] was used to filter the low-quality reads of the raw data, and the Q value was defined as Sanger. Then, all the clean reads were mapped to the database on the basis of their coverage and similarity. Burrows–Wheeler Aligner (BWA-MEM, Wellcome Trust Sanger Institute, Wellcome Genome Campus, Cambridge, UK) was used in chloroplast genome assembly to generate the bam files. The depth was calculated using Samtools (Medical Population Genetics Program, Broad Institute, Cambridge, MA, USA) and plotted using Rscript (with the smoothScatter function). The accuracy of the assembly of the four boundaries (SSC, LSC and IR regions) of the chloroplast sequences was confirmed through PCR and Sanger sequencing using the validated primers listed in Table S5. The assembled complete chloroplast genome sequence of *M. cochinchinensis*, *M. tricolor* and *M. bibracteolatus* were submitted to the NCBI, and the accession numbers were MH161424, MH161425 and MH161423, respectively. The raw data of three species were submitted to the NCBI. The Bioproject ID of this study is PRJNA587349. The SRA accession ID of *M. tricolor* is SRR10442639, that of *M. bibracteolatus* is SRR10442640, and that of *M. cochinchinensis* is SRR10442641.

#### *4.3. Genome Comparison and Phylogenetic Analyses*

The whole-genome alignment for the chloroplast genomes of three *Macrosolen* species were performed and plotted using the mVISTA program (http://genome.lbl.gov/vista/mvista/submit. shtml) [42]. Gene content comparison was analyzed by CPGAVAS2 (Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing,

China) [43] and identified by manual correction. To determine the phylogenetic positions of three *Macrosolen* species within Santalales, we analyzed the chloroplast genomes of 16 species, encompassing 11 other taxa within this lineage, *Viscum album* (KT003925), *V. coloratu* (NC\_035414), *V. crassula* (KT070881), *V. minimum* (KJ512176), *Osyris alba* (KT070882), *Schoepfia jasminodora* (KX775962), *Champereia manillana* (NC\_034931), *T. chinensis* (KY996492), *T. sutchuenensis* (KY996493), *T. delavayi* (MH161426), and *T. thibetensis* (MH161427). The chloroplast genomes of *Panax ginseng* (AY582139) and *N. tabacum* (NC\_001879) were used as outgroups.
