**3. Discussion**

Numerous variations occur in the chloroplast genomes of parasitic plants. However, only a small number of plants within Santalales have been studied. In this study, the complete chloroplast genomes of *M. cochinchinensis*, *M. tricolor* and *M. bibracteolatus* from Santalales were assembled, annotated and analyzed. Compared with the chloroplast genomes of the model plant *Nicotiana tabacum*, all the *ndh* genes of the chloroplast genomes were lost amongst the three species, and the *infA* gene, which codes for a translation initiation factor, was also missing in these species. These cases were similar to those of *T. chinensis* and *T. sutchuenensis* [7]. The *rpl16* and *ycf15* genes were lost in the three species, but they were still present in *T. chinensis* as pseudogenes (Figure 9). However, compared with the results reported by Shin et al. [36], different gene contents of the chloroplast genome were observed in *M. cochinchinensis*. These studies have shown that *M. cochinchinensis* contains the exon 1 fragment of the *ndhB* gene and a fragment of the *infA* gene, whereas the *rpl36* gene is completely lost. However, the *rpl36* gene is still present in the chloroplast genome according to our results. *M. cochinchinensis* has lost the *infA* gene and all *ndh* genes. The number of tRNA genes also differed between the two studies. We annotated 35 tRNA genes, but previous studies only obtained 30 tRNA genes. The evolution of the chloroplast genome in parasitic plants, particularly nonphotosynthetic holoparasites, can lead to significantly reconfigured plastomes [21]. The losses of *ndh* genes are associated with nutritional status or extensive rearrangements of chloroplast structures [37], and they have occurred in the reported chloroplast genomes of parasitic plants [7]. Our study also showed that *ndh* genes were lost in the transformation from autotrophy to heterotrophy [38].

**Figure 9.** Comparison of the chloroplast genome gene content of six parasitic plants and one model plant (*Nicotiana tabacum*). The common existing genes in the complete chloroplast genome of the seven species are not listed. Red boxes indicate each gene present, and green boxes indicate that each gene is considered as a pseudogene. The yellow boxes indicate an absent gene.

The Santalales order consists of a small number of autotrophic species and a large number of parasitic species which are root or aerial (stem) parasites [39]. According to the Engler system, Santalales consists of seven families. We downloaded five families belonging to Santalales, which were available in the National Center for Biotechnology Information (NCBI) at that time, and two

species as outgroups to analyze the phylogenetic relationships of *Macrosolen* in Santalales. The present study showed that Loranthaceae is closely related to Olacaceae, whereas Viscaceae is closely related to Santalaceae and Opiliaceae. These results are similar to those of previous studies [13,14]. All the phylogenetic results strongly support that Loranthaceae and Viscaceae diverged independently from each other.

As folk medicine in China, *M. cochinchinensis*, *M. tricolor* and *M. bibracteolatus* have been used to treat diseases for a long time, and their dried stems and branches with leaves are used as medicinal parts. However, *Macrosolen* species are similar in appearance, especially when they are processed into medicinal slices, thereby causing difficulty in their identification. The identification of parasitic medicinal materials has rarely been reported. Though phytochemical approaches have played an important role in species identification [26], they are inadequate because they are limited to the environment and harvest period. Molecular characterization has shown an improved specificity for plants [23,26]. In our study, mutational hotspots such as the *ycf1* gene, the *ccsA* gene and the *trnF-trnM* intergenic region are potential sites for identification of *Macrosolen* species.
