Gene Losses and Plastome Degradation in the Hemiparasitic Species Plicosepalus acaciae and Plicosepalus curviflorus: Comparative Analyses and Phylogenetic Relationships among Santalales Members

Abstract

The Plicosepalus genus includes hemiparasitic mistletoe and belongs to the Loranthaceae family, and it has several medicinal uses. In the present study, we sequenced the complete plastomes of two species, Plicosepalus acaciae and Plicosepalus curviflorus, and compared them with the plastomes of photosynthetic species (hemiparasites) and nonphotosynthetic species (holoparasites) in the order Santalales. The complete chloroplast genomes of P. acaciae and P. curviflorus are circular molecules with lengths of 120,181 bp and 121,086 bp, respectively, containing 106 and 108 genes and 63 protein-coding genes, including 25 tRNA and 4 rRNA genes for each species. We observed a reduction in the genome size of P. acaciae and P. curviflorus and the loss of certain genes, although this reduction was less than that in the hemiparasite and holoparasitic cp genomes of the Santalales order. Phylogenetic analysis supported the taxonomic state of P. acaciae and P. curviflorus as members of the family Loranthaceae and tribe Lorantheae; however, the taxonomic status of certain tribes of Loranthaceae must be reconsidered and the species that belong to it must be verified. Furthermore, available chloroplast genome data of parasitic plants could help to strengthen efforts in weed management and encourage biotechnology research to improve host resistance.

Keywords: Plicosepalus acaciae; Plicosepalus curviflorus; comparative analysis; loranthaceae; mistletoe; phylogenetic relationship; plastome; plastome structure.

Figure 1

(a) Gene map of the Plicosepalus acaciae plastid genome. (b) Gene map of the Plicosepalus curviflorus plastid genome. Small single copy (SSC), large single copy (LSC) and inverted repeats (IRa and IRb) are indicated. Thick lines on the outer complete circle identify the inverted repeat regions (IRa and IRb). The genes outside the circle are transcribed counterclockwise, whereas those inside the circle are transcribed clockwise. Genes belonging to different functional groups are highlighted in different colours. The dark grey area in the inner circle indicates the CG content of the plastome, using OGDRAW tool Version 1.3.1.

Figure 1

(a) Gene map of the Plicosepalus acaciae plastid genome. (b) Gene map of the Plicosepalus curviflorus plastid genome. Small single copy (SSC), large single copy (LSC) and inverted repeats (IRa and IRb) are indicated. Thick lines on the outer complete circle identify the inverted repeat regions (IRa and IRb). The genes outside the circle are transcribed counterclockwise, whereas those inside the circle are transcribed clockwise. Genes belonging to different functional groups are highlighted in different colours. The dark grey area in the inner circle indicates the CG content of the plastome, using OGDRAW tool Version 1.3.1.

Figure 2

Amino acid frequencies of the protein-coding sequences of Plicosepalus acaciae (blue) and Plicosepalus curviflorus (orange) chloroplast genomes using MEGA software Version 11.0; the most and least frequent amino acids are shown.

Figure 3

Number of each repeat type—F, forward; P, palindromic; R, reverse; and C, complement repeats—in the plastid genome of Plicosepalus acaciae and Plicosepalus curviflorus and six species from Loranthaceae, using REPuter 2 software. Taxillus chinensis had the highest frequency of palindromic repeats, Nuytsia floribunda had the highest frequency of forward repeats, and Plicosepalus curviflorus had the highest frequency of reverse repeats. Complement repeats were the least common type of repeat.

Figure 4

Number of different simple sequence repeat (SSR) types in the plastid genomes of Plicosepalus acaciae and Plicosepalus curviflorus and six species from Loranthaceae using MISA software v2.1. The majority of SSRs in the cp genome were monorepeats.

Figure 5

Number of SSR types in the complete chloroplast genome, protein-coding regions, and non-coding regions of (a) Plicosepalus acacia, (b) Plicosepalus curviflorus.

Figure 6

Whole chloroplast genome alignments for Loranthaceae species via the mVISTA program, using the annotation of Plicosepalus acaciae as reference. The x-axis represents the coordinates in the cp genome, while the y-axis indicates percentage identity from 50% to 100%. The top grey arrows indicate the position and direction of each gene. Pink indicates non-coding sequences (NCS), blue indicates protein-coding genes, and light green indicates tRNAs and rRNAs.

Figure 7

Comparison of the large single copy (LSC), a small single copy (SSC) and two inverted repeats (IRa and IRb) region borders among the chloroplast genomes of eight Loranthaceae species using IRSCOPE. Variations in the region’s length and gene locations are observed.

Figure 8

Synonymous (Ks) and Ka/Ks ratio values of 59 protein-coding genes of the Plicosepalus acacia vs. Loranthaceae plastomes (Plicosepalus curviflorus, Scurrula chingii, Taxillus chinensis, Loranthus europaeus, Dendrophthoe pentandra, Nuytsia floribunda and Elytranthe), using the KaKs Calculator 2.0 to detect substitution, selection, and beneficial mutation genes under selective pressure (>1).

Figure 9

Heatmap displaying a comparison of the plastid genome gene content of 11 parasitic plants and 1 autotrophic plant (Erythropalum scandens) using Plotly software. The common existing genes in the plastid genome of the 12 species are not listed. Orange colour indicates each gene present and seafoam indicates a pseudogene. The yellow indicates an absent gene.

Figure 10

Phylogenetic tree construction inferred from the complete chloroplast genomes of 21 taxa including Plicosepalus acaciae and Plicosepalus curviflorus, using Maximum Likelihood (ML) methods and Megatool. The tree shows the relationships between tribes and sub-tribes of Loranthaceae and related families of the order Santalales (Viscaceae, Santalaceae, and Schoepfiaceae), Nicotiana tabacum was used as an outgroup. The numbers in the branch nodes represent bootstrap support (BS). All branches of the tree were highly supported with 100% bootstrap values. Monophyly of the Loranthaceae family was strongly supported and P. acacia and P. curviflorus belong to subtribe Tapinanthinae.