Complete sequence and comparative analysis of the mitochondrial genome of the rare and endangered Clematis acerifolia, the first clematis mitogenome to provide new insights into the phylogenetic evolutionary status of the genus

Abstract

Clematis is one of the large worldwide genera of the Ranunculaceae Juss. Family, with high ornamental and medicinal value. China is the modern distribution centre of Clematis with abundant natural populations. Due to the complexity and high morphological diversity of Clematis, the genus is difficult to classify systematically, and in particular, the phylogenetic position of the endangered Clematis acerifolia is highly controversial. The use of the mitochondrial complete genome is a powerful molecular method that is frequently used for inferring plants phylogenies. However, studies on Clematis mitogenome are rare, thus limiting our full understanding of its phylogeny and genome evolution. Here, we sequenced and annotated the C. acerifolia mt genome using Illumina short- and Nanopore long-reads, characterized the species first complete mitogenome, and performed a comparative phylogenetic analysis with its close relatives. The total length of the C. acerifolia mitogenome is 698,247 bp and the main structure is multi-branched (linear molecule 1 and circular molecule 2). We annotated 55 genes, including 35 protein-coding, 17 tRNA, and 3 rRNA genes. The C. acerifolia mitogenome has extremely unconserved structurally, with extensive sequence transfer between the chloroplast and mitochondrial organelles, sequence repeats, and RNA editing. The phylogenetic position of C. acerifolia was determined by constructing the species mitogenome with 24 angiosperms. Further, our C. acerifolia mitogenome characteristics investigation included GC contents, codon usage, repeats and synteny analysis. Overall, our results are expected to provide fundamental information for C. acerifolia mitogenome evolution and confirm the validity of mitochondrial analysis in determining the phylogenetic positioning of Clematis plants.

Keywords: Clematis acerifolia; clematis; mitochondrial genome; organelle genome; phylogenetic relationship; repeats.

FIGURE 1

Branched conformation of C. acerifolia mitogenome. (A): Sketch of the mitogenome of C. acerifolia, containing 136 nodes. (B): The mitogenome sketch obtained after selecting the best path supported by the long read data of ONT and resolving the individual repeat regions. (C): Mitogenome mapping of C. acerifolia, with circular DNA molecules comprising contig1 and linear DNA molecules consisting of contig2, contig3, contig4, contig5, contig6 and contig7.

FIGURE 2

C. acerifolia mitogenome gene map. Genes shown on the outside and inside of the circle are transcribed clockwise andcounterclockwise, respectively. The dark gray region in the inner circle depicts GC content.

FIGURE 3

C. acerifolia mitogenome relative synonymous codon usage (RSCU). (A) Before RNA editing. (B) After RNA editing. Codon families are shown on the x-axis. RSCU values are the number of times a particular codon is observed relative to the number of times that codon would be expected for a uniform synonymous codon usage.

FIGURE 4

Number of RNA editing sites predicted by individual PCGs in mitochondria.

FIGURE 5

C. acerifolia mitogenome repeatedsequence diagram. Two mitochondrial molecules were analyzed separately forrepetitive sequences (one linear and one circular), taking the linear molecule 1 as an example. For example, the colored lines on the innermost circle connect the two repetitive sequences of TEs, the green line represents Palindromic Match (P), the yellow line represents Forward Match (F), and the purple line represents Reverse Match (R). The black line on the second circle represents TRs, and the black line on the outermost circle represents SSRs. molecule 2 and so on.

FIGURE 6

Schematic representation of gene transfers between chloroplast and mitogenome in C. acerifolia. The blue arcs in the diagram represent the mitogenome, the green arcs represent the chloroplast genome, and the rosy red lines between the arcs correspond to the genomic fragments that are homologous.

FIGURE 7

Phylogenetic tree of 24 angiosperms based on the sequences of 39 conserved mitochondrial PCGs. Zygophyllum fabago and Tribulus terrestris were selected as outgroups. The number at each node is the bootstrap probability.

FIGURE 8

C. acerifolia mitogenome synteny. Bars indicated the mitogenomes, and the ribbons showed the homologous sequences between the adjacent species. The red areas indicate where the inversion occurred, the gray areas indicate regions of good homology. Common blocks less than 0.5 kb in length are not retained, and regions that fail to have a common block indicate thatthey are unique to the species.

FIGURE 9

Dot plot and collinearity analysis of the mitogenomes of C. acerifolia, A. maxima and A. kusnezoffii. (A, B) are dot plots of A. maxima and A. kusnezoffii with C. acerifolia, respectively. (C) is the collinearity analysis between mitogenomes, with homologous regions represented by the same color blocks and connected by lines.