## 3 Results and Discussion
### Genomic Divergence Comparison through Rearrangements and Divergent Hotspots  
To investigate gene rearrangements within Campanulaceae species, LCB patterns were visualized. Connected color lines illustrate a highly rearranged structure across six species, and IR regions had more stable LCBs than LSC and SSC regions (Figure 4). To better characterize LCBs at the sequence level, the whole sequence of _A. japonicum_ was compared to that of _H. asiatica_, _A. remotiiflora_, and _C. carpatica_ species. The results indicate that sequence orders had largely moved between _A. japonicum_ and the other species (Figure S2). Both LCB rearrangements and sequence orders showed that considerable variation occurred intensively in the SSC region at positions of 116,880-152,791 bp (Figures 4 and S2).  
To clarify the divergent hotspots in hypervariable regions predicted by the LCBs, multi-layer alignment was performed using mVISTA with four genus species. Alignment for homology comparison showed that many non-conserved genes were found in whole positions (Figure 5). These genes were mainly discovered in single-copy regions, which were analyzed to have a high level of rearrangement of LCBs (Figures 4 and 5). The presence of these genes indicates that _A. japonicum_ experienced high structural variability in the plastome.  
Figure 3: Visualization of the junction characteristics in four boundaries using IRscope with _A. japonicum_ as a reference. The colored boxes show the four identified regions, and JLB, JSB, JSA, and JLA indicate a junction boundary between the two regions.  
Generally, conserved genes in IR regions are used as evolutionary stability indicators because these genes maintain plastome stability in most plants [48]. We found ten protein  
Figure 4: Features of multiple alignment with local collinear blocks (LCBs) using Mauve progressive algorithm. Color-coded LCBs present conserved sequences of collinear blocks, and color lines represent connecting relationships of each LCB.  
Figure 5: Visualization of plastome alignments for homology comparison using mVISTA with _A. japonicum_ as a reference. Arrows show location/direction of gene. X- and y-axes indicate genome sequence length and pairwise percent of identity, respectively. Blue-coded box shows conserved genes (exon), and red-coded box shows conserved non-coding sequences (CNSs).  
coding genes and nine RNA-related genes in IR regions (Table S4). Of the detected IR genes, the _clpP_ gene has three exons, three genes (_ndhA_, _trnA-UGC_, and _trnI-GAUI_) have two exons, and the remaining genes have one exon. Interestingly, the _clpP_ gene has two introns and three exons consisting of 146 bp for exon-1, 284 bp for exon-2, and 242 bp for exon-3. Previous studies have suggested that the _clpP_ gene may have affected plastome structures with structural variations in mutation hotspots [49].  
The LCB and divergent hotspot results differ from previous studies [50] in that coding regions have mostly conserved genes in most plant species. However, large-scale rearrangement events have been reported in coding regions of Campanulaceae species [13; 14]. Considering that rearrangement variations occur very rarely [51], many non-conserved genes of coding regions suggest that _Asyneuma_ species have undergone a large evolutionary divergence.