## 2 Results
### Comprehensive Comparative Analysis of Phytolaca Chloroplast Genomes  
Comparative analysis of IR expansion and contraction in the six _Phylotaloca_ species, using _Adgestis clematidea_ as a reference, revealed that five species (_P. acinosa_, _P. insularis_, _P. polyandra_, _P. japonica_, and _P. latbenia_) exhibited an IR expansion of approximately 210-218 bp, whereas _P. americana_ showed a contraction of 150 bp (Figure S4). The _rps19_ gene, located at the LSC/IRb boundary, extended into the IRb region by 179 bp in five _Phylotaloca_ species, with the exception of _P. latbenia_. The _ndh_F gene, located at the IRb/SSC boundary, was uniformly included (1 bp) in the IRb region across all _Phylotaloca_ species. The _ycfl_ gene, located at the SSC/IRa boundary, is approximately 3874 bp in the SSC region for all _Phylotaloca_ species, except for _P. americana_, where it differs slightly at 3910 bp. The _trnH_ gene, located at the LSC/IRa boundary, is 2-3 bp away from the IRa in most species; however, in _P. americana_, it is 28 bp away. Compared to the cp genome of _A. clematidea_, IR contraction was observed in _P. americana_, whereas IR expansion was observed in the other five _Phylotaloca_ species. However, the gene order and position within _Phylotaloca_ species were similar. The sequences of the six _Phylotaloca_ species were compared using mVISTA, with _P. acinosa_ as the reference, to visualize sequence similarities and differences (Figure 4). As a result of the comparison, most of the variations were found in the IGS regions, except for the _ycfl_ gene of _P. americana_. Among the six sequences, _P. insularis_ showed the highest similarity with _P. acinosa_, whereas _P. americana_ exhibited the greatest variation. _P. americana_-specificvariations have been identified in several genes and IGS regions, including _rps16-trnQ-UUC_, _trnS-GCU-trnG-UICC_, _pdfl-trnS-GGA_, _pdfl-cemA_, _psal-rpl33_, and _ycf1_. Additionally, to assess genetic variation, we calculated the nucleotide diversity (Pi) values for the genes and IGS regions of the cp sequences from the six _Phytolaca_ species (Figure 5). Among the six _Phytolaca_ species, the regions with the highest Pi values were _ccsA-ndhD_ (0.0335), _trnM-atpE_ (0.02405), _trnT-trnL_ (0.02051), _rps16-trnQ_ (0.01805), _ndhF-rpl32_ (0.01783), and _rpl32-trmL_ (0.01655). When excluding regions with a Pi value of 0, the lowest Pi value was 0.00047, which was observed in the _trnG_ intron. These results indicate that the cp genome sequences contained various regions of variation among the six species, with several hotspot regions of high Pi values, particularly in the LSC and SSC regions. This variation highlights the areas of increased Pi within the _Phytolaca_ cp genomes. Figure 3: Distribution of repeat sequences in the chloroplast genome of _Phytolaca acinosa_. (**A**) Number of SSR repeats by large single-copy (LSC), small single-copy (SSC), and inverted repeat (IR) regions. (**B**) Number of SSRs distributed across exon, intron, and intergenic spacer (IGS) regions. (**C**) Frequency of six different SSR types. (**D**) Distribution of tandem repeats in LSC, SSC, IR, exons, intron regions, and IGSs. (**E**) Distribution of tandem repeats by length in six _Phytolaca_ species. (**F**) Frequency of different repeat types in the chloroplast genome. Figure 4: Comparison of the complete chloroplast genomes of six _Phylolaca_ species using mVISTA. Sequence variations between genomes are highlighted by white bars, while regions of sequence identity are indicated on a scale from 50% to 100%. Forward and reverse transcription of genes are indicated by gray arrows. Exonic regions are shown by purple bars, conserved non-coding sequences by pink bars, and green markings below the bars indicate the inverted repeat (IR) regions. Figure 5: Comparison of nucleotide diversity (Pi) values among six _Phylolaca_ species. The graphs depict the average nucleotide diversity (Pi) values for genes and intergenic spacers (IGS) across the chloroplast genomes of six _Phylolaca_ species.