## 3 Discussion  
The complete cp genome of P. acinosa was successfully assembled with a total length of 156,433 bp and a typical quadripartite structure comprising an LSC region, an SSC region, and two IR regions. The overall GC content was found to be 36.8%, with the IR regions displaying a notably higher GC content (42.6%) than the LSC (34.6%) and SSC (30.3%) regions. This pattern of elevated GC content in IR regions relative to single-copy regions aligns with the cp genomes of other angiosperms, indicating a conserved structural feature across diverse plant lineages [43; 44]. The P. acinosa cp genome contains 113 unique genes, including 79 protein-coding genes, 4 rRNAs, and 30 tRNAs, which are typical of other angiosperms [26; 45]. The conservation of gene content across species suggests that the cp genome of P. acinosa has remained largely unchanged over evolutionary time, underscor  
Figure 8: Phylogenetic tree with divergence times estimated by the Bayesian model based on 76 protein-coding genes from 14 species of Phytolaccoidae and Agdestidoideae. At all nodes, the poster prior value is 1, and each node displays the average divergence time. The purple bar corresponds to the 95% highest posterior density (HPD). ing the critical roles these genes play in essential functions such as photosynthesis, ATP synthesis, and transcription [46]. Codon usage is closely related to gene expression efficiency and accuracy, and offers insights into the evolutionary patterns of cp genomes [47]. In this study, _P. acinosa_ and related species, including _P. insularis_, _P. polyandra_, _P. japonica_, _P. latbenia_, and _P. americana_, exhibited nearly identical codon numbers and relative synonymous codon usage (RSCU) values, suggesting a strong preference for certain synonymous codons. Analysis of the RSCU values showed that approximately half of the codons exhibited a notable bias (RSCU > 1), with those featuring an A or T at the third nucleotide position being especially favored. This pattern aligns with observations in various angiosperms, suggesting a conserved evolutionary trend in the cp genomes of these species [48; 49]. Certain codons such as CGC (arginine), CTC, and CTG (both leucine) have relatively low RSCU values, whereas GCT (alanine), TAA (stop codon), and AGA (arginine) had higher RSCU values. This pattern likely reflects biases in codon usage influenced by mutational pressure and translational efficiency [50; 51]. The distribution of SSRs across the six _Phytplacca_ species showed a consistent pattern, with the majority concentrated in the LSC and SSC regions. A notable proportion of these SSRs are located in noncoding regions, such as the IGS. In these regions, relaxed selective pressure allows mutations to persist without disrupting essential functions, unlike in coding regions, where harmful mutations are often eliminated by natural selection [52; 53; 54]. This permissive environment fosters the accumulation of repetitive sequences such as SSRs through mechanisms such as replication slippage, recombination events, and associations with retrotransposposons [55; 56]. These mechanisms contribute to sequence expansion and genetic variation in these regions [57; 58]. Interestingly, _P. americana_ exhibited a lower overall number of SSRs than other species, suggesting species-specific genomic variation. This variation could be attributed to factors such as genome size, random mutations, or differential selection pressures, although this may not indicate significant evolutionary divergence [59; 60; 61]. Similarly, tandem repeats predominantly accumulated in noncoding regions across the LSC and SSC in all species. Although SSRs are shorter and simpler, tandem repeats exhibit a greater length and complexity, potentially contributing to genome stability or the formation of secondary DNA structures in specific contexts [62]. The analysis of repeat types across all species revealed that palindromic repeats were the most abundant, followed by forward repeats, with _P. americana_ exhibiting similar patterns. However, _P.