# Conserved Plastid Genomes of _Pourthiaea_ Trees: Comparative Analyses and Phylogenetic Relationship
## 2 Materials and Methods
### Evolutionary and Phylogenomic Analyses  
To investigate the selection patterns of the 79 plastid PCGs, the synonymous (dS) and nonsynonymous (dN) nucleotide substitution rates, and their ratio (\(\omega\) = dN/dS) were computed using a site-specific model executed in the Codeml program (NSsites = 0, 1, 2, 3, 7, 8, model = 0, seqtype = 1) [42] of PAML4.9 [43] with F3 \(\times\) 4 codon frequency and cleandata = 1. To run the Codeml program, two files are required, namely seqfile and treefile. After extracting using PhyloSuite v1.2.2 [44], aligning using MAFFT v7 [45], and removing the stop codons manually, 79 plastid protein-coding sequences were used as the input segfiles. The ML tree constructed using RAxML v8.2.8 [46] based on 79 plastid protein-coding sequences was used as the input treefile. A likelihood ratio test (LRT) was used to calculate a _chi_-square approximation to determine the model fit. The Bayes  
\begin{table}
\begin{tabular}{l l l l l} \hline \hline
**Species** & **GenBank Numbers** & **Raw Reads** & **Clean Reads** & **Mean Coverage** \\ \hline _Pourthiaea bautverdiana_ & PQ197389 & 24,239,328 & 24,062,356 & 114.48\(\times\) \\ _Pourthiaea parifolia_ & PQ197390 & 22,662,706 & 22,404,606 & 61.31\(\times\) \\ _Pourthiaea villosa_ & PQ197391 & 24,516,204 & 24,214,064 & 109.37\(\times\) \\ _Photinia glomer_ & PQ197388 & 23,435,210 & 23,321,484 & 214.50\(\times\) \\ \hline \hline \end{tabular}
\end{table}
Table 1: Assembly information for three _Pourthiaea_ species and _Photinia glomera_.  
Empirical Bayes (BEB) analysis was used to recognize the sites under positive selection with posterior probabilities \(\geq\) 0.95.  
Phylogenetic trees were constructed using 29 complete plastome sequences and corresponding 29 rrDNA sequences to explore the phylogenetic relationship of _Pourthiaea_, with _Photinia glomerata_ as the outgroup. Bayesian inference (BI) and maximum likelihood (ML) methods were used for phylogenomic analyses. The PhyloSuite v1.2.2 [44] was used to extract the 78 plastid protein-coding sequences shared by 29 species. The 78 shared plastid protein-coding sequences were aligned using MAFFT v7 [45] with the codon mode, trimmed using trimAl v1.2 [47] with automated1, and then concatenated using PhyloSuite v1.2.2 [44]. The nrDNA sequences were aligned using MAFFT v7 [45] with the Auto algorithm. Then, the alignment was manually adjusted to minimize nucleotide mismatches. The ML analysis was performed using RAxML v8.2.8 [46] with 1,000 bootstrap replicates. Other parameters were set to \"rxxmlHPC -p 12,345 -f a - m GTRGAMMA \(\times\) 12,345 -s input -n output\". The BI analysis was performed using MrBayes v3.1.2 [48] with the best-fit substitution model (TVM + I + G for 78 shared plastid protein-coding sequences and GTR + I + G for nrDNA sequences) determined by Modeltest v3.7 [49] with the Akaike Information Criterion [50]. The two independent Markov chain Monte Carlo algorithms were run for 5,000,000 generations with one cold chain and three heated chains. The trees were sampled every 100 generations and the first 25% were discarded. Stationarity was achieved when the average standard deviation of the split frequencies < 0.01.