Chlorophyta is one of the two majors lineages of green plants, and the class Palmophyllophyceae has recently been identified as the deepest branch of Chlorophyta. This study used a custom-built amino acid substitution model for chloroplast genome data and the node-discrete composition heterogeneity model (NDCH2) to infer the phylogenetic relationship of the Palmophyllales and Prasinococcales, plus the internal groups of class Palmophyllophyceae. A chloroplast data set of 67 coding-genes of 28 green plant taxa was constructed. Analyses of the nucleotide data set demonstrated that the degenerate-codon method performed better than simply removing the third codon positions for eliminating the effects of synonymous substitutions. The custom amino acid model was estimated from the protein data set by a maximum likelihood procedure, FastMG, and named as prasREV (prasinophyte reversible model). Both the prasREV and NDCH2 models were shown to be a better fit than other substitution models. Phylogenetic analyses using these models indicate that the macroscopic Palmophyllales is the sister-group of the unicellular Prasinococcales, which correspond with their common feature of having gelatinous matrix embedding cells. Palmophyllales is one independent origin of multicellularity in green plants, and along with Prasinococcales, forms the earliest-diverging clades of the Chlorophyta, the Palmophyllophyceae.
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