Phylogeny of genetic codes and punctuation codes within genetic codes

Punctuation codons (starts, stops) delimit genes, reflect translation apparatus properties. Most codon reassignments involve punctuation. Here two complementary approaches classify natural genetic codes: (A) properties of amino acids assigned to codons (classical phylogeny), coding stops as X (A1, antitermination/suppressor tRNAs insert unknown residues), or as gaps (A2, no translation, classical stop); and (B) considering only punctuation status (start, stop and other codons coded as −1, 0 and 1 (B1); 0, −1 and 1 (B2, reflects ribosomal translational dynamics); and 1, −1, and 0 (B3, starts/stops as opposites)). All methods separate most mitochondrial codes from most nuclear codes; Gracilibacteria consistently cluster with metazoan mitochondria; mitochondria co-hosted with chloroplasts cluster with nuclear codes. Method A1 clusters the euplotid nuclear code with metazoan mitochondria; A2 separates euplotids from mitochondria. Firmicute bacteria Mycoplasma/Spiroplasma and Protozoan (and lower metazoan) mitochondria share codon-amino acid assignments. A1 clusters them with mitochondria, they cluster with the standard genetic code under A2: constraints on amino acid ambiguity versus punctuation-signaling produced the mitochondrial versus bacterial versions of this genetic code. Punctuation analysis B2 converges best with classical phylogenetic analyses, stressing the need for a unified theory of genetic code punctuation accounting for ribosomal constraints.