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Peptide compositions constructed out of whole sets of protein sequences can be used as species signatures for phylogenetic analysis. To account for point mutations, an amino acid substitution model is integrated into the complete composition vectors through a novel peptide clustering algorithm. Such a refined signature is expected to highlight deeper evolutionary relationships among the species and employed into the whole genome phylogenetic analysis to define a new evolutionary distance measure. Computational experiments have been set up to validate the effectiveness of this new measure and a vertebrate evolutionary tree using a dataset of 832 proteins for 64 vertebrates is reported.