Phylogenetic analysis

The data obtained from the sequencing analysis were used as the target sequence in a Basic Local Alignment Search Tool search to determine the identity. The sequences were subsequently aligned using the ClustalW program http://www.ebi.ac.uk/Tools/msa/clustalw2 (11). The GenBank accession numbers of the sequences used in the current study are presented in Table I. A phylogenetic tree was constructed from the mRNA sequences using MEGA version 6 software (12) with the neighbor-joining method (13) with 1,000 bootstrap replications (14). To identify the evolutionary forces underlying the divergence in the primate MIOX genes, the synonymous (dS; encoded amino acid does not change) and non-synonymous (dN; the encoded amino acid changes) substitutions were quantified using the modified Nei-Gojobori method (15), and the MIOX CDs from apes, Old World monkeys (OWMs) and New World monkeys (NWMs), with the lemur counterpart sequence as the out-group. The hypothesis of positive or adaptive evolution (dN>dS), purifying selection (dN<dS) and neutrality (dN=dS) was then tested using a codon-based Z test of selection (16), which is included in the MEGA version 6 software (12). P<0.05 was considered to indicate a statistically significant difference.

Primates MIO-X sequences from NCBI GenBank.