The five divergent amino acids rule and evolution analysis of cytochrome c oxidase as new methods for Scolopendra Linnaeus, 1758 species differentiation (Chilopoda. Scolopendromorpha).

Scolopendra Linnaeus, 1758 is a genus of centipede showing reasonably good morpho-molecular correlation. This often allows the acceptable species differentiation and phylogenetic positioning by just using the partial sequence of the cytochrome c oxidase (COI) gene. However, for biological, statistical, or technical reasons, several exceptions to that fact have been observed, which together with a lack of a solid criterion to differentiate species at the molecular level, make difficult the interpretation of some taxonomic and systematic outcomes. With the scope of providing a standardized system improving the molecular Scolopendra species delimitation and surpassing some issues related with the mtDNAs use, a total of 45 representative COI sequences belonging to 22 taxa are tested, but for the first time, performing distinct amino acids chains evolution analyses. To illustrate this, a general evaluation of the genus is firstly provided, while to exemplify some of the deeper analysis, the cases of S. paradoxa Domenech, 2018 and S. spinosissima Kraepelin, 1903 are then here re-explored using some alternative tools. As a result, the partial COI protein sequences analysis identified, at least, ten amino acid residues positions as useful for Scolopendra species differentiation, being generally five divergent residues enough to distinguish taxa ["the five divergent amino acid rule"]. When this premise wasnt fulfilled, the amino acid frequency, exclusivity, or electrochemical properties and especially the r substitution probability test, helped solve the cases. Among S. paradoxa and S. spinosissima five diverging residues were found, with one of them being exclusive to the entire genus for the former taxon. Also, the substitution probability (r) analysis showed strong positive selection for three of these five divergent amino acids. As exceptions, S. dawydoffi Kronmuller, 2012 has been found indistinguishable from S. multidens Newport, 1844 by this method, suggesting its eventual synonymy, while S. cataracta Siriwut, Edgecombe & Panha, 2016 showed an intraspecific maximum divergence of 5 amino acids. Finally and at nomenclatural level, the species name S. hainanum Kronmuller, 2012 is emended as S. hainanensis while the taxon S. mojiangica Zhang & Chi, is here declared nomen nudum, due its original description remains unavailable.