Neofunctionalization is an event in the evolutionary history of a protein family in which the function changes from the function of its parent sequence. Through neofunctionalization, pairs of proteins that originate from the sequence by speciation (orthology) or by duplication (paralogy) can diverge in function. Two families of urea ABC transporter permease subunits, UrtB and UrtC, show themselves to be neofunctionalization donor families (PMID: 20102603). They show locally conserved sites of ancient origin, associated specifically with urea transport in an otherwise highly divergent protein family. Yet individual members the of urea ABC transporter permease subunit equivalog families often show much more recent branching in molecular phylogenetic trees from paralogs that now differ in function. One consequence of neofunctionalization in such families is that SIMBAL heat maps can identify short regions of sequence as hot spots that significantly outscore full-length sequences as predictors of conserved protein function.

Inferring which of two paralogs has undergone neofunctionalization may be attempted by comparing branch lengths in a molecular phylogenetic tree. The longer branch often reflects a burst of positive selection to optimize the neofunctionalized protein for its new function. In the absence of a paralog with conserved function for comparison, neofunctionalization for a protein sometimes may be inferred by an elevated ratio of non-synonymous to synonymous codon changes in the nucleotide sequence of the gene.