Multi-locus analysis of Giardia duodenalis intra-Assemblage B substitution patterns in cloned culture isolates suggests sub-Assemblage B analyses will require multi-locus genotyping with conserved and variable genes

► The substitution rate of a gene determines its ability to resolve different relationships formed at different times. ► Mixed-substitution-rate multi-locus-genotyping ensures accurate phylogenetic inference in divergent populations. ► Allelic sequence heterozygosity substitution patterns follow the same trend as regular substitution patterns (those at all alleles).