Clonal diversity in Giardia duodenalis isolates from Thailand: Evidences for intragenic recombination and purifying selection at the beta giardin locus

The beta giardin locus of Giardia duodenalis encodes a structural component of ventral disc and exhibits sequence variation among isolates rendering it a useful marker for genotypic analysis. To determine the distribution of genotypes of G. duodenalis in Thailand and to explore the extent of sequence variation in this locus, we deployed the PCR-RFLP method and sequence analysis of recombinant subclones from 30 clinical isolates. In total, assemblage B was more prevalent than assemblage A. Sequence analysis revealed that 13 isolates had clonal mixtures, comprising three to five distinct sequences per isolate. Nucleotide diversity of assemblage B was greater than that of assemblage A. A striking transitional bias was noted at the first and the third positions of codons in both assemblages; however, they differed in the patterns of nucleotide diversity at 0-fold and 4-fold-degenerate sites. Most amino acid exchanges were conservative in terms of polarity, charge and volume. Both assemblages have evolved under purifying selection as evidenced by a significantly greater number of mean synonymous substitutions per synonymous site (dS) than that of nonsynonymous substitutions per nonsynonymous site (dN) as well as significant negative Tajima's D values and its related statistics. The significant negative Tajima's D test at nonsynonymous sites further suggests that elimination of slightly deleterious mutations at these sites by purifying selection is ongoing as predicted in the nearly neutral theory. Furthermore, a minimum number of seven recombination sites was detected by the four gamete test in assemblage B, consistent with previous reports on meiotic recombination in G. duodenalis. Therefore, accurate subassemblage assignment of clinical isolates that has practical consequence for disease control could be complicated by the presence of intra-isolate clonal diversity and interallelic recombination.