Significant multilocus linkage disequilibrium (mLD) for bothP. falciparum and P. vivax post-LLIN
For P. falciparum , matching haplotypes (allowing missing loci) were seen in all post-LLIN datasets and the pre-LLIN ESP2 2005 dataset. However, for P. vivax , matching haplotypes (allowing missing loci) were rarely seen and only in post-LLIN data sets. Among the 332 complete P. falciparum multilocus haplotypes (9-loci successfully genotyped) from all study sites, 16 repeated haplotypes were found, with 11 haplotypes represented two times, three represented three times, and two represented four times. Clonal haplotypes were always found within the same year and province, and in all cases except one in the same catchment area, but not always in the same village (7/16 haplotypes found in neighbouring villages). In ESP2 2005, one clonal haplotype was found in two villages (Yenigo and Sengo, Figure 1) from different catchment areas, roughly 40km apart. Among the 303 complete P. vivax multilocus haplotypes (10-loci), two haplotypes were repeated, with one haplotype represented two times (in two different villages in ESP 2012), and one represented four times (three in one village, one in a neighbouring village in MAD 2010).
To investigate whether inbreeding was present in these populations (Smith, Smith, O’Rourke, & Spratt, 1993), non-random associations among the microsatellite loci (mLD) were calculated for all complete and unique haplotypes. Whilst mLD was absent from most pre-LLIN populations, significant mLD was observed in P. falciparum and P. vivaxinfections post-LLIN (Table 1). In ESP 2012-13, mLD was high with unique infections indicating the circulation of closely related haplotypes in the population, suggesting near clonal transmission (low recombination between diverse clones and high levels of inbreeding) or the presence of high proportions of meiotic siblings among isolates (Bright et al., 2014; Smith et al., 1993), as observed in the village of Sunuhu (Table S3). Low, but significant mLD was found for P. falciparum in Madang in 2006 (Table 1), however this population was structured (Schultz et al., 2010), resulting in a phenomenon called the Wahlund effect, confirmed by the fact that linkage equilibrium was restored when mLD was analysed separately for subpopulations (Jennison et al., 2015; Wahlund, 1928) (Table S4). In post-LLIN Madang, the observed mLD forP. falciparum is not the result of subpopulation structure, as significant mLD remained in the subpopulations (Table S4). In the Mugil area in 2014, significant mLD for P. falciparum remains due to the circulation of a few very closely related haplotypes in the Megiar village (Table S4, Dataset 1).