Contrasting and dynamic patterns of population structure for both P. falciparum and P. vivax
As previously described, for P. falciparum, low to moderate genetic differentiation was seen between the Wosera 2005 (ESP1) and Madang 2006 studies (Jennison et al., 2015; Schultz et al., 2010) (Figure 3). Comparisons were not done between Pf 2005/6 and otherPf populations as it was not possible to calibrate data through combining allele calls before binning (see Materials and Methods). Post-LLIN, there remains low to moderate genetic differentiation between ESP (2012-2013) and Madang (2014) (Figure 3). However, there was little genetic differentiation of East Sepik P. falciparum populations pre-LLIN (ESP2 2005) compared to post-LLIN (2012-13), nor between Madang 2010 and 2014 populations (Figure 3). For P. vivax , a different pattern can be seen, with low genetic differentiation between provinces pre-LLIN, which increases in the post LLIN-studies (Figure 3). Similar to P. falciparum , within province genetic differentiation between the different time points does not increase post-LLIN.
Population genetic structure was further investigated by Bayesian cluster analysis using STRUCTURE (Pritchard et al., 2000). Haplotypes or populations with ancestry in more than one cluster are considered admixed and indicates that substantial gene flow occurs between defined geographic areas. Our analysis identified three P. vivax and three P. falciparum clusters (Figure 4, S7, S8 and S9). The clustering patterns show that the P. falciparum populations in later years are more mixed than the populations of 2005/2006, where populations clustered according to geographical locations including amongst the three catchment areas within Madang Province (Figure 4) (Schultz et al., 2010){Schultz, 2010 #16}. On the contrary, P. vivax populations were very diverse and displayed little population structure in all time points, despite the increase in differentiation between ESP and MAD post-LLIN populations (Figures 4 and 5).
For P. vivax , as we observed significant mLD post-LLIN in ESP, local phylogenetic analysis was conducted. This supports focal transmission as shown by the clustering of haplotypes from the same village: Sunuhu (Figure 5). The STRUCTURE analysis also shows some evidence of this inbred cluster (Figure 4B). Interestingly, this village had the highest prevalence in the region in the 2012-13 survey (36% infected with P. vivax by qPCR compared to 0.5-9% in other villages (Kattenberg et al., 2020). In Sunuhu, clonal and closely related haplotypes (≤2 unmatched alleles) were observed in 48% (11/23) of the haplotypes from that village (see supplementary file 1). The 11 closely related haplotypes were observed throughout the village, were not clustered in neighbouring households, and were not associated with participant characteristics (p>0.05), such as age and sex (Table S3).