24Malaria is the most significant parasitic disease affecting humans, with 212 25 million cases and 429,000 deaths in 2015 1 , and resistance to existing drugs endangers 26 the global malaria elimination campaign. Atovaquone (ATO) is a safe and potent 27 antimalarial drug that acts on cytochrome b (cyt. b) of the mitochondrial electron 28 transport chain (mtETC) in Plasmodium falciparum, yet treatment failures result in 29 resistance-conferring SNPs in cyt. b. Herein we report that rather than the expected de 30 novo selection of resistance, previously unknown mitochondrial diversity is the genetic 31 mechanism responsible for resistance to ATO, and potentially other cyt. b targeted 32 drugs. We found that P. falciparum harbors cryptic cyt. b. Y268S alleles in the multi-33 copy (~22 copies) mitochondrial genome prior to drug treatment, a phenomenon known 34 as mitochondrial heteroplasmy. Parasites with cryptic Y268S alleles readily evolve into 35 highly resistant parasites with >95% Y268S copies under in vitro ATO selection. Further 36 we uncovered high mitochondrial diversity in a global collection of 1279 genomes in 37 which heteroplasmic polymorphisms were >3-fold more prevalent than homoplasmic 38 SNPs. Moreover, significantly higher mitochondrial genome copy number was found in 39Asia (e.g., Cambodia) versus Africa (e.g., Ghana). Similarly, ATO drug selections in 40 vitro induced >3-fold mitochondrial copy number increases in ATO resistant lines. 41Hidden mitochondrial diversity is a previously unknown mechanism of antimalarial drug 42 resistance and characterization of mitochondrial heteroplasmy will be of paramount P275T, K272R, G280D, L283I, V284K, L144S and F267V 18-20 (Extended Data Table 1). 62Second, drug susceptibility studies with ATO resistant Y268S mutants demonstrate a 63 broad range of potency rather than a dichotomous response that would be expected 64 from a single SNP. By using paired parasites collected upon patient admission and 65 subsequent treatment failure from Phase II trials of ATO (Extended Data Table 2), we 66 discovered three distinct in vitro ATO resistance phenotypes (Table 1). A recrudescent 67 isolate (TM90-C6B) initially typed as Y268 (WT) exhibited low level ATO resistance, 68 other isolates with Y268S from recrudescent isolates (e.g., TM90-C2B) demonstrated 69 . CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/232033 doi: bioRxiv preprint first posted online Dec. 10, 2017; moderate resistance to ATO and myxothiazol, and two isolates from ATO-70 pyrimethamine treatment failures showed extreme resistance (TM92-C1086 and TM92-71 C1088) (Table 1). Surprisingly, the extreme ATO resistance phenotype produced 72 greatly reduced susceptibility to a broad range of mtETC inhibitors (Table 1), even 73 though the parasites expressed the common Y268S/N SNPs and no apparent 74 resistance-associated SNPs in candidate mtETC encoding gene...

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