Malaria life cycle intensifies both natural selection and random genetic drift Hsiao-Han Changa,1, Eli L. Mossb, Daniel J. Parka,b, Daouda Ndiayec, Souleymane Mboupc, Sarah K. Volkmanb,d,e, Pardis C. Sabetia,b, Dyann F. Wirthb,d, Daniel E. Neafseyb, and Daniel L. Hartla,1 aDepartment of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138; bBroad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, MA 02142; cFaculty of Medicine and Pharmacy, Université Cheikh Anta Diop de Dakar, BP 5005 Dakar Fann, Senegal; dDepartment of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115; and eSchool for Nursing and Health Sciences, Simmons College, Boston, MA 02115 Contributed by Daniel L. Hartl, October 22, 2013 (sent for review July 22, 2013) Analysis of genome sequences of 159 isolates of Plasmodium fal- population size is constant, generations are nonoverlapping, and ciparum from Senegal yields an extraordinarily high proportion each new generation is formed by sampling parents with re- (26.85%) of protein-coding genes with the ratio of nonsynony- placement from the current generation. The major differences mous to synonymous polymorphism greater than one. This pro- between the malaria life cycle and the WF model are that each portion is much greater than observed in other organisms. Also malaria life cycle includes two transmissions, multiple gen- unusual is that the site-frequency spectra of synonymous and non- erations of asexual reproduction, and population expansions and synonymous polymorphisms are virtually indistinguishable. We bottlenecks. Before population genetic inferences can be con- ducted through analysis based on WF assumptions, it is necessary hypothesized that the complicated life cycle of malaria parasites fi might lead to qualitatively different population genetics from that to determine whether the malaria life cycle is suf ciently well described by the WF model. If the life cycle impacts features of predicted from the classical Wright-Fisher (WF) model, which fi population genetics, then inferences based on conventional assumes a single random-mating population with a nite and con- interpretations of the WF model may need to be adjusted. stant population size in an organism with nonoverlapping gener- In a previous study based on only 25 parasite isolates, we ations. This paper summarizes simulation studies of random observed two unusual patterns in the P. falciparum genome that genetic drift and selection in malaria parasites that take into ac- had not been reported in any other organism (4). First, we ob- count their unusual life history. Our results show that random served synonymous and nonsynonymous site-frequency spectra genetic drift in the malaria life cycle is more pronounced than un- that were more similar than expected, given that nonsynonymous der the WF model. Paradoxically, the efficiency of purifying selec- sites likely experience stronger selection. Second, almost 20% of tion in the malaria life cycle is also greater than under WF, and the the genes showed a ratio of nonsynonymous to synonymous fi relative ef ciency of positive selection varies according to condi- polymorphism (πN/πS) greater than 1. In Drosophila melanogaster tions. Additionally, the site-frequency spectrum under neutrality is (14), fewer than 2% of the genes have πN/πS greater than 1. also more skewed toward low-frequency alleles than expected Because nonsynonymous mutations result in changes to amino with WF. These results highlight the importance of considering acids, they are likely to have a deleterious effect and exist in low the malaria life cycle when applying existing population genetic frequencies in the population or be completely eliminated. In tools based on the WF model. The same caveat applies to other other organisms, the nonsynonymous site-frequency spectrum is species with similarly complex life cycles. more skewed toward low-frequency alleles than the synonymous site-frequency spectrum; examples include humans (15–17), alaria, which is caused by the parasite Plasmodium falci- fi Mparum, is one of the major causes of death worldwide. To Signi cance aid the development of vaccines and drug treatments for malaria, researchers have studied the P. falciparum genome and identified Genomic sequences of 159 isolates of the malaria parasite genes that are essential to malaria parasites as well as genes that Plasmodium falciparum exhibited highly unusual patterns of are related to drug-resistance phenotypes using population ge- single-nucleotide polymorphism. We hypothesized that these – netic tools (1–6). Researchers have also focused on particular patterns might result from the repeated bottlenecks in host – genes related to drug resistance and characterized the evolu- vector and vector host transmission as well as the intense tionary pathways of emerging drug resistance using Escherichia competition between parasites within a single host. Computer – simulations of the malaria life cycle recapitulated the unusual coli and Saccharomyces cerevisiae as model systems (7 10). – Malaria parasites have a complex life cycle with two types patterns of polymorphism observed. In the classical Wright of host organisms: humans and female Anopheles mosquitoes. Fisher model in population genetics, random changes in gene fi Malaria parasites are transmitted from mosquito to humans frequency caused by nite population size (random drift) di- fi through the bite of an infected mosquito. In the human host, the minish the ef ciency of natural selection. The trade-off be- parasite reproduces asexually multiple times, and the within- tween drift and selection has been widely assumed to be robust human population size increases from 10 to 102 at the time of to details of the life cycle. In the malaria parasite, however, infection to 108–1013 within a few weeks. When another female both selection and drift are simultaneously enhanced. mosquito feeds on the blood of the infected human, 10–103 malaria gametocytes are transmitted back to the mosquito host, Author contributions: H.-H.C., D.E.N., and D.L.H. designed research; H.-H.C. performed EVOLUTION research; D.N., S.M., S.K.V., P.C.S., D.F.W., D.E.N., and D.L.H. contributed new reagents/analytic and these immature gametes undergo maturation, fuse to form tools; H.-H.C., E.L.M., and D.J.P. analyzed data; and H.-H.C. and D.L.H. wrote the paper. zygotes, and undergo sexual recombination and meiosis, and The authors declare no conflict of interest. the resulting haploid cells reproduce asexually and form spor- Data deposition: The sequences reported in this paper have been deposited in the Na- ozooites that migrate to the salivary glands to complete the life tional Center for Biotechnology Information Sequence Read Archives under the accession cycle (11). These features of the malaria life cycle pose potential nos. SRP003502, and SRP012397. problems when attempting to analyze population genetic data 1To whom correspondence may be addressed. E-mail: [email protected] or using simpler models of life history and reproduction. [email protected]. Much of population genetics theory is based on the concept of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. a Wright–Fisher (WF) population (12, 13). In the WF model, the 1073/pnas.1319857110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1319857110 PNAS | December 10, 2013 | vol. 110 | no. 50 | 20129–20134 Downloaded by guest on September 27, 2021 Oryctolagus cuniculus (18), D. melanogaster (19), and Capsella is described in detail in Materials and Methods (also see Fig. 1). grandiflora (20). The key parameters in the model are the selective advantage or Potential explanations for these unusual patterns including disadvantage of a mutant allele within the human host per cycle sequencing error and annotation error could be ruled out, and of asexual reproduction (sh), the selective advantage or disad- dramatically relaxed or diversifying selection for almost 20% vantage of a mutant allele within the mosquito vector per cycle of of protein-coding genes seems unlikely. Although selection on asexual reproduction (sm), the transmission advantage or disad- antigens could possibly explain the high prevalence of genes with vantage of a mutant allele from the human host to the mosquito π greater than π , the nonsynonymous site-frequency spectrum N S vector (tm) and from the mosquito vector to the human host (th), is skewed toward low-frequency alleles, which is not what the number of human hosts (N), the average number of mos- one would expect if frequency-dependent balancing selection quito vectors per human host (a), the number of sporozoites and explains the phenomenon. Because of the complexities of the gametocytes transmitted between the vector and the human host malaria life cycle, we wondered whether the malaria life cycle (D), the probability that a parasite undergoes replication in itself could explain part of these unusual patterns. More recent a given asexual cycle (P), and the number of asexual generations work in Plasmodium vivax, a close relative with a similar life history to P. falciparum, also revealed large numbers of genes at which the parasite population remains at its maximum size (i.e., peak parasitemia) in the human host (e). Using this model, with πN/πS greater than 1 (21), supporting the idea that factors common to Plasmodium species but different from most other we examined random genetic drift during the malaria life cycle by species may cause allele-frequency patterns that deviate from comparing the probability of loss of a selectively neutral mutant WF expectations. allele after one complete life cycle (regarded as one generation in Although the behavior of the WF model is relatively robust to the malaria model) with that after one generation in the WF model. In the malaria model, the probability of loss of a new deviations from many underlying assumptions, there are exam- − neutral allele is as high as 74%, whereas it is approximately e 1 ≈ ples in which the WF model is known to perform poorly.