1 Vol.:(0123456789) Scientifc Reports | (2022) 12:226 | https://doi.org/10.1038/s41598-021-03943-z www.nature.com/scientificreports Spontaneous mutation rate estimates for the principal malaria vectors Anopheles coluzzii and Anopheles stephensi Iliyas Rashid 1,2,3,7 , Melina Campos 1,7 , Travis Collier 1 , Marc Crepeau 1 , Allison Weakley 4 , Hans Gripkey 1 , Yoosook Lee 5 , Hanno Schmidt 6 & Gregory C. Lanzaro 1* Using high-depth whole genome sequencing of F0 mating pairs and multiple individual F1 ofspring, we estimated the nuclear mutation rate per generation in the malaria vectors Anopheles coluzzii and Anopheles stephensi by detecting de novo genetic mutations. A purpose-built computer program was employed to flter actual mutations from a deep background of superfcially similar artifacts resulting from read misalignment. Performance of fltering parameters was determined using software-simulated mutations, and the resulting estimate of false negative rate was used to correct fnal mutation rate estimates. Spontaneous mutation rates by base substitution were estimated at 1.00 × 10 −9 (95% confdence interval, 2.06 × 10 −10 —2.91 × 10 −9 ) and 1.36 × 10 −9 (95% confdence interval, 4.42 × 10 −10 —3.18 × 10 −9 ) per site per generation in A. coluzzii and A. stephensi respectively. Although similar studies have been performed on other insect species including dipterans, this is the frst study to empirically measure mutation rates in the important genus Anopheles, and thus provides an estimate of µ that will be of utility for comparative evolutionary genomics, as well as for population genetic analysis of malaria vector mosquito species. Te process of evolution depends on the occurrence of new mutations which provide genetic variation and infuence phenotypic traits 1 . Te rate of de novo mutations is a key determinant of the rate of evolution of an organism under a molecular clock model 2 . DNA repair mechanisms normally ensure that genetic material is copied with fdelity during meiosis and transferred from one generation to the next 3 . However, at some small frequency, the transmission of damaged and improperly repaired DNA, or of DNA with replication errors, to the next generation causes a germline mutation in the ofspring, which may then be subject to natural selection 4 . Te precise estimation by empirical methods of the de novo germline mutation rate in multicellular organisms with large genome sizes has remained a great challenge even with the advent of next-generation DNA sequencing technologies because of inherent limitations, biases, and errors 5 . Previous studies have been conducted to estimate the mutation rate per generation in organisms ranging from prokaryotes to eukaryotes including invertebrates and vertebrates 6 . Te rate of the mutation per site per generation in vertebrates was estimated as 4.6 × 10 –9 in a bird species (the fycatcher Ficedula albicollis) 7 , 4.5 × 10 –9 in wolves (Canis lupus) 8 , and 5.4 × 10 −9 in mouse (Mus musculus) 9 . Te spontaneous mutation per base pair per generation for African green monkeys (Chlorocebus sabaeus) 10 was estimated at a rate of 0.94 × 10 –8 which is slightly lower than the rate of 1.2 × 10 –8 per base pair per generation that was reported in chimpanzees (Pan troglodytes) 11 . Te mutation rate per base per generation estimated in humans was 1.2 × 10 −8 in two separate studies 12,13 and was associated with a higher mutation rate in exons compared to introns 14 . Recent studies reveal that many factors may afect mutations rates, including genomic heterogeneity, population diferences, and both cis- and trans-acting factors that infuence mutagenic processes 15–17 . OPEN 1 Vector Genetics Laboratory, Department of Pathology, Microbiology and Immunology, UC Davis, 1089 Veterinary Medicine Dr, 4225 VM3B, Davis, CA 95616, USA. 2 Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA, USA. 3 Tata Institute for Genetics and Society, Center at inStem, Bangalore, Karnataka 560065, India. 4 Department of ChEM-H Operations, Stanford University, 450 Serra Mall, Stanford, CA 94305, USA. 5 Florida Medical Entomology Laboratory, University of Florida, 200 9th St SE, Vero Beach, FL 32962, USA. 6 Anthropology, Institute of Organismic and Molecular Evolution (iomE), Johannes Gutenberg University of Mainz, Saarstraße 21, 55122 Mainz, Germany. 7 These authors contributed equally: Iliyas Rashid and Melina Campos. * email: gclanzaro@ucdavis.edu