bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.301572; this version posted September 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Title: 2 Persistence and invasiveness of high-level heteroplasmy through 3 biparental transmission of a selfish mitochondrion in Drosophila 4 5 Running title: Biparental transmission of a selfish mitochondrion in Drosophila 6 7 Authors: Guilherme C. Baião1,§, Anton Strunov2,§, Eleanor Heyworth1,§, Daniela I. 8 Schneider2,*, Julia Thoma2, Lisa Klasson1,#, Wolfgang J. Miller2,# 9 10 Author affiliations: 1 Molecular Evolution, Department of Cell and Molecular Biology, Science 11 for Life Laboratory, Uppsala University, Husargatan 3, 751 24, Uppsala, Sweden; 2 Lab 12 Genome Dynamics, Department Cell & Developmental Biology, Center for Anatomy and Cell 13 Biology, Medical University of Vienna, Schwarzspanierstraße 17, 1090, Vienna, Austria. 14 § The authors contributed equally 15 # Corresponding authors, and equal contribution 16 * Current address: Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, FL 33612, USA. 17 Emails: 18 [email protected] 19 [email protected] 20 [email protected] 21 [email protected] 22 [email protected] 23 [email protected] 24 [email protected] 25 26 Keywords: Heteroplasmy, mitochondria, biparental & paternal transmission, selfish genetic 27 elements, Drosophila, evolution, genomics, introgression, development. 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.301572; this version posted September 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 28 ABSTRACT 29 Heteroplasmy is the coexistence of more than one type of mitochondria in an organism. 30 Although widespread sequencing has identified several cases of transient or low-level 31 heteroplasmy that primarily occur through mutation or paternal leakage, stable, high-titer 32 heteroplasmy remains rare in animals. In this study we present a unique, stable and high-level 33 heteroplasmy in male and female flies belonging to the neotropical Drosophila paulistorum 34 species complex. We show that mitochondria of D. paulistorum are polyphyletic and form two 35 clades, a and b, with two subclades each. Mitochondria of the a2 subclade appear functional 36 based on their genomic integrity but are exclusively found in heteroplasmic flies and never in 37 homoplasmy, suggesting that they are a secondary mitotype with distinct functionality from 38 the primary mitochondria. Using qPCR, we show that a2 titer do not respond to energetic 39 demands of the cell and are generally higher in males than females. By crossing hetero- and 40 homoplasmic flies, we find that a2 can be transmitted to their offspring via both parents and 41 that levels are dependent on nuclear background. Following a2 mitotype levels during 42 embryogenesis, we demonstrate that this secondary mitotype replicates rapidly just after 43 fertilization of the egg in a period when primary mitochondria are dormant. This so-called 44 “Replication precox“ mitochondrial phenotype likely prevents the a2 mitotype from being 45 outcompeted by the primary mitotype – and thereby secures its persistence and further spread 46 as a selfish mitochondrion, we hereby designate “Spartacus”. Finally, we reconstruct the 47 evolutionary history of mitochondria in the willistoni subgroup uncovering signs of multiple 48 mitochondrial losses and introgressions. Our data indicate an a-like mitochondrial ancestor in 49 the willistoni subgroup, with the b mitotype likely acquired via introgression from an 50 unidentified donor. We hypothesize that the selfish characteristics of a2 might have emerged 51 as a response to competition for inheritance with the introgressed b mitotype. 52 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.301572; this version posted September 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 53 INTRODUCTION 54 It is generally assumed that individuals of most animal species carry a single, maternally 55 inherited mitochondrial haplotype (a mitotype) [1, 2]. However, recent advances in sequencing 56 technology and genomic analyses show that mitochondrial heteroplasmy, the presence of 57 multiple mitotypes in one individual, is found in a number of vertebrate and invertebrate 58 species [3, 4]. 59 Most often, heteroplasmy is the result of either mitochondrial mutations that 60 accumulate over an individual’s lifetime, or paternal leakage. In both cases, the additional 61 mitotype is usually transient and persists at low levels within an individual [5]. Most host- 62 mitochondria relationships are ancient, with both parties coevolving over extended periods of 63 time, and disturbances caused by the introduction and persistence of new mitotypes may 64 reduce fitness or cause disease [6, 7]. Stable, long-term heteroplasmy is therefore relatively 65 uncommon and may be evolutionarily disadvantageous (Reviewed in [8]). Accordingly, 66 selection against heteroplasmy has been proposed as an explanation for the evolution of 67 uniparental inheritance in mitochondria [9]. Mechanistically, transmission of male 68 mitochondria in animals is prevented by both pre- and postmating defense and destruction 69 mechanisms of the host ([5, 10-15] and references therein), and if these fail, the rare paternal 70 mitotype is usually outcompeted by maternal mitochondria during early development or within 71 subsequent generations [16]. 72 The best-known case of animal heteroplasmy is that of freshwater mussels of the 73 family Unionidae, which display double uniparental mitochondrial inheritance (DUI). In these 74 species, two different mitochondrial haplotypes, M and F, are transmitted exclusively by males 75 and females, respectively [17, 18]. Stable heteroplasmy is also found in several isopods in 76 which mitochondria encode different tRNA variants [19-21]. Among insects, prevalence (the 77 frequency of heteroplasmic individuals in a population) at various levels (the proportions of 78 different mitotypes within an individual) has been described in natural populations of the bed 79 bug Cimex lectularius L. [22], the neotropical ants of the Ectatomma ruidum complex [23], the 80 brown dog tick Rhipicephalus spp. [24], and the leaf beetles Gonioctena intermedia [25]. For 81 the genetic model system Drosophila, natural heteroplasmy has been detected in D. 82 mauritiana [26], D. simulans [27-29], D. melanogaster [30], and D. subobscura [31, 32]. 83 Slightly higher prevalence and levels have been found in interspecies F1 hybrids of different 84 closely related Drosophila species ([33-35] and references therein). In spite of a growing 85 number of reports on heteroplasmy in animals including humans (recently reviewed in [36]), 86 little is known as to how heteroplasmic systems have evolved and what the functional 87 differences between mitotypes could be (recently reviewed in [37]). 88 However, when discussing causes and consequences of mitochondrial heteroplasmy, 89 it might be useful to consider mitotypes as primary or secondary endosymbionts, similarly to 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.301572; this version posted September 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 90 what is done for bacterial endosymbionts of insects where multiple infections and obligate 91 mutualism are common phenomena. Hence, primary bacterial endosymbionts as well as 92 mitochondria are normally maternally inherited, long-term co-evolved with the host and have 93 a well-defined function that make them obligate for the eukaryotic host cell, while secondary 94 bacterial endosymbionts and recently acquired or evolved mitotypes are non-fixed, can have 95 variable, often condition-specific and sometimes harmful effects on their hosts [38-40]. While 96 primary endosymbionts might evolve from secondary endosymbionts as a way for the host to 97 secure higher fitness over long evolutionary time, secondary symbionts can be maintained 98 either because they provide a benefit under specific conditions or because they have the ability 99 to cheat the host and be transmitted selfishly without any positive effect [40]. Similarly, 100 secondary mitochondria might thus be maintained in the host for the same reason and result 101 in heteroplasmy (reviewed in [37]). Additionally, obligate dependencies on multiple 102 endosymbionts evolved in several insect. In such cases, one symbiont has often lost genes 103 so that the genome of the other contain complementary genes involved in a function that was 104 previously performed by only one of them [41, 42]. Hence, similarly, stable heteroplasmy might 105 also be maintained if mitotypes with complementary gene sets are present in the same host. 106 The Neotropical Drosophila