ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2020

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 1914

Genomic and transcriptomicinvestigation of reproductiveincompatibility in Drosophila

GUILHERME COSTA BAIÃO

ISSN 1651-6214ISBN 978-91-513-0897-5urn:nbn:se:uu:diva-406758

Dissertation presented at Uppsala University to be publicly examined in Room A1:111a,BMC, Husargatan 3, Uppsala, Friday, 24 April 2020 at 13:15 for the degree of Doctor ofPhilosophy. The examination will be conducted in English. Faculty examiner: ProfessorAlistair Darby (University of Liverpool, Institute of Integrative Biology).

AbstractBaião, G. C. 2020. Genomic and transcriptomic investigation of reproductive incompatibilityin Drosophila. Digital Comprehensive Summaries of Uppsala Dissertations from theFaculty of Science and Technology 1914. 69 pp. Uppsala: Acta Universitatis Upsaliensis.ISBN 978-91-513-0897-5.

Both nuclear and cytoplasmic elements can contribute to the emergence of reproductiveincompatibilities that influence evolution and speciation. In the projects that compose this thesis,we use genomics and transcriptomics to study some of those elements in Drosophila.

In the first study, we show that Wolbachia, an endosymbiotic bacterium known to causereproductive alterations in its hosts, influences gene expression in D. paulistorum. Affectedgenes were associated with biological functions such as metabolism, immunity, reproduction,and chemical communication. Our results indicate that Wolbachia accentuates the differencesin expression profiles between semispecies and suggest that the symbiont influences host pre-and postmating isolation.

In the second paper, we uncover widespread persistent heteroplasmy in D. paulistorum. Wereveal that D. paulistorum mitochondria are polyphyletic, with two divergent mitotypes, and thatthe heteroplasmy likely originated through introgression. One of the mitotypes shows biparentalinheritance, non-responsiveness to host energy demands and rapid titer increase in the earlyembryo. We hypothesize that such selfish traits evolved in response to competition betweenmitotypes.

In the third project, we show that differentially expressed genes between D. paulistorumsemispecies are associated with a variety of biological processes, especially broad regulatoryfunctions that occur via variability in transcription, translation and ubiquitination of post-translational modification. We reveal that the expression profile of F1 inter-semispecies hybridsis markedly similar to that of the maternal line, and that Wolbachia has a small but potentiallysignificant interaction with genes that are differentially expressed in semispecies and F1 hybrids.

Finally, we use comparative genomics to study the evolution of closely related Wolbachiastrains with known reproductive phenotypes. We confirm previous observations that Wolbachiagenomes are very dynamic and that phage-associated regions are particularly variable and likelyinvolved in horizontal transfer of genes linked to reproductive phenotypes. An in-depth screenfor genetic elements potentially involved in Wolbachia-induced cytoplasmic incompatibilityrecovers genes previously known to be involved in the phenotype and novel candidates.

In conclusion, this thesis contributes to our understanding of genetic factors that affectDrosophila evolution, particularly those leading to reproductive incompatibility in D.paulistorum and associated with Wolbachia.

Keywords: Wolbachia, Drosophila, Drosophila paulistorum, Differential Gene Expression,Reproductive Incompatibility, Reproductive Isolation, Comparative Genomics,Transcriptomics, RNA-Seq, Heteroplasmy, Mitochondria, Genomic conflict

Guilherme Costa Baião, Department of Cell and Molecular Biology, Molecular Evolution,Box 596, Uppsala University, SE-752 37 Uppsala, Sweden.

© Guilherme Costa Baião 2020

ISSN 1651-6214ISBN 978-91-513-0897-5urn:nbn:se:uu:diva-406758 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-406758)

To my parents, who first showed methe wonders of the living world

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Baião, G. C., Schneider, D., Miller, W. J., Klasson, L. (2019) The effect of Wolbachia on gene expression in Drosophila pau-listorum and its implications for symbiont-induced host specia-tion. BMC Genomics, 20(1):465

II Baião, G. C.*, Strunov, A*. Heyworth, E. R.*, Schneider, D.,

Thoma, J., Klasson, L., Miller, W. J (2020) Persistence of high-level heteroplasmy through biparental transmission of a selfish mitochondrion in Drosophila paulistorum. Manuscript

III Baião, G. C., Schneider, D., Miller, W. J., Klasson, L. (2020)

Differential gene expression in semispecies and hybrids of Dro-sophila paulistorum. Manuscript

IV Janice, J., Baião, G. C., Galinou, M., Bourtzis, K., Klasson, L.

(2020). Comparative genomics of closely related Wolbachia strains infecting Drosophila. Manuscript

(*) Shared first authorship.

Reprints were made with permission from the respective publishers.

Papers by the author not included in the thesis

1. Darwell, C. T., Fischer, G., Sarnat, E. M., Friedman, N. R., Liu, C., Baião, G. C., Mikheyev, A. S., Economo, E. P. (2020) Genomic and phenomic analysis of island ant community assembly. Molec-ular Ecology, 00:1–17

2. Baião, G. C., Forshage, M. (2018) Revision of the West Palaearctic

species of Rhoptromeris Förster, 1869 (Hymenoptera: Figitidae: Eucoilinae). Journal of Natural History, 52(17-20):1201–1224

3. Wahlberg, E., Baião, G. C., Häggqvist, S., Martinsson, S., Pistone,

D., Pape, T. (2014) Ancistrocerus waldenii waldenii (Viereck 1906) (Hymenoptera: Vespidae, Eumeninae), a new addition to the fauna of Greenland. Zootaxa, 3838(1):143–150

4. Forshage, M., Baião, G. C. (2014) Revision of Mani’s Figitidae

types (Hymenoptera: Cynipoidea). Zootaxa, 3784(4):498–500

Contents

Introduction ................................................................................................... 13 

Chapter I – An introduction to species thinking ........................................... 14 Naming and classifying biodiversity ........................................................ 14 Linnaeus and the taxonomic revolution ................................................... 15 What is a species? .................................................................................... 16 

Species are dynamic ............................................................................ 16 Species concepts .................................................................................. 16 

Why is it difficult to define species? ........................................................ 17 Do we need to define species? ................................................................. 18 

Chapter II – Speciation ................................................................................. 19 The genetic basis of evolution .................................................................. 19 What is speciation? ................................................................................... 19 Mechanisms leading to evolutionary change ........................................... 20 

Natural selection .................................................................................. 20 Genetic drift ......................................................................................... 21 

Reproductive incompatibilities................................................................. 22 Prezygotic isolation ............................................................................. 22 Postzygotic isolation ............................................................................ 22 

Speciation genes ....................................................................................... 23 Gene expression and reproductive isolation ........................................ 23 

Intragenomic conflicts .............................................................................. 24 Nuclear conflicts .................................................................................. 24 Mitonuclear conflicts ........................................................................... 25 Symbiont-host conflicts ....................................................................... 25 

Chapter III – The Drosophila paulistorum system ....................................... 26 The Drosophila host ................................................................................. 26 

The Drosophila willistoni group .......................................................... 26 Drosophila paulistorum ....................................................................... 28 

The endosymbiont Wolbachia .................................................................. 30 General aspects .................................................................................... 30 Wolbachia genomics ............................................................................ 31 Wolbachia-induced phenotypes ........................................................... 31 Wolbachia as a driver of host speciation ............................................. 33 Wolbachia in Drosophila paulistorum ................................................ 33 

Aims .............................................................................................................. 35 

Methods – Bioinformatic tools for genomic and transcriptomic studies ...... 36 Sample preparation ................................................................................... 36 

Obtaining genetic material from symbionts ......................................... 36 Sequencing ............................................................................................... 36 

Short- and long-read sequencing technologies .................................... 37 Genomic analyses ..................................................................................... 38 

Genome assembly ................................................................................ 38 Genome annotation .............................................................................. 38 Comparative genomics ........................................................................ 39 Ortholog clustering .............................................................................. 39 Phylogenetic analyses .......................................................................... 39 

Transcriptomic analyses ........................................................................... 40 Methods for studying the transcriptome .............................................. 40 Transcriptome assembly ...................................................................... 41 Differential expression analysis ........................................................... 41 

Results ........................................................................................................... 43 Paper I. The effect of Wolbachia on gene expression in Drosophila paulistorum and its implications for symbiont-induced host speciation .. 43 Paper II. Persistence of high-level heteroplasmy through biparental transmission of a selfish mitochondrion in Drosophila paulistorum ....... 44 Paper III. Differential gene expression in semispecies and hybrids of Drosophila paulistorum ........................................................................... 45 Paper IV. Comparative genomics of closely related Wolbachia strains infecting Drosophila ................................................................................ 46 

Discussion – Reproductive incompatibilities in Drosophila ........................ 48 

Conclusions and Perspectives ....................................................................... 51 

Svensk Sammanfattning ................................................................................ 52 

Resumo em Português ................................................................................... 55 

Acknowledgements ....................................................................................... 58 

References ..................................................................................................... 60 

Abbreviations

AM Amazonian (semispecies of Drosophila paulistorum) AMP Antimicrobial peptide BDMI Bateson-Dobzhansky-Muller incompatibility CA Centro-American (semispecies of Drosophila paulistorum)CI Cytoplasmic incompatibility DE Differentially expressed GFR Gut flora restored GO Gene ontology OR Orinocan (semispecies of Drosophila paulistorum) PCR Polymerase chain reaction

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Introduction

I have been fascinated by nature for as long as I can remember. As a kid, I would spend hours and hours reading books about plants and animals and I would carefully examine every living creature that I could find. I was con-stantly amazed by them, and the very notion that something could be alive seemed almost magical. This curiosity towards nature has followed me throughout my life, led me to study biology, and eventually got me to follow the chain of events that resulted in this work.

In the following chapters, I aim to provide you with an overview of the biological context of my thesis. Hopefully, this summary will also allow the reader who is not so familiar with the subject to become acquainted with the main questions and challenges associated with it. I start with an introduction about why we classify living organisms and what are the main difficulties as-sociated with this apparently simple task. Next, I discuss what species are, how they evolve, and how reproductive incompatibilities are important for the process of speciation. Finally, I focus on one particular system, a Drosophila fruit fly and its endosymbiotic companion. It is in this apparently tiny — but in reality immensely vast— universe of a fruit fly that my colleagues and I ask questions about symbiosis, genetic conflicts and speciation, among other top-ics. I also provide a summary of our main findings and a discussion about how they relate to previously published literature. For the most curious, you can read all the details in the four papers contained in this thesis.

I hope that you find something that interests you and, most of all, I hope that this book makes you look at fruit flies with at least a slightly larger sense of wonder.

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Chapter I – An introduction to species thinking

Having words for these forms makes the differences between them so much more obvious. With words at your disposal, you can see more clearly. Finding the words is another step in learning to see.

Robin Wall Kimmerer, Gathering moss

Naming and classifying biodiversity Naming and classifying biodiversity has always been an essential part of hu-man life. Communicating which species are edible, dangerous or suitable for various purposes was a matter of life and death to our ancestors and continues to play an important role in communities that live in close contact with nature. Even in industrialized urban societies, precise identification of organisms is essential for disease control, conservation and biotechnological projects.

It should not come as a surprise, then, that descriptions and classifications of biodiversity appear in early writings from various parts of the world. Chi-nese, Egyptian and Assyrian records dated centuries or even millennia B.C.E. already described medicinal plants and discussed their uses (Core 1955; Radford 1974). In Europe, the first known biodiversity classifications are from ancient Greece, where Aristotle (384-322 B.C.E) arranged the natural world in what he called the scala naturae. Around the same time, Theophrastus (370-285 B.C.E.), wrote De Historia plantarum, an influential work which was used in plant taxonomic studies until the Middle Ages. A few centuries later, Dioscorides (40-90 C.E.) compiled and described the use of hundreds of me-dicinal plants from Roman and Greek territories, while Plinius (23-79 C.E.) named and discussed the fauna and flora of the Roman Empire (Core 1955; Paterlini 2007; Radford 1974).

These Greek and Roman works were the basis of our scientific biodiversity classification. Some were used in Europe for over a thousand years, and it was only around the 16th century that new systems effectively replaced them (Stace 1980). At that time, scientists referred to plants and animals by long Latin phrase names. A honeybee, for example, could have been called: Apis pu-bescens, thorace subgriseo, abdomine fusco, pedibus posticus glabis, un-trinque margine ciliates, which is certainly descriptive but not particularly practical or concise. It is easy to imagine that scientific publications dealing

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with multiple organisms could rapidly become confusing, especially if a spe-cies happened to be known by multiple names. The situation became even more complicated when navigators started bringing samples from around the globe back to Europe, dramatically increasing the number of known species and of names associated with them. Soon, scientists were facing a taxonomic chaos, and there was a great need for a more efficient system of biological classification (Paterlini 2007)

Linnaeus and the taxonomic revolution Scientists such as Caesalpino (1519 – 1603), Bauhin (1560 – 1624), John Ray (1627 – 1705) and Tournefort (1656 – 1708) made important contributions to taxonomy and classification, developing new systems and compiling names of plant species and their synonyms (Radford 1974; Stace 1980). It was not until the Swedish physician and naturalist Carl Linnaeus, though, that a new scientific standard for how living beings were named and classified was es-tablished.

In his work, Linnaeus described thousands of species using both phrase names and what he called “trivial names” — a combination of a genus and a single specific name. These binomials were originally meant as quick refer-ences, but they proved much more practical than the longer phrase names and eventually replaced them completely. Linnaeus also grouped organisms into kingdoms, classes and orders according to similarity, making classifications easier to manage. Although similar solutions had been previously proposed by other scientists, Linnaeus was the first to combine and consistently apply them to most organisms known at the time, creating a standardized reference that others could apply in their own work. As a consequence, despite some cri-tiques, Linnaeus’ system gained wide acceptance in the scientific community. His books Species Plantarum (1753) and the tenth edition of Systema Naturae (1758) mark the publication of a large number of binomial names and are con-sidered by many as an initial mark of modern taxonomy (Paterlini 2007; Stace 1980).

Since then, Linnaeus’ system has been revised and adapted multiple times, with new rules and mechanisms created to improve classification and nomen-clatural stability. One example is the adoption of type specimens, which link a species name and description to a particular specimen and provide an im-portant reference for posterior studies (Soltis 2007). In spite of the changes, many of the fundamental insights proposed by Linnaeus remain at the core of modern taxonomy more than 250 years later, making his system one of the most important contributions to modern biology (Paterlini 2007).

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What is a species? Species are arguably the basic units of biological diversity. In the next sec-tions, I will discuss how they are defined, and whether such definitions con-tribute to our scientific understanding of nature.

Species are dynamic The development of a modern classification system helped us organize our knowledge about biodiversity. It also allowed scientists to better visualize similarities and differences between organisms, which led them to propose hypotheses about how species relate to each other (Paterlini 2007). A revolu-tion in our scientific understanding about the subject came with the work of Darwin and Wallace (Darwin and Wallace 1858), who presented convincing evidence that species evolve through “descent with modifications”. In The Origin of Species (1859), Darwin describes the variability of traits among in-dividuals and notes that these are at least partially inheritable. He also ob-serves that not all individuals in a population reproduce, and that this makes each generation slightly different from the previous. Based on that, he con-cludes that species change through time, and that two populations can diverge from a common ancestor if the individuals which reproduce in each of them carry different traits.

Darwin’s and Wallace’s ideas are the basis of modern evolutionary theory and constitute one of the most important discoveries in biology. Since its pub-lication, the theory of evolution has received innumerous contributions from natural and exact sciences, and its mechanisms and implications are constantly being tested (Hey et al. 2005). And if taxonomy and classification laid ground for the development of hypotheses that led to the theory of evolution, it is equally true that evolution shaped how we perceive, define and delimit spe-cies.

Species concepts Few topics in biology have been discussed as much as the definition of spe-cies. Out of more than 20 different definitions found in modern literature (Mayden 1997), I will present three which are particularly relevant for us and then discuss why it is so difficult to reach a consensus.

Morphological concept Perhaps the most intuitive way of classifying organisms is by their morphol-ogy. The idea that individuals of a species should look more like each other than those that belong to another group is tempting. However, it comes with considerable challenges. It is, for example, often difficult to identify which morphological traits are informative, recognize and account for convergent

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evolution, and define the extent of morphological variation which is accepta-ble within a species (Mayden 1997). Setting these issues apart, the morpho-logical concept is also criticized for not taking evolutionary aspects into ac-count, for its inability to deal with cryptic species, and for the limited applica-bility to organisms with few traditional morphological characters, such as mi-croorganisms. In spite of these critiques, this concept is still often cited and some of its elements have been incorporated into other definitions (Mallet 2008; Mayden 1997).

Biological concept According to the biological species concept, species are not defined by mor-phological similarities, but rather by the ability to reproduce and generate fer-tile offspring. Or as Mayr defines it in his book Systematics and the Origin of Species (1942), “Species are groups of actually or potentially interbreeding natural populations which are reproductively isolated from other such groups.”(Orr 2005). Criticisms against this definition include its non-applica-bility to asexual organisms, the difficulty of assessing reproductive compati-bility between populations that do not coexist in the same space and time, and several documented cases of intra-species infertility and fertile interspecies hybrids (Mallet 2010; Mayden 1997). Nonetheless, the biological species con-cept is widely used and is the core of modern speciation theory, as will be discussed in the next chapter.

Phylogenetic concept The development of phylogenetics as a method for reconstructing evolution-ary relationships between organisms led to the idea that species should corre-spond to monophyletic — or at least clearly diagnosable — biological lineages (Mallet 2010; Mayden 1997). One of the challenges associated with this is that determining the accurate phylogeny of a group is not always easy, especially when different characters or genes produce conflicting results. There is also the rather arbitrary decision of which phylogenetic clusters should receive the status of species. Even so, phylogenetic species are gaining popularity as DNA sequencing becomes cheaper and the interest in understanding evolutionary relationships grows.

Why is it difficult to define species? Different species concepts are based on distinct biological aspects of the or-ganisms they deal with. Consequently, biologists working on comparative morphology, paleontology, ecology or evolutionary genomics are likely to reach different species delimitations. And since all concepts are based on im-portant biological properties, it is easy to conclude that all have merit within their own context (De Queiroz 2007).

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So far, we do not have a species definition or classification which captures the complexity of biodiversity. It is hard to fit the continuous variation pro-duced by evolution into discreet categories, and it is difficult or perhaps im-possible to capture all dimensions of an organism into one or a few criteria. In the last decades, we have also come to expect more from species concepts and biological classifications, which now must reflect our knowledge about evo-lution. However, the Linnaean system was designed to be functional, and not necessarily to reflect a “natural order” between organisms (Stevens 2002). While the idea is certainly tempting, the current model might not be able to fulfill all of our expectations.

Do we need to define species? Considering the difficulty of reaching a consensus on how to define species, one might wonder whether we need such definition at all. Before we answer that, it is worth going back to the reason why humans started naming and clas-sifying organisms in the first place: we need a way of communicating pre-cisely about different organisms and their properties.

In that sense, a system for naming, identifying and classifying biodiversity is absolutely necessary. But whether this will be based on the current taxo-nomic ranks and the concept of “species” is a matter of debate. We must adapt our current system to our new technologies and the data they provide, includ-ing the huge amount of genetic information already available, which will cer-tainly increase manifold in the coming years. Some authors propose new methodologies for species delimitation (Yang and Rannala 2010), others sug-gest a more integrated and accessible taxonomic system (Godfray 2002), while some say we should separate species as “units of classification” from species as “units of evolution” (Dupré 2001). Now it is a matter of time until these possibilities are tested, improved, and one day perhaps adopted. But while a new system is not implemented, we can definitely make the best of the one we have. The fact that biological diversity does not fit perfectly in our concepts does not necessarily make them less useful. In fact, these flaws high-light where our understanding of nature fails and guide us to new hypotheses. Our current species concepts allow us to investigate a wide range of biological questions as long as we keep their limitations in mind. This is the case, for example, of how the biological species concept promoted the development of our theory of speciation.

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Chapter II – Speciation

There is grandeur in this view of life, with its several powers, having been originally breathed into a few forms or into one; and that, whilst this planet has gone cycling on according to the fixed law of gravity, from so simple a begin-ning endless forms most beautiful and most wonderful have been, and are be-ing, evolved.

Charles Darwin, The Origin of Species

The genetic basis of evolution When Darwin proposed that evolution resulted from “descent with modifica-tions”, there were still no good explanation for how traits were inherited across generations. The basic principles of inheritance and allele segregation fol-lowed the rediscovery, in the beginning of the 20th century, of Gregor Men-del’s observations on trait inheritance in pea plants. Shortly after, a series of scientific developments complemented Mendel’s work and marked the begin-ning of genetics as a study field. Remarkable findings of the time include in-vestigations by Frederick Griffith and the Avery-MacLeod-McCarty experi-ment, which led to the conclusion that DNA encodes and transmits genetic information; de Vries’ work showing that variation in the genetic code was created by mutations; and Thomas Hunt Morgan’s finding that genetic mate-rial is organized in chromosomes (Richmond 2006). These and other discov-eries were combined with theories on population dynamics and evolution by Dobzhansky, Mayr and others to produce the modern evolutionary synthesis. The result was a great development in our theoretical and empirical knowledge on evolutionary processes, including speciation and the mecha-nisms behind it (Coyne 2016).

What is speciation? In a broad sense, speciation is the process through which populations diverge to form new species. Modern speciation theory is tightly linked to the biolog-ical species concept and, consequently, to the notion of reproductive compat-ibility (Coyne 2016). Thus, speciation can be more specifically understood as

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the emergence of reproductive incompatibilities which can maintain pheno-typic and genetic divergence between populations even when they coexist in sympatry (Seehausen et al. 2014). This is the most common understanding in contemporary literature (Orr 2005) and is the one adopted in the present work. It is interesting to have in mind, though, that other interpretations for specia-tion could be possible if we chose to work with a different definition of spe-cies.

Mechanisms leading to evolutionary change We have mentioned that evolution works on genetic variation, which is intro-duced by mutations. But what is it that makes some mutations persist in a lineage while others are eliminated? And why do some populations diverge while others remain in unity? To answer these questions, we must examine the two mechanisms which drive evolution: natural selection and genetic drift.

Natural selection In The origin of species, Darwin not only proposed that lineages evolve, but also presented natural selection as a mechanism for this process. He reasoned that differences in phenotypic traits should result in variable degrees of adapt-ability to the environment, and that better adapted individuals have higher chances of survival and reproduction. Thus, the environment selects for indi-viduals with phenotypes that give them higher fitness. We can then conclude that genes associated with fit individuals persist and are likely to increase their frequency in the population.

Selective forces can manifest in a variety of ways, including environmental pressure by pathogens and predators, ability to find nutrients and resources, sexual selection or intragenomic conflicts. Selection can change the gene pool of a groups of individuals in different ways (Fig. 1), and populations subjected to divergent or disruptive selection will tend to differentiate and potentially speciate. Natural selection thus provides a mechanism for lineages to evolve and diverge. Well known examples of species radiations caused by a combi-nation of sexual and ecological selection are the Hawaiian Drosophila (Templeton 1979) and African cichlids (Kocher 2004).

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Figure 1. Types of natural selection. In this schematic representation, we see how the approximately normal distribution of a trait in a population can be affected by different types of selection. Directional selection will favor one of the extremes of the distribution, shifting the mean value of the trait towards it. Stabilizing selection favors intermediate trait values, narrowing the curve and reducing its extremes. Fi-nally, disruptive selection favors both extremes of the curve, eliminating intermedi-ates and promoting divergence within the population.

Genetic drift Genetic drift is the variation in allelic frequencies in a population due to ran-dom sampling of individuals. It has more pronounced effects in small groups, where chance effects tend to affect a proportionally larger portion of the indi-viduals and allele copies. Examples of drift are bottlenecks, characterized by drastic random reduction in population size — for example due to an environ-mental disaster — and the founder effect, which occurs when a population is started by a small subset of another. Since drift is a random process, it has equal chances of increasing or decreasing the frequency of beneficial, neutral and deleterious alleles. This is an important contrast to natural selection, which favors adaptive traits and tends to eliminate or reduce the frequency of deleterious phenotypes and alleles.

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The relative importance of selection and drift for speciation has been object of much debate. Mayr strongly defended that the founder effect plays an im-portant role in island speciation, but evidence for that are still scarce (Orr 2005; Turelli et al. 2001). Studies so far suggest that it is unlikely that drift alone can lead to speciation, although it can perhaps affect its probability and speed when combined with natural selection (Sobel et al. 2010; Templeton 2008).

Reproductive incompatibilities In order for genetic drift or natural selection to cause divergence and specia-tion they need to promote the emergence of reproductive isolation. This ap-pears in multiple forms which, for simplicity, can be broadly grouped accord-ing to the reproductive stage in which they occur.

Prezygotic isolation Prezygotic isolation is often driven by factors extrinsic to the individual, such as ecological and sexual selection (Seehausen et al. 2014). They are usually divided into pre- and postmating barriers according to when they manifest. Premating barriers include differences in geographic distributions, phenolog-ical isolation, exploitation of distinct ecological niches and assortative mating (Seehausen et al. 2014). Postmating prezygotic barriers, on the other hand, comprise for example gamete incompatibilities and conflicts between the fe-male reproductive tract and components of the male ejaculate (Bernasconi et al. 2004; Turissini et al. 2017). Prezygotic isolation mechanism such as mate discrimination in animals and pollination specificity in plants, are very com-mon, and for this reason they are sometimes considered earlier-evolving or even more important than postzygotic isolation (Butlin et al. 2012).

Postzygotic isolation Extrinsic postzygotic isolation usually manifests as low hybrid mating success in a particular environment, while intrinsic isolation corresponds to genetic problems independent from exterior influence (Seehausen et al. 2014). Per-haps the most common form of postzygotic isolation is Bateson-Dobzhansky-Muller incompatibilities (BDMI) (Coyne 2016; Orr 1997; Seehausen et al. 2014). In this model, alleles which evolve independently in distinct popula-tions lead to partial or complete reproductive incompatibility when combined in the hybrid offspring. The negative phenotype only manifests when the al-leles are mixed, which means that they can have neutral or beneficial effects in their original genetic backgrounds. This explains how selection can drive

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the emergence of postzygotic isolation even though it is obviously deleterious. (Orr 1997).

In many cases, pre- and postzygotic isolation coexist and interact with each other. Notably, postzygotic barriers are known to create selective pressure for the emergence of prezygotic isolation. This phenomenon, known as reinforce-ment, is driven by selection against resources being wasted on fruitless mating or on the production of not-so-fit hybrids (Noor and Feder 2006)

Speciation genes The identification of genes associated with reproductive isolation is essential for our understanding of the genetic mechanisms that lead to speciation. The discovery of these “speciation genes” has greatly benefited from the advent of methods for assessing genomic divergence, identifying signatures of intro-gression and selection, mapping genes associated with specific phenotypes, and analyzing gene expression (Castillo and Barbash 2017; Seehausen et al. 2014) As a result, we now have a growing list of speciation genes from Dro-sophila, mice, fish, plants, and other organisms (Nosil and Schluter 2011; Rieseberg and Willis 2007). The study of these genes suggests that reproduc-tive isolation is only rarely caused by a single genetic elements, and that the additive effects of multiple genes are usually involved (Orr 1991, 1997). Spe-ciation genes are associated with a variety of functions and appear to be fast evolving, with divergence driven by different selective pressures (Nosil and Schluter 2011; Orr et al. 2004). Comparative analyses between species and populations show that they accumulate substitutions in both coding and regu-latory sequences, which suggests that structural as well as expression variation contribute to reproductive isolation (Mack and Nachman 2017; Wolf et al. 2010).

Gene expression and reproductive isolation Gene expression seems to play a role in both pre- and postzygotic isolations in a variety of organisms, and sterile and infertile hybrids often show misex-pression in comparison to the parental lineages (Mack and Nachman 2017; Wolf et al. 2010). In Drosophila, several speciation genes have putative roles in transcriptional regulation, and some are themselves misexpressed in hy-brids (Mack and Nachman 2017). Misexpression of spermatogenesis genes, for example, is linked to male sterility in crosses between subspecies of D. willistoni (Gomes and Civetta 2014), while differential expression of odorant binding proteins affects premating isolation in D. sechellia (Matsuo et al. 2007). In a more indirect way, high plasticity in gene expression can also con-tribute to speciation by increasing adaptability to distinct habitats, which in the long term can lead to ecological divergence (Pavey et al. 2010).

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Intragenomic conflicts Intragenomic conflicts are antagonistic interactions between vertically inher-ited genetic elements within an individual. They result from a competition for inheritance and often involve male- and female-linked elements, meiotic driv-ers, mobile selfish elements, and conflicting interests between nuclear and cy-toplasmic genomes (J. Werren 2011). Genomic conflicts are very common and often lead to a “genetic arms race” between elements which selfishly pro-mote their own transmission — sometimes at the cost of the organisms’ fitness — and suppressors that counteract those effects (Rice 2013). Since genomic conflicts are often associated with reproductive phenotypes, they are thought to play a substantial role in the emergence of reproductive isolation (Crespi and Nosil 2013; Presgraves 2010). BDMIs, for example, can be caused by elements evolving through genomic conflict and lead to postzygotic isolation (Lindtke and Buerkle 2015) while prezygotic barriers can result from the ef-fect of symbionts that manipulate the development or behavior of an organ-ism, such as Cardinium and Wolbachia (Sara L. Goodacre and Martin 2012; J. H. Werren et al. 2008). A characteristic of genomic conflicts is that unlike ecological pressures, they do not necessarily favor adaptive phenotypes. Con-flicts often do not result in fitness gains and may instead lead to the fixation of deleterious traits (Crespi and Nosil 2013).

Nuclear conflicts Genomic conflicts can arise between genetic elements within the nuclear ge-nome. They are common in sex-linked genetic elements and are often cryptic due to the evolution of suppressors (Presgraves 2008). Meiotic drivers, for example, increase their own chance of segregation by manipulating meiosis or selectively killing gametes that do not carry the driving allele (J. Werren 2011). Sperm competition has been implicated in speciation of Drosophila montana (Ala-Honkola et al. 2016), and genes linked to the X-chromosome are associated with sex ratio distortion and male hybrid sterility in crosses be-tween D. albomicans and D. nasuta (Zhang et al. 2015). In the last case, auto-somal suppressors have also been detected.

Transposable elements have also been implicated in reproductive isolation. Over-replication of transposable elements has been shown to directly cause defects in hybrids, and indirect effects linked to speciation have also been sug-gested (Serrato-Capuchina and Matute 2018). Transposable elements are im-plicated in speciation between D. mojavensis and D. arizonae (Lopez-Maestre et al. 2017) and an insertion in the desat1 gene is associated with alterations in pheromone profile and incipient speciation in D. melanogaster (Marcillac et al. 2005).

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Mitonuclear conflicts Mitonuclear conflicts are generally assumed to play an important role in spe-ciation (Hill 2015, 2016) and to have strong impact on important biological functions. They are suggested, for example, to be involved in the emergence of BDMIs (Burton and Barreto 2012) as well as in the evolution of germlines and sex (Hadjivasiliou et al. 2012; Radzvilavicius et al. 2016). The evolution of selfish mitotypes has been shown to negatively affect fitness in a variety of organisms (Clark et al. 2012; Havird et al. 2019; Jasmin and Zeyl 2014; Schnable and Wise 1998). Strict maternal inheritance may have evolved as a mechanism for reducing mitochondrial genetic diversity and consequently the rate of evolution of selfish traits (Greiner et al. 2015). However, female-spe-cific inheritance leads to a different set of sex-linked conflicts including a phe-nomenon known as the “mother’s curse” (Gemmell et al. 2004). Since selec-tion on the mitochondrial genome happens primarily in females, mutations that harm males may not be purged and accumulate, especially if they also provide fitness benefits to females (Frank and Hurst 1996; Smith and Connallon 2017). It has been shown, for example, that human mitochondrial diseases are more prevalent in men than in women, and that many male-dele-terious mitochondrial mutations are neutral in females (Dowling and Adrian 2019). These negative effects are counteracted by the evolution of autosomal suppressors and through kin selection, when male-benefiting mutations are preserved if they indirectly benefit females for example through favorable so-cial interactions or fitter progeny (Dowling and Adrian 2019) As a result of this balance between the mother’s curse and suppression mechanisms, the net impact in natural populations remains poorly understood.

Symbiont-host conflicts There is increasing evidence that symbiotic interactions have a significant role in speciation (Brucker and Bordenstein 2012). Gut microbes, for example, ap-pear to affect host behavior and are possibly implicated in premating isolation (Ezenwa et al. 2012; Sara L. Goodacre and Martin 2012; Sharon et al. 2010). However, it is the vertically inherited reproductive symbionts which are known to have the strongest impact on host pre- and postmating isolation (Bennett and Moran 2015). As in the case of mitochondria, nearly strict ma-ternal inheritance leads to selfish symbiont-driven phenotypes that favor in-fected females, and hosts often evolve supressors to balance some of these effects (Hornett et al. 2008; Koehncke et al. 2009; Shan et al. 2019; Vala et al. 2002). Symbionts such as Cardinium and Wolbachia have evolved a surpris-ing number of reproductive phenotypes which are discussed in the next chap-ter, in the section about Wolbachia.

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Chapter III – The Drosophila paulistorum system

The Drosophila host For over a century, Drosophila have been used as models for studies in a va-riety of biological and medical fields, including genetics, developmental biol-ogy, animal behavior and evolution. The small size, short generation time and ease of handling make these insects very suitable for lab cultures, and the ac-cumulated knowledge about their biology brings several advantages. The well curated genome and large set of genetic tools, for example, allow the investi-gation of biological questions in a level of detail that would not be possible in almost any other organism (Markow and O'Grady 2007, Hales, Korey et al. 2015). When it comes to speciation studies, the combination of molecular and genetic tools developed for D. melanogaster and the diversity of species which show varying degrees of reproductive compatibility offer many experimental possibilities (Markow and O'Grady 2007).

The Drosophila willistoni group The Neotropical Drosophila willistoni group comprises a total of 24 species distributed in three subgroups: alagitans, bocainensis and willistoni (Bächli 2019). The alagitans subgroup includes six poorly known species from south-ern Mexico and Central America, several of which are recorded from single localities (Wheeler and Magalhães 1962; Zanini et al. 2015a). The bocainensis has a total of 12 species in a mix of rare and relatively common taxa. They are spread across the Americas and at least one them, D. nebulosa, has a very wide distribution from Argentina to relatively northern parts of the U.S.A. (Zanini et al. 2015a). The third subgroup, willistoni, comprises six closely related trop-ical species. Among these, D. equinoxialis, D. paulistorum, D. tropicalis and D. willistoni are widely distributed in South and Central Americas (Spassky et al. 1971), while D. insularis is endemic to the Antilles and D. pavlovskiana is recorded from Guyana and Venezuela (Spassky et al. 1971; Zanini et al. 2015b).

Phylogenetically, the willistoni group is sister to the also Neotropical sal-tans radiation (O'Grady and Kidwell 2002). Analyses based on nuclear and mitochondrial markers show that the bocainensis subgroup is basal to willis-toni (Gleason et al. 1998; Robe et al. 2010), and although no phylogenetic

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work has been done with alagitans, male morphology indicates that it is closely related to bocainensis (Wheeler and Magalhães 1962). The willistoni subgroup is thus the most derived and corresponds to a recent radiation of approximately 7.3 million years (Zanini et al. 2018). Within that subgroup, D. equinoxialis and D. paulistorum are the most recently diverged taxa (Fig. 2)

Figure 2. Representation of phylogenetic relationships within the willistoni group. D. paulistorum (in bold) and D. equinoxialis are the most recently diverged species within the willistoni subgroup. Relationships between the alagitans and bo-cainensis subgroups are not clearly defined, but both occupy basal positions in com-parison to the willistoni subgroup. Based on Based on (Wheeler and Magalhães 1962; Zanini et al. 2018)

Scientific interest in the willistoni subgroup developed after Dobzhansky and colleagues realized that flies previously classified as D. willistoni actually rep-resented a cluster of cryptic taxa (Burla et al. 1949). Although morphologi-cally very similar, the different species could be distinguished through mating compatibility assays and by the analysis of the male genitalia, karyotyping and allozyme electrophoresis (Burla et al. 1949; Spassky et al. 1971). Soon it was noted that the species showed varying levels of inter- and intraspecific repro-ductive incompatibilities (L Ehrman and Powell 1982; Winge 1965), which contributed to making the willistoni subgroup a model for studies on repro-ductive isolation, speciation and hybridization (Burla et al. 1949; Civetta and Gaudreau 2015; Theodosius Dobzhansky and Pavlovsky 1967; L Ehrman and Powell 1982; Mardiros et al. 2016; Perez-Salas and Ehrman 1971; Schneider et al. 2019; Winge and Cordeiro 1963; Winge 1965). Among the six species of the subgroup, one stands out as perhaps the most complex and interesting case from a speciation point of view: D. paulistorum.

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Drosophila paulistorum

Those forms which possess in some considerable degree the character of spe-cies, but which are so closely similar to some other forms, or are so closely linked to them by intermediate gradations, that naturalists do not like to rank them as distinct species, are in several aspects the most important to us.

Charles Darwin, The Origin of Species

Drosophila paulistorum is a species cluster in statu nascendi (T. Dobzhansky and Spassky 1959). Early experiments with dozens of populations from dif-ferent localities showed that while some crosses readily produce fertile off-spring, others fail completely or result in sterile male hybrids (T. Dobzhansky and Spassky 1959; Theodosius Dobzhansky and Pavlovsky 1967; Lee Ehrman 1965). As a result, six semispecies were recognized: Amazonian (AM), An-dean Brazilian, Centro American (CA), Interior, Orinocan (OR) and Transi-tional (Perez-Salas et al. 1970). A seventh semispecies, the “Guianan”, was discussed in several early publications but ultimately described as D. pavlov-skiana based on chromosomic differences and a stronger reproductive isola-tion (Kastritsis and Dobzhansky 1967).

D. paulistorum semispecies have distinct but partially overlapping geo-graphical distribution and in most cases show strong reproductive incompati-bility with each other (T. Dobzhansky and Spassky 1959). In that sense, they behave like full-fledged species. However, a few lines from each semispecies can hybridize and produce fertile offspring, and the same occurs in crosses involving the Transitional semispecies (T. Dobzhansky et al. 1964; Malogolowkin 1962). Consequently, each semispecies is only a few crosses away from exchanging genes with any other, which makes D. paulistorum a superspecies as defined by Mayr (Box 1) (T. Dobzhansky et al. 1964). Alt-hough it is unclear how often hybridization occurs in the wild, the fact that multiple semispecies are often found in sympatry shows that interbreeding is not frequent enough to blur the distinctions between them (Perez-Salas et al. 1970).

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Box 1: Taxonomic terms associated with the willistoni group and D. paulistorum.

Reproductive isolation in D. paulistorum Hybridization between D. paulistorum semispecies is prevented by both pre- and postmating incompatibilities. Females discriminate against males of other forms, particularly in sympatric populations (Lee Ehrman 1965; Miller et al. 2010), and postmating isolation is observed as infertility or complete male sterility in F1 hybrids (T. Dobzhansky et al. 1964). Interestingly, early exper-iments discovered that postmating isolation could be induced in at least some intraspecific crosses if the females were injected with the solution obtained by macerating a fly from another semispecies (Williamson and Ehrman 1967). After the procedure, the injected female would start producing sterile sons when bred with males from its own kind (Williamson and Ehrman 1967). This observation led to the hypothesis that incompatibility between some D. pau-listorum semispecies was caused by an infectious agent. The idea was sup-ported by microscopy studies and by experiments showing that antibiotics and heat treatments affected the degree of hybrid sterility (Lee Ehrman 1968; Kernaghan and Ehrman 1970). It was only decades later, though, that it was determined that the microorganism involved was the symbiotic bacterium Wolbachia (Miller et al. 2010). Since then, Wolbachia has been linked to a number of unusual reproductive phenotypes in D. paulistorum.

Subspecies: Phenotypically similar populations which inhabit a geographic subdivi-sion of the range of the species. Usually show a low degree of behavioral isolation and assortative mating. Semispecies: Population that have acquired some but not all properties of species and which show more restricted gene flow than conspecific populations. Usually show a higher degree of behavioral isolation and assortative mating. Superspecies: A monophyletic group of species which are morphologically different or somehow reproductively isolated. Taxa that constitute a superspecies are usually considered semispecies Adapted from (Mayr 1970; Robe et al. 2010) and presented as a guide to the reader. Definitions and applicability can vary according to different authors.

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The endosymbiont Wolbachia Insects harbor a broad range of symbiotic interactions with microbes (Moran et al. 2008). Apart from ubiquitous gut bacteria (Engel and Moran 2013), many insects carry intracellular symbiont which provide them with nutrients (Baumann 2005; Douglas 2009) or protect them against parasites, pathogens and predators (Flórez et al. 2015). However, perhaps the most widespread in-sect symbionts are those that affect and manipulate host reproduction (Duron et al. 2008; Hilgenboecker et al. 2008; Zug and Hammerstein 2012). Among these, Wolbachia, stand out as having the highest prevalence, widest host range, and largest known number of reproductive and non-reproductive phe-notypes (Hurst and Frost 2015).

General aspects Wolbachia was discovered in 1924 by Marshall Hertig and Samuel Wolbach, who observed them as “Rickettsia-like” microorganisms in tissues of Culex pipiens mosquitoes (Hertig and Wolbach 1924). Since then, these Alphapro-teobacteria have been found in an impressive number of invertebrate hosts, including a majority of insect species (Hilgenboecker et al. 2008) some arach-nids (S. L. Goodacre et al. 2006), crustaceans (Cordaux et al. 2012) and filarial nematodes (Taylor et al. 2005). Wolbachia might be the most widespread in-tracellular bacteria on Earth, with estimates that up to 66% of insect species could be infected (Hilgenboecker et al. 2008). Several reports of low titer in-fections which are below the detection limit of common screening techniques suggest that the prevalence could be even higher (Müller et al. 2013; Schneider et al. 2018).

Wolbachia are very diverse in their biology. They colonize primarily the reproductive organs of the host and are well known for their ability to manip-ulate arthropod reproduction (J. H. Werren et al. 2008). However, Wolbachia are also found in tissues such as muscles, gut, fat body and the brain, and these somatic infections are likely associated with several less known phenotypes (Pietri et al. 2016). Wolbachia are usually vertically inherited through the ma-ternal line, but comparative analyses of host-symbiont phylogenies indicate frequent horizontal transmission between arthropod hosts. What facilitates horizontal transmission between hosts is not well known, with different stud-ies suggesting introgression from closely related lineages and transfers through parasitoids, predation, and sharing of food sources (Reviewed in Pietri et al. 2016). Arthropods are also often infected with multiple, sometimes dis-tantly related strains (James et al. 2002). Nematode-associated Wolbachia, on the other hand, have an obligate relationships with their hosts and much higher concordance of host-symbiont phylogenies, indicating almost strict reliance on vertical inheritance (Ferri et al. 2011).

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Wolbachia genomics Phylogenetically, Wolbachia are closely related to other obligate intracellular bacteria including Anaplasma, Ehrlichia and Neorickettsia (Dumler et al. 2001). In spite of considerable genetic and phenotypic diversity, only one spe-cies is currently recognized: Wolbachia pipientis. Instead, strains are orga-nized into supergroups based on genetic similarity, and long-standing discus-sions exist about whether some of these should be described as distinct taxa (Chung et al. 2018{Ramirez-Puebla, 2015 #2079; Lindsey et al. 2016)}.

At least 13 complete and several draft Wolbachia genomes have been pub-lished to date (Sinha et al. 2019). Genomes of arthropod-associated strains are larger (1.2 – 1.6 Mbp), have many repeats and contain a variable number of prophages (Klasson et al. 2008; Wu et al. 2004). Mutualistic, nematode-asso-ciated Wolbachia, on the other hand, have smaller (0.9 – 1.1 Mbp) genomes which lack both repeats and phages. The genome reduction of nematode Wolbachia might also be a consequence of the loss of genes associated with infection and reproductive manipulation (Brownlie and O'Neill 2005).

Wolbachia genomes go through extensive recombination within and across supergroups, and phage-associated regions are particularly variable (Baldo et al. 2006; Ellegaard et al. 2013b; Klasson et al. 2009). Wolbachia prophages are often laterally transferred between strains and carry genes with eukaryotic domains (Bordenstein and Bordenstein 2016; Chafee et al. 2010). This could indicate that lateral transfer of genes has occurred between phage and eukar-yotic hosts. Interestingly, there are also several reports of Wolbachia DNA being integrated into host genomes, although the evolutionary implication of these insertions is not known (Hotopp et al. 2007; Klasson et al. 2014). Phage-associated genes are also important for Wolbachia phenotypes. As an exam-ple, the Octomom region is implicated in Wolbachia titer regulation and sym-biont-mediated virus protection to the host (Chrostek and Teixeira 2015), while other phage genes have a central role in the CI phenotype (J. F. Beckmann et al. 2017; Chen et al. 2019; LePage et al. 2017). Wolbachia ge-nomes also carry type IV secretion systems which are likely important for the maintenance of an intracellular lifestyle and induction of reproductive pheno-types (Masui et al. 2000; Rances et al. 2008).

Wolbachia-induced phenotypes Non-reproductive phenotypes Wolbachia can induce a number of non-reproductive phenotypes sometimes in combination with reproductive manipulation. These include, for example, providing vitamins (Moriyama et al. 2015) and increasing host resistance to pathogens (Kamtchum-Tatuene et al. 2017; Teixeira et al. 2008). Wolbachia-induced resistance to virus has become an important research field and is being used as a strategy for controlling mosquito-transmitted viral diseases

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(Kamtchum-Tatuene et al. 2017). Other less understood phenotypes include effects on host translation (Grobler et al. 2018), production of reactive oxygen species (Zug and Hammerstein 2015), proteolysis (White et al. 2017) and me-tabolism (Pietri et al. 2016).

Reproductive manipulation Wolbachia effects on host reproduction were first detected in 1973 by Yen and Barr, who observed cytoplasmic incompatibility (CI) in Culex pipiens mosquitos infected with the symbiont (Yen and Barr 1973). The four main reproductive phenotypes associated with Wolbachia are feminization of ge-netic males, induction of parthenogenesis, male killing, and cytoplasmic in-compatibility (J. H. Werren et al. 2008) The first three bias sex ratio in favor of (infected) females, while CI induces sterility in infected males when crossed with non-infected females (Hurst and Frost 2015). This creates a re-productive advantage for infected females in a population of mixed infection status. Interestingly, the specific phenotype caused by a Wolbachia strain de-pends both on symbiont and host properties. The same strains have been shown, for example, to cause either CI or male killing depending in different species of Drosophila (Jaenike 2007) and moth (Sasaki et al. 2005).

Cytoplasmic incompatibility is the most common reproductive phenotype induced by Wolbachia and works as a modification-rescue system, with in-fected males carrying a toxin in the sperm which must be neutralized by an antidote in the egg (J. H. Werren 1997). If the female is not infected or carries the “wrong” antidote, the toxin will remain active and embryonic development will stop due to asynchrony between paternal and maternal chromosomes dur-ing the first mitotic division (Tram and Sullivan 2002). CI can occur either between infected males and non-infected females (unidirectional CI) or be-tween individuals carrying distinct and incompatible strains of Wolbachia (bi-directional CI) (J. H. Werren et al. 2008). Recent studies showed that the phage-associated genes cifA-cifB and its homologs are implicated in the CI phenotype in D. melanogaster (LePage et al. 2017) and Culex pipiens (J. F. Beckmann et al. 2017; Chen et al. 2019). Evidence suggests that both A and B proteins induce sperm modification (J. F. Beckmann et al. 2017; Chen et al. 2019; LePage et al. 2017), while the A protein is likely also involved in rescue (J. F. Beckmann et al. 2017; Shropshire et al. 2018). While exact mechanisms are not known, the effect of cifB is associated with a deubiquitylase domain and one of its paralogs (cinB, in C. pipiens) is a nuclease. The cifB ortholog in C. pipiens has also been suggested to target nuclear import and protamine-histone exchange factors in the host (J. F. Beckmann et al. 2017; John Frederick Beckmann et al. 2019b; Chen et al. 2019). It is interesting to note that modification and rescue can function independently, and different Wolbachia strains can perform one, both or none of them (Poinsot et al. 2003; Zabalou et al. 2008).

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Wolbachia-host dependence In several systems, the usually facultative Wolbachia-host association has shifted to a situation in which the host depends on the symbiont for performing vital functions which were likely ancestrally independent of the symbiosis. In the wasp Asobara tabida, for example, oogenesis will fail if the symbiont is eliminated (Dedeine et al. 2001). In some systems with parthenogenesis-in-ducing Wolbachia, male production is restored if the symbiont is eliminated, but the emerging adults show drastic fitness reduction due to a breakdown of sexual functions (Pannebakker et al. 2005; R. Stouthamer et al. 2010). Nema-tode-associated Wolbachia have also been suggested as a case of host-symbi-ont dependence, with Wolbachia playing an essential role in host metabolic and defensive functions as well as in embryogenesis and larval development (Casiraghi et al. 2002; Darby et al. 2012). Interestingly, a study of closely related Asobara species showed independent cases of Wolbachia-dependence based on distinct mechanisms, suggesting that such dynamics can evolve rel-atively often (Dedeine et al. 2005; Kremer et al. 2009).

Wolbachia as a driver of host speciation Reproductive phenotypes induced by Wolbachia have been suggested to lead to reproductive isolation and consequently contribute to host speciation. The role of postmating isolation caused by bidirectional CI in host divergence is supported by theoretical models (Telschow et al. 2005) and empirical evi-dence, as in the examples of Nasonia (Bordenstein et al. 2001) and some Dro-sophila (Shoemaker et al. 1999). Wolbachia-induced parthenogenesis has also been implicated in interruption of gene flow and consequent incipient specia-tion between parthenogenetic and sexual population of a weevil (Elias-Costa et al. 2019). Wolbachia is also likely to affect host premating isolation through influence on several reproductive behaviors, including oviposition, mate choice, mating rate and male-male competition, among others (Sara L. Goodacre and Martin 2012).

Wolbachia in Drosophila paulistorum Wolbachia has been detected in all semispecies of D. paulistorum and is sug-gested to have reached fixation in all tested lines (Miller et al. 2010). However, infection titer varies dramatically and the OR semispecies is the only ones in which Wolbachia is detected through standard PCR screening. Infection in other semispecies can only be observed through more sensitive methods (Miller et al. 2010; Müller et al. 2013).

Wolbachia is implicated in various reproductive phenotypes in D. paulis-torum and may play a role in host oogenesis (Miller et al. 2010). Antibiotics-treated females were reported to develop anomalous nurse cells, a phenotype

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analogous to that observed in some Asobara wasps (Dedeine et al. 2001) (Miller et al. 2010). The symbiont is also linked to mate choice and premating isolation, with females carrying experimentally reduced Wolbachia titer showing less discrimination against males of other semispecies than their wild type counterparts (Miller et al. 2010). Males depleted of Wolbachia, on the other hand, are discriminated by wild type females of their own semispecies (Schneider et al. 2019). These effects are possibly linked to symbiont influ-ence on D. paulistorum pheromone profile, which is supported by the detec-tion of Wolbachia in oenocytes cells linked to pheromone production (Schneider et al. 2019). Interestingly, Wolbachia has a specific lateralized in-fection pattern in the D. paulistorum brain which has also be implicated in host behavior effects (Strunov et al. 2017). An effect of Wolbachia on D. pau-listorum postmating incompatibility has also been associated with symbiont overreplication possibly leading to male sterility in inter-semispecies hybrids (Miller et al. 2010)

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Aims

The general aim of this thesis has been to investigate nuclear and cytoplasmic genetic factors associated with reproductive incompatibility in Drosophila, particularly in association with the endosymbiont Wolbachia. Specific goals of each project were to: Characterize the impact of Wolbachia on gene expression and associated

biological function in D. paulistorum, investigating whether the observed effects contribute to pre- and postzygotic isolation between semispecies (Paper I);

Investigate the newly discovered persistent biparentally-inherited hetero-

plasmy in D. paulistorum, with focus on understanding its evolutionary history and exploring genomic and biological features of the mitotypes (Paper II);

Characterize the differences in gene expression and associated biological functions between semispecies and hybrids of D. paulistorum, investigat-ing potential roles in reproductive isolation and associations with Wolbachia (Paper III);

Investigate genomic differences between closely related Wolbachia

strains, with focus on identifying and understanding the evolutionary pat-terns of genetic elements linked to host phenotypes, particularly cytoplas-mic incompatibility (Paper IV).

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Methods – Bioinformatic tools for genomic and transcriptomic studies

In this chapter, I present an overview of the tools and methods used in this thesis as well as a discussion about their main capabilities and limitations.

Sample preparation In general, high-quality sequence data leads to better results and greatly facilitates bioinformatic analyses. In order for good quality sequences to be produced, suf-ficient amount of genetic material must be obtained and samples need to be free from chemical and biological contaminants. Proper handling of samples before extraction is particularly critical for transcriptomic studies, as organisms respond rapidly to stimuli and this may cause undesired changes in their RNA profile. Since RNA is also not as stable as DNA, care must be taken so it does not degrade.

Obtaining genetic material from symbionts Sequencing of unculturable microorganisms is often challenging due to the diffi-culty of obtaining enough relatively pure genetic material. When working with symbionts such as Wolbachia, one often needs to use enrichment protocols that increases the relative amount of symbiont DNA in the sample, otherwise host DNA will predominate in the sequences (C. M. Stouthamer et al. 2018). Multiple displacement amplification can also be used in combination with enrichment of symbiont cells to increase the amount of DNA prior to sequencing. However, it can introduce errors and biases which must be accounted for during downstream analysis. The enrichment protocol described in Ellegaard et al. 2013a was used for obtaining sequences for Papers II and IV.

Sequencing High throughput sequencing has dramatically changed the amount of data available for genomic and transcriptomic studies. As an example, producing the first human genome took approximately 13 years and costed 0.5-1.0 billion dollars. By 2006, the price of sequencing a similar genome was down to $20-

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$25 million dollars, and ten years later it reached $1500. Nowadays, a draft human genome can be produced in 24 hours for approximately $1000, and some companies aim to bring the price down to $100 in the next 10 years (Herper 2017). It is clear that technology efficiency and cost have changed substantially, and obtaining genomic data soon will not be a bottleneck for most research projects. Analyzing and interpreting the vast amount of data, however, is likely to remain a challenge for a longer time.

Short- and long-read sequencing technologies Sequencing technologies can be broadly divided according to the length of the sequence fragments that they generate. Short-read technologies such as those offered by Illumina and BGI produce very high data output with relatively low error rates. This allows even large genomes to be sequenced with good cover-age and for a reasonable price. However, the 100-300 bp read length repre-sents a challenge for downstream assembly and does not allow the resolution of repeat regions in the genome. Long-read sequencing technologies by com-panies such as PacBio and Oxford Nanopore have somewhat lower output and higher error rates, but generate fragments of up to several thousand contiguous base pairs which facilitates assembly and the resolution of repetitive regions. Data for Papers II and IV were based on a combination of short (Illumina) and long (PacBio) DNA reads. The short and relatively simple mitochondrial ge-nomes studied in Paper II could be fully assembled only with short reads, alt-hough long reads, when available, were useful for making sure that no large repeats existed in the control region. High-quality assembly of the more com-plex Wolbachia genomes, on the other hand, requires long reads that can re-solve the multiple repetitive parts (Paper IV).

Papers I and III were based on short-read Illumina RNA sequences. In tran-scriptomic studies, it is often necessary to treat samples with a ribosomal RNA depletion protocol. Otherwise, rRNA, which can make up to 98% of the total RNA in a cell, will prevent proper quantification of other types of RNA (Bryant and Manning 1998). Problems in the rRNA depletion reaction can result in accidental removal of mRNA transcripts and consequently bias the sequence profile in the sample. Similarly, care must be taken that all samples are treated in the exact same way during library preparation and sequencing, otherwise systematic biases might be introduced in the data.

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Genomic analyses Genome assembly Genome assembly can be a technically and computationally challenging task, especially when tens or hundreds of millions of short reads need to be cor-rectly sorted, ordered and combined. SPAdes (Bankevich et al. 2012) is an assembler which uses k-mer-based de Bruijn graphs and which usually pro-duces good results for short prokaryotic genomes like Wolbachia and mito-chondria. It was used for assembling all mitochondrial genomes in Paper II. If an assembly run does not produce satisfactory results, it is often useful to vary the k-mer length used by the assembler, the amount of data provided as input, or to try assemblers based on different algorithms. The use of long reads also greatly simplifies the assembly process and allows the bridging of gaps be-tween contigs assembled with short reads. By combining both types of reads in a same study, one can take advantage of the lower error rates of short reads during contig assembly and use long reads for accurately combining them into longer sequences. Several modern software can perform hybrid short- and long-read assemblies, including SPAdes.

Genome annotation Identifying the features present in a genome and gathering functional information about them is a crucial step in most analyses. Structural annotation is based on sequence signatures and patterns that identify protein-coding genes, RNAs, in-tron-exon junctions and other genomic features of interest. Functional annotation, on the other hand, is usually based on sequence similarity to previously annotated sequences. Pipelines such as DIYA (Stewart et al. 2009) and Prokka (Seemann 2014) can be used for producing draft annotations which can be posteriorly com-plemented and curated as needed. Several specialized tools also exist for predict-ing particular features such as tRNAs or pseudogenes. HMMER (Eddy et al. 2015) can be used for annotation of protein domains and RNA features by com-paring sequences against databases such as Pfam and Rfam, while BLAST can be used for example with NCBI’s nr database. Comparisons to previously annotated genomes of closely related taxa can provide valuable information about features specific to the organism of interest.

It is important to consider, though, that databases and genomes are not al-ways curated and many contain incorrect information. Furthermore, our knowledge about gene functions is still limited and largely biased in respect to a few model organisms. Thus, obtaining high-quality functional annotation for non-model organisms is usually time consuming and requires careful man-ual curation. However, good annotations are the basis for several other anal-yses, including comparative studies like the one presented in Paper IV. The annotation of the Wolbachia genomes assembled in Paper IV greatly benefited

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from the comparison with previously published high-quality genomes (Ellegaard et al. 2013b; Klasson et al. 2009). However, manual curation was still done for confirming predictions and verifying low similarity hits associ-ated with rapidly evolving proteins, pseudogenes and unique features.

Comparative genomics Comparative genomic analyses can provide important information about the evolution of genomes and the organism they belong to. GATK (McKenna et al. 2010) allows the identification of SNPs and indels between genomes (Pa-per IV), which provides an ideal of sequence conservation, while tools such as Geneconv (Sawyer 1989), PhiPack (Bruen et al. 2006) and ClonalFrame (Didelot and Falush 2007) allow testing for recombination between sequences (Paper II). The comparison of codon usage in protein coding sequences and calculation of metrics such GC content, GC percentage in different codon po-sitions, and ratio of synonymous substitutions per synonymous site to non-synonymous substitutions per non-synonymous site (dN-dS) also provide in-sight into sequence relatedness and evolution, including the detection of se-lective pressures affecting codon usage and sequence conservation (Paper II).

Ortholog clustering Clustering tools can be used for identifying orthologous proteins in a dataset. OrthoMCL (L. Li et al. 2003), for example, generates ortholog clusters with basis on sequence similarity estimated from reciprocal blast hits. Ideally, clus-ters should contain all orthologs of a given gene and no other, more distantly related, copies. Parameters can be adjusted to make clustering more or less stringent, and some testing might be necessary to identify what works best for the dataset at hands. OrthoMCL was used for clustering protein coding tran-scripts in one of the analysis of Paper I for creating gene clusters used in dif-ferential expression analysis in Paper III. In Paper IV, clustering was used for the identification of ortholog proteins across different Wolbachia genomes.

Phylogenetic analyses Phylogenetic inferences are used to reconstruct the evolutionary history of or-ganisms (or sometimes genes, or genomes) based on the analysis of shared heritable traits under an evolutionary model. DNA sequences are a great source of information for phylogenetics, since they provide a large quantity of variable heritable traits (nucleotide positions). However, before reconstructing a phylogeny from a set of sequences, it is important that they are aligned so that only orthologous sites are compared. The comparison of non-orthologous sites will lead to erroneous inferences, which is why heteroplasmy and intro-gression events such as described in Paper II can be problematic if not properly

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identified and addressed. There are many alignment tools to perform either nucleotide or protein sequence alignments, as for example MAFFT (Katoh and Standley 2013). It is also common to use a trimming tool such as trimAl (Capella-Gutierrez et al. 2009) for removal of poorly aligned regions from the alignment. The resulting file can then be used for downstream analyses.

The main phylogenetic inference methods are maximum likelihood, imple-mented for example by RAxML (Stamatakis 2014) and Bayesian inference, used in MrBayes (Ronquist and Huelsenbeck 2003). An evolutionary substi-tution model must be chosen for the analysis, and most modern tools can infer an appropriate model from the data. When interpreting a phylogeny, it is im-portant to remember that different genes or fragments within a genome can reveal distinct evolutionary histories if they were, for example, recombined or acquired through lateral gene transfer or introgression. In those cases, one should investigate what is the cause of the phylogenetic conflict and make an informed decision about how to interpret the result. In Papers II and IV, the procedures above were used with either maximum likelihood and Bayesian inferences to generate trees based on sequence sets varying from whole mito-chondrial genomes to single or concatenated genes

Transcriptomic analyses The transcriptome is the sum of all RNA transcripts in a cell or population of cells, including both coding and non-coding RNAs. Unlike the genome, the transcriptome is dynamic and reflects all factors that influence gene expres-sion in a particular moment. Thus, it provides a snapshot of an organism’s response to its environment in a given moment. In controlled experiments, the transcriptome allows us to detect which genes are active in different experi-mental condition, which also provides an idea of gene function.

Methods for studying the transcriptome Methods for studying the transcriptome have advanced rapidly in the last dec-ades. Nowadays, most studies use RNA-seq and, sometimes, microarrays. Mi-croarrays are based on the detection of RNA transcripts which hybridize with fluorescence-tagged complimentary probes immobilized on an array. Thus, an important limitation of the technique is the need of previous knowledge about the sequences of interest so that adequate probes can be designed. While mi-croarrays are still cost-effective for investigating a known set of transcripts, their popularity is steadily decreasing (Lowe et al. 2017).

RNA-seq is based on high throughput sequencing and allows the capture of nearly the whole RNA pool of a sample. Since it works with random pri-mers, RNA-seq performs well with novel organisms for which no genome is

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available. Sequenced transcripts provide an overview of which genes are tran-scribed in a given moment, and quantification of the reads provides an esti-mate of gene expression level. RNA-seq can be used for profiling coding and non-coding RNAs, for discovery of novel genes and splice variants, and for improvement of gene annotation, among others applications (Ozsolak and Milos 2011). Short read sequencing is still the most common technique, but as long-read sequencing becomes more efficient and affordable, its use in tran-scriptome studies is also increasing (Tachibana 2015). RNA-seq was used in Papers I and III.

Transcriptome assembly Depending on the study, transcriptome assembly may or may not be neces-sary. If a genome is available for the species of interest and all that is wanted is a quantification of the expression of different genes, then reads can be mapped directly to the transcriptome and no assembly is necessary. However, if a genome is not available or if full transcripts are needed for any purpose, the data needs to be assembled.

Transcriptome de novo assemblies carry unique challenges in comparison to their genomic counterparts, especially in the case of eukaryotes. The pres-ence of multiple isoforms per gene and the sequencing of immature mRNAs which still contain intron sequences introduce a lot of complexity to the data. As a result, the assembly process is complicated and the result can be rela-tively messy, with a lot of redundancy and some misassembled or chimeric transcripts. The use of long read sequencing greatly simplifies transcriptome assemblies, as most transcripts are likely to be sequenced in single reads. However, it is still relatively uncommon compared to short- read sequencing.

Trinity (Grabherr et al. 2011) is probably the most popular transcriptome assembler and was our choice for performing the de novo assembly of tran-scriptomes of the AM, CA and OR semispecies of D. paulistorum used in Papers I and III. After the assembling, we used bioinformatic pipelines to filter the transcriptome and remove redundancy that could complicate or introduce bias in downstream analyses.

Differential expression analysis One of the main applications of transcriptomic data is the analysis and com-parison of gene expression across different conditions, as for example differ-ent sexes or tissues or experimental treatments. These studies allow the iden-tification of genes whose expression is significantly different between condi-tions as well as a quantification of the fold change difference them. The main steps of a differential expression analysis are:

- Aligning RNA reads to a reference, which can be either a genome or de novo assembled transcriptome.

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- Quantifying reads mapped to each feature of interest (genes, tran-scripts, exons).

- Comparing expression between conditions and determining which features are significantly differentially expressed (DE).

There are several tools which can performing each of these steps. In Paper I, we used STAR (Dobin et al. 2013) to align reads to the transcriptomes. STAR is a splice-aware aligner, which means that it can recognize intron-exon junc-tions and can be used for mapping RNA reads either to a genome or transcrip-tome. In Paper III we switched to using BWA (H. Li 2013), as it mapped a higher percentage of reads to our reference in a much faster time. BWA is not splice aware, but since the transcriptome does not contain introns, this is not a problem.

Read quantification was done with FeatureCounts (Liao et al. 2014) for both Paper I and III. It is possible to configure the software to count reads mapping to different features (i.e. exons, genes or clusters of genes, as in Paper III) depending on the research question that is being investigated. One must also choose how to handle multimapping reads, which can be either excluded, mapped to a single location or assigned to all possible matching sites.

Differential expression analysis in Papers I and III was performed with the R package DEseq2 (Love et al. 2014), which performs all necessary normali-zation and statistical testing of the data. Since different tools employ distinct methods and algorithms, they always produce slightly different results. It seems to be the case, though, that strong signals in the data are likely to be detected by all methods, and it is mostly genes near the significance threshold that vary between different pipelines.

Once a list of DE genes is obtained, it is common to annotated them with standardized gene ontology (GO) terms. Various tools can then be used for identifying GO-terms which are overrepresented (enriched) among the DE genes. GO-term enrichment analysis provides information on biological pro-cesses, molecular functions and cellular compartments associated with the an-notated genes. The expression level of a gene and the fold change between conditions can also provide clues about the significance of the results. In Pa-pers I and III, we used the software topGO (Alexa and Rahnenfuhrer 2016) to perform GO enrichment of the genes DE in different condition and explore enriched functions associated with them.

Overall, in spite of a number of challenges, differential expression analysis is a powerful tool for exploring questions which cannot be answered through purely genomic approaches. It provides insight into putative gene functions and allows the identification of candidate genes likely to be involved in spe-cific responses.

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Results

Paper I. The effect of Wolbachia on gene expression in Drosophila paulistorum and its implications for symbiont-induced host speciation We investigate the effect of Wolbachia on gene expression of D. paulistorum and discuss whether the observed effects contribute to the emergence of pre- and postmating isolation in host populations. The study was based on Illumina RNA-seq data from wild type and antibiotics-treated, gut flora restored (GFR) flies. Samples from heads and abdomens of males and females of the AM, CA and OR semispecies were used. We assembled high quality transcriptomes for AM, CA and OR, mapped reads of each semispecies to its respective tran-scriptome, quantified reads associated with each contig, and performed differ-ential expression analysis. We found 175 - 1192 genes DE by Wolbachia in the three semispecies. Most DE genes were detected in OR, which is the only D. paulistorum semispecies with high titer infection. The OR transcriptome was also the only one containing Wolbachia transcripts.

Genes DE in the three semispecies were associated with several global, sex- and tissue-specific biological functions. Wolbachia was shown to have a strong global effect on host metabolism. We saw indications of increased ca-tabolism in wild type flies, with upregulation of tricarboxylic acid cycle genes, and signs of higher anabolism in GFR, with upregulated pentose-phosphate pathway genes. We suggest that the high metabolic cost of Wolbachia for the host has systemic effects which possibly lead to other phenotypic effects as-sociated with the symbiont. We see an effect of Wolbachia in host immunity, with global upregulation of anti-microbial peptides (Diptericin B, attacins) and condition-specific differential expression of other defense-related genes. Such effects could be either a host response to control the infection or a result of symbiont modulation of the immune system, as Wolbachia is linked to in-creased resistance against pathogens.

A large number of reproductive genes are downregulated by Wolbachia in abdomens of both sexes. In females, many cell-cycle and oocyte development genes are affected, some of which could play a role in postmating incompati-bilities. Loss of function mutants of deadhead, for example, show defects in chromatin segregation which resemble the phenotype of Wolbachia-induced CI. Reproductive genes DE in males are mostly unannotated, although some

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are linked to postmating modulation of female behavior, like antares and aquarius.

Genes involved in pheromone production are also DE and could potentially affect premating isolation. In particular, FASN1, FASN2 and desat2, which are downregulated in male abdomens, have been linked to incipient speciation in other Drosophila species. The downregulation of odorant binding proteins in males could also affect how suitable mates are perceived and lead to assorta-tive mating. We saw an unexpected strong upregulation of muscle-associated genes in abdomens of both sexes. The implications of this effect are unclear, but an impact on locomotion could perhaps interfere with courtship rituals. Wolbachia infection also had other, less understood phenotypes, including downregulation of translation and cytochrome P450 genes in heads of both sexes. We see that the gene expression patterns in wild type D. paulistorum heads allow more clear distinction between semispecies than in their GFR counterparts, which again suggests a symbiont role in semispecies distinction. Although experimental evidence might be needed to confirm the impact of some phenotypes, overall our results support a possible role of Wolbachia in pre- and postmating isolation between D. paulistorum semispecies.

Paper II. Persistence of high-level heteroplasmy through biparental transmission of a selfish mitochondrion in Drosophila paulistorum We investigate a case of stable, high-level heteroplasmy associated with bipa-rental mitochondrial inheritance in the Drosophila paulistorum species com-plex. Using a combination of whole-genome mitochondrial sequencing and lab techniques, we sampled a total of 23 Drosophila lines belonging to six species of the willistoni group and three of the saltans group. Heteroplasmy was detected in 25-100% of the individuals in 40% of the 17 tested D. paulis-torum lines. Depending on the line, the least abundant mitotype corresponded to 2-50% of the total mitochondria.

We discover that D. paulistorum heteroplasmy is associated with two dis-tinct mitotypes — and — which have approximately 3% nucleotide diver-sity. Based on their phylogenies, the mitotypes were further divided into sub-groups 1 and 2. Heteroplasmic lines are observed to always carry the 2 mi-totype, which is also never found in homoplasmy. Further analysis of 2 re-vealed that it has proportionally higher titer in males than in females and that it is consistently biparentally inherited in both intra- and inter-semispecies crosses. We discover that 2 replicates rapidly during early embryo develop-ment, a period when mitochondria are usually quiescent. We name this behav-ior “replication precox” and hypothesize that it is a strategy for increasing titer of the relatively few sperm-derived mitochondria and thus prevent them from

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being outcompeted by the initially much more abundant maternal mitochon-dria in the egg. Usually, mitochondrial titer is correlated with the energy de-mand of the tissue in which it resides, a pattern observed in 2 mitochondria. However, 2 shows constant titer throughout different parts of the fly, indi-cating that it may not fulfill the usual energy-supplying role of mitochondria.

These results suggest that 2 may be a selfish element which maximizes its own chances of inheritance independently of the energetic needs of the host. In spite of this, the 2 genome is under purifying selection and appar-ently fully functional, with similar size to the genome of other mitotypes and no loss-of-function mutations. Genomic coverage and sequence conservation also clearly distinguish it from nuclear mitochondrial sequences, which sug-gests that 2 might provide yet unknown functions for the host or perhaps produce energy under specific conditions.

Phylogenetic analyses suggest an -like ancestral mitotype in the willistoni subgroup and indicate that was probably acquired through introgression from an unknown or extinct species of the willistoni group. A comparison of mitochondrial and nuclear phylogenies suggests that mitochondrial losses and gains via introgression have happened multiple times within both the willis-toni subgroup and D. paulistorum. Based on our results we hypothesize that the selfish features of 2 evolved in response to competition for inheritance with the introgressed mitotype. This is, to our knowledge, the first report of stable heteroplasmy originating from mitochondrial introgression.

Paper III. Differential gene expression in semispecies and hybrids of Drosophila paulistorum In this paper, we use RNA-seq to investigate differences in gene expression between the AM, CA and OR semispecies of Drosophila paulistorum as well as between AMxOR F1 hybrids and their parental lineages. Separate samples from heads and abdomens of males and females were used in the analysis. The concatenated transcriptomes of the three semispecies were used as a reference for read mapping, and differential expression was performed on gene clusters containing orthologs from the three transcriptomes.

Nearly 70% of the gene clusters in our reference were identified as DE in at least one pairwise comparison, and around a fifth of these were DE simul-taneously in all three pairwise comparisons. Several gene clusters were up- or downregulated in one semispecies in comparison to both others, most of which were either DE globally (i.e. in all conditions) or in a specific condition. A smaller number of genes had significantly different expression levels in all three semispecies. In all of these sets, male abdomen was the condition with the highest number of DE genes, which suggests that male-linked functions evolve fast in D. paulistorum.

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We find that considering the divergence time between the semispecies, AM shows a disproportionally high number of up- and downregulated genes, which may be the consequence of semispecies-specific selection. Many glob-ally DE genes in all semispecies are involved in regulatory function governed by transcription, translation and post-translational modifications, among other processes. These affect a broad range of biological functions such as repro-duction, development and neural functions. Several genes which were up- or downregulated in specific conditions are associated with processes that have a potential role on reproductive isolation. Examples include male accessory gland proteins, reproductive functions, and the odorant binding protein lush, which has a known role in pheromone reception. Desaturases previously im-plicated in speciation in other Drosophila systems were globally downregu-lated in CA.

Our analysis shows that the expression pattern of F1 hybrids strongly re-sembles that of the maternal line. We see only a few genes which are up- or downregulated in hybrids in comparison to the parental lines, most of which are in female abdomens. Among these are downregulated genes associated with cuticle development and upregulated genes linked to cell cycle and re-production, which could be involved in hybrid reproductive disfunctions. We also observe downregulation of antimicrobial peptides (AMPs) in male hy-brids, which could be linked to previously reported overreplication of Wolbachia in D. paulistorum hybrids and potentially linked to male hybrid sterility.

We see small but potentially relevant interaction between Wolbachia and genes DE between semispecies, among which are desaturases in the CA fe-male abdomen and an effect of AM and OR Wolbachia on immune genes. AM Wolbachia is seen to have a stronger effect on heads than on abdomens, which correlates with published observations that Wolbachia influence mating be-havior in the AM semispecies. Overall, we see a large number of DE genes between semispecies, some of which are potentially implicated in reproduc-tive isolation and represent good candidates for further experimental studies.

Paper IV. Comparative genomics of closely related Wolbachia strains infecting Drosophila We perform comparative analysis of five newly sequenced and four previ-ously published Wolbachia genomes. All strains used in this work had been previously transferred into the Drosophila simulans STC line and character-ized either as mod+ resc+, mod- resc+ or mod- resc- according to their CI modification and rescue capabilities in the host. The standardized host genetic background allowed us to associate differences in CI phenotype with the ge-netic features of the genomes, providing us with an opportunity for detailed

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screening of CI-associated genes. Using whole genome sequencing of the same strains from their native hosts, we also verify that the transfer to D. sim-ulans is associated with only minimal nucleotide changes and is unlikely have affected their CI phenotypes.

We perform a detailed analysis of two groups of very closely related strains wSan, wYak and wTei (collectively referred to as wSYT) and wNo and wMa, describing genes and regions which are unique to one or some of them. We observe that even though wSYT only differ by a few dozen nucleotides, they still show considerable genomic rearrangements. Overall, most differences detected between the genomes are concentrated in phage-associated regions. A phylogenetic reconstruction based on concatenated single copy orthologs confirms published results that wTei is the sister taxon to wSan and wYak, which recapitulates the phylogeny of their hosts. The three strains are closely related to other Supergroup A Wolbachia included in the dataset, particularly wMel. We analyze the evolutionary history of the wMel WD0513 gene, which is located in the phage-associated Octomom region, and find evidence that it was likely laterally transferred between arthropods and the intracellular bac-teria Rickettsiella and Cardinium. The same gene had previously been shown to have been laterally transferred between Wolbachia and mosquitos.

By associating genomic features of the different strains with their CI phe-notypes, we identify several candidate genes involved in the modification and rescue functions of CI. The analysis recovers the cifA-cifB genes, recently shown to play a major role in CI, and presents previously undescribed candi-dates for future experimental testing. We also discuss how our data fits the hypothesis that Wolbachia titer might compensate for a less-than-perfect fit between modification and rescue genes from different strains. We analyze the evolution of the CI genes, particularly in Supergroup A Wolbachia, and high-light the importance of phages for the lateral transfer of these genes across Wolbachia lineages.

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Discussion – Reproductive incompatibilities in Drosophila

In the projects that make up this thesis, we used a combination of genomic and transcriptomic tools to investigate genetic factors which influence the evolu-tion of Drosophila lineages, especially the emergence and maintenance of re-productive incompatibility.

One such factor is the endosymbiont Wolbachia, well known for its ability to manipulate arthropod reproduction. Wolbachia-induced CI can play an im-portant role in host reproductive isolation (Bordenstein et al. 2001; Shoemaker et al. 1999; Telschow et al. 2005; Telschow et al. 2007), and understanding the genes and mechanisms associated with it has been a longstanding goal of the scientific community. The recent discovery of two genes with major roles in CI was an important step in this direction, but several questions remain to be answered before we can explain the whole complexity of the phenotype in different systems (John F. Beckmann et al. 2019a).

We add to this knowledge by performing an in-depth comparative genomic analysis of closely related Wolbachia strains which show variable CI pheno-types in the same host background (Paper IV). Our screening for candidate genes linked to the phenotype recovered the previously known CI genes and presented novel candidates for both modification and rescue functions. We also collect further evidence of the dynamic nature of Wolbachia genomes, which show extensive rearrangements even among very closely related strains. Phage-associated regions, in particular, recombine frequently and seem to contribute to phenotypic innovations through lateral transfer of sev-eral genetic elements including most CI-associated genes. Interestingly, some Wolbachia phage genes have eukaryotic-like domains (Bordenstein and Bordenstein 2016), and we show evidence of multiple ancestral lateral trans-fers of such genes between arthropod hosts and different intracellular bacterial lineages (Paper IV). These results highlight the importance of phages in the evolution of Wolbachia and potentially other intracellular bacteria.

However, CI is not the only Wolbachia phenotype implicated in host re-productive isolation. The symbiont is known to affect host behavior in multi-ple ways, some of which are likely to generate reproductive isolation (Sara L. Goodacre and Martin 2012). In the D. paulistorum complex, Wolbachia af-fects mating behavior, especially in the AM semispecies (Miller et al. 2010).

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The symbiont has been shown to reduce female receptivity to inter-sem-ispecies mates and to play a role in male attractiveness to females of their own kind (Miller et al. 2010). This effect is likely associated with a symbiont effect on host pheromone profile (Schneider et al. 2019), and is supported by the Wolbachia effect on genes associated with pheromone production and recep-tion (Paper I). Interestingly, AM Wolbachia has a proportionally stronger ef-fect on genes which are misexpressed in heads rather than abdomens of AMxOR F1 hybrids, again suggesting a putative symbiont effect on host be-havior and pheromone signaling (Paper III). D. paulistorum semispecies are known to have distinct male chemical profiles (Chao et al. 2010). Our work supports a difference also in signal reception by showing that AM flies have, in comparison to AM and OR, distinct expression levels of gustatory receptors and of an odorant receptor (lush) strongly associated with pheromone signal-ing (Paper III).

Although CI has so far not been detected in D. paulistorum, we see a strong effect of Wolbachia on the expression of many genes associated with repro-duction and cell cycle in both males and females (Paper I). This suggests that the symbiont must have an influence on D. paulistorum reproduction and pos-sibly contributes to postmating isolation. Further studies are necessary to elu-cidate the exact nature of this effect, but one possibility is that the changes are associated with a previously reported link between Wolbachia and oogenesis in D. paulistorum (Miller et al. 2010).

Postmating isolation in D. paulistorum has also been linked to pathogenic overreplication of Wolbachia in hybrids due to breakdown of host regulatory mechanisms (Miller et al. 2010). Host control of symbiont populations is sometimes linked to the production of AMPs (Login et al. 2011), and a similar mechanism is supported in D. paulistorum by the higher expression of AMPs associated with Wolbachia infection (Paper I). We find that AMP production in AMxOR hybrids is downregulated in comparison to the parental lines (Pa-per III), again supporting a possible dysfunction in symbiont control in inter-semispecies offspring.

Overall, our results corroborate previous evidence that Wolbachia affects multiple aspects of D. paulistorum biology and is likely implicated in repro-ductive isolation. However, patterns of incompatibility between semispecies are complex, and nuclear factor are likely to also play a role. We observe that semispecies differ in the expression of many genes linked to regulatory func-tions that occur via, for example, transcription, translation and post-transla-tional regulation (Paper III). Although the precise mechanisms being affected by those genes are not known, they clearly have potential to cause significant differences in the biology between the different semispecies. We also see dif-ferences in the expression of some genes associated with reproduction, with AM showing a distinct pattern from CA and OR with regards to the expression of genes linked to male functions such as spermatogenesis and accessory gland proteins (Paper III). This fact, together with the previous observations

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of pheromone signaling and premating isolation, corroborates earlier sugges-tions that AM is close to becoming a fully diverged species (Chao et al. 2010).

The interplay between nuclear and symbiont factors causing reproductive incompatibility can also manifest through reinforcement. In the closely related species D. subquinaria and D. recens, nuclear-associated premating isolation has evolved in response to selective pressures imposed by Wolbachia-induced postmating incompatibility (Shoemaker et al. 1999). It seems plausible, given Wolbachia’s ability to influence host behavior, that the opposite could also happen, that is, Wolbachia-driven premating isolation evolving as a response to nuclear-associated postmating isolation.

Wolbachia is expected to be in linkage disequilibrium with other mater-nally inherited genetic elements, and events of mitochondrial sweeps and re-placement are often associated with the symbiont (Jiggins 2003; Turelli et al. 1992). Mitochondrial replacement following Wolbachia introgression through hybridization may have important consequences for the host, as more mitonu-clear conflicts are expected if the new mitotype has not coevolved with the nuclear genome (Rand et al. 2006). In D. paulistorum, persistent heteroplasmy involving two divergent mitotypes is associated with consistent biparental in-heritance of a potentially selfish mitochondrion (Paper II). Selfish mitochon-dria are associated with negative fitness in a number of organisms (Havird et al. 2019), and heteroplasmy itself appears to be mostly selected against. Inter-estingly, though, consistent biparental inheritance is likely to balance the in-fluence of both sexes on the mitochondrial genome and may reduce conflicts due to strict maternal co-evolution associated with the mother’s curse (Kuijper et al. 2015). Heteroplasmy can also constitute a challenge for phylogenetic reconstructions. Previous studies of the evolutionary history of the willistoni group were not aware of the heteroplasmic state of D. paulistorum and mix markers from both mitochondrial clades, which can lead to erroneous conclu-sions.

Overall, our studies expand our knowledge about genome evolution in the endosymbiont Wolbachia and on how it manipulates host reproduction, con-tribute to our understanding of reproductive incompatibility in Drosophila — particularly D. paulistorum — and uncover a novel system of biparentally in-herited heteroplasmy in D. paulistorum.

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Conclusions and Perspectives

This thesis adds to our understanding of how nuclear and cytoplasmic ele-ments contribute to reproductive isolation in Drosophila systems. In particu-lar, we explore genomic features that help make Wolbachia the most success-ful endosymbiont in the world, and we investigate how this bacterium inter-acts with D. paulistorum in early stages of speciation. Our results also charac-terize the divergence in gene regulation between semispecies of D. paulistorum and highlight yet another complex feature of this system, wide-spread paternally inherited mitochondrial heteroplasmy.

The new scientific information that was generated not only brings answers to existing questions, but also provides guidance for future investigations. As an example, the various candidate genes which we uncover can be used as input for experimental work on both Wolbachia phenotypes and D. paulisto-rum reproductive isolation. Long-read and single-cell transcriptomics can also build on our results, reducing technical difficulties associated with transcrip-tome assemblies and providing precise information on symbiont effect in spe-cific cells. These techniques have great potential for symbiont studies in gen-eral, but even more in cases when large biological effects are caused by a few symbiont cells infecting very specific parts of the host, as in most D. paulis-torum semispecies. Genome sequencing of Wolbachia from D. paulistorum and other willistoni group species would also allow us to investigate genetic elements associated with particular phenotypes which are seen in this system but which are not common in other Drosophila species.

A larger scale sampling of wild populations of D. paulistorum and related species would most likely also contribute to our understanding of the dynam-ics and diversity of Wolbachia and mitochondria in the system. That would allow us to verify how general our current observations are with regard to the whole species and could bring new insight on the role that different elements play in causing reproductive incompatibilities in nature.

Finally, I believe that every study based on organisms originating from threatened environments, like some of these Drosophila species from Neo-tropical forests, should be a reminder of how much we can learn from these creatures, how fragile they are, and that their fate is largely in our hands.

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Svensk Sammanfattning

Jorden är hem åt en otrolig mångfald av organismer som har de mest varie-rande former, storlekar och livsstilar, och som alla utvecklades från en enda gemensam förfader. Att förstå den process som ger upphov denna artrikedom och hur den fortsätter att generera nya arter är en av de viktigaste och största frågorna inom evolutionsbiologin.

För att artbildning ska kunna ske krävs reproduktiva barriärer som förhind-rar genutbyte mellan individer. Dessa barriärer kan över tid leda till att gene-tiska och fenotypiska skillnader mellan populationerna uppstår och ger upp-hov till reproduktiv isolering. Sådan isolering kan visa sig på många olika sätt. Den typ av reproduktiva isolering som agerar innan någon parning sker inbe-griper geografisk eller temporal åtskildhet, preferenser när det gäller partner-val eller distinkta parningsbeteenden. Reproduktiv isolering som inträffar ef-ter parning mellan två individer kan å andra sidan bero på att könscellerna är inkompatibla eller att de har genetiska olikheter som resulterar i låg fitness eller sterilitet när de kombineras i en gemensam avkomma. Inkompatibiliteter som ger upphov till reproduktiv isolering kan evolvera genom både selektiva eller slumpmässiga processer, och en möjlig orsak till hur de uppstår är ge-nomiska konflikter mellan vertikalt nedärvda genetiska element som existerar i samma organism. En sådan konflikt kan uppstå på grund av konkurrens om nedärvning och kan involvera gener som finns på kärngenomet, själviska ge-ner och cytoplasmatiska element såsom mitokondrier och mikrobiella en-dosymbionter. I det senare fallet introducerar strikt maternell nedärvning också en könsbunden konflikt, eftersom maternellt nedärvda element tenderar att gynna honor i populationen.

Flugor av släktet Drosophila är bra modeller för att studera reproduktiv isolering och artbildning. De är små, lätta att odla i labbet och olika Dro-sophila-grupper visar olika grader av reproduktiv isolering. Många arter är också infekterade av den endosymbiotiska bakterien Wolbachia, som kan på-verka reproduktionen inte bara hos Drosophila utan hos många olika insekter. Wolbachia inducerar flera olika former av reproduktiva fenotyper som alla gynnar infekterade kvinnor, och flera studier tyder på att de kan spela en viktig roll vid artbildning i värden. Den vanligaste fenotypen som Wolbachia indu-ceras hos sin värd är cytoplasmatisk inkompatibilitet (CI), ett tillstånd som genererar sterilitet hos infekterade hanar när de korsas med honor som an-tingen är oinfekterade eller som är infekterad av en annan icke-kompatibel Wolbachia-stam. CI fungerar som ett toxin-antitoxin system, där infekterade

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hanar bär ett toxin, d.v.s. ett gift, i spermierna som måste motverkas av en antitoxin, d.v.s. ett motgift, i ägget. Om toxinet som förs över med spermierna inte neutraliseras av antitoxinet dör embryot på grund av de defekter som upp-står under ett visst stadie i utvecklingen. I årtionden har forskare försökt iden-tifiera vilka gener som orsaker CI och vad mekanismen bakom CI är. Det var dock bara för några år sedan som de första generna med en bekräftad roll i fenotypen identifierades. I vårt arbete har vi jämfört den kompletta arvsmas-san mellan nära besläktade Wolbachia-stammar för att få insikt i hur Wol-bachia-genom evolverar och för att identifiera gener som skulle kunna vara involverade i CI. Vi bekräftar att Wolbachia har ett mycket dynamiska genom, med väldigt många strukturella förändringar även mellan närbesläktade stam-mar. Vi såg att de flesta av skillnaderna i geninnehåll mellan stammarna är förknippade med regioner i genomet som härstammar från bakteriella virus, i.e. fager, som ofta överförs mellan avlägset besläktade Wolbachia-stammar och därmed bidrar till förflyttningen av gener kopplade till de fenotyper Wol-bachia inducerar, t.ex. de gener som orsaker CI. Förutom de kända generna för CI, så kunde vi genom denna analys också identifiera ytterligare gener som är associerade med både toxin- och antitoxin-funktionerna hos denna fenotyp. Med hjälp av transkriptomik undersökte vi hur Wolbachia påverkar genut-trycket i Drosophila paulistorum, ett artkomplex bestående av sex underarter där artbildningsprocess är i sin begynnelse. I denna studie observerade vi att Wolbachia har inflytande på många olika processer i värden t.ex. dess meta-bolism, immunitet, reproduktion och kemiska kommunikation. Dessutom såg vi att Wolbachia verkar bidra till att göra de olika underarternas genuttryck mer distinkt. Våra resultat stödjer tidigare teorier om att Wolbachia bidrar till att skapa reproduktiv isolering i D. paulistorum.

Med samma typ av metodik undersökte även skillnader i genuttryck mellan underarter av D. paulistorum och mellan hybrider och deras föräldrar. Genom denna analys upptäckte vi att underarterna till stor del skiljer sig åt i uttrycket av gener som är förknippade med regleringsfunktioner och att hybrider har ett uttrycksmönster som mest liknade deras mödrar. Vi ser också en interaktion mellan Wolbachia och några av de gener som har olika uttryck mellan under-arter och i hybrider. Några av dessa skillnader i genuttryck kan eventuellt ligga bakom den reproduktiva isoleringen som existerar mellan de olika underar-terna i D. paulistorum och ge upphov till sterilitet hos hybrider mellan under-arter.

Slutligen upptäckte vi att D. paulistorum ofta bär på två distinkta mitokondrier, mitotyper, som samexisterar i en individ. Vi visar att en av dessa mitokondrier ärvs från båda föräldrar till skillnad från i de flesta andra djur, där mitokondrien enbart ärvs modern, och att detta troligtvis beror på att den kopierar sig själv tidigt under embryoutvecklingen när vanliga mitokondrier inte gör det. Vi såg även att en av de två mitotyperna troligtvis förvärvats från en okänd men besläktad art, och att konkurrensen om nedärvning mellan dessa

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båda mitotyperna kan ha lett till uppkomsten av de själviska egenskaper som vi observerade hos en av dem.

Sammanfattningsvis bidrar vårt arbete till en ökad förståelse av olika mek-anismer som ger upphov till reproduktiv isolering i Drosophila, särskilt i när-varo av endosymbionten Wolbachia.

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Resumo em Português

Nosso planeta abriga uma diversidade incrível de organismos com os mais variados tamanhos, formas e estilos de vida. E, por incrível que pareça, toda essa biodiversidade evoluiu de um único ancestral comum. Entender como isso aconteceu e como novas espécies continuam a ser geradas é um dos gran-des objetivos da biologia evolutiva. Esse processo, conhecido como especia-ção, normalmente envolve o aparecimento de incompatibilidades reprodutivas que impedem indivíduos de populações distintas de reproduzir. Dessa forma, diferenças genéticas e fenotípicas entre as populações podem persistir e au-mentar.

Incompatibilidades reprodutivas se manifestam de várias maneiras. O iso-lamento pré-zigótico é aquele que acontece antes da fecundação, podendo ocorrer tanto antes quanto depois do acasalamento. Exemplos de isolamento pré-zigótico antes do acasalamento incluem separação geográfica ou tempo-ral, acasalamento preferencial ou comportamentos de corte distintos. Já a in-compatibilidade gamética é uma forma de isolamento que ocorre após o aca-salamento, mas ainda antes da fecundação. O isolamento pós-zigótico pode se manifestar através de um valor adaptativo reduzido em híbridos ou na forma de incompatibilidades genéticas que causam inviabilidade ou esterilidade da prole. Incompatibilidades reprodutivas podem surgir através de seleção natu-ral ou deriva genética, e uma fonte comum de pressões seletivas capazes de causar incompatibilidade são os conflitos entre elementos genéticos transferi-dos verticalmente. Esses conflitos são uma consequência da competição entre diferentes elementos genéticos por segregação. Eles podem envolver genes nucleares, elementos genéticos móveis e elementos citoplasmáticos como mi-tocôndrias e endossimbiontes. Neste último caso, a herança estritamente ma-terna também introduz conflitos ligados a sexo, já que elementos herdados pela mãe tendem a favorecer fêmeas na população.

Moscas do gênero Drosophila são ótimos modelos para o estudo de incom-patibilidade genética e especiação. Elas são pequenas, fáceis de manter em laboratório, e diferentes grupos de espécies exibem variados graus de isola-mento reprodutivo. Muitas espécies também carregam uma bactéria endos-simbiótica chamada Wolbachia, que é transmitida de uma geração para a outra através da linhagem materna e afeta a reprodução de uma grande quantidade de insetos. A Wolbachia é capaz de causar diferentes formas de manipulação reprodutiva, e todas elas beneficiam as fêmeas infectadas na população. Vá-

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rios estudos sugerem que incompatibilidades reprodutivas causadas por Wol-bachia podem desempenhar um papel importante no processo de especiação do hospedeiro.

O fenótipo mais comum causado pela Wolbachia é chamado incompatibi-lidade citoplasmática (IC). Esse fenômeno gera uma esterilidade condicional em machos infectados quando eles cruzam com fêmeas não infectadas ou com fêmeas que carregam uma variedade de Wolbachia não compatível com a que eles próprios possuem. A IC funciona como um sistema toxina-antídoto. Ma-chos infectados produzem uma toxina que causa uma modificação no esperma e que deve ser resgatada por um antídoto presente no óvulo. Se a toxina não for neutralizada, o embrião morre devido a defeitos na primeira divisão celu-lar. Cientistas tentaram por décadas identificar genes associados à IC e procu-raram decifrar seu mecanismo de atuação. Porém, foi apenas há poucos anos que os primeiros genes com papel confirmado no fenótipo foram identifica-dos. Em nosso trabalho, nós comparamos os genomas completos de cepas pró-ximas de Wolbachia a fim de entendermos melhor como esses simbiontes evo-luem. Também procuramos por genes associados à IC e encontramos vários candidatos para as funções de modificação e resgate, dentre os quais estavam tanto os genes previamente associados a IC como novos candidatos ainda des-conhecidos.

Nossos resultados confirmam que a Wolbachia tem um genoma muito di-nâmico, com rearranjos frequentes mesmo entre cepas próximas. A maioria das diferenças entre cepas está concentrada em regiões de profagos. Esses pro-fagos são frequentemente transferidos lateralmente e contribuem para o mo-vimento de genes ligados a diferentes fenótipos, incluindo a IC. Utilizando técnicas de transcriptoma, nós também investigamos o impacto de Wolbachia na expressão gênica de Drosophila paulistorum, um grupo de seis semi-espé-cies em estágio insipiente de especiação. Nós observamos uma influência da Wolbachia em várias funções do hospedeiro, incluindo metabolismo, imuni-dade, reprodução e comunicação química. Os resultados sugerem que a Wol-bachia desempenha um papel no surgimento de isolamento reprodutivo nessas drosófilas. Nós também investigamos diferenças de expressão gênica entre semi-espécies e entre híbridos e suas linhas parentais. Descobrimos que as semi-espécies diferem em genes associados a funções reguladoras e que os híbridos têm um padrão de expressão que se assemelha ao de suas mães. Tam-bém vimos que Wolbachia interage com alguns dos genes diferencialmente expressos entre semi-espécies e híbridos.

Finalmente, descobrimos que D. paulistorum frequentemente carrega dois tipos distintos de mitocôndria que coexistem de forma estável. Mostramos que uma dessas mitocôndrias provavelmente foi adquirida através de introgressão com uma espécie relacionada, e que a competição por segregação entre os dois mitótipos provavelmente levou ao surgimento de características egoístas em

57

um deles. Em resumo, nosso trabalho amplia nosso conhecimento sobre in-compatibilidades reprodutivas em Drosophila, especialmente na presença do endossimbionte Wolbachia.

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Acknowledgements

My years in Uppsala as a PhD student have been filled with intense learning. Working with science is almost never boring, and the experience of living in a new country, meeting people from different places and exploring the differ-ences between Brazil and Sweden has been extremely enriching and stimulat-ing. Of course, living abroad also brought many challenges, but thankfully I met amazing people who helped me go through it all.

One person, in particular, has had a main role in this whole adventure. If it weren’t for my supervisor, Lisa, the work that resulted in this thesis would never have been possible. Most likely, I wouldn’t even be in Sweden. When we first met I had almost no knowledge about bioinformatics, but she believed in my willingness to learn and in what I could accomplish. She has supported me continuously during this journey and taught me an immense lot about bi-ology, bioinformatics and how to be a good scientist. I am really grateful for everything!

At Uppsala University I found not only a great supervisor, but also amazing friends and colleagues. The whole MoleEvo crew, past and present, have been with me through good and bad times and provided countless moments of learning and fun. Jan, Siv and Thijs, as our PIs, and Ajith, Anna, Anja, Christian, Courtney, Disa, Emil, Erik H, Erik P, Feifei, Felix, Gaëlle, Henning, Jennah, Jessin, Jun-Hoe, Jonathan H, Jonathan L, Joran, Karl, Kasja, Kirsten, Laura, Lina, Mayank, Max, Minna, Moritz, Serra, Tom, Wei, Will, Zeynep; you were all a part of this journey in one way or another, and my work and life in Uppsala would not have been nearly as interesting and enjoyable without you. Dani, my Perl knowledge and ping pong skills wouldn’t be half what they are if it weren’t for you! Ellie, you were the best office-mate ever, and we had so many great project ideas together! Don’t worry, we will still put them all into practice! Eva F., 7-eleven, 7-eleven!!! Anders L., first horse gut microbiome, then the world!

Andrea and Alejandro, we started this journey (almost!) together and we are finishing it (almost!) together! You are both amazingly kind and smart people who taught me a lot and who I really admire. Thank you for being not only awesome friends (presential friends!!!) but also my family in Sweden. Mahsita, you are an incredible person! Thank you for all the laughs, dances, conversations about life and for always being there for me (Oh, and for pro-nouncing my name correctly, of course!!). Guillaume, one day we were danc-ing kizomba in Stockholm and the next we were drinking beer and eating

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andouillette in the French Riviera! That’s how life should be! You and Mahsa have been wonderful friends, and my only wish is that we find more time to hang out!

Ana Rosa and Giulia P., you are two of the funniest, most creative and most stylish people that I know (and style is very important in this world, you know!). You are very much missed in Uppsala! Maria, thank you for all the laughs, talks, cookies and chocolates during these years! Emilio, Gianni, Carmen, Guillermo, Ana, somehow the number of Spanish (speaking!) peo-ple on this list keeps increasing!

Kelvin, I don’t know what my life would be without tea and Vietnamese food! Thanks for all the great moments as flatmates and all the evenings in Stockholm. Eva G., Fred, Anders H. Ana M., thank you for all the great times we spent together and the many evenings on the dance floor. Vince! Valeu por trazer um pedacinho de Brasil pra essa vida Sueca daqui (e por me dar um teto quando eu precisava!). Giulia T., it feels like it was yesterday that we started our masters together! We don’t meet nearly as often as we should, but know that you have a special place in my heart! Anna, Maria, Moham-mad, Efstratia, Harris, Fotini, Moos (and Alexandros!); who would have thought that a teacher training course could be so much fun? You are all amaz-ing and I have only the best memories from our time together.

Alice, thank you for being so caring and supportive during all this time, it made a world of a difference, especially in the most stressful moments!

Aos meus pais, muito obrigado pelo apoio incondicional em todos os mo-mentos. Nada disso teria sido possível sem vocês. À minha família e a todos os amigos no Brasil, obrigado pelo apoio presencial ou à distancia durante todos esses anos. Estamos longes mas de certa forma sinto que estamos mais próximos do que nunca.

Thank you, Andrea, Alejandro and Giulia P., for reading and comment-ing on different parts of this thesis. Obrigado, Anninha, pela ilustração da capa! There are many more people who should be listed above, but time, space and memory are too short! If you think that you are one of them, believe me, I think so too — and in my heart you definitely are!

Science is fun, but it is you who make it all worth it! Thank you with all my heart.

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