Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1914 Genomic and transcriptomic investigation of reproductive incompatibility in Drosophila GUILHERME COSTA BAIÃO ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-513-0897-5 UPPSALA urn:nbn:se:uu:diva-406758 2020 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 of Philosophy. The examination will be conducted in English. Faculty examiner: Professor Alistair Darby (University of Liverpool, Institute of Integrative Biology). Abstract Baião, G. C. 2020. Genomic and transcriptomic investigation of reproductive incompatibility in Drosophila. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty 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 reproductive incompatibilities 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 cause reproductive alterations in its hosts, influences gene expression in D. paulistorum. Affected genes were associated with biological functions such as metabolism, immunity, reproduction, and chemical communication. Our results indicate that Wolbachia accentuates the differences in 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. We reveal that D. paulistorum mitochondria are polyphyletic, with two divergent mitotypes, and that the heteroplasmy likely originated through introgression. One of the mitotypes shows biparental inheritance, non-responsiveness to host energy demands and rapid titer increase in the early embryo. We hypothesize that such selfish traits evolved in response to competition between mitotypes. In the third project, we show that differentially expressed genes between D. paulistorum semispecies are associated with a variety of biological processes, especially broad regulatory functions that occur via variability in transcription, translation and ubiquitination of post- translational modification. We reveal that the expression profile of F1 inter-semispecies hybrids is markedly similar to that of the maternal line, and that Wolbachia has a small but potentially significant interaction with genes that are differentially expressed in semispecies and F1 hybrids. Finally, we use comparative genomics to study the evolution of closely related Wolbachia strains with known reproductive phenotypes. We confirm previous observations that Wolbachia genomes are very dynamic and that phage-associated regions are particularly variable and likely involved in horizontal transfer of genes linked to reproductive phenotypes. An in-depth screen for genetic elements potentially involved in Wolbachia-induced cytoplasmic incompatibility recovers genes previously known to be involved in the phenotype and novel candidates. In conclusion, this thesis contributes to our understanding of genetic factors that affect Drosophila 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 1651-6214 ISBN 978-91-513-0897-5 urn:nbn:se:uu:diva-406758 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-406758) To my parents, who first showed me the 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 ..................................................................................