bioRxiv preprint doi: https://doi.org/10.1101/2020.09.21.305854; this version posted September 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Large-scale characterization of sex pheromone communication sys- tems in Drosophila Mohammed A. Khallaf 1‡, Rongfeng Cui 2, Jerrit Weißflog 3, Aleš Svatoš 3, Hany K.M. Dweck 1,4, Dario Ric- cardo Valenzano 2, Bill S. Hansson 1†, and Markus Knaden 1†‡ Insects use sex pheromones as a reproductive isolating mechanism to attract conspecifics and repel het- erospecifics. Despite the profound knowledge of sex pheromones, little is known about the coevolutionary mechanisms and constraints on their production and detection. Using whole-genome sequences to infer the kinship among 99 drosophilids, we investigate how phylogenetic and chemical traits have interacted at a wide evolutionary timescale. Through a series of chemical syntheses and electrophysiological recordings, we identify 51 sex-specific compounds, many of which are detected via olfaction. Behavioral analyses reveal that many of the 42 male-specific compounds are transferred to the female during copulation and mediate female receptivity and/or male courtship inhibition. Measurement of phylogenetic signals demonstrates that sex pheromones and their cognate olfactory channels evolve rapidly and independently over evolutionary time to guarantee efficient intra- and inter-specific communication systems. Our results show how sexual isolation barriers between species can be reinforced by species-specific olfactory signals. Main blends of shared chemical compounds as a result of genetic similarities and biosynthetic pathways shared rganisms communicate with each other through by ancestry 7,8. This diversity in sex pheromone Oexchanging signals that include visual, acoustic, communication can become further affected by tactile and chemical (smell and taste) senses. The factors like geographical or host variations. For chemical sense is common in all organisms, from example, sympatric species develop pronounced bacteria to mammals, and therefore, regarded from an divergent communication systems to overcome the evolutionary perspective as the oldest one. Animals risk of hybridization, while the unimpeded divergence are surrounded by a world full of odors emitted from due to geographic barriers may lead to relaxed conspecific or heterospecific individuals, as well as accumulation of differences 9. Moreover, colonization from the environment. The ability to exchange and of a different host – an ecological adaptation – could decipher these signals has significant impact on a also lead to differential sex pheromones and new species’ success as odors help to avoid imminent ways of signal transmission and perception 10,11. threats and localize and judge food or potential Although many studies have reported the diversity mates. Olfactory systems have, therefore, evolved in of sex pheromones among related species, the a sophisticated way to meet the challenge of detecting evolutionary phylogenetic history of these traits and and discriminating a countless number of odorants. their detection systems remains obscure. While it is well established that, and how animals use Flies, like most animals, rely on odors for intra- and interspecific communication, the chemical cues to locate and choose an appropriate evolution of olfactory systems with respect to signal mating partner 1,12-14. For several reasons, flies within production and perception is poorly understood. the genus Drosophila represent ideal species to study One of the most crucial channels that the evolution and diversity of sex pheromones, as have been suggested to contribute to speciation are well as their associated behaviors. First, Drosophila the sex pheromone-sensing channels 1. Volatile sex species live in an extensive range of diverse habitats pheromones – airborne chemicals that stimulate across all climatic conditions, from deserts and caves sexual behaviors in the opposite sex – are the to mountains and forests 15. In these environments, primary signals that reinforce the isolation barriers drosophilids feed and breed on varied hosts such as between different species. These species-specific decaying fruits, slime fluxes, mushrooms, flowers, as signals often provide a full biography written in scent well as frog spawn 16. Second, sexual behaviors of molecules about the sender, such as information about drosophilid flies differ quantitatively and qualitatively reproductive and internal status. Diversification of sex 17, which may include nuptial gift donation 18, pheromones among species arises either via neutral partners’ song duet 19,20, territorial dating 20, or the drift, or via sexual, and/or natural selection 2-6. Closely release of an anal fluidic droplet 21. Third, the neural related species tend to use different pheromone processing of pheromones in the brain of some 1Department of Evolutionary Neuroethology, Max Planck Institute for Chemical Ecology, D-07745, Jena, Germany. 2Max Planck Institute for Biology of Ageing and, CECAD Research Center at University of Cologne, D50931, Cologne, Germany. 3Group of Mass Spectrometry and Proteomics, Max Planck Institute for Chemical Ecology, D-07745, Jena, Germany. 4Present address: Department of Molecular, Cellular, and Developmental Biology, Yale University, CT 06520, New Haven, USA. †These authors share senior authorship; ‡Correspondence: [email protected] (M.K.); [email protected] (M.A.K.) bioRxiv preprint doi: https://doi.org/10.1101/2020.09.21.305854; this version posted September 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. drosophilids, especially D. melanogaster, is well we recovered four main clusters within the genus understood 22. Fourth, pheromone receptors are Drosophila. First, the Drosophila subgenus that narrowly tuned to fly odors 23 and expected to evolve contains five main groups (repleta, virilis, melanica, at fast rates to match the dramatic diversity of cardini, and immigrans groups); second, the Zaprionus pheromones among closely related species 5,24. Lastly, subgenus that includes Zaprionus indianus; third, out of the 52 classes of olfactory sensory neurons the Dorsilopha subgenus that includes D. busckii; (OSNs) in D. melanogaster 25, only four respond to fourth, the Sophophora subgenus that includes fly odors and are localized in a specific sensillum melanogaster, obscura, willistoni, and saltans groups type 26. Hence, the restricted number of orthologues, (Fig. 1a). that are always expressed in an easily identifiable roup melanica group a lis g and accessible sensillum type, represent promising viri ca RAxML bootstrap support r D.pseudotalamancana din = 100 i g candidates to study the coevolution of Drosophila ro up D.montana D.littoralis D.americana pheromones and their corresponding receptors. D.nannoptera D.lummei D.virilis D.bromeliae D.melanica D.euronotus D.gaucha D.paramelanica D.micromelanica D.robusta D.polychaeta D.repletoides Olfactory sexual communication in D. D.hydei Z.indianus D.mettleri D.acutilabella D.stalkeri D.arawakana p D.dunni u D.mercatorum D.ornatifrons o D.repleta melanogaster is arguably one of the best studied r D.tripunctata g D.pallidipennis a D.buzzatii i 27 t D.testacea m e D.hamatofila l D.putrida m p systems in animals , and is by a large part carried i e D.macrospina g r D.mulleri r D.quinaria a D.wheeleri D.palustris n s out through a single molecule, cis-vaccenyl acetate D.navojoa D.albomicans g r D.nasuta o 28 D.arizonae u Zaprionus 95 D.sulfurigaster p D.moj.mojavensis sophila (cVA) . This compound is produced exclusively by Dro 87 47 99 D.immigrans D.moj.wrigleyi 97 D.hypocausta D.moj.baja Root 99 D.sternopleuralis males and transferred to females during copulation, 97 D.funebris D.moj.sonorensis Colocasiomyini Dorsilopha D.busckii S.latifasciaeformis D.simulans which then reduces the attractiveness of the freshly 99 D.mauritiana S.lebanonensis a or D.sechellia 29 oph C.pararufithorax Soph D.melanogaster mated females . Moreover, cVA regulates multiple 92 C.procnemis 98 D.yakuba 96 95 D.sturtevanti 96 D.santomea behaviors: it induces sexual receptivity in virgin s D.saltans D.erecta a 98 98 l D.eugracilis t a D.suzukii 30-32 33 n D.neocordata s D.biarmipes females , elicits aggression in males , modulates g D.emarginata D.takahashii r o D.nebulosa D.fuyamai u 34 p D.willistoni D.elegans D.ficusphila oviposition behaviors , and acts as aggregation D.tsacasi w D.sucinea D.greeni il D.equinoxialis D.birchii li D.serrata s D.kikkawai 30,35 t D.asahinai o D.imaii D.lacteicornis D.rufa n D.auraria D.triauraria pheromone in presence of food . Despite the i D.paulistorumD.bifasciata g ro u p p D.azteca u D.guanche D.affinis ro D.subobscura g profound knowledge of cVA-induced behaviors in D. er st D.persimilis ga D.ananassae o D.bipectinata n ob la melanogaster, little is known about analogous stimuli sc e u D.pseudoobscura D.malerkotliana m ra gro 0.04 that regulate social and sexual behaviors in other up drosophilids. Here, we identify the sex pheromones b b’ and their roles in 99 species within the family 1 0.5 Drosophilidae, explore the evolution of pheromone 0 signaling systems with respect to phylogenetic −0.5 Correlation coefficient relationships, and highlight how sexual isolation −1 barriers between species are reinforced by olfactory signals. Whole-genome information-based phylogeny Fig. 1. Phylogenetic relationships and chemical varia- of 99 drosophilids tions among drosophilids (a) Phylogeny of 99 species within the family Drosophilidae inferred The genus Drosophila is arguably one of the most from 13,433,544 amino acids sites that represent 11,479 genes (See Supplementary Table 1 and Materials and Methods for details).