bioRxiv preprint doi: https://doi.org/10.1101/2020.06.25.172619; this version posted February 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Deep ancestral introgression shapes evolutionary history of dragonflies and 2 damselflies 3 Anton Suvorov1$, Celine Scornavacca2,*, M. Stanley Fujimoto3, Paul Bodily4, Mark 4 Clement3, Keith A. Crandall5, Michael F. Whiting6,7, Daniel R. Schrider1,* and Seth M. 5 Bybee6,7,* 6 7 1Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 8 2Institut des Sciences de l’Evolution Université de Montpellier, CNRS, IRD, EPHE CC 064, 9 Place Eugène Bataillon, 34095 Montpellier Cedex 05, France. 10 3Department of Biology, Brigham Young University, Provo, UT 11 4Department of Computer Science, Idaho State University, Pocatello, ID 12 5Computational Biology Institute, Department of Biostatistics and Bioinformatics, Milken 13 Institute School of Public Health, George Washington University, Washington, DC 14 6Department of Biology, Brigham Young University, Provo, UT 15 7M.L. Bean Museum, Brigham Young University, Provo, UT 16 $Correspondence: [email protected] 17 *Equal contributions 18 19 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.25.172619; this version posted February 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 20 SUMMARY 21 22 Introgression is arguably one of the most important biological processes in the evolution of 23 groups of related species, affecting at least 10% of the extant species in the animal kingdom. 24 Introgression reduces genetic divergence between species, and in some cases can be highly 25 beneficial, facilitating rapid adaptation to ever-changing environmental pressures. Introgression 26 also significantly impacts inference of phylogenetic species relationships where a strictly binary 27 tree model cannot adequately explain reticulate net-like species relationships. Here we use 28 phylogenomic approaches to understand patterns of introgression along the evolutionary history 29 of a unique, non-model insect system: dragonflies and damselflies (Odonata). We demonstrate 30 that introgression is a pervasive evolutionary force across various taxonomic levels within 31 Odonata. In particular, we show that the morphologically “intermediate” species of 32 Anisozygoptera (one of the three primary suborders within Odonata besides Zygoptera and 33 Anisoptera), which retain phenotypic characteristics of the other two suborders, experienced high 34 levels of introgression likely coming from zygopteran genomes. Additionally, we found evidence 35 for multiple cases of deep inter-superfamilial ancestral introgression. 36 37 INTRODUCTION 38 39 In recent years there has been a large body of evidence amassed demonstrating that multiple 40 parts of the Tree of Life did not evolve according to a strictly bifurcating phylogeny [1, 2]. 41 Instead, many organisms experience reticulate network-like evolution that is caused by an 42 exchange of inter-specific genetic information via various biological processes. In particular, 43 lateral gene transfer, incomplete lineage sorting (ILS) and introgression can result in gene trees 44 that are discordant with the species tree [3, 4]. Lateral transfer and introgression both involve 45 gene flow following speciation, thereby producing “reticulate” phylogenies. Incomplete lineage 46 sorting (ILS), on the other hand, occurs when three or more lineages coalesce in their ancestral 47 population. This often arises in an order that conflicts with that of the true species tree, and thus 48 involves no post-speciation gene flow and therefore does not contribute to reticulate evolution. 49 Phylogenetic species-gene tree incongruence observed in empirical data can provide insight into 50 underlying biological factors that shape the evolutionary trajectories of a set of taxa. The major bioRxiv preprint doi: https://doi.org/10.1101/2020.06.25.172619; this version posted February 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 51 source of reticulate evolution for eukaryotes is introgression where it affects approximately 25% 52 of flowering plant and 10% of animal species [1, 5]. Introgressed alleles can be fitness-neutral, 53 deleterious [6] or adaptive [7, 8]. For example, adaptive introgression has been shown to provide 54 an evolutionary rescue from polluted habitats in gulf killifish (Fundulus grandis) [7], yielded 55 mimicry adaptations among Heliconius butterflies [9] and archaic introgression has facilitated 56 adaptive evolution of altitude tolerance [10], immunity and metabolism in modern humans [11]. 57 Additionally, hybridization and introgression are important and often overlooked mechanisms of 58 invasive species establishment and spread [12]. 59 Odonata, the insect order that contains dragonflies and damselflies, lacks a strongly 60 supported backbone tree to clearly resolve higher-level phylogenetic relationships [13, 14]. 61 Current evidence places odonates together with Ephemeroptera (mayflies) as the living 62 representatives of the most ancient insect lineages to have evolved wings and active flight [15]. 63 Odonates possess unique anatomical and morphological features such as a specialized body 64 form, specialized wing venation, a distinctive form of muscle attachment to the wing base [16] 65 allowing for direct flight and accessory (secondary) male genitalia that support certain unique 66 behaviors (e.g., sperm competition). They are among the most adept flyers of all animals and are 67 exclusively carnivorous insects relying primarily on vision [17, 18] to capture prey. During their 68 immature stage they are fully aquatic and spend much of their adult life in flight. 69 Biogeographically, odonates exhibit species ranges varying from worldwide dispersal [19] to 70 island-endemic. Odonates also play crucial ecological roles in local freshwater communities, 71 being a top invertebrate predator as both adults and immatures [20]. Due to this combination of 72 characteristics, odonates are quickly becoming model organisms to study specific questions in 73 ecology, physiology and evolution [21, 22]. However, the extent of introgression at the genomic 74 scale within Odonata remains largely unknown. 75 Two early attempts to tackle introgression/hybridization patterns within Odonata were 76 undertaken in [23, 24]. The studies showed that two closely related species of damselflies, 77 Ischnura graellsii and I. elegans, can hybridize under laboratory conditions and that genital 78 morphology of male hybrids shares features with putative hybrids from I. graellsii–I. elegans 79 natural allopatric populations [23]. The existence of abundant hybridization and introgression in 80 natural populations of I. graellsii and I. elegans has received further support from an analysis of 81 microsatellite data [25]. Putative hybridization events have also been identified in a pair of bioRxiv preprint doi: https://doi.org/10.1101/2020.06.25.172619; this version posted February 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 82 calopterygoid damselfly species, Mnais costalis and M. pruinosa based on the analyses of two 83 molecular loci (mtDNA and nucDNA) [26], and between Calopteryx virgo and C. splendens 84 using 16S ribosomal DNA and 40 random amplified polymorphic DNA (RAPD) markers [27]. A 85 more recent study identified an inter-specific hybridization between two cordulegasterid 86 dragonfly species, Cordulegaster boltonii and C. trinacriae using two molecular markers 87 (mtDNA and nucDNA) and geometric morphometrics [28]. Furthermore, in various biological 88 systems, the empirical evidence shows that hybridization can potentially lead to intermediate 89 phenotypes [29] observed at molecular level (e.g. semidominant expression in inter-specific 90 hybrids [30]) as well as organismal morphology (e.g. [31-33]). The Anisozygoptera suborder, 91 which contains only three extant species, retains traits shared with both dragonflies and 92 damselflies (hence its taxonomic name), ranging from morphology and anatomical structures 93 [34] to behavior and flight biomechanics [35]. These characteristics could suggest a hybrid origin 94 of this suborder. The potential introgression scenario for Anisozygoptera is yet to be formally 95 tested using genome-wide data. 96 Here we present a comprehensive analysis of transcriptomic data from 83 odonate 97 species. First, we reconstruct a robust phylogenetic backbone using up to 4341 genetic loci for 98 the order and discuss its evolutionary history spanning from the Carboniferous period (~360 99 mya) to present day. Furthermore, in light of the “intermediate” phenotypic nature of 100 Anisozygoptera, we investigate phylogenetic signatures of introgression within Odonata. Most