Predation Drives Specialized Host Plant Associations In
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bioRxiv preprint doi: https://doi.org/10.1101/2020.06.16.150730; this version posted June 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Predation drives specialized host plant associations in preadapted milkweed 2 bugs (Heteroptera: Lygaeinae) 3 4 Georg Petschenka*1, Rayko Halitschke2, Anna Roth3, Sabrina Stiehler3, Linda Tenbusch3, Tobias 5 Züst4, Christoph Hartwig5, Juan Francisco Moreno Gámez6, Robert Trusch7, Jürgen Deckert8, 6 Kateřina Chalušová9, Andreas Vilcinskas3,5, Alice Exnerová9 7 8 *correspondence to: [email protected] 9 10 1Department of Applied Entomology, Institute of Phytomedicine, Faculty of Agricultural 11 Sciences, University of Hohenheim, 70599 Stuttgart, Germany 12 2Department of Molecular Ecology, Max Planck Institute for Chemical Ecology, 07745 Jena, 13 Germany 14 3Institute for Insect Biotechnology, Justus Liebig University Giessen, Heinrich-Buff-Ring 26–32, 15 35392 Giessen, Germany 16 4Institute of Plant Sciences, University of Bern, Altenbergrain 21, CH-3013 Bern, Switzerland 17 5Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Branch for 18 Bioresources, Ohlebergsweg 12, 35392 Giessen, Germany 19 6Sociedad Andaluza de Entomología, 41702 Dos Hermanas (Sevilla), Spain 20 7State Museum of Natural History Karlsruhe, 76133 Karlsruhe, Germany 21 8Museum für Naturkunde, Leibniz Institute for Evolution and Biodiversity Science, 10115 Berlin, 22 Germany 23 9Department of Zoology, Faculty of Science, Charles University, 12843 Prague, Czech Republic 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.16.150730; this version posted June 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 41 Abstract 42 Host plant specialization across herbivorous insects varies dramatically, but the underlying 43 evolutionary mechanisms are little-known. The milkweed bugs (Heteroptera: Lygaeinae) are 44 ancestrally associated with plants of the Apocynaceae from which they commonly sequester 45 cardiac glycosides for defense, facilitated by resistant Na+/K+-ATPases and adaptations for 46 transport, storage and discharge of toxins. Here, we show that three Lygaeinae species 47 independently colonized four novel non-apocynaceous hosts, convergently producing cardiac 48 glycosides. A fourth species shifted to a new source of toxins by tolerating and sequestering 49 alkaloids from meadow saffron (Colchicum autumnale, Colchicaceae). Across three species 50 tested, feeding on seeds containing toxins did not improve growth, but sequestration mediated 51 protection against predatory lacewing larvae and birds. We conclude that physiological 52 preadaptations and convergent phytochemistry facilitated novel specialized host associations. 53 Therefore, selection by predators on sequestration of defenses, rather than the exploitation of 54 novel dietary resources, can lead to obligatory specialized host associations in generalist insects. 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.16.150730; this version posted June 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 83 Introduction 84 Herbivorous insects show tremendous variation with regard to dietary specialization. While it is a 85 long-standing assumption that phytochemicals may restrict and direct the evolution of host plant 86 use1, the explicit role of phytochemicals as drivers of host plant associations has been revealed in 87 only a few systems2–5. Proposed mechanisms of how plant secondary compounds could mediate 88 insect-plant interactions include physiological trade-offs in the efficiency of host plant use 89 between generalists and specialists1,6–9. Alternatively, it has been shown that novel host plant 90 associations can create enemy-free spaces for herbivores10,11 either by providing defense11 or 91 refuge from natural enemies10. However, even though it is widely recognized that many insects 92 not only use plants as a dietary resource but also sequester (i.e. absorb and store) plant toxins to 93 defend themselves against predators12–14, the extent to which sequestration could drive the 94 evolution of insect-host plant associations has rarely been addressed4,15. 95 While it has been hypothesized that dietary specialization and sequestration of plant 96 toxins can lead to an evolutionary dead end4,16, there is evidence that ecological specialization 97 does not necessarily prevent host range expansion4. Nevertheless, sequestration and dietary 98 specialization seem to be evolutionarily linked13,17–20, and predators driving the occupation of 99 enemy-free-spaces are typically considered to select for specialization9,21. Recent research 100 indicated that sequestration requires different resistance traits than are required to merely cope 101 with dietary toxins22, suggesting that selection by predators or parasitoids (i.e. the third trophic 102 level) opens a second arena for coevolutionary escalation13,22. Consequently, a rigorous analysis 103 of coevolution between plants and specialized insects requires the integration of adaptations 104 underlying bitrophic interactions with adaptations driven by higher trophic levels13,22,23. 105 Here, we used milkweed bugs (Heteroptera: Lygaeinae) as a model system to test 106 hypotheses about the evolutionary drivers leading to specialized associations with particular plant 107 species. The Lygaeinae comprise about 600 primarily seed-eating species that are well known for 108 their predilection of plants in the Apocynaceae worldwide24–27. Milkweed bugs typically exhibit a 109 red-and-black aposematic coloration and, in addition to defensive scent glands typical for 110 Heteroptera28, several species have been shown to acquire defenses against predators from their 111 host plants29–32. Upon attack, many milkweed bug species release sequestered toxins in a 112 defensive secretion from a specialized storage compartment of the integument (the dorsolateral 113 space)33,34. The large milkweed bug (Oncopeltus fasciatus (Dallas, 1852)) in particular has been 114 studied in detail with regard to sequestration of cardiac glycosides, which it derives from seeds of 115 milkweeds in the Apocynaceae genus Asclepias35. 116 Cardiac glycosides are important defense metabolites of plants in the Apocynaceae, and 117 evolved convergently in at least 11 additional botanical families36. Both compound subtypes, the 118 cardenolides and the bufadienolides, are specific inhibitors of the ubiquitous animal enzyme 119 Na+/K+-ATPase. Specialized insects from at least six taxonomic orders, including several 120 lygaeine species37–40, tolerate cardenolides by expressing Na+/K+-ATPases with several amino 121 acid substitutions that mediate a high degree of cardenolide resistance in vitro (target site 122 insensitivity)40,41. Duplication of the gene coding for the α-subunit of Na+/K+-ATPases in the 123 lygaeine bugs O. fasciatus and Lygaeus kalmii Stal, 1874 resulted in three different copies with 124 up to four amino acid substitutions in regions of the protein critical for cardiac glycoside 125 binding38,39,42, rendering the enzyme resistant to cardiac glycosides42. In addition to resistance, 126 sequestration requires accumulation of toxins from the dietary resource, and milkweed bugs 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.16.150730; this version posted June 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 127 possess an as-of-yet unidentified mechanism for the transport of toxins across the gut epithelium. 128 In summary, milkweed bugs possess a suite of traits related to sequestration and defense that 129 includes aposematic coloration, resistant Na+/K+-ATPases, and mechanisms for accumulation, 130 storage, and release of toxins. This suite of traits may also function as a physiological 131 preadaptation facilitating the sequestration of novel toxin compounds. For example, the milkweed 132 bug Neacoryphus bicrucis (Say, 1825) sequesters pyrrolizidine alkaloids31, a class of compounds 133 unrelated to cardiac glycosides. 134 Sequestration of cardiac glycosides by lygaeine bugs was initially described for O. 135 fasciatus and L. kalmii feeding on Asclepias species43, as well as for Caenocoris nerii (Germar, 136 1847) and Spilostethus pandurus Scopoli, 1763 on oleander (Nerium oleander), both belonging to 137 the Apocynaceae44. A broad survey based on dried museum specimens demonstrated the presence 138 of cardiac glycosides in many genera of Lygaeinae24, suggesting that sequestration of cardiac 139 glycosides is a common trait of milkweed bugs. Furthermore, an evolutionary analysis revealed 140 that sequestration of cardiac glycosides, target site insensitivity