The genetic basis of a plant–insect coevolutionary key innovation Christopher W. Wheat*†‡, Heiko Vogel*, Ute Wittstock*§, Michael F. Braby¶ʈ, Dessie Underwood**, and Thomas Mitchell-Olds*†† *Max Planck Institute for Chemical Ecology, Beutenberg Campus, Hans Knoell Strasse 8, 07745 Jena, Germany; ¶School of Botany and Zoology, Australian National University, Canberra ACT 0200, Australia; **California State University, 1250 Bellflower Boulevard, Long Beach, CA 90840; and ††Department of Biology, Duke University, Durham, NC 27708 Edited by May R. Berenbaum, University of Illinois at Urbana–Champaign, Urbana, IL, and approved October 23, 2007 (received for review July 5, 2007) Ehrlich and Raven formally introduced the concept of stepwise co- emplified by the cabbages and Arabidopsis), and mistletoes. evolution using butterfly and angiosperm interactions in an attempt Phylogenetic reconstruction of almost 90% of the Pieridae to account for the impressive biological diversity of these groups. genera (74 recognized genera plus six subgenera, based on 1,066 However, many biologists currently envision butterflies evolving 50 bp of the EF-1␣ gene) was recently completed (13). These results to 30 million years (Myr) after the major angiosperm radiation and indicate that Fabales feeding is the ancestral state of Pieridae thus reject coevolutionary origins of butterfly biodiversity. The un- (Fig. 1). The Fabales feeders are the Dismorphiinae and nearly resolved central tenet of Ehrlich and Raven’s theory is that evolution all Coliadinae, whereas the sister to the Coliadinae, the Pierinae, of plant chemical defenses is followed closely by biochemical adap- primarily feed on Brassicales (Fig. 1) (12). Within Pierinae, there tation in insect herbivores, and that newly evolved detoxification are two subsequent derived shifts away from glucosinolate mechanisms result in adaptive radiation of herbivore lineages. Using feeding onto mistletoes and other species. Thus, the Pierinae one of their original butterfly-host plant systems, the Pieridae, we represent a single origin of glucosinolate feeding (Fig. 1). identify a pierid glucosinolate detoxification mechanism, nitrile-spec- The evolutionary appearance of the plant order Brassicales ifier protein (NSP), as a key innovation. Larval NSP activity matches (Eurosid II, Dicotyledons) presented a radical new chemical chal- the distribution of glucosinolate in their host plants. Moreover, by lenge for insect herbivores, known as the glucosinolate-myrosinase using five different temporal estimates, NSP seems to have evolved system (14). All Brassicales have this system, which is one of the best shortly after the evolution of the host plant group (Brassicales) (Ϸ10 and most widely studied chemical plant defenses (15–17). Its Myr). An adaptive radiation of these glucosinolate-feeding Pierinae effectiveness as an anti-herbivore defense becomes apparent upon followed, resulting in significantly elevated species numbers com- tissue damage, such as insect feeding. Tissue damage brings the pared with related clades. Mechanistic understanding in its proper formerly compartmentalized myrosinase enzyme into contact with historical context documents more ancient and dynamic plant–insect nontoxic glucosinolates, which it hydrolyzes into breakdown prod- interactions than previously envisioned. Moreover, these mechanistic ucts such as isothiocyanates (18–20). Whereas Homo sapiens may insights provide the tools for detailed molecular studies of coevolu- find these breakdown products enjoyable condiments (e.g., mus- EVOLUTION tion from both the plant and insect perspectives. tard, wassabi), they are well known to be highly toxic to many insect herbivores (15, 21–23). adaptive radiation ͉ Brassicales ͉ Pieridae ͉ diversification ͉ We have identified two independent lepidopteran detoxifica- Bayesian relaxed molecular clock tion mechanisms for the glucosinolate-myrosinase defense sys- tem at biochemical and molecular levels by means of functional he relative timing of adaptive radiations in host plants and genomics approaches, beginning with glucosinolate sulfatase Ttheir butterfly herbivores is controversial. Although the (GSS) in the diamondback moth Plutella xylostella (Plutellidae) major angiosperm radiation occurred Ϸ140 to 100 million years (24). GSS desulfates glucosinolates, producing metabolites that ago (Mya), fossil data suggest that diversification of ‘‘primitive’’ no longer act as substrates for myrosinases. The second, called Lepidoptera occurred before this time and butterflies radiated nitrile-specifier protein (NSP), has recently been identified for long after these host plants (Ͻ75 Mya) (1–3). Many espouse this the pierid butterfly Pieris rapae (20). NSP, expressed solely in the recent butterfly origin, which necessarily implies a very limited larval midgut, promotes the formation of nitrile breakdown role, if any, for coevolution in butterfly diversification (1–4). products instead of toxic isothiocyanates upon myrosinase- However, others posit a much older age of butterflies (Ͼ100 catalyzed glucosinolate hydrolysis (20). The GSS and NSP Mya), with speciation influenced by angiosperm evolution and detoxification mechanisms are distinctly different from each the breakup of the supercontinent Gondwana (5, 6). This lack of other, as well as the other identified host plant detoxification consensus on both the timing of butterfly diversification, which resulted in the Ϸ17,000 extant species today, and the role of coevolution arises from the notably poor fossil record of Lepi- Author contributions: C.W.W. and T.M.-O. designed research; C.W.W., H.V., and U.W. performed research; U.W., M.F.B., D.U., and T.M.-O. contributed new reagents/analytic doptera (5, 7). Consequently, whereas some have described likely tools; C.W.W., H.V., and U.W. analyzed data; and C.W.W., H.V., and U.W. wrote the paper. scenarios, no studies have tested for the effects of key innova- The authors declare no conflict of interest. tions on butterfly diversification even though coevolution This article is a PNAS Direct Submission. through key innovations was first introduced to science using †Present Address: Pennsylvania State University, Department of Biology, 208 Mueller butterflies and their angiosperm host plants as exemplars (8– Laboratories, University Park, PA 16802. 12). To explore the potential role of coevolution in butterfly ‡To whom correspondence should be addressed. E-mail: [email protected]. diversification, we focus on the family Pieridae, composed of the §Present Address: Institute of Pharmaceutical Biology, Braunschweig University of Tech- commonly known white and sulfur butterflies. Recent significant nology, 38106 Braunschweig, Germany. advances in functional genomics and phylogenetics in this family ʈPresent Address: Biodiversity Conservation Division, Department of Natural Resources, provide a unique opportunity to resolve the controversies briefly Environment and the Arts, P.O. Box 496, Palmerston NT 0831, Australia. reviewed above (see also ref. 5). This article contains supporting information online at www.pnas.org/cgi/content/full/ Pieridae use three major host plant groups: the Fabales 0706229104/DC1. (Legumes), the Brassicales (glucosinolate-containing plants ex- © 2007 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0706229104 PNAS ͉ December 18, 2007 ͉ vol. 104 ͉ no. 51 ͉ 20427–20431 Downloaded by guest on October 1, 2021 Eroessa chiliensis Anthocharidini Cunizza hirlanda Hesperocharis crocea Mathania leucothea Euchloe ausonides Zegris eupheme Anthocharis belia Anthocharis cardamines Saletara liberia Appias paulina Aoa affinis Appiadina Appias drusilla Appias aegis Talbotia naganum Pieris rapae Ascia monuste Ganyra josephina Phulia nymphula Infraphulia ilyodes Pierina Pierphulia rosea Theochila maenacte Tatochila autodice Hypsochila wagenknechti Baltia butleri Pontia callidice Pontia helice e Leptophobia aripa Pieris rapae an Pieriballia viardi ir Perrhybris pamela ei Itaballia demophile P Belenois java Leuciacria acuta Leuciacria olivei Delias belladonna Delias aganippe Aporia crataegi Aporia agathon Aporiina Melete lycimnia Leodonta tellane Pereute charops Neophasia menapia Eucheira socialis Archonias brassolis Catasticta teutila Charonias eurytele Aporia crataegi Catasticta cerberus Mylothris bernice Mylothris agathina Cepora perimale Prioneris philonome Elodina angulipennis Dixeia charina Colotis group Hebomoia glaucippe Colotis hetaera Eronia cleodora Ixias pyrene Gideona lucasi Pinacopteryx eriphea Leptosia nina Pareronia valeria Colotis evagore Nepheronia thalassina Nathalis iole Kricogonia lyside Eurema hecabe Pyristia proterpia Eurema mexicana Leucidia brephos Coliadinae Teriocolias zelia Gandaca harina Phoebis sennae Aphrissa statira Gonepteryx rhamni Anteos clorinde Colias philodice Zerene cesonia Colias hyale Dercas gobrias Catopsilia pomona 80 60 40 20 0 MYA. Late Cretaceous Paleocene Eocene Oligocene Miocene Plio. CRETACEOUS PALEOGENE NEOGENE Fig. 1. Chronogram of the Pieridae, showing host plant relationships and the NSP activity of representative species. Circles represent presence (filled) or absence (empty) of NSP activity in larvae from relevant genera. Vertical labels indicate higher taxa and informal groups. Branches are scaled relative to a divergence time of 85 Mya, shown on the x axis. Yellow branches refer to terminal taxa that are predominately Fabales feeding, whereas green branches are glucosinolate feeding, and blue are derived non-glucosinolate feeding