Aggressive mimicry coexists with mutualism in an aphid Adrián Salazara, Benjamin Fürstenaub, Carmen Queroc, Nicolás Pérez-Hidalgod, Pau Carazoa,e, Enrique Fonta, and David Martínez-Torresa,1 aInstitut Cavanilles de Biodiversitat i Biologia Evolutiva, Universitat de València, Apartado de Correos 22085, 46071 Valencia, Spain; bInstitute of Biology, Applied Zoology/Animal Ecology, Freie Universität Berlin, 12163 Berlin, Germany; cInstitute of Advanced Chemistry of Catalonia, Consejo Superior de Investigaciones Científicas, 08034 Barcelona, Spain; dDepartamento de Biodiversidad y Gestión Ambiental, Universidad de León, 24071 León, Spain; and eEdward Grey Institute, Department of Zoology, University of Oxford, Oxford OX1 3PS, United Kingdom Edited by Nancy A. Moran, University of Texas at Austin, Austin, TX, and approved December 3, 2014 (received for review July 23, 2014) Understanding the evolutionary transition from interspecific ex- produced by the same genome) (Fig. 1). Like other members of ploitation to cooperation is a major challenge in evolutionary this tribe, P. cimiciformis alternates not only between oviparous biology. Ant–aphid relationships represent an ideal system to this sexual reproduction and viviparous parthenogenesis, but also be- end because they encompass a coevolutionary continuum of inter- tween two alternative hosts, and exhibits both wingless and winged actions ranging from mutualism to antagonism. In this study, we morphs. Most unusually, during its root-dwelling phase (Fig. 1), report an unprecedented interaction along this continuum: aggres- P. cimiciformis has two morphologically distinct wingless morphs sive mimicry in aphids. We show that two morphs clonally produced where other closely related Fordini species have just one (9). Until by the aphid Paracletus cimiciformis during its root-dwelling phase recently, the only known wingless root-dwelling morph in this establish relationships with ants at opposite sides of the mutual- species was a flat yellowish-white aphid (hereafter “flat morph”) ism–antagonism continuum. Although one of these morphs exhibits usually reported (as in other Fordini species) to be ant-attended the conventional trophobiotic (mutualistic) relationship with ants of (7, 10, 11). However, Ortiz-Rivas et al. (9) recently demonstrated the genus Tetramorium, aphids of the alternative morph are trans- the existence of an alternative round, olive-green wingless morph ported by the ants to their brood chamber and cared for as if they (hereafter “round morph”) resembling the single root-dwelling EVOLUTION were true ant larvae. Gas chromatography-mass spectrometry anal- wingless morph occurring in related Fordini species. Importantly, yses reveal that the innate cuticular hydrocarbon profile of the mimic each of these two morphs of P. cimiciformis is able to produce morph resembles the profile of ant larvae more than that of the both morphs through parthenogenesis (9) although the factors alternative, genetically identical nonmimic morph. Furthermore, we triggering the production of one morph or the other remain un- show that, once in the brood chamber, mimic aphids suck on ant known and their phenology is incompletely understood (SI Text). larva hemolymph. These results not only add aphids to the limited The aim of this study was to explore the potential functional list of arthropods known to biosynthesize the cuticular chemicals of significance of this polyphenism occurring during the root- their deceived hosts to exploit their resources but describe a remark- dwelling phase of the P. cimiciformis life cycle. We characterized able case of plastic aggressive mimicry. The present work adds a pre- the nature of each aphid morph’s interactions with ants of the viously unidentified dimension to the classical textbook paradigm of genus Tetramorium, their main tending ants (7, 10, 11), and – aphid ant relationships by showcasing a complex system at the evo- particularly with Tetramorium semilaeve, one of the most lutionary interface between cooperation and exploitation. Significance aggressive mimicry | aphids | ants | mutualism | polyphenism The best known relationship between ants and aphids consists major challenge in evolutionary biology is to understand the in aphids providing ants with honeydew while receiving hy- Afactors governing the evolutionary transitions between in- gienic services and protection in return. We report an un- terspecific exploitation and cooperation. Interactions between precedented aphid–ant interaction in which one of the two ants and aphids can be ranked along a continuum from mutu- clonally produced root-dwelling morphs of the aphid Paracletus alism to antagonism (1, 2), thus providing an excellent system to cimiciformis imitates the cuticular hydrocarbons of Tetramorium address this issue. In the best known relationship between ants ant larvae, inducing ants to transport the aphids to their brood and aphids, ants eat the sugar-rich honeydew excreted by the chamber, where they suck on ant larva hemolymph. To our aphids and, in return, provide them with protection and hygienic knowledge, this strategy constitutes the first known case of services (2, 3). This kind of interaction is termed trophobiosis aggressive mimicry in aphids. Moreover, because the alterna- and is considered to be mutualistic (3). Although most ant–aphid tive morph maintains a “conventional” relationship with ants, trophobiotic associations are facultative, some ant species bring our findings are unusual in that they report, within the same aphids or even aphid eggs into their nests in winter, ensuring the species (and within a single clone), the coexistence of two continuity of the relationship (4–6). Here, we report an unprece- evolutionary strategies at disparate points in the mutualism– dented ant–aphid relationship at the evolutionary interface be- antagonism continuum. tween cooperation and exploitation involving the coexistence of two aphid clonal morphs: a trophobiotic morph and an alternative Author contributions: A.S., P.C., E.F., and D.M.-T. designed research; A.S., B.F., C.Q., N.P.-H., ’ and D.M.-T. performed research; B.F., C.Q., and N.P.-H. contributed new reagents/analytic morph that acts as an aggressive mimic to infiltrate ants brood tools; A.S., B.F., C.Q., P.C., E.F., and D.M.-T. analyzed data; and A.S., P.C., E.F., and D.M.-T. chamber and suck on their larvae. wrote the paper. Paracletus The aphid species participating in this interaction is The authors declare no conflict of interest. cimiciformis , a member of the tribe Fordini in the gall-inducing This article is a PNAS Direct Submission. P. cimiciformis aphid subfamily Eriosomatinae. is widely dis- Data deposition: The sequences reported in this paper have been deposited in the Gen- tributed across Europe and has been recorded in Asia and North Bank database (accession nos. KM101062–KM101073). Africa (7). As is generally true for aphids, but even more so for 1To whom correspondence should be addressed. Email: [email protected]. P. cimiciformis gall-inducing aphids (8), the life cycle of is highly This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. plastic, exhibiting numerous polyphenisms (alternative phenotypes 1073/pnas.1414061112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1414061112 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 Primary host Secondary host the honeydew (honeydew consumption) for as long as the flow () (Poaceae roots) continued. This sequence was repeated several times (range = 8–28) during the observation periods, but, occasionally (in 3 out Summer 1 End of of 12 interactions), ants initiated an escalation of aggressive be- summer 1 Afg haviors that ended up with the aphid being killed and consumed. Wfg Flat aphids. In 12 out of 12 staged interactions, flat aphids re- Wfg Fx Autumn 1 sponded to being contacted by ants by retracting their limbs and Gall generations Gall lying motionless. Ants performed antennation, to which aphids Spring 1 responded by excreting honeydew in 5 out of 12 interactions. In E RM sharp contrast to interactions with the round morph, a tropho- Winter 2 Root-dwelling generations biotic sequence was never observed with flat morph aphids. Autumn 2 Winter 1 Instead, ants always proceeded by picking up the aphid and End of summer 2 ? carrying it into the nest, where it was licked by several workers. Spring 2 During licking, flat aphids occasionally excreted honeydew (4 out Ant of 12 interactions). Finally, flat aphids were transported to the Sf FM brood chamber, where they were subjected to additional licking M Summer 2 and deposited on the pile of larvae (12 out of 12 interactions) (Fig. 2B and Movie S2). Once on the pile of larvae, flat aphids Summer 2 Wvg exhibited larva probing (a behavior exclusive to this morph), by Sxp which aphids pierced ant larvae with their stylet (3 out of 12 interactions) (Fig. 2 C and D and Movie S3). Fig. 1. Simplified diagram of the biannual life cycle of P. cimiciformis. In a second round of observations performed to analyze quan- Sexual reproduction takes place on P. terebinthus trees, its primary host, titative differences in particular behaviors (Materials and Methods), where up to five different morphs occur. Of these generations, three de- T. semilaeve velop inside distinct galls that they induce in their host’s leaves. Toward the we found highly significant differences in behavior end of summer, the last generation born inside the galls consists of winged toward