JOURNAL OF EXPERIMENTAL ZOOLOGY (MOL DEV EVOL) 308B:454–463 (2007) Integration of Wings and Their Eyespots in the Speckled Wood Butterfly Pararge aegeria CASPER J. BREUKER1,2Ã, MELANIE GIBBS3, HANS VAN DYCK3 PAUL M. BRAKEFIELD4, CHRISTIAN PETER KLINGENBERG5, 2 AND STEFAN VAN DONGEN 1Centre for Ecology and Conservation, University of Exeter, Cornwall Campus, Penryn, TR10 9EZ, UK 2Department of Biology, Group of Evolutionary Biology, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium 3Biodiversity Research Centre, Behavioural Ecology and Conservation Group, Catholic University of Louvain, (UCL), Croix du Sud 4, B-1348 Louvain- la-Neuve, Belgium 4Department of Evolutionary Biology, Institute of Biology, Leiden University, P.O. Box 9516, 2300 RA Leiden, The Netherlands 5Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK ABSTRACT We investigated both the phenotypic and developmental integration of eyespots on the fore- and hindwings of speckled wood butterflies Pararge aegeria. Eyespots develop within a framework of wing veins, which may not only separate eyespots developmentally, but may at the same time also integrate them by virtue of being both signalling sources and barriers during eyespot development. We therefore specifically investigated the interaction between wing venation patterns and eyespot integration. Phenotypic covariation among eyespots was very high, but only eyespots in neighbouring wing cells and in homologous wing cells on different wing surfaces were developmentally integrated. This can be explained by the fact that the wing cells of these eyespots share one or more wing veins. The wing venation patterns of fore- and hindwings were highly integrated, both phenotypically and developmentally. This did not affect overall developmental integration of the eyespots. The adaptive significance of integration patterns is discussed and more specifically we stress the need to conduct studies on phenotypic plasticity of integration. J. Exp. Zool. (Mol. Dev. Evol.) 308B:454– 463, 2007. r 2007 Wiley-Liss, Inc. How to cite this article: Breuker CJ, Gibbs M, Van Dyck H, Brakefield PM, Klingenberg CP, Van Dongen S. 2007. Integration of wings and their eyespots in the speckled wood butterfly Pararge aegeria. J. Exp. Zool. (Mol. Dev. Evol.) 308B:454–463. It is often hypothesised that developmental and is as such subject to selection in the context of integration evolves adaptively to match functional interactions with predators (Lyytinen et al., 2003; integration, which would allow for faster adaptive Srygley, 2004; Stevens, 2005), the resting back- evolutionary change (Wagner and Altenberg, ’96; ground (Nijhout, 2001), with potential mates Griswold, 2006), but how well are they really (Warzecha and Egelhaaf, ’95; Breuker and Brake- matched in biological systems (for an overview, see Breuker et al., 2006)? The pattern elements on Lepidopteran wings are an excellent system to Grant sponsor: Research Program of the Research Foundation study this (Beldade and Brakefield, 2002; McMil- Flanders (FWO); Grant number: G.0155.05. ÃCorrespondence to: Dr. Casper J. Breuker, Centre for Ecology and lan et al., 2002; Brakefield, 2006). The wing Conservation, University of Exeter, Cornwall Campus, Penryn, TR10 pattern as a whole is clearly an important 9EZ, UK. E-mail: [email protected] Received 4 March 2007; Accepted 1 April 2007 functional morphological component of the phe- Published online 31 May 2007 in Wiley InterScience (www. notype (reviewed in Brakefield and French, ’99), interscience.wiley.com). DOI: 10.1002/jez.b.21171 r 2007 WILEY-LISS, INC. INTEGRATION OF BUTTERFLY WINGS 455 field, 2002; Robertson and Monteiro, 2005) or with phological studies by Schwanwitsch and Su¨ffert the thermal environment (like degree of melanisa- conducted in the 1920s and 30s. They established a tion, e.g. Kingsolver and Wiernasz, ’91; Van Dyck framework of butterfly wing patterning, the so- and Matthysen, ’98). Although it has been rarely called ‘‘nymphalid groundplan’’ (Schwanwitsch, explicitly tested, individual wing pattern elements ’24, ’35; Su¨ffert, ’27; Nijhout, ’91). According to are therefore often assumed to be to a large degree this groundplan, a butterfly wing may consist of functionally integrated (Brakefield, 2001). It has three paired vertical bands, called the symmetry been suggested, however, that wing pattern ele- systems, while (horizontal) wing veins further ments might not show the same degree of subdivide the wing into distinct wing cells, which developmental integration, despite the often large then can contain one or more pattern elements (positive) phenotypic (and genetic) covariances (Fig. 1). It is these wing cells and the wing pattern found among them and despite the fact that they elements they contain that are hypothesised to be are often serial homologues and produced by the developmentally separate from other such wing same developmental mechanisms (Nijhout, ’91; cells (see review in Nijhout et al., 2003). Paulsen, ’94; Beldade and Brakefield, 2002; The best studied and understood wing pattern Beldade et al., 2002a,b; McMillan et al., 2002). elements are those in the wing cells from the border The objective of this study is to investigate this ocelli symmetry system, and it has been confirmed hypothesis of weak developmental integration of that there is a large degree of wing cell indepen- eyespots despite large phenotypic covariation. dence (Beldade and Brakefield, 2002; McMillan We consider traits to be developmentally inte- et al., 2002). The border ocelli will also be the focus grated when they together act as an integrated of our study. Although the functional role of a large and context-insensitive component of develop- number of upregulated developmental genes in the ment, even when subjected to (random) develop- wing cells, most notably Distal-less, and successive mental perturbations (Schlosser, 2004; Schlosser reaction-diffusion and diffusion-threshold steps in and Wagner, 2004). If the development of two eyespot development have been inferred (Brake- traits is independent, then a random deviation in field, ’98; Sekimura et al., 2000; Brunetti et al., one of them will not be consistently associated 2001; Koch and Nijhout, 2002; McMillan et al., with a deviation in the other trait. A popular 2002; Nijhout et al., 2003), the role of the wing cell means of evaluating the response to developmen- borders (i.e. the wing veins and wing margins) in tal perturbations is by quantifying the difference eyespot development is somewhat poorly under- between the right and a left side of a bilaterally stood. Wing veins do not determine the presence or symmetrical trait (Van Valen, ’62; Palmer and absence of individual eyespots, but it seems that Strobeck, ’86). In a sample of individuals, there- they may contribute to the inductive signalling for fore, the signed asymmetries (R-L) of two inde- the position and morphology of each wing pattern pendently developing traits will be uncorrelated. element within the wing cell. Furthermore, in a Signed asymmetry not only refers to how much a number of butterfly species, they can also act as right and left side differ, but also whether right boundaries for developing wing pattern elements, or left was the larger side. If the two traits are, either by acting as a sink, destroying morphogenetic however, developmentally linked, then the effects substances, or because they don’t allow for cell-to- of the perturbations can be transmitted directly cell communication of signals (Koch and Nijhout, between the traits. This would produce a statis- 2002; Nijhout et al., 2003; Reed and Gilbert, 2004; tical relationship between the signed asymmetries Reed and Serfas, 2004; Reed et al., 2007). In these of the traits. The covariances between signed butterfly species, eyespots are incapable of develop- asymmetries of traits result therefore from their ing across wing veins, which is easily observable. developmental connections, but not from parallel Although the wing veins may compartmentalise variation of independent pathways, and can there- and separate the development of eyespots, wing fore be used to infer developmental integration veins may actually also integrate eyespots by (Klingenberg et al., 2001; Klingenberg, 2003). We acting as both inductive signalling sources and consider traits to phenotypically covary, or in barriers for the diffusion of morphogenetic sub- other words to be phenotypically integrated, when stances. As eyespots are laid down in this network a particular size or shape of a trait consistently of wing veins it is feasible that a change in the corresponds with that of another trait. overall wing venation pattern will affect all wing The idea that wing pattern elements are devel- pattern elements simultaneously, which could opmentally separate stems from comparative mor- potentially increase both phenotypic and develop- J. Exp. Zool. (Mol. Dev. Evol.) DOI 10.1002/jez.b 456 C.J. BREUKER ET AL. Fig. 1. The dorsal (A) and ventral (B) wing surface of a Pararge aegeria butterfly. The corresponding numbers on forewing and hindwing indicate homologous landmarks (i.e. wing vein intersections or where a wing vein meets the edge of the wing). Nomenclature of the symmetry systems and wing pattern elements (eyespots) is after Schwanwitsch (’35) and Nijhout (’91). FW, forewing; HW, hindwing; OC, ocellus; d, dorsal; v, ventral. The numbers refer to the wing cell. Only the black area of the eyespots was measured.