Downloaded from http://rsbl.royalsocietypublishing.org/ on February 12, 2015 Biol. Lett. (2011) 7, 670–673 selection is renewed, and therefore may accelerate an doi:10.1098/rsbl.2011.0268 evolutionary response to the selection pressure. Published online 14 April 2011 We examined the extent to which a behavioural Animal behaviour defence persists in the absence of selection from avian brood parasitism. The interactions between avian brood parasites and their hosts are ideal for Persistence of host determining the fate of adaptations once selection has been relaxed, owing to shifting distributions of defence behaviour in the hosts and parasites [5,6] or the avoidance of well- defended hosts by parasites [7,8]. Host defences such absence of avian brood as rejection of parasite eggs may be lost in the absence parasitism of selection if birds reject their oddly coloured eggs [9,10], but are more likely to be retained because Brian D. Peer1,2,3,*, Michael J. Kuehn2,4, these behaviours may never be expressed in circum- Stephen I. Rothstein2 and Robert C. Fleischer1 stances other than parasitism [2,3]. Whether host 1Center for Conservation and Evolutionary Genetics, defences persist in the absence of brood parasitism is Smithsonian Conservation Biology Institute, National Zoological Park, critical to long-term avian brood parasite–host coevolu- Smithsonian Institution, PO Box 37012, MRC 5503, Washington, tion. If defences decline quickly, brood parasites can DC 20013-7012, USA alternate between well-defended hosts and former 2Department of Ecology, Evolution, and Marine Biology, University of California, Santa Barbara, CA 93106, USA hosts that have lost most of their defences, owing to the 3Department of Biological Sciences, Western Illinois University, Macomb, costs of maintaining them once parasitism has ceased, IL 61455, USA 4 or follow what has been termed the ‘coevolutionary Western Foundation of Vertebrate Zoology, 439 Calle San Pablo, cycles’ model of host–brood parasite coevolution [3]. Camarillo, CA 93012-8506, USA *Author for correspondence ([email protected]). By contrast, if defences persist for long periods of time, as in the ‘single trajectory model’, brood parasites will The fate of host defensive behaviour in the have to evolve adaptations such as egg mimicry for one absence of selection from brood parasitism is critical to long-term host–parasite coevolution. or a small number of hosts, which will force them to We investigated whether New World Bohemian become specialized [3]. waxwings Bombycilla garrulus that are allopatric Cedar waxwings, Bombycilla cedrorum, are hosts of the from brown-headed cowbird Molothrus ater and parasitic brown-headed cowbird, Molothrus ater in North common cuckoo Cuculus canorus parasitism America and eject most cowbird eggs added to their have retained egg rejection behaviour. We found nests [11,12]. Bohemian waxwings Bombycilla garrulus that egg rejection was expressed by 100 per cent are closely related to cedar waxwings but breed north of Bohemian waxwings. Our phylogeny revealed of the cowbird’s range, with the exception of some over- that Bohemian and Japanese waxwings Bomby- lap in western Canada [13]. This overlap would have cilla japonica were sister taxa, and this clade little effect on the Bohemian waxwing gene pool because was sister to the cedar waxwing Bombycilla it is extremely rare in this area [13,14 ], making the cedrorum. In addition, there was support for a split between Old and New World Bohemian Bohemian waxwing an ideal host for testing the validity waxwings. Our molecular clock estimates suggest of the single trajectory model and the extent to which that egg rejection may have been retained for defences are retained in the absence of selection. Here, 2.8–3.0 Myr since New World Bohemian waxwings we tested whether Bohemian waxwings express egg rejec- inherited it from their common ancestor with the tion under conditions of relaxed selection, and therefore rejecter cedar waxwings. These results support probably inherited it from a common ancestor with cedar the ‘single trajectory’ model of host–brood para- waxwings. We also estimate how long this defence may site coevolution that once hosts evolve defences, have persisted under relaxed selection, using an estimate they are retained, forcing parasites to become of clade age based on a molecular clock. more specialized over time. Keywords: brood parasitism; coevolution; egg rejection; molecular clock; relaxed selection 2. MATERIAL AND METHODS Material and methods can be found in the electronic supplementary material. 1. INTRODUCTION When selection pressures are relaxed, adaptations may 3. RESULTS decay because they incur costs [1]. However, when (a) Experimental parasitism adaptations are expressed only in the presence of Bohemian waxwings rejected 100 per cent (n ¼ 9 nests) specific stimuli, they may have no effect on fitness of cowbird eggs after an average of 1.6 days following and such neutral traits may persist for long periods parasitism (range 1–4 days). No host eggs were damaged after selection has been relaxed [2,3]. Information on or missing following ejections. We videotaped one nest how long adaptations are retained or conversely how following experimental parasitism and the bird returned quickly adaptations are lost in the absence of benefits to the nest, peered inside, grasped the egg between its is relatively scanty (but see [2,4]). Whether adaptations mandibles, and flew away with the egg. are lost or retained is significant, because an adaptation that is retained or declines slowly may be present if (b) Phylogenies and date estimates Electronic supplementary material is available at http://dx.doi.org/ Both Cyt b and ND2 genes, under maximum-parsimony 10.1098/rsbl.2011.0268 or via http://rsbl.royalsocietypublishing.org. and maximum-likelihood criteria, produced similar Received 8 March 2011 Accepted 25 March 2011 670 This journal is q 2011 The Royal Society Downloaded from http://rsbl.royalsocietypublishing.org/ on February 12, 2015 Persistence of host defence behaviour B. D. Peer et al. 671 Bombycilla cedrorum AF285792 Bombycilla cedrorum AF285791 Bombycilla cedrorum FFJ177315 100/100 Bombycilla cedrorum AY329448 Bombycilla cedrorum EEU834869 Bombycilla cedrorum AF285786 91/ Bombycilla garrulus AF285793 NW 85 100/100 Bombycilla garrulus AF285794 100 Bombycilla garrulus AY329449 96/99 Bombycilla garrulus FFJ177316 100/ Bombycilla garrulus AY228049 59/90 100 Bombycilla garrulus AF285796 OW Bombycilla garrulus AF285795 bootstraps ML 200/ MP 1000 Bombycilla japonica AF151394 50/99 Bombycilla japonica FFJ177317 Bombycilla japonica AF285798 Bombycilla japonica AF285797 100/69 Phainopepla nitens AY329469 Phainopepla nitens 9897 Phainopepla nitens AF285788 Phainopepla nitens FFJ177322 0.1 Figure 1. The best maximum-likelihood (ML) phylogenetic tree produced by RAXML [15]. Numbers above branches are support values from 200 bootstraps of ML tree/support values from 1000 bootstraps of maximum-parsimony (MP) tree. Table 1. Divergence rates (per cent sequence change per million years), dates (in millions of years) and credibility intervals. (Rates are used as priors and were obtained for each gene from an external rate calibration based on a related clade of Hawaiian honeyeaters (S. Sonsthagen and R. C. Fleischer 2011, unpublished data). Dates are estimated in BEASTusing these rate priors.) age estimates in Myr (95% CI) rate prior (95% CI) gene % divergence Myr21 Bombycilla clade B. garrulus clade B. cedrorum clade B. garrulus versus B. japonicus Cyt b 2.26 (1.44–3.08) 2.96 (1.22–5.13) 1.50 (0.46–2.80) 1.67 (0.43–3.18) 2.29 (0.91–4.10) ND2 2.43 (1.59–3.45) 2.79 (1.20–4.52) 1.10 (0.06–2.22) 0.68 (0.07–1.62) 1.81 (1.20–4.52) topologies with generally high support for clades repre- waxwings may have originated in the New World, and senting traditional species (B. cedrorum, B. garrulus and this is supported by the observation that their closest Bombycilla japonica; figure 1). In all analyses, B. garrulus relatives are New World silky flycatchers [17]. They and B. japonica were sister taxa, and this clade was sister split into the Old World Bohemian waxwing after colo- to B. cedrorum. In addition, there was support in all nizing the Old World, which then split into the analyses for a split between Old World and New World Japanese waxwing and the New World Bohemian waxw- samples of B. garrulus. Combined gene analysis also ing after recolonizing North America. The alternative produced strong support via both inference methods scenario is that Old World Bohemian waxwings colo- for the topologies found in single gene analyses. nized North America and split into cedar waxwings; Dates for key nodes in the waxwing tree were highly Old World Bohemian waxwings also split from Japanese consistent between the two genes, and these indicate waxwings; and there was a second colonization of the that the radiation (basal split) of bombycillid species New World by Old World Bohemian waxwings that began roughly 2.79 (ND2)to2.96(Cyt b) Myr ago resulted in the New World Bohemian waxwings. (table 1). The split between New World and Old World Accordingly, egg rejection may have evolved in populations of B. garrulus was estimated at 1.1–1.5 Myr response to cowbird parasitism in the Nearctic or ago based on ND2 and Cyt b analyses, respectively. cuckoo parasitism in the Palaearctic. However, there are several factors that indicate that the first scenario of cowbird parasitism was the probable selection 4. DISCUSSION pressure. Based on current ecologies and ranges, Bohemian waxwings rejected 100 per cent of cases of there is greater overlap between cowbirds and cedar experimental parasitism despite being allopatric from waxwings than cuckoos and the two Old World wax- brown-headed cowbirds. The biogeographic history wing species [13,18,19]. There are also no records of of the Bombycillidae lineage is unclear [16], making parasitism on Old World waxwings by any cuckoo it difficult to ascertain the dynamics of egg rejection species [20], whereas the cedar waxwing is a regular evolution in waxwings; however, there appear to be host of the cowbird [21].