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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 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 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 –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]. 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 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 gene pool because was sister to the 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 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 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

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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 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]. Cowbirds parasitize cedar at least two equally parsimonious scenarios. Cedar waxwings despite the waxwing’s unsuitable frugivorous

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672 B. D. Peer et al. Persistence of host defence behaviour diet, because cowbirds are generalist parasites and use K. & Foster, S. A. 2009 Relaxed selection in the wild: many hosts [21]. By contrast, cuckoos are specialists contexts and consequences. Trends Ecol. Evol. 24, 487– [18] and it is unlikely that they were able to specialize 496. (doi:10.1016/j.tree.2009.03.010) on hosts with such an inappropriate diet. Dating esti- 2 Peer, B. D., Rothstein, S. I., Delaney, K. S. & Fleischer, R. mates indicate that cedar waxwings and New World C. 2007 Defence behaviour against brood parasitism is deeply rooted in mainland and island scrub-jays. Anim. Bohemian waxwings split 2.8–3.0 Myr ago, suggesting Behav. 73,55–63.(doi:10.1016/j.anbehav.2006.06.005) that egg rejection may have been retained in Bohemian 3 Rothstein, S. I. 2001 Relic behaviours, coevolution and waxwings through speciation for the same length of the retention versus loss of host defences after episodes time in the absence of parasitism. However, an extinct of avian brood parasitism. Anim. Behav. 61, 95–107. brood parasite species may have parasitized Bohemian (doi:10.1006/anbe.2000.1570) waxwings in the past. Cowbirds may also have been 4 Blumstein, D. T., Daniel, J. C. & Springett, B. P. 2004 more widespread at the end of the Pleistocene [22] A test of the multi-predator hypothesis: rapid loss of anti- and Bohemian waxwings may have been parasitized predator behavior after 130 years of isolation. Ethology during this time. If true, rejection still has been 110, 919–934. (doi:10.1111/j.1439-0310.2004.01033.x) retained at fixation in this species for at least 8000– 5 Lahti, D. C. 2006 Persistence of egg recognition in the 10 000 years. Regardless, these data clearly indicate absence of cuckoo brood parasitism: pattern and mech- anism. Evolution 60, 157–168. (doi:10.1111/j.0014- that host defences such as egg rejection can be retained 3820.2006.tb01090.x) for long periods of time in the absence of selection. 6 Nakamura, H., Kubota, S. & Suzuki, R. 1998 Coevolu- Cedar waxwings reject 69 per cent of cowbird eggs tion between the common cuckoo and its major hosts (n ¼ 58 nests) and are unique among cowbird hosts in in Japan: stable versus dynamic specialization on hosts. that they reject eggs at a higher frequency during the In Parasitic birds and their hosts: studies in coevolution (eds laying period of the nesting cycle (87.5%) compared S. I. Rothstein & S. K. Robinson), pp. 94–112. with the incubation stage (40%) [12]. Bohemian wax- Oxford, UK: Oxford University Press. wings rejected eggs regardless of when they were added 7 Davies, N. B. & de L. Brooke, M. 1989 An experimental to nests. Cedar waxwings damage their eggs when study of co-evolution between the cuckoo, Cuculus attempting to puncture-eject cowbird eggs because of canorus, and its hosts. II. Host egg markings, chick discrimination and general discussion. J. Anim. Ecol. smaller bills, but the Bohemian waxwing we observed 58, 225–236. (doi:10.2307/4996) grasp-ejected the artificial egg and removed it. 8 Marchetti, K. 1992 Costs to host defence and the persist- Only four selection pressures are known to favour ence of parasitic cuckoos. Proc. R. Soc. Lond. B 248, egg rejection [23] and the only one, other than inter- 41–45. (doi:10.1098/rspb.1992.0040) specific parasitism, that could account for rejection in 9 Lotem, A., Nakamura, H. & Zahavi, A. 1995 Constraints on such as Bohemian waxwings is conspecific egg discrimination and cuckoo–host co-evolution. Anim. brood parasitism. However, conspecific parasitism Behav. 49, 1185–1209. (doi:10.1006/anbe.1995.0152) rarely selects for rejection because of the similarity 10 Peer, B. D. & Rothstein, S. I. 2010 Phenotypic plasticity between the eggs of conspecifics, and this form of para- in common grackles (Quiscalus quiscula) in response to sitism has never been reported in Bohemian waxwings repeated brood parasitism. Auk 127, 293–299. (doi:10. or the better studied cedar waxwing [13,19]. 1525/auk.2009.09063) 11 Rothstein, S. I. 1975 An experimental and teleonomic Our findings and others [2,3,5,23,24] (but see [10]) investigation of avian brood parasitism. Condor 77, suggest that anti-parasite behaviours persist in many 250–271. (doi:10.2307/1366221) hosts in the absence of parasitism for long periods of 12 Rothstein, S. I. 1976 Experiments on defenses cedar wax- time and support the single trajectory model of host– wings use against cowbird parasitism. Auk 93, 675–691. avian brood parasite coevolution in which whole host 13 Witmer, M. C. 2002 Bohemian waxwing (Bombycilla communities become increasingly resistant to brood garrulus). In The birds of North America online (ed. parasitism. As more hosts evolve and retain defences, A. Poole). Ithaca, NY: Cornell Laboratory of Ornithology. parasites are forced to circumvent them through the Retrieved from the birds of North America online. (See evolution of egg mimicry; however, because host species http://bna.birds.cornell.edu.bnaproxy.birds.cornell.edu/ have eggs that differ, parasites will only be able to mimic bna/species/714). 14 Sauer, J. R., Hines, J. E. & Fallon, J. 2008 The North one or a small number of hosts. Anti-brood parasite American breeding bird survey, results and analysis 1966– behaviours are yet another of an increasing number 2007. Laurel, MD: USGS Patuxent Wildlife Research of examples of long-term retention of behaviours after Center. the relaxation of selection pressures [1] that have 15 Stamatakis, A., Hoover, P. & Rougemont, J. 2008 A rapid potential consequences on present day behaviour. bootstrap algorithm for the RAXML web-servers. Syst. Biol. 75, 758–771. (doi:10.1080/10635150802429642) This research was approved by the United States Fish and 16 Spellman, G. M., Cibois, A., Moyle, R. G., Winker, K. & Wildlife Service, State of Alaska Department of Fish and Barker, F. K. 2008 Clarifying the systematics of an Game, and the University of California IACUC Committee. enigmatic avian lineage: what is a bombycillid? Mol. Phy- logenet. Evol. 49, 1036–1040. (doi:10.1016/j.ympev. This research was supported by N.S.F. grant 0078139 2008.09.006) awarded to S.I.R., R.C.F. and B.D.P. 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19 Witmer, M. C., Mountjoy, D. J. & Elliot, L. 1997 22 Rothstein, S. I. & Peer, B. D. 2005 Conservation sol- Cedar waxwing (Bombycilla cedrorum). In The birds of utions for threatened and endangered cowbird North America online (ed. A. Poole). Ithaca, NY: (Molothrus spp.) hosts: separating fact from fiction. Cornell Laboratory of Ornithology. Retrieved from Ornithol. Monogr. 57, 98–114. the birds of North America online. (See http:// 23 Peer, B. D. & Sealy, S. G. 2004 Fate of grackle (Quiscalus bna.birds.cornell.edu.bnaproxy.birds.cornell.edu/bna/ spp.) defenses in the absence of brood parasitism: impli- species/309). cations for long-term parasite–host coevolution. Auk 20 Lowther, P. E. 2010 Host list of avian brood parasites. 121, 1172–1186. (doi:10.1642/0004-8038(2004) 2. Cuculiformes; Cuculidae. See http://fm1.fieldmu- 121[1172:FOGQSD]2.0.CO;2) seum.org/aa/Files/lowther/OWcList.pdf. 24 Peer, B. D., McIntosh, C. E., Kuehn, M. J., Rothstein, 21 Rothstein, S. I. 1976 Cowbird parasitism of the cedar S. I. & Fleischer, R. C. In press. Complex biogeographic waxwing and its evolutionary implications. Auk 93, history of Lanius shrikes and its implications for the evol- 498–509. ution of defenses against avian brood parasitism. Condor.

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