vs. body differed: (1) during the pre-calling period, i.e. the period of time before the start of the first owl bout; (2) when owls were calling, i.e. the period of owl call activity; and (3) during the post- calling period, i.e. the period of time after the end of the last owl bout, when owls were silent. The statistical analyses were performed with the SAS macro program, GLIMMIX (version 8.2 [28]), which iterates the MIXED procedure (PROC MIXED in the SAS software). The dependent variable was represented by all the brightness scores calculated as the differences between the mean brightness values of white badge vs. background and white badge vs. body. Mixed-effects models permit both the fixed (i.e. the above cited three calling periods) and random effects (i.e. male owls) to be fitted in a single analysis. Fixed effects model the mean values of the response variable as a function of covariates, while random effects model any patterns in the residuals around these fixed effects, e.g. those generated by repeated observations on the same individual. Because we had repeated measures of the same individuals and wanted to avoid pseudoreplication problems, we considered individuals as a random effect, while the within- individual effects were defined as a fixed factor. Differences among periods were calculated by the LSMEANS statement in SAS. For all analyses, means are given 6SE and statistical significance was set at P,0.05. References 1. Staicer CA, Spector DA, Horn AG (1996) The dawn chorus and other diel patterns in acoustic signaling. In: Kroodsma DE, Miller EH, eds. Ecology and evolution of acoustic communication in birds. Ithaca: Cornell University Press. pp 426–453. 2. Burt JM, Vehrencamp SL (2005) Dawn chorus as an interactive communication network. In: McGregor PK, ed. Animal communication networks. Cambridge: Cambridge University Press. pp 320–343. 3. Catchpole CK, Slater PJB (1995) Bird song. Biological themes and variations. Cambridge: Cambridge University Press. 248 pp. 4. Hutchinson JMC (2002) Two explanations of the dawn chorus compared: how monotonically changing light levels favour a short break from singing. Anim Behav 64: 527–539. 5. Endler JA, The´ry M (1996) Interacting effects of lek placement, display behavior, ambient light and color patterns in three neotropical forest-dwelling birds. Am Nat 148: 421–452. 6. McNaught MK, Owens IPF (2002) Interspecific variation in plumage color among birds: species recognition or light environment? J Evolution Biol 15: 505–514. 7. Gomez D, The´ry M (2004) Influence of ambient light on the evolution of colour signals: comparative analysis of a Neotropical rainforest bird community. Ecol Lett 7: 279–284. 8. Endler JA (1993) The color of light in forests and its implications. Ecol Monogr 63: 1–27. 9. Bradbury JW, Vehrencamp SL (1998) Principles of animal communication. Sunderland: Sinauer Associates. 780 pp. 10. Penteriani V (2002) Variation in the function of eagle owl vocal behaviour: territorial defence and intra-pair communication? Ethol Ecol Evol 14: 275–281. 11. Penteriani V (2003) Breeding density affects the honesty of bird vocal displays as possible indicators of male/territory quality. Ibis 145 (on-line): E127–E135. 12. Delgado MM, Penteriani V (2007) Vocal behaviour and neighbour spatial arrangement during vocal displays in eagle owls. J Zool 271: 3–10. 13. Penteriani V, Delgado MM, Alonso-A´ lvarez C, Sergio F (2007a) The importance of visual cues for nocturnal species: eagle owls signal by badge brightness. Behav Ecol 18: 143–147. 14. Penteriani V, Delgado MM, Alonso- A´lvarez C, Viqueira N, Sergio F, et al. (2007b) The importance of visual cues for nocturnal species: eagle owl fledglings signal with white mouth feathers. Ethology 113: 934–943. Eagle Owl Dusk Chorus Supporting Information Figure S1 The different contrasts among the eagle owls’ white throat badges, the surrounding background and owl body during the time brackets of the experiment. Found at: doi:10.1371/journal.pone.0004960.s001 (1.58 MB DOC) Video S1 Eagle owls showing the white badge during vocal displays. The different contrasts among the eagle owls’ white throat badges, the surrounding background and owl body during the time brackets of the experiment. Found at: doi:10.1371/journal.pone.0004960.s002 (120.33 MB MPG) Acknowledgments The first draft of the manuscript was deeply improved based on the criticisms and suggestions of G.J.L. Beckers, G. Bortolotti, H. Pietia¨inen, G.A. Sonerud, and D. Spector. Prof. L. Monje helped us with the Photoshop analyses of brightness contrast. Author Contributions Conceived and designed the experiments: VP. Performed the experiments: VP MdMD. Analyzed the data: VP MdMD. Wrote the paper: VP. Contributed to conceive and design the experiment: MMD. Contributed to write the paper: MMD. 15. Penteriani V, Delgado MM (2008) Owls may use faeces and prey feathers to signal current reproduction. PLoS ONE 3: e3014. doi:10.1371/journal.- pone.0003014. 16. Penteriani V, Alonso- A´lvarez C, Delgado MM, Sergio F (2006) Brightness variability in the white badge of the eagle owl. J Avian Biol 37: 110–116. 17. Villafuerte R (1994) Riesgo de predacio´n y estrategia defensivas del conejo, Oryctolagus cuniculus, en el Parque nacional de Do n˜ana. PhD Thesis. Cordoba: University of Cordoba. 18. Delibes M, Hiraldo F (1981) The rabbit as prey in the Iberian Mediterranean ecosystem. In: Myers K, MacInnes CD, eds. Proc. of the World Lagomorph Conference Guelph University Press. pp 614–622. 19. Dona´zar JA (1987) Geographic variations in the diet of the eagle owls in western Mediterranean Europe. In: Nero RW, Clark RJ, Knapton RJ, Hamre RH, eds. Biology and conservation of northern forest owl: symposium proceedings. Fort Collins: General Technical Report RM-142, USDA, Forest Service. pp 220–224. 20. Henwood K, Fabrick A (1979) A quantitative analysis of the dawn chorus: temporal selection for communicatory optimisation. Am Nat 114: 260–274. 21. Brown TJ, Handford P (2003) Why birds sing at dawn: the role of consistent song transmission. Ibis 145: 120–129. 22. Dabelsteen T, Mathevon N (2002) Why do songbirds sing intensively at dawn? A test of the acoustic transmission hypothesis. Acta Ethol 4: 65–72. 23. Ho¨glund J, Eriksson M, Lindell L (1992) Females of the lek breeding great snipe, Gallinago media, prefer males with white tails. Anim Behav 40: 23–32. 24. Jiguet F, Bretagnolle V (2001) Courtship behaviour in a lekking species: individual variations and settlement tactics in male little bustard. Behav Proc 55: 107–118. 25. Aragone´s J, De Reyna LA, Recuerda P (1999) Visual communication and sexual selection in a nocturnal bird species, Caprimulgus ruficollis, a balance between crypsis and conspicuousness. Wilson Bull 111: 340–345. 26. Penteriani V, Delgado MM, Maggio C, Aradis A, Sergio F (2005) Development of chicks and pre-dispersal behaviour of young in the eagle owl Bubo bubo. Ibis 147: 155–168. 27. McCullagh P, Nelder JA (1989) Generalised linear modelling. London: Chapman and Hall. 511 pp. 28. SAS Institute (2001) SAS/STAT software: changes and enhancements, version 8.2. North Carolina: SAS Publishing. 62 pp. PLoS ONE | www.plosone.org 4 April 2009 | Volume 4 | Issue 4 | e4960