Naturwissenschaften (2008) 95:293300 DOI 10.

1007/s00114-007-0328-x

ORIGINAL PAPER

Parasites and health affect multiple sexual signals in male common wall lizards, Podarcis muralis
Jos Martn & Luisa Amo & Pilar Lpez

Received: 4 June 2007 / Revised: 30 October 2007 / Accepted: 4 November 2007 / Published online: 5 December 2007 # Springer-Verlag 2007

Abstract Multiple advertising sexual traits may either advertise different characteristics of male condition or be redundant to reinforce reliability of signals. Research has focused on multiple visual traits. However, in animals that use different multiple additional sensory systems, such as chemoreception, different types of traits might have evolved to signal similar characteristics of a male quality using different sensory channels. We examined whether ventral coloration and chemicals in femoral gland secretions of male common wall lizards, Podarcis muralis, are affected by their health state (blood-parasite load and cell-mediated immune response). Our results indicated that less parasitized lizards had brighter and more yellowish ventral colorations and also femoral secretions with higher proportions of two esters of octadecenoic acid. In addition, lizards with a greater immune response had more saturated coloration and secretions with higher proportions of octadecenoic acid methyl ester. We suggest that these signals would be reliable because only healthier males seemed able to allocate more carotenoids to coloration and presumably costly chemicals to secretions. The use of multiple sensory channels may provide more opportunities to signal a male quality under
J. Martn (*) : L. Amo : P. Lpez Departamento de Ecologa Evolutiva, Museo Nacional de Ciencias Naturales, C.S.I.C., Jos Gutirrez Abascal 2, 28006 Madrid, Spain e-mail: Jose.Martin@mncn.csic.es Present address: L. Amo Department of Animal Population Biology, Centre for Terrestrial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Boterhoeksestraat 48, P.O. Box 40, 6666ZG Heteren, The Netherlands

different circumstances, but also may reinforce the reliability of the signal when both types of traits may be perceived simultaneously. Keywords Sexual signals . Parasites . Immune response . Coloration . Femoral glands

Introduction The evolution of multiple sexual signals is one of the most controversial and least understood features of sexual selection. Some theoretical models, but not others, have suggested that multiple traits may convey honest information on the genetic and phenotypic quality of males (Johnstone 1995, but see Grafen 1990; Iwasa and Pomiankowski 1994). Empirical research has found that different traits such as tail length and carotenoid-dependent color badges in widowbirds (Euplectes ardens) may be honest signals that function in intra- and intersexual selection, respectively (Andersson et al. 2002). Female bowerbirds (Ptilonorhynchus violaceus) use both plumage UV coloration of males and bower characteristics to choose mates because they signal different aspects of health state of males (Doucet and Montgomerie 2003). Also, in male peacocks, tail feathers and behavioral displays may signal general and current health state, respectively, to females (Loyau et al. 2005). Male tree lizards Urosaurus ornatus have two status signaling color badges, but throat badges are used in short-term encounters for short-distance communication and dorsal badges in longterm encounters for long distance communication (Zucker 1994). These results suggest that multiple traits may either signal different characteristics of male condition or be redundant as a way to reinforce the reliability of signals (Sullivan 1994; Rowe 1999).

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However, a requisite for multiple signals to be evolutionarily stable as sexual signals is that they are all similarly honest and condition dependent. The parasite-mediated sexual selection hypothesis predicts that females should prefer males with the most elaborate sexual ornaments because the degree of ornament elaboration may be limited by a males ability to resist disease infection (Hamilton and Zuk 1982). The energetic costs of producing and maintaining the immune response to fight against parasites may create a trade-off with the elaboration of sexual ornaments (Wedekind 1992; Wedekind and Folstad 1994). Therefore, only individuals in good condition can mount strong defenses and produce extravagant sexual ornaments (Westneat and Birkhead 1998). Most of this research on multiple sexual traits has focused on signals that are perceived through visual sensory systems alone, despite use of other sensory systems, such as chemosensory ones by many animals (Wyatt 2003). The use of multiple signals based on multiple sensory systems may aid assessment of male quality via different circumstances. For example, colorful visual signals might be more important in long-distance communication, whereas chemical signals may be more important when two individuals are close together and also provide information when the signaler left the area (e.g., Lpez et al. 2002, 2003). Multiple types of signals may also be useful to increase the likelihood that all individuals correctly perceived the information, if some individuals might be more sensitive to visual than to chemical signals, and inversely Also, environmental conditions (light levels, humidity, etc.) may render some type of signals more effective than others in different microhabitats (Endler 1992). Therefore, we could expect that in animals that use different multiple sensory systems, different types of traits may have evolved to signal similar characteristics of a male quality using different sensory channels. Lizards may be considered as visually guided animals in many contexts. Accordingly, some species have evolved a variety of colorful sexually dimorphic ornaments that are used in intraspecific relationships (reviewed in Cooper and Greenberg, 1992). For example, the size and color characteristics of male visual badges may determine success in malemale competition (Thompson and Moore 1991; Olsson 1994) or mate acquisition (Baird et al. 1997; Kwiatkowski and Sullivan 2002; Anderholm et al. 2004). However, chemical signals also play prominent roles in social relationships of many lizards (reviewed in Mason 1992; Alberts 1993). For example, chemical secretions from the femoral glands of males may reliably convey information about social status or about a males quality (Lpez and Martn 2002; Martn and Lpez 2006b; Olsson et al. 2003). Both visual and chemical signals may be important in different contexts within the same lizard species (Lpez et al. 2002, 2003).

The common wall lizard (Podarcis muralis) is a small lacertid lizard (6076 mm adult snout-to-vent length, SVL) widespread in central Europe and mountain areas of the northern and central Spain (Gruschwitz and Bhme 1986). This is a sexually dimorphic lizard, with adult males having, in the study population, orange throat and ventral coloration, which is usually whitish in juveniles and females. This ventrolateral coloration seems to have a role in intraspecific relationships as what occurs in other lizards (Cooper and Burns 1987; Olsson 1994; Lpez et al. 2002). Male P. muralis have well-developed femoral pores that produce chemical secretions during the breeding season, whereas females have vestigial femoral pores lacking secretion (Martn and Lpez 2006a). Wall lizards can discriminate conspecific chemical cues, and scent is used as a territory marker and as an assessment criterion for territorial status (Edsman 1990). In addition, P. muralis lizards support high blood parasite loads (Amo et al. 2005), which are affected by testosterone levels (Oppliger et al. 2004). In other related lacertid lizard species, health and parasite loads affect proportion of chemicals in femoral secretions (Lpez et al. 2006; Martn and Lpez 2006b, c). Therefore, this lizard species is a good candidate to explore the relationships between parasites, health state, and the expression of multiple types of sexual signals. In this paper, we examined whether characteristics of the throat and ventral coloration and the lipophilic fraction (i.e., composition and relative proportions of each compound) of femoral gland secretions of male common wall lizards P. muralis may be affected by their health state (blood parasite load and cell-mediated immune response). We hypothesized that interindividual variation in health state may affect the expression of both visual and chemical sexual dimorphic traits of males, which may allow to use these traits as signals of male quality in intraspecific communication.

Material and methods Lizards health measurements We captured by noosing 28 adult male P. muralis during April 2004, which coincided with the mating season of lizards, in different places in a Scots pine forest over a large area near Cercedilla (Madrid province, Spain) at an elevation of 1,500 m. Lizards were individually housed at El Ventorrillo Field Station, 5 km from the capture site, in outdoor opaque plastic cages (8050 cm) containing rocks for cover. We measured lizard snout-to-vent length (SVL) with a ruler as an indicator of body size. To assess blood parasite load, we extracted blood from the postorbital sinus and made a smear on a microscope

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slide, which was later fixed in methanol and stained in Giemsa. We counted on mounted slides the number of intraerythrocytic hemogregarines (mean SE = 7.8 1.9 infected cells/2,000 erythrocytes; the only parasites found, see details in Amo et al. 2005). Prevalence of hemogregarines was of 78.6%. To assess T-cell-mediated immune (CMI) response in vivo, we used the phytohemagglutinin (PHA) injection test (Svensson et al. 2001). However, although PHA-induced swelling is related to heightened immune cell activity, it also may involve both innate and adaptive components of the immune system (Martin et al. 2006). Thus, this test does not appear to be an unambiguous index of T-cell-mediated immunity per se, but rather a multifaceted index of cutaneous immune activity. However, in this study, we used this test because we were interested on a standardized index of immunocompetence, independently of the type of immune cells involved on it. We measured with a pressuresensitive spessimeter the thickness (to the nearest 0.01 mm) of the right hindlimb foot pad at a marked point, before and after 24 h of injecting 0.02 mg of PHA dissolved in 0.01 ml of phosphate-buffered saline (PBS) at the marked point. We calculated the CMI response as the difference between preand postinjection thickness measures (Smits et al. 1999). Lizard did not show any sign of stress for these tests and were released to their capture sites. Color measurements

similar P values, and we therefore present only the results of the analysis based on the mean values. Analyses of chemicals in femoral gland secretions Immediately after a male was captured, we pressed with forceps around its femoral pores to collect secretion directly in glass vials, which was stored at 20C. We analyzed secretions with a ThermoQuest Trace 2000 gas chromatograph-mass spectrometer (GC-MS) fitted with a 30-m column (Supelco, Equity-5). A total of 50 chemical compounds were initially identified by comparison of their mass spectra in the NIST 1998 computerized mass spectral library, and later confirmed, when possible, using authentic standards (for details of methods and chemicals in secretions of this lizard see Martn and Lpez 2006a). The relative amount of each compound was determined as the percent of the total ion current (TIC). Then, we selected the peaks that represented >02% relative peak area (which altogether represent 97% of total TIC area) to reduce the number of variables to be used in multivariate analysis (Dietemann et al. 2003). The relative areas of the 38 selected peaks were transformed following the formula of Aitchison (1986) and used as variables in a principal components analysis (PCA) that extracted six principal components (PCs), which together accounted for 76% of the variance (Table 1; for similar analyses see Lpez et al. 2006). Data analyses

We measured color of the throat and ventral scales of male lizards using a spectrophotometer (Minolta CM2600d). Color was characterized according to the tristimulus color variables: hue, saturation, and brightness (HSB; McGraw and Ardia 2003; Montgomerie 2006). The hue define the color. Thus, for example, hue is negatively correlated to redness. Saturation (also called chroma) refers to the saturation or purity of the dominant frequency of the light. The farther away from zero, the more saturated the color is. Brightness (also called lightness) of a reflected light signal is a function of both the range of wavelengths reflected and the surface structure of the skin (Bradbury and Vehrencamp 1998). We made measures of the HSB tristimulus color variables at four different standardized spots in the throat and ventral scales. Intraclass correlation coefficients based on variance components derived from one-way ANOVAs (Lessells and Boag 1987) showed that these four measurements were significantly highly repeatable within each individual (hue, r=0.81, F26,81 =18.45, P<0.0001; saturation, r = 0.92, F26,81 = 11.34, P < 0.0001; brightness, r = 0.68, F26,81 =2.90, P=0.00014). Thus, we calculated the mean values of the four measures. Nevertheless, we repeated all statistical analyses (see below) using each of these four values. These analyses resulted in the same conclusions, with

To examine the relationship between health state of lizards (blood parasite load and CMI response; both log-transformed) and coloration or proportions of chemicals, we used backward stepwise general regression models (GRM) with means of hue, saturation, and brightness of coloration or the PCs for chemicals as potential predictor variables. Because parasite and health measures might be related to body size (SVL), we also included SVL as a covariate in the initial models. Analyses were performed with Statistica 6.0 software (StatSoft, Tulsa, OK).

Results Visual signals Blood parasite load was significantly and negatively correlated with brightness (=0.35, t=2.06, P=0.048) and hue (=0.45, t=2.66, P=0.013) values (Stepwise GRM model: R2 =0.48, F2,25 =11.72, P=0.0003). Thus, lizards with lower blood parasite loads had brighter colorations that were more yellowish (Fig. 1a,b). The CMI response was significantly and positively correlated

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Table 1 Principal components analysis for relative proportion of main chemicals in femoral gland secretions of male lizards PC-1 Dodecanoic acid Tetradecanoic acid Hexadecanoic acid Hexadecenoic acid Octadecanoic acid Octadecenoic acid Octadecadienoic acid Eicosanoic acid Erucic acid Octadecenoic acid, methyl ester Octadecenoic acid, hexadecyl ester Eicosatetraenoic acid, ethyl ester Tetradecanol Nonadecanone -Tocopherol Squalene Cholesterol Cholesta-5,7-dien-3-ol Cholesta-3,5-diene Cholesta-5,7,9(11)-trien-3-ol Cholesta-4,6-dien-3-ol 24-Propylidene-cholest-5-en-3-ol Ergosterol Campesterol Cholest-4-en-3-one Ergosta-5,8-dien-3-ol Cholesta-4,6-dien-3-one -Sitosterol Ergostanol 4,4-Dimethyl-cholesta-5,7-dien-3-ol Stigmasterol Unidentified steroid 1 Unidentified steroid 2 Unidentified steroid 3 Unidentified steroid 4 Unidentified steroid 5 Unidentified steroid 6 Unidentified steroid 7 Eigenvalue % Variance Correlations in bold are significant at P<0.001 0.75 0.32 0.05 0.13 0.39 0.21 0.26 0.21 0.37 0.05 0.40 0.82 0.53 0.80 0.69 0.39 0.66 0.05 0.15 0.06 0.49 0.06 0.27 0.03 0.26 0.46 0.32 0.04 0.45 0.05 0.10 0.03 0.51 0.25 0.56 0.13 0.72 0.42 11.80 31.0 PC-2 0.22 0.19 0.88 0.43 0.82 0.45 0.44 0.03 0.28 0.61 0.38 0.22 0.04 0.03 0.36 0.62 0.54 0.35 0.44 0.19 0.43 0.09 0.17 0.03 0.26 0.14 0.06 0.04 0.49 0.18 0.21 0.29 0.20 0.09 0.34 0.14 0.42 0.44 5.34 14.1 PC-3 0.04 0.05 0.02 0.52 0.05 0.12 0.30 0.10 0.01 0.09 0.04 0.03 0.02 0.28 0.03 0.35 0.03 0.72 0.50 0.90 0.07 0.27 0.02 0.34 0.09 0.30 0.08 0.19 0.20 0.01 0.16 0.86 0.53 0.01 0.15 0.17 0.24 0.34 4.75 12.5 PC-4 0.14 0.37 0.10 0.07 0.02 0.02 0.15 0.79 0.57 0.06 0.04 0.02 0.07 0.18 0.16 0.07 0.01 0.08 0.15 0.20 0.26 0.82 0.18 0.89 0.10 0.38 0.02 0.03 0.06 0.13 0.06 0.20 0.34 0.22 0.15 0.40 0.24 0.60 2.87 7.6 PC-5 0.26 0.24 0.06 0.24 0.07 0.02 0.18 0.33 0.40 0.58 0.65 0.13 0.08 0.19 0.05 0.02 0.11 0.03 0.24 0.03 0.29 0.14 0.24 0.01 0.18 0.14 0.01 0.21 0.06 0.18 0.11 0.08 0.01 0.28 0.19 0.17 0.07 0.05 2.32 6.1 PC-6 0.09 0.12 0.26 0.39 0.10 0.19 0.04 0.18 0.16 0.03 0.27 0.12 0.39 0.19 0.23 0.13 0.10 0.08 0.10 0.01 0.06 0.18 0.08 0.05 0.73 0.30 0.14 0.27 0.54 0.32 0.53 0.01 0.40 0.05 0.22 0.04 0.26 0.04 1.78 4.7

with color saturation values (R 2 = 0.27, F 1,26 = 9.86, P=0.004). Thus, lizards with a greater CMI response had more saturated colorations (Fig. 1c). Body size was not significantly related to parasite load or CMI and was not included in the final models. Chemical signals The lipophilic fraction of femoral secretion of P. muralis is a mixture of steroids (83.7%) and carboxylic acids ranging between C9 and C20 and their esters (12.9%) as predominant

components, but we also found -tocopherol (2.0%), squalene (1.0%), four alcohols (0.3%), and a ketone (0.1%). The five most abundant chemicals were cholesterol (55.8%), cholesta-5,7-dien-3-ol (7.1%), 4,4-dimethylcholesta-5,7-dien-3-ol (6.8%), hexadecanoic acid (4.9%), and ergosta-5,8-dien-3-ol (3.9%). Blood parasite load was significantly and positively correlated with PC-5 (Stepwise GRM: R2 =0.32, F1,26 = 12.29, P=0.002). Thus, lizards with lower blood parasite loads had secretions with relatively higher proportions of octadecenoic acid methyl ester and octadecenoic acid

Naturwissenschaften (2008) 95:293300 Fig. 1 Relationships between a, b blood parasite load or c the cell-mediated immune (CMI) response and the HSB tristimulus color variables (hue, brightness, saturation) characterizing the ventral coloration of male lizards

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hexadecyl ester (Table 1; Fig. 2a). The CMI response was significantly and negatively correlated with PC-2 (R2 =0.31, F1,26 =11.41, P=0.002). Thus, lizards with a better health state, as indicated by their greater CMI response, had relatively lower proportions of hexadecanoic and octadecanoic acids, and of squalene, and higher proportions of octadecenoic acid methyl ester in their femoral secretions (Table 1; Fig. 2b). Body size was not related to parasite load or CMI and was not included in the final models.

Discussion Our results indicate that interindividual variability in parasite load and immunocompetence of male P. muralis lizards may affect both ventral coloration and the relative proportions of lipophilic chemical compounds in femoral gland secretions. Therefore, it is likely that coloration and chemical secretions, two sexually dimorphic characters, may be used as intraspecific signals providing reliable information on health state of the signaler. Less parasitized lizards had brighter and more yellowish ventral colorations, and secretions with higher proportions of two esters of octadecenoic acid. In addition, lizards with a greater immune response had more saturated coloration and secretions with higher proportions of octadecenoic acid methyl ester. In lizards, hemogregarines destroy erythrocytes, which results in depressed hematocrit levels (Wintrobe

1991), reduced hemoglobin concentrations, and reduced capacity for oxygen transportation (Oppliger et al. 1996; Veiga et al. 1998). These detrimental effects, together with the activation of the immune system to fight against the infection, may affect different aspects of lizards physiology, which may directly affect the ability to allocate some pigments to coloration and certain chemicals to femoral secretions. With respect to coloration and health, in some birds, there is a similar relation between color intensity health and immunocompetence (Figuerola et al. 1999; Saks et al. 2003a). Malaria-infected male lizards (Sceloporus occidentalis) exhibit significantly more black and less pale coloration on their ventral surface than do noninfected males (Ressel and Schall 1989). Similarly, female lizards (Sceloporus virgatus) with lower body condition and higher ectoparasite load have less saturated throat coloration (Weiss 2006). In other populations of P. muralis, there are different color morphs, which also differ in average immune responses (Sacchi et al 2007). Male color variability may be important because, for example, color brightness seems to be preferred by females in several lizard species (e.g., Baird et al. 1997; Kwiatkowski and Sullivan 2002), which may use coloration as a signal of male condition. On the other hand, the parasite-related variation in chemicals in secretions makes it likely that parasitized lizards have different scent signatures. In fact, octadece-

298 Fig. 2 Relationships between a blood parasite load or b the cell-mediated immune (CMI) response and the PC axis extracted in a PCA for relative proportion of chemicals in femoral gland secretions of male lizards

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noic (=oleic) acid induced strong chemosensory responses in the related lizard Podarcis lilfordi (Cooper et al. 2002). Female mice and rats can distinguish between the odors of infected and noninfected males and are less attracted to the scent of infected males (e.g., Kavaliers and Colwell 1995; Penn and Potts 1998; Penn et al. 1998; Zala et al. 2004). Alterations in odor due to parasite infections may be a pathological by-product of infection, a consequence of parasite manipulation of the host or an adaptive response to cope with infection (Zala et al. 2004). A relationship between the immune response and the scent characteristics, which may affect female preferences, has been found in other two lacertid lizard species (Lpez and Martn 2005; Martn and Lpez 2006b) and in mealworm beetles (Tenebrio molitor) (Rantala et al. 2002, 2003). Signals used to indicate quality of a male should be costly to produce or to maintain (Zahavi 1975; Pomiankowski 1988; Grafen 1990). Our results suggest that both visual and chemical signals of P. muralis lizards are honest and costly to produce, as only males with a better health state can afford to allocate higher proportions of some pigments to ventral coloration and of octadecenoic esters to their femoral secretions to produce exaggerated and honest multiple ornaments. Carotenoid and pteridine pigments contained

in specialized integumental cells (chromatophores) produce red, orange, and yellow coloration in lizards (Ortiz et al. 1962; Cooper and Greenberg 1992; Macedonia et al. 2000). Carotenoids and other pigments often participate in several physiological mechanisms as regulators of the immune function, free radical scavenging, and detoxification (Mller et al. 2000; Blount 2004; McGraw 2005). As animals may suffer a trade-off between allocation of pigments to physiological functions and ornamentation (Faivre et al. 2003; McGraw and Ardia 2003), the expression of coloration may serve as an honest signal of individual quality (Mller et al. 2000; Blount et al. 2003; Blount 2004). Male birds that can afford to allocate more carotenoids to produce more elaborate visual displays are healthier individuals with superior immunocompetence (Blount et al. 2003). In birds, hue and saturation of feathers are positively correlated with the total carotenoid content of the feathers (Saks et al. 2003b). In chuckwalla lizards, Sauromalus obesus, male color likely functions as an indicator of food resources to females because male color is based partly on carotenoid pigments (Kwiatkowski and Sullivan 2002). It is likely that only those male lizard P. muralis with less parasites and a greater immune response were able to find or defend more food resources and allocate more yellow carotenoids to their

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299 Anderholm S, Olsson M, Wapstra E, Ryberg K (2004) Fit and fat from enlarged badges: a field experiment on male sand lizards. Proc R Soc Lond B 271(Suppl):142144 Andersson S, Pryke SR, Ornborg J, Lawes MJ, Andersson M (2002) Multiple receivers, multiple ornaments, and a tradeoff between agonistic and epigamic signaling in a widowbird. Am Nat 160:683691 Baird TA, Fox SF, McCoy JK (1997) Population differences in the roles of size and coloration in intra- and intersexual selection in the collared lizard, Crotaphytus collaris: influence of habitat and social organization. Behav Ecol 8:506517 Blount JD (2004) Carotenoids and life-history evolution in animals. Arch Biochem Biophys 430:1015 Blount JD, Metcalfe NB, Birkhead TR, Surai PF (2003) Carotenoid modulation of immune function and sexual attractiveness in zebra finches. Science 300:125127 Bradbury JW, Vehrencamp SL (1998) Principles of animal comunication. Sinauer, Sunderland, MA Cooper WE, Burns N (1987) Social significance of ventrolateral coloration in the fence lizard, Sceloporus undulatus. Anim Behav 35:526532 Cooper WE, Greenberg N (1992) Reptilian coloration and behavior. In: Gans C, Crews D (eds) Biology of the reptilia, vol 18. University of Chicago Press, Chicago, pp 298422 Cooper WE, Prez-Mellado V, Vitt LJ (2002) Lingual and biting responses to selected lipids by the lizard Podarcis lilfordi. Physiol Behav 75:237241 Dietemann V, Peeters C, Liebig J, Thivet V, Hlldobler B (2003) Cuticular hydrocarbons mediate discrimination of reproductives and nonreproductives in the ant Myrmecia gulosa. Proc Natl Acad Sci USA 100:1034110346 Doucet S, Montgomerie R (2003) Multiple sexual ornaments in satin bowerbirds: ultraviolet plumage and bowers signal different aspects of male quality. Behav Ecol 14:503509 Edsman L (1990) Territoriality and competition in wall lizards. PhD Thesis. Univ Stockholm, Sweden Endler JA (1992) Signals, signal conditions, and the direction of evolution. Am Nat 139:125153 Faivre B, Grgoire A, Prault M, Czilly F, Sorci G (2003) Immune activation rapidly mirrored in a carotenoid-based secondary sexual trait. Science 300:103 Figuerola J, Muoz E, Gutierrez R, Ferrer D (1999) Blood parasites, leucocytes and plumage brightness in the cirl bunting, Emberiza cirlus. Funct Ecol 13:594601 Funari SS, Barcelo F, Escriba PV (2003) Effects of oleic acid and its congeners, elaidic and stearic acids, on the structural properties of phosphatidylethanolamine membranes. J Lipid Res 44:567575 Grafen A (1990) Biological signals as handicaps. J Theor Biol 144:517546 Gruschwitz M, Bhme W (1986) Podarcis muralis, Mauereidechse. In: Bhme W (ed) Handbuch der Reptilien und Amphibien Europas. Aula Verlag, Wiesbaden, Germany, pp 20923 Hamilton WD, Zuk M (1982) Heritable true fitness and bright birds: a role for parasites. Science 218:384387 Iwasa Y, Pomiankowski A (1994) The evolution of mate preferences for multiple sexual ornaments. Evolution 48:853867 Johnstone RA (1995) Honest advertisement of multiple qualities using multiple signals. J Theor Biol 177:8794 Kavaliers M, Colwell DD (1995) Discrimination by female mice between the odours of parasitized and non- parasitized males. Proc R Soc Lond B 261:3135 Kwiatkowski MA, Sullivan BK (2002) Geographic variation in sexual selection among populations of an Iguanid lizard, Sauromalus obesus (=ater). Evolution 56:20393051 Lessells CM, Boag PT (1987) Unrepeatable repeatabilities: a common mistake. Auk 104:116121

ventral skin coloration, resulting in higher hue and saturation values. Similarly, chemicals used in secretions of lizards may be important in many physiological functions and can only be allocated to secretions if they are diverted from general metabolism. Thus, only high quality males that obtain an adequate supply of these chemicals are able to elaborate such chemical ornaments (Martn and Lpez 2006b, c). In P. muralis, a higher proportion of waxy esters (=esters of octadecenoic acid) in secretions would confer more stability to scent marks under conditions where high levels of humidity increase evaporation (Alberts 1992; Martn and Lpez 2006a). However, this allocation to secretions might be costly because octadecenoic acid (=oleic acid) is the main unsaturated fatty acid in lipid reserves, and it helps to maintain cell membranes in a fluid state necessary for biological functioning (Funari et al. 2003). Thus, it is likely that only healthier males could deviate enough octadecenoic acid from lipid reserves and metabolism to produce more efficient chemical signals. Previous studies suggested that multiple traits may signal different characteristics or be redundant (Zucker 1994; Andersson et al. 2002; Doucet and Montgomerie 2003; Loyau et al. 2005). Our results indicate that variations in different types of signals (coloration and chemicals in femoral secretions) of male lizard P. muralis may provide similar reliable information on blood parasite load and health state of a male. The signals are received by different sensory systems, but similar physiological mechanisms may ensure reliability. Nevertheless, within each sensory channel, different health characteristics are signaled by slightly different characteristic of the same traits. The use of multiple sensory channels may provide more opportunities to signal a male quality via different circumstances, but also may reinforce the reliability of the signal when both types of traits may be perceived simultaneously.
Acknowledgements We thank three anonymous reviewers for helpful comments, El Ventorrillo MNCN Field Station for use of their facilities, and J. Moreno for allowing us to use the spectrophotometer. Financial support was provided by the MEC project CGL200500391/ BOS, and by an El Ventorrillo CSIC and a MEC postdoctoral fellowship grants to L. Amo. The experiments comply with the current laws of the country (Spain) in which they were performed.

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