DIPLOMARBEIT. Titel der Diplomarbeit. Does Facial Attractiveness Convey an Individual s Immunological Constitution? angestrebter akademischer Grad

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1 DIPLOMARBEIT Titel der Diplomarbeit Does Facial Attractiveness Convey an Individual s Immunological Constitution? angestrebter akademischer Grad Magistra der Naturwissenschaften (Mag.rer.nat.) Verfasserin / Verfasser: Matrikel-Nummer: Studienrichtung (lt. Studienblatt): Betreuerin / Betreuer: Katharina Zimmer Anthropologie (A442) Prof. Dr. Karl Grammer Wien, am

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3 Does Facial Attractiveness Convey an Individual s Immunological Constitution? Masterthesis for achieving the magister s degree at the faculty of Life Sciences of the University of Vienna accomplished at the Ludwig-Boltzmann-Institute for Urban Ethology submitted by Katharina Zimmer May 2007

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5 5 1. Abstract Introduction Health and Parasite Resistance Mate Preferences Concerning Facial Attractiveness...8 Facial Symmetry...9 Skin Homogeneity and Coloration The Major Histocompatibility Complex...13 MHC Heterozygosity Hypotheses Methods Sample Composition MHC Typing Facial Stimuli Image Scoring Facial Asymmetry, Skin Texture and Skin Coloration Statistical Analysis Results Study MHC Heterozygosity and Facial Attributes...29 Facial Attractiveness and Health Study Attractiveness and Health in Masked Facial Images Discussion Attractive Health Information in Facial Traits Attractiveness of MHC Heterozygosity Health Care and Variation in Facial Traits Reasons for the Variation in Facial Traits Sustenance of MHC Polymorphism...40 Heterozygote Advantage Hypothesis...40 Rare Allele Advantage Hypothesis...40 Allele Specific Overdominance Hypothesis...41 MHC-dependent Mate Preferences...42 Supertypes Mediation of Individual MHC Genotypes...44 Body Scent...44 Bilateral Symmetry...45 Skin Condition Conclusion References Appendix Curriculum Vitae Acknowledgements...61

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7 7 1. Abstract Sexual attraction is supposed to be an adaptation responding to cues of a potential mates health constitution. Facial features like symmetry, skin texture and skin coloration are reported to affect the perception of attractiveness and health and thus, are probably influenced by the immune system. Since heterozygosity of genes of the major histocompatibility complex (MHC) which encode antigen presenting proteins, is reported to be of advantage when facing diseases, it may account for individual variation of these facial traits. With a sample of 84 men and 105 women we tested whether heterozygosity (at 3 as well as at 5 loci of the MHC) positively influences the development of facial symmetry, skin homogeneity and skin coloration as well as ratings of attractiveness and health. We applied computational methods for measuring asymmetry, skin texture and color and examined the effect of facial asymmetry, homogeneity and coloration of skin on the perception of attractiveness and health. We could not detect any influence of MHC heterozygosity on the mentioned facial traits or on the ratings of attractiveness and health. These results indicate no direct association between MHC heterozygosity and the development of these facial characteristics. However, we found that facial asymmetry was negatively correlated with perceived health in both sexes and with perceived attractiveness in women. Redness of skin was positively, whereas blueness was negatively correlated with perceived health in men. There were no effects of skin homogeneity on ratings of attractiveness or health. Thus, we still suggest that some information about an individual s health constitution is conveyed by the human face, though the mechanisms are yet unknown.

8 8 2. Introduction 2.1. Health and Parasite Resistance Parasite resistance plays a crucial role in evolutionary processes, where longlived individuals acting as hosts find themselves in an arms race against rapidly coevolving parasites and pathogens (van Valen, 1973; Hamilton, 1980). Parasites can evolve a number of generations in the life span of a single host organism adapting thereby themselves to the host s internal milieu and spread to others, especially if the individuals to which they spread provide much the same milieu as the original host. Hence, host organisms in populations where individuals are very much alike are at disadvantage, because parasites can evolve to the point of defeating their immune defense mechanisms. Sexual reproduction creates genetically different offspring and thereby provides different host environments in every new generation to which parasites have to adapt newly. Moreover, in the face of this hostparasite coevolutionary arms race healthy, parasite resistant individuals should be preferred in mate choice decisions, since they could pass advantageous genes on offspring (Fisher, 1915; Hamilton and Zuk, 1982) Mate Preferences Concerning Facial Attractiveness Mate preferences are considered to be shaped by selection processes whereby sexual attraction is regarded as an adaptation responding to cues of health in potential mates due to parasite pressure. Since individuals vary in their susceptibility to diseases attributable to differences in immunological constitution, traits that reliably indicate individual health are expected to be attractive (Zahavi, 1975; Symons, 1979; Thornhill and Gangestad, 1993, 1999; Grammer and Thornhill, 1994). The human face probably plays a major role in mate choice decisions (see Thornhill and Gangestad, 1999) and cross-cultural studies found a consensus

9 9 between individuals about what makes a face attractive (Perrett et al., 1994; Cunningham et al., 1995). Hence, one might expect that information about the immunological constitution is communicated through the human face. Facial Symmetry Facial symmetry is regarded to advertise parasite resistance to potential mates (e.g. Grammer and Thornhill, 1994; Thornhill and Gangestad, 1999), since the stable development of bilaterally symmetrical traits is supposed to reliably reflect the ability of an organism to cope with genetic (e.g. mutations, chromosomal anomalies, inbreeding, homozygosity) and environmental (e.g. food quality and quantity, toxins, parasites and pathogens) perturbations that occur during ontogeny (Livshits and Kobyliansky, 1991; Møller and Thornhill, 1998; Møller, 1999). These perturbations may be responsible for small random deviations from perfect bilateral symmetry if an individual is not able to undergo stable development in given environmental conditions. In other words, symmetrical faces should display immunological fitness and thus, may also be favored in mate choice decisions (Thornhill and Gangestad, 1993). Since 1994, when Grammer and Thornhill for the first time were able to show that facial symmetry was positively associated with judgements of health and attractiveness rated by both sexes in opposite-sex individual faces, several studies could replicate that symmetrical faces are regarded as more healthy than asymmetric ones (Rhodes et al., 2001; Jones et al., 2001; Fink et al., 2006a). Moreover, the assumption that facial symmetry should reliably indicate immune resistance is supported by two studies that found more symmetrical individuals actually healthier than less symmetrical ones (Shackelford and Larsen, 1997; Waynforth, 1998). In line with these results, numerous studies were able to demonstrate a preference for symmetric facial traits being positively related to judgements

10 10 of attractiveness (Grammer and Thornhill, 1994; Scheib, 1999; Perrett et al., 1999; Mealey et al., 1999; Little et al., 2001; Jones et al., 2001; Hume and Montgomery, 2001; Penton-Voak et al., 2001; Fink et al., 2006a). Additionally, Thornhill and Gangestad (1994) found that facial symmetry was positively associated with life time number of sex partners in both sexes, which indicates an actual advantage in intra- and intersexual competition. If the assessment of asymmetry is an adaptation to ease the discrimination between potential mates on the basis of apparent health (Thornhill and Gangestad, 1993), one might predict an opposite-sex bias in sensitivity to facial asymmetry. Consistent with that position, Jones et al. (2001) and Penton-Voak et al. (2001) reported that judgements of health and attractiveness regarding faces differing in their level of symmetry were more distinct in opposite-sex rating conditions than in own-sex rating conditions. There are a few studies that were not able to replicate the relationship between facial symmetry and judgements of health and attractiveness. Swaddle and Cuthill (1995) investigated facial symmetry by creating composites following the method of Grammer and Thornhill (1994). Composites are made by blending gray values between two or more pictures where the gray value of the blended pixel corresponds to the arithmetic mean of the gray values of the original pair of pixels. Perfect symmetric faces were made by blending the original face with its mirror reflection (the right side appears on the left side and vice versa) averaging thereby facial features on both sides. They hypothesized that unmanipulated faces should be rated as less attractive compared to the perfect symmetric counterparts, but found that more symmetrical faces were perceived as less attractive. The difference between this study and that of Grammer and Thornhill (1994) lies in the masking of the facial stimuli applied by Swaddle and Cuthill (1995). Obviously, they wanted to eliminate the potential influence of hair style and

11 11 adornments by placing an ellipse onto the face, but thereby cut off facial contours. This may have eliminated the reference for an optimal assessment of facial asymmetry or otherwise made faces looking somewhat unnatural. Further, Perrett et al. (1999) showed that blending mirror images of faces can increase skin blemishes. After controlling for this, they could repeat the finding that faces manipulated to a perfectly symmetrical version were rated as more attractive than the original, untreated ones. Noor and Evans (2003) also investigated the perception of attractiveness in more or less symmetric faces and found no effect of symmetry on ratings of attractiveness. They also showed original faces, their symmetrical versions made of blended mirror reflections and additionally asymmetrical versions created by reducing or enlarging pupils, nostrils and mouth corners in opposite directions on each side of the face. Again, as the sample size was large enough the reasons for their result might be increased skin irritations in the composites and unnatural looking features emerged from averaging an original face with its mirror reflection. Due to the methodological discrepancies of the studies that were not able to show an influence of facial symmetry on the perception of attractiveness and the majority of published studies that could, it seems reasonable to still assume an effect of symmetry on mate preferences and mate choice. Skin Homogeneity and Coloration Another facial feature that is reported to affect the perception of attractiveness and might provide information about the quality of the immune system is skin texture. Inhomogeneous skin due to lesions, eruptions, wrinkles and other disturbances is supposed to be mainly caused by hormonal imbalances and vascular dysfunctions. Several studies reported that skin irritations are related to disturbed sex hormone ratios in men and women

12 12 (Schiavone et al., 1983; Hall and Phillips, 2005; Placzek et al., 2005). Essah et al. (2006) found that hyperandrogenism in women often causes acne and hirsutism. Kanda and Watanabe (2005) showed positive effects of estrogen on female skin: Estrogen stimulates the proliferation of keratinocytes and enhances collagen synthesis which both inhibit the development of wrinkles. Moreover, estrogen suppresses apoptosis and thus prevents epidermal atrophy. Baker (1998) found dry skin and wrinkles in men with low levels of testosterone. Since sex hormones are immunosuppressive by inhibiting certain factors in the immune response (see Kanda and Watanabe, 2005), clear skin should indicate a fit immune system that can handle high levels of sex hormones (Grossmann, 1985; see Grammer et al., 2002, p.95). In line with this assumption Fink et al. (2001) showed that homogeneity of skin texture positively affected ratings of health and attractiveness in male and female faces. Also skin coloration might give some information about an individual s health status and thus, influence the perception of attractiveness. Fink et al. (2001) found that blue and green values of skin color measured in a red, green and blue (RGB) color space were negatively, whereas red values and color saturation were positively correlated with judgments of attractiveness in female faces. They concluded that slightly red-colored cheeks may indicate a normal blood circulation in peripheral vessels, and thus, a reduction of the red value might reveal a physiological imbalance. Several other studies support the suggestion that skin texture as well as coloration might act as cues for health and affect thereby attraction to potential mates. However, these studies did not differentiate between skin texture and coloration as Fink et al. (2001) did. Jones et al. (2004a and 2004b) found that health ratings of facial skin were positively correlated with ratings of facial attractiveness in male faces. Additionally, Fink et al. (2006b)

13 13 demonstrated with shape-standardized stimulus faces that homogenous skin color distribution was perceived as more attractive and healthy The Major Histocompatibility Complex The major histocompatibility complex (MHC) in the human genome is known to play a major role concerning health and parasite resistance. It encodes glycoproteins known as human leukocyte antigens (HLAs) which are located on the surface of all nucleated somatic cells binding antigens derived from pathogens or parasites and presenting them to T cells that trigger the appropriate immune response. Thus, the MHC is essential for the initiation of immune responses and also for immunological self/non-self recognition (Klein, 1986; see Penn and Ilmonen, 2005a; Sommer, 2005). The differentiation between self peptides and foreign peptides (antigens) is actually mediated by T cell recognition. During fetal growth undifferentiated T lymphocytes develop in the bone marrow and migrate to the thymus. There, an enormous number of monospecific T lymphocytes develops through random recombination of T cell receptor genes. Afterwards, these T lymphocytes are sorted out in a twostep process called thymic selection. In the thymic cortex T lymphocytes are presented with self-antigens bound to HLA. Only those T lymphocytes that bind to the HLAs survive, all others die by apoptosis. This mechanism ensures that T cells will be able to recognize the HLAs. T lymphocytes that survive this positive selection migrate to the thymic medulla and are again presented with HLA/self-antigen complexes. There, T lymphocytes that bind the antigens get eliminated. This process is called negative selection and guarantees that T cells will not bind to self-antigens. About 98% of monospecific T lymphocytes die during the development processes in the thymus by failing either positive or negative selection, while the other 2% leave the thymus as mature T cells. The immune system learns to differentiate between self and non-self

14 14 antigens about the time of birth. Peptides with which the child has contact during fetal growth, are usually lifelong regarded as its own, whereas all other peptides that were encountered by T cells later in life are perceived as foreign (see Penn and Potts, 1999). The loci of the MHC are located on chromosome number 6 and are divided into three regions: MHC class I, II and III. At the MHC class I region, there are three classical MHC loci (MHC-A, -B, -C) and three non-classical loci (MHC-E, -F, -G) that encode molecules which are involved in the inhibition of the activity of natural killer-cells. At the MHC class II region, there are another three classical MHC loci (MHC-DP, -DQ, -DR) and several nonclassical loci (LMP2, LMP7, TAP1, TAP2, Tapasin) that encode molecules which catalyze or inhibit the ligation of peptides to MHC class I molecules. MHC class III loci encode molecules of the complement system (C4, Bf, C2). The MHC is termed complex, because its loci are very closely seated next to each other. Therefore, the probability that individual combinations of alleles get separated by recombination is comparably low. Thus, individual combinations are inherited as so called haplotypes (Penn and Ilmonen, 2005a). In this study the classical MHC genes are of interest, because they exhibit more polymorphism than the non-classical MHC loci or any other gene section in the human genome. So far researchers could detect up to 800 different alleles for a single classical human MHC locus (see online data base on This leads to the suggestion that the classical MHC loci are exposed to selection processes (Sommer, 2005). There is much evidence that the MHC is shaped by evolutionary processes due to its particularly high polymorphism. First of all, neutral alleles that are not exposed to selection tend to fixation in the genome. Second, the MHC shows more regional patterns than other gene sections. MHC alleles as well as entire

15 15 haplotypes vary between populations in that some alleles or haplotypes are common whereas others are rare. Hence, in local populations the number of different alleles is considerably lower than the number of known alleles for each locus, but nevertheless higher than the number of different alleles of other gene sections in the human genome (Penn and Ilmonen, 2005a). Finally, there are less homozygous individuals than expected by random mate selection (Penn and Ilmonen, 2005b). Due to these matters of fact, the MHC seems to be qualified as appropriate defense mechanism in the host-parasite coevolutionary arms race. MHC Heterozygosity High allelic variability (heterozygosity) on the loci of this gene section increases the probability of carrying beneficial alleles that may be passed on offspring. For instance, Carrington et al. (1999) showed that individuals heterozygous on MHC class I loci progressed slower to AIDS after an HIV infection compared to others who were homozygous for one or more loci. Additionally, hepatitis B viruses were more efficiently eliminated in individuals that were heterozygous on MHC class II loci compared to homozygotes of this region (Thursz et al., 1997). Moreover, Penn et al. (2002) found that heterozygous house mice were more resistant than homozygotes facing an infection with multiple strains of pathogens. However, individual heterozygosity is an attribute that cannot be inherited and hence, is out of question as a potential mate choice criterion producing heterozygous offspring. Nevertheless, there might exist other merits from choosing heterozygous mates due to their advantage against certain diseases. Direct benefits for offspring could be a potential function of the preference for MHC heterozygous mates. If heterozygosity promotes survival, it might enhance investment capabilities. Thus, potential merits from choosing

16 16 heterozygous mates are a prolonged period of high-quality parental care and a reduced risk of contracting diseases for offspring (Kirkpatrick and Ryan, 1991). Moreover, as already mentioned before, MHC heterozygosity increases the probability of carrying beneficial alleles that could be passed on offspring, thereby enhancing their viability. Since facial attractiveness is supposed to convey immunocompetence through symmetry, skin texture and skin coloration, MHC genes might also be involved in facial preferences by contributing to individual variation in these facial traits. Only recently two studies investigated the potential link between MHC-heterozygosity and facial attractiveness. Thornhill et al. (2003) could not detect any associations between facial symmetry, attractiveness and MHC heterozygosity. However, they found men s heterozygosity to be positively correlated with women s preferences for their body odor. That s quite interesting, since body odor is reported to be influenced by the MHC (review in Wedekind and Penn, 2000). Further, Thornhill et al. (2003) could repeat the finding of Rikowski and Grammer (1999) who showed a positive correlation between facial attractiveness and sexiness of body odor in both sexes. Roberts et al. (2005a) raised this issue again and demonstrated that women preferred the faces of men who were heterozygous at all analyzed loci compared to faces of men who were homozygous at one or more loci of the MHC. Still, no significant effect of heterozygosity could be found on facial symmetry. Roberts et al. (2005a) also conducted health-ratings of facial skin patches and showed that skin of heterozygotes was perceived as more healthy. Hence, the MHC actually seems to be involved in mate choice decisions.

17 Hypotheses With this work we reassume the topic of previous studies from Thornhill et al. (2003) and Roberts et al. (2005a) by relating perceived facial attractiveness and healthiness as well as facial symmetry and skin condition to MHC heterozygosity to clarify the inconsistent findings of these two investigations. We predict that MHC heterozygosity positively affects the development of facial symmetry, homogeneity of skin texture and coloration of skin by using computational methods for measuring symmetry and skin texture as well as color following Fink et al. (2001). Individuals that are heterozygous at all analyzed loci of the MHC should have lower values of facial asymmetry and higher values of skin homogeneity compared to individuals that are homozygous at one or more loci of the MHC. Additionally, heterozygotes should have lower values of green and blue, but higher values of red in skin color than homozygotes. If MHC heterozygosity positively affects these facial traits, we further expect that heterozygosity on genes of the MHC is positively associated with judgements of facial attractiveness and health. Moreover, we test the influence of facial symmetry, homogeneity of skin and skin coloration on the perception of attractiveness and health. Faces with low values of asymmetry and high values of skin homogeneity should be regarded as more attractive and healthy. High values of blue and green in facial skin color should have negative effects on ratings, while slightly reddish skin should be considered more attractive and healthy.

18 18 Does Facial Attractiveness Convey an Individual s Immunological Constitution? 3. Methods 3.1. Sample Composition A total number of 84 men (mean age ± SD=44 ± 17, range=18-78) and 105 women (44 ± 18, 18-91) took part in this study actually investigating MHC and its influence on body odors. For the purpose of keeping the number of different MHC haplotypes small, members of big families (at least 10 family members) were asked to participate in the study. The sample consisted of thirteen families with on average 16.5 participating members (max. 30 family members). All 189 participants originated from a close region around Greifenburg, Carinthia, Austria a village with about two thousand inhabitants (Fig. 1). They were all informed about the aims of this study, gave their consent to participate and received 100 for compensation. Ethical approval for this study was obtained from Ethics Committees in Austria and the USA. Figure 1. Greifenburg in Carinthia, Austria All participants originated from a close proximity of this village.

19 MHC Typing 5 ml of blood were collected from all participants using vacuettes lined with EDTA (Ethylene-diamine-tetraacetic acid) to prevent clotting. Low resolution typing of MHC-A, -B, -Cw, -DRB1 and DQB1 resulting in 2 digits per locus that identify a group of alleles encoding a single HLA ( was performed by reverse sequence specific oligonucleotide (SSO) typing. In case of ambiguities samples were additionally typed by a polymerase chain reaction (PCR) based technique that uses sequence specific primers (SSP typing). High resolution typing resulting in 4 digits per locus which identify a single allele ( was performed by nucleotide sequencing of exons 2 and 3 of MHC class I and exon 2 of MHC class II loci. To assure the privacy of participants personal data collaborators who did the typing at the Clinical Department for Blood Group Serology, Medical University of Vienna were unaware of the names of blood donors. Previous studies concerned with MHC heterozygosity in a behavioral context did not carry out high resolution typing, but low resolution typing, which potentially might have heightened the MHC variation in this study. Therefore, we checked for differences between high and low resolution typing in the sample. Values for individual heterozygosity defined as the sum of heterozygous MHC loci of an individual stayed exactly the same using high or low resolution. We proceeded with high resolution data for further calculations. Since previous studies (Thornhill et al., 2003; Roberts et al., 2005a) employed only MHC-A, -B and -DRB1 in their studies, we conducted all analyses concerned with heterozygosity on the basis of the five analyzed loci as well as on the basis of the three previous used loci to allow for comparability.

20 20 19 different alleles were detected at MHC locus A, 27 at locus B, 16 at locus Cw, 26 at locus DRB1 and 13 at locus DQB1. Analysis of five loci found 59 men to be heterozygous at all loci compared to 25 men who were homozygous at one or more loci. Within women, 85 were heterozygous at all five loci and 20 were homozygous at one or more loci. Analysis of three loci identified 66 men to be heterozygous at all loci whereas 18 men were homozygous at one or more loci. Within women, 90 were heterozygous at all three loci and 15 were homozygous at one or more loci (for detailed information see table 1). Table 1. Participant s MHC heterozygosity at 5 loci and at 3 loci. heterozygosity at 5 loci frequency heterozygosity at 3 loci frequency men heterozygous at 5 loci 59 heterozygous at 3 loci 66 heterozygous at 4 loci 10 heterozygous at 2 loci 15 heterozygous at 3 loci 12 heterozygous at 1 locus 3 heterozygous at 2 loci 3 women heterozygous at 5 loci 85 heterozygous at 3 loci 90 heterozygous at 4 loci 11 heterozygous at 2 loci 13 heterozygous at 3 loci 7 heterozygous at 1 locus 2 heterozygous at 2 loci 1 heterozygous at 1 locus Facial Stimuli Participants faces were color photographed with a high-resolution digital camera at a distance of five meters under constant light conditions with a light source on each side of the face in order to prevent shading. The camera was positioned in eye height of the participants and photographs were taken without flash light. Participants were asked to remove glasses and strands of hair from the face and to look straight into the camera with a neutral expression. For later assignment with other data, participants had to held a

21 21 small blackboard with their subject-code beneath their faces. Pictures were edited with Adobe Photoshop CS Version for cropping the background and rotating slightly tilt heads. Finally, all facial images had the same orientation at a size of 350x525 pixels with a resolution of 72 dpi Image Scoring Participants facial images were shown to 65 male students (mean age ± SD=23 ± 3, range=17-33) and 66 female students (22 ± 3, 18-33) of the Biocenter, University of Vienna by an interactive computer program on 4 color-standardized screens at a resolution of 1024x768 pixels. Every student was presented with 20 facial pictures randomly chosen from all 189 participants images. Each face was rated for attractiveness and health by moving a controller on a continuous rating scale ranged from 0 to 100 (Fig. 2). Figure 2 shows the rating interface, on which students rated the participants.

22 22 We aggregated female ratings of male stimuli and male ratings of female stimuli, thus controlling for differences between the sexes in the perception of attractiveness regarding potential mates. Interrater reliability was sufficiently high (female ratings: attractiveness: Cronbach s α=0.62, health: α=0.54; male ratings: attractiveness: α=0.79, health: α=0.68). Menstrual cycle phase of female raters was recorded, because women s facial preferences are reported to differ with cycle stage (Penton-Voak et al., 1999; 2001). Only 8 women were in their fertile phase, which is reported to be the critical period when changes in facial preferences happen. In the face of this small proportion of women in their fertile phase, no changes in the means of aggregated ratings were assumed. Since age, hair style or condition, adornments and clothing are considered to influence the perception of attractiveness we conducted a second rating with masked images of a subsample of participants aged under 40. Masking was done with Adobe Photoshop CS Version by setting an ellipse with blurred edges around the faces hiding the background and by removing piercings with the stamp function (Fig. 3). Figure 3. Differences in picture styles between rating study 1 and 2. On the left, there is an example of an original, untreated portrait used in rating study 1. On the right sight, one can see an example of a masked portrait used in rating study 2.

23 23 The resulting 37 men (mean age ± SD=29 ± 7, range=18-39) and 45 women (27 ± 7, 18-39) were all rated by 30 male students (23 ± 2, 19-30) and 30 female students (21 ± 3, 18-30). In this subsample, analysis with five loci found 23 men to be heterozygous at all loci compared to 14 men who were homozygous at one or more loci. Within women, 36 were heterozygous at all five loci and 9 were homozygous at one or more loci. Analysis with three loci detected 27 men to be heterozygous at all loci, whereas 10 men were homozygous at one or more loci. Within women, 40 were heterozygous at all three loci and 5 were homozygous at one or more loci (for detailed information see table 2). Table 2. Participant s MHC heterozygosity at 5 loci and at 3 loci in the rating study 2. heterozygosity at 5 loci frequency heterozygosity at 3 loci frequency men heterozygous at 5 loci 23 heterozygous at 3 loci 27 heterozygous at 4 loci 4 heterozygous at 2 loci 9 heterozygous at 3 loci 9 heterozygous at 1 locus 1 heterozygous at 2 loci 1 women heterozygous at 5 loci 36 heterozygous at 3 loci 40 heterozygous at 4 loci 7 heterozygous at 2 loci 4 heterozygous at 3 loci 1 heterozygous at 1 locus 1 heterozygous at 1 locus 1 In this second study, interrater reliability was higher (female ratings: attractiveness: Cronbach s α=0.78, health: α=0.73; male ratings: attractiveness: α=0.79, health: α=0.80). All ratings were z-transformed to eliminate individual differences in the use of the rating scale.

24 24 Does Facial Attractiveness Convey an Individual s Immunological Constitution? 3.5. Facial Asymmetry, Skin Texture and Skin Coloration All analyses of facial asymmetry, skin texture and color were done with Facial Explorer 1.0 (Grammer et al., 1998). This program measures the coordinates of selected pixels and computes measures for asymmetry, texture and coloration. (Details of these analyses can be found in Fink et al., 2001 and Grammer et al., 2002, pp ). For asymmetry analysis first all images had to be standardized in size and orientation using a simplified version of the procrustes analysis developed by Wolberg (1990) and Bookstein (1997). All digital images were coded by manually marking 72 predefined feature points ( landmarks, source coordinates) in each face (Fig. 4). Figure 4 shows the 72 landmarks that were used for image standardization in size, location and orientation. The landmark definitions can be found in the appendix.

25 25 Afterwards, the program calculates the mean coordinates (destination coordinates) for all faces. Next, the center of gravity (CG) of the source coordinates for each face and the CG of the destination coordinates for all faces are determined. i.e. x-coordinate: x CG =! x1 + x x Nlandmarks n Each face is then moved on the picture plane until its CG matches the CG of the destination coordinates. Finally, each face is expanded to 150% of its original size and then down-scaled in one-pixel steps until the square sum of difference between the source and destination coordinates reaches a minimum (least squares method). After scaling, each face is rotated about its CG for 45 and then stepwise rotated back the same way as above (least squares method). As a result, all faces have the same size, orientation and location in the image. For measuring asymmetry of facial traits the Facial Explorer 1.0 uses a digital image analysis algorithm. First, an image section for the analysis has to be defined. We chose a rectangle described by the outer corner of the eyes, the highest point of the eyelids and the lowest point of the lower lip. Eyebrows were left out, because one can easily manipulate them by twitching, so that they are not reliably identifiable (Fig. 5).

26 26 Figure 5. Defined area for symmetry analysis. For symmetry analysis a rectangle described by the outer corner of the eyes, the highest point of the eyelids and the lowest point of the lower lip was chosen. Eyebrows were left out, because one can easily manipulate them. This window is divided into one-pixel-wide, horizontal slices. Every slice is disposed to the left and moved back in one-pixel steps. The symmetry point for every slice is located where the difference between pixels of the two halves of each slice reaches the minimum. In a perfectly symmetrical face the vertical line through the symmetry points of each slice is straight and equals the distance between the top and the bottom of the defined window. Individual values of asymmetry are obtained by dividing the line of symmetry points by the height of the window, which results in values greater than 1 for asymmetry, since 1 represents perfect symmetry. For analyzing the homogeneity of skin texture the program applies cooccurrence matrices. Again, a measurement section has to be defined in every facial picture. For this analysis, we used a square patch on the right cheek comparably with previous rating studies that used parts of facial skin (Jones et al., 2004b; Roberts et al. 2005a) (Fig. 6).

27 27 Figure 6. Defined area for skin analysis. For skin analysis we used a square patch on the right cheek defined by the outer corner of the right eye and the most lateral point of the right nostril. The top of the patch started 25 pixels under the outer corner of the eye and the bottom was limited by 15 pixels beneath the point on the nostril. Co-occurrence matrices count how often pairs of equal gray values of neighboring pixels along a certain direction occur in the defined window. Out of originally 14 features of these matrices, we chose homogeneity as it performed best in previous studies (Grammer et al., 2002; Fink et al., 2001). For measuring skin coloration we used the same skin patch on the right cheek in every facial picture as for the textural analysis. The program determines skin color in a red, green and blue (RGB) color space, color saturation and color brightness. Colors in the RGB-color space are represented as values between 0 and for red, green and blue. Values fo color saturation distinguish weak color (a value of 0 is colorless) from strong color (maximum values), whereas values of color brightness describe the luminance of a color (a value of 0 represents black; a maximum value means brightest color). For the analysis of skin coloration some pictures had to be excluded since they

28 28 were taken under different light conditions. In study 1 faces of 64 men and 69 women could be analyzed for their skin color and in study 2 faces of 29 men and 30 women Statistical Analysis Ratings of attractiveness and health, facial asymmetry, skin texture and skin color are supposed to be influenced by individuals age (Fink et al., 2006b; Furnham et al., 2004; Kowner, 1996). Therefore, all measurements including the ratings of attractiveness and health, facial asymmetry, skin texture and skin color were regressed on participants age. Residuals were used for further analyses to eliminate potential confounding effects of age (e.g. Kozieł and Pawłowski, 2003). As these measures were normally distributed we used Pearson s r for correlations and independent samples t tests for calculating differences between heterozygotes (at all loci) and homozygotes (at one or more loci) regarding measures of facial traits and ratings of attractiveness and health. All statistical analyses were done with SPSS version 12.0 with two-tailed significance levels, for which the.05 level was defined as critical.

29 29 4. Results 4.1. Study 1 MHC Heterozygosity and Facial Attributes No significant differences could be found between completely heterozygotes and homozygotes (at one or more loci) calculated on the base of 5 loci and 3 loci regarding perceived facial attractiveness and perceived health in faces of both sexes (Tab. 3). Table 3. Effects of MHC heterozygosity on facial traits in study 1 and 2. Heterozygotes (at all analyzed loci) did not differ in their level of facial asymmetry and skin homogeneity and in their perceived attractiveness and health from homozygotes (at one or more loci). study 1 study 2 men women men women attractiveness 5 loci t=-0.83, p=0.41 t=-1.16, p=0.25 t=-1.06, p=0.30 t=-0.57, p= loci t=0.26, p=0.79 t=-1.41, p=0.16 t=-0.18, p=0.86 t=-0.59, p=0.56 health 5 loci t=-0.60, p=0.58 t=-1.28, p=0.20 t=-0.75, p=0.46 t=-1.38, p= loci t=0.34, p=0.74 t=-0.97, p=0.33 t=0.14, p=0.89 t=-1.02, p=0.32 asymmetry 5 loci t=-0.06, p=0.95 t=0.79, p= loci t=-0.27, p=0.79 t=0.39, p=0.70 homogeneity 5 loci t=0.37, p=0.71 t=-1.01, p= loci t=1.18, p=0.24 t=-0.07, p=0.94

30 30 Further, there was no significant effect of MHC heterozygosity on facial asymmetry and skin homogeneity neither at the base of 5 loci nor at 3 loci (Tab. 3). Finally, measures of skin color were also not influenced by MHC heterozygosity neither at 5 loci nor at 3 loci (Tab. 4). Table 4. Differences between heterozygotes and homozygotes regarding skin coloration. No significant differences between completely heterozygotes and homozygotes (at one or more loci) could be found in both sexes regarding different measures of skin coloration neither on the basis of 5 loci nor on the basis with 3 loci. men women heterozygosity at 5 loci heterozygosity at 3 loci t df p t df p red 0, ,438 0, ,872 green -0, ,400 0, ,492 blue -0, ,469 0, ,724 saturation 0, ,476-0, ,554 brightness -0, ,485 0, ,981 red -0, ,582-0, ,955 green 0, ,831-0, ,741 blue -0, ,950-0, ,465 saturation 0, ,589 1, ,209 brightness 0, ,590 0, ,904

31 31 Facial Attractiveness and Health Facial asymmetry was not significantly correlated with perceived facial attractiveness (men: r=-0.128, p=0.252; women: r=-0.160, p=0.104). However, there was a negative correlation between facial asymmetry and perceived health in both sexes (men: r= , p=0.026; women: r=-0.192, p=0.049) (Fig. 7). Homogeneity of skin texture was not significantly correlated with perceived facial attractiveness (men: r=-0.055, p=0.619; women: r=0.129, p=0.189) and health (men: r=0.023, p=0.834; women: r=0.043, p=0.663), and none of the different measures of skin color were associated with perceived attractiveness and health (Tab. 5). Figure 7. Correlation between facial asymmetry and perceived health in both sexes of study 1. The blue dots and the blue regression line describe the male sample, while the red dots and the red regression line signal the female sample of rating study 1. Asymmetry and rated health in faces were significantly negatively related to each other in both sexes (men: r=-0.246, p=0.026; women: r=-0.192, p=0.049).

32 Study 2 Attractiveness and Health in Masked Facial Images In this subsample again, no significant effect of MHC heterozygosity neither at 5 loci nor at 3 loci could be found neither on perceived facial attractiveness nor on perceived facial health (Tab. 3). In contrast to the first rating, we could not find a significant correlation between facial asymmetry and perceived attractiveness (r=-0.141, p=0.412) and health (r=-0.164, p=0.338) in male faces. However, there was a negative correlation between facial asymmetry and rated attractiveness (r=-0.418, p=0.004) as well as healthiness (r=-0.337, p=0.023) in women (Fig. 8). Figure 8. Correlation between facial asymmetry and perceived attractiveness and health in female faces of study 2. The blue dots and the blue regression line describe perceived attractiveness, while the red dots and the red regression line signal perceived health. In rating study 2, asymmetry in female faces was negatively correlated with judgements of attractiveness (r=-0.418, p=0.004) and health (r=-0.337, p=0.023).

33 33 Skin homogeneity did not significantly correlate with perceived facial attractiveness (men: r=0.209, p=0.215; women: r=0.142, p=0.351) or health (men: r=0.043, p=0.799; women: r=0.115, p=0.453). Still, there were significant correlations between some measures of skin color and perceived health in male faces (red: r=0.391, p=0.035; blue: r=-0.421, p=0.022; brightness: r=-0.459, p=0.012) (Fig. 9). All other measures of skin color did not significantly correlate with perceived facial attractiveness and health in both sexes (Tab. 5). Figure 9. Correlation of red and blue values of skin and color brightness with perceived health in male faces of study 2. The red and blue dots correspond to red and blue values of skin and color brightness is represented by the gray dots. In ratings study 2, redness of skin was positively (r=0.391, p=0.035), whereas blueness (r=-0.421, p=0.022) and color brightness of skin (r=-0.459, p=0.012) were negatively associated with perceived health in male faces.

34 34 Table 5. Correlation between skin color and perceived facial attractiveness and health. In study 1, correlations between skin coloration and rated attractiveness and health were not significant in both sexes, whereas in study 2, there were some significant correlations between measures of skin color and perceived health in male faces (red, blue, brightness). study 1 study 2 men attractiveness health attractiveness health red r=0,058 p=0,645 r=0,120 p=0,343 r=0,351 p=0,061 r=0,391 p=0,035 green blue r=-0,040 p=0,749 r=-0,070 p=0,581 r=-0,196 p=0,306 r=-0,317 p=0,093 r=-0,107 p=0,395 r=-0,176 p=0,164 r=-0,303 p=0,109 r=-0,421 p=0,022 saturation r=0,073 p=0,562 r=0,158 p=0,210 r=0,183 p=0,341 r=0,309 p=0,101 brightness r=-0,075 p=0,551 r=-0,105 p=0,408 r=-0,353 p=0,060 r=-0,458 p=0,012 women red r=-0,095 p=0,430 r=0,038 p=0,752 r=-0,228 p=0,224 r=-0,053 p=0,778 green r=0,137 p=0,257 r=-0,069 p=0,569 r=0,175 p=0,352 r=0,018 p=0,921 blue r=0,148 p=0,219 r=-0,036 p=0,763 r=0,141 p=0,454 r=-0,022 p=0,907 saturation r=-0,152 p=0,206 r=0,033 p=0,780 r=-0,128 p=0,499 r=0,038 p=0,839 brightness r=0,103 p=0,394 r=-0,034 p=0,774 r=0,210 p=0,265 r=0,050 p=0,790

35 35 5. Discussion This survey examined if MHC heterozygosity that is reported to be of certain advantage against parasites and pathogens (Thursz et al., 1997; Carrington et al., 1999) positively affects the ontogenetical development of facial symmetry, homogeneity of skin and skin coloration, which are three traits that are found to influence the perception of attractiveness and health (review in Thornhill and Gangestad, 1999). Thereby, results address the hypothesis that attraction is an adaptation responding to cues of potential mates health constitution (Symons, 1979) Attractive Health Information in Facial Traits In the first study, facial asymmetry was negatively correlated with perceived health in both sexes, whereas in the second study, facial asymmetry was negatively correlated with ratings of attractiveness and health in women. Further, in study 2 some measures of skin color were significantly associated with perceived health in men: The red component of the RGB-color space was positively, the blue component and color brightness were negatively correlated with perceived health in male faces. (The slight differences between the two studies should be mainly due to the drawn samples of raters and differences in picture style). All mentioned measures were controlled for participants age to eliminate potential confounding age effects. These results are in line with our hypotheses and correspond to previous studies that found an effect of facial asymmetry and skin coloration on ratings of attractiveness and health (Grammer and Thornhill, 1994; Fink et al., 2001; Fink et al., 2006a; Fink et al., 2006b). Moreover, two studies found more symmetric individuals to be actually healthier than less symmetric ones (Shackelford and Larsen, 1997; Waynforth, 1998) and Thornhill and Gangestad (1994) showed

36 36 that facial symmetry was positively associated with life time number of sex partners in both sexes, which indicates actual advantage of more symmetric individuals in intra- and intersexual competition. Hence, regarding the hostparasite coevolutionary arms race there should be some information about the quality of the immune system in human faces. However, contrary to Fink et al. (2001) who used the same method as we did, we were not able to detect any correlations between homogeneity of skin and perceived attractiveness or health. For analyzing textural homogeneity we used a small skin patch on the right cheek of every participant as applied by Roberts et al. (2005a) with their health ratings of skin patches. For representing the textural condition of an entire face the used patch might have been too small. Often the skin on the cheeks is thin in comparison to, for instance, the skin of the forehead. Hence, cheeks could be relatively even whereas the forehead could be quite inhomogeneous. Fink et al. (2001) used a bigger analysis section than we did measuring skin texture of almost the entire lower face, where features like the nose and the mouth contribute to the texture analysis and may introduce confounding effects. Hence, it would be reasonable to analyze several sections of skin texture in the face (e.g. on the front, on each side of the cheeks and on the chin) and calculate the mean value for skin homogeneity in further studies Attractiveness of MHC Heterozygosity We were not able to detect any effects of MHC heterozygosity on the observed facial traits (controlled for age) or on the ratings of attractiveness and health (controlled for age). This result indicates that there seems to exist no direct connection between heterozygosity of genes of the MHC and the development of facial characteristics like symmetry, skin texture or color. This is in line with Thornhill et al. s (2003) study, where no association could

37 37 be discovered between MHC heterozygosity and facial symmetry or facial attractiveness. Roberts et al. (2005a) also failed to identify an influence of MHC heterozygosity on facial symmetry. However, they were able to detect a positive effect of MHC heterozygosity on ratings of facial attractiveness and health judgements of the skin patches. Thus, MHC heterozygosity could potentially influence characteristics of skin while it has no influence on facial symmetry. However, as Roberts et al. (2005a) proposed, we measured skin texture as well as skin color with computational methods and could not find any effect of MHC heterozygosity on these traits. There are some methodological differences between the studies of Thornhill et al. (2003) and Roberts et al. (2005a) that could account for the differences in results, though sample sizes were almost identical in both studies. First, there was a wider variation of participants age in Thornhill et al. s sample that might have influenced judgements of attractiveness. Second, unlike Thornhill et al., Roberts et al. masked facial images to diminish confounding effects of hair style and condition as well as clothing. Both a smaller age variation and masked images are likely to reduce variance in judgements in Roberts et al. s study. Our study is well comparable with these two studies as the sample size was quite the same and two ratings were conducted with differences in participants age variation and differences in picture style. In the first study, we used similar conditions like Thornhill et al. (2003) with a wide variation in the age of participants and unmasked pictures. In the second study, we reduced the sample size for a smaller age variation and showed masked images like Roberts et al. (2005a). Moreover, since we used more MHC loci than these previous studies, which potentially increases variation in heterozygotes, all statistical tests were calculated based on the five analyzed loci as well as the three loci used in previous studies. Results showed no differences in the use of 3 or 5 loci regarding any effects of MHC

38 38 heterozygosity on facial traits or the perception of attractiveness and health. Moreover, there were no differences in results between rating study 1 and 2 concerning any influence of MHC heterozygosity on ratings of attractiveness or health Health Care and Variation in Facial Traits In contrast to our hypotheses we conclude that there does not seem to exist a direct association between MHC heterozygosity and the observed facial characteristics as well as the perception of facial attractiveness and health. One possible explanation for why there was no detectable, direct effect of MHC heterozygosity is that nowadays medicine is able to inhibit and prevent most harmful diseases and parasite pressure is comparably low in this part of the world. Moreover, our participants originated from a rural region, where population density is lower than in urban areas. In comparably sparsely populated regions parasites cannot spread that fast and diseases are not as easily transmitted. Hence, variation in facial traits due to functional differences of the immune system might be reduced. Consistent with the notion that attractiveness is an adaptation responding to honest health signals, Gangestad and Buss (1993) found that across many societies attractiveness is most important as a mate choice criterion in areas with high parasite prevalence. Therefore, an obvious preference for heterozygous mates may appear in regions with less health care or high parasite pressure. Although no evidence has been found that MHC heterozygosity is associated with developmental stability or facial symmetry and only one study yet reported an association between MHC heterozygosity and health judgements of skin patches (Roberts et al., 2005a), heterozygosity on genes of the MHC is associated with disease resistance (Thursz et al., 1997; Carrington et al., 1999). Hence, choosing heterozygous mates though individual

39 39 heterozygosity is not heritable could be beneficial for offspring by prolonged periods of high-quality parental care and a reduced risk of contracting diseases (Kirkpatrick and Ryan, 1991). Moreover, the probability of possessing beneficial alleles is higher in heterozygotes. Therefore, another potential merit from choosing heterozygous mates is the transmission of advantageous alleles to offspring Reasons for the Variation in Facial Traits A further explanation for why we found no direct effects of MHC heterozygosity in this study could be that it simply does not influence the ontogenetical development of facial symmetry or the homogeneity and coloration of skin. Maybe other parts of the immune system account for developmental stability or nutrition plays a major role for individual deviations from bilateral symmetry. Regarding skin irritations it is also conceivable that skin bacteria cause certain inhomogeneities. It is thought that the microbial flora of the skin is influenced by the MHC in that some ectoparasitic strains get eliminated by certain alleles (Howard, 1977; see Wedekind and Penn, 2000). Hence, resistant strains in the absence of beneficial alleles could potentially cause irritations of skin. In that case, skin inhomogeneities would be indirectly associated with MHC heterozygosity or specific alleles, and this indirect causality would not be easily testable. Nevertheless, Roberts et al. (2005a) found faces of heterozygous men to be preferred by women. This could be explained by so-called hormone markers in faces. These traits like prominent eyebrows and big jaws in men or prominent cheeks and a small lower face compared to the upper face in women are supposed to be developed under the influence of sex hormones (Grammer et al., 2002, pp ). Since high levels of sex hormones are costly to maintain, because they are immune suppressive (see Kanda

40 40 and Watanabe, 2005), hormone markers in the face could potentially mediate the quality of the immune system or more precisely MHC heterozygosity. Since sex hormones are also considered to be involved in generating clear skin, further studies should turn attention to individual levels of sex hormones when investigating effects of MHC heterozygosity on facial traits Sustenance of MHC Polymorphism In the face of the host-parasite coevolutionary arms race sustenance of MHC variability is highly important due to its essential role in the immune system. There are some potential mechanisms which could account for the maintenance of the high diversity of the MHC. Heterozygote Advantage Hypothesis This study was only concerned with the heterozygote advantage hypothesis due to limitations in sample composition. It states that heterozygous individuals should be able to present a wider range of antigens to the immune system than homozygotes due to the higher diversity of MHC alleles and resulting higher diversity of HLAs, given that MHC alleles show codominance. Hence, heterozygous individuals should be resistant against more different pathogens and parasites than homozygotes (see Penn and Potts, 1999; Sommer, 2005). Rare Allele Advantage Hypothesis Another account for the maintenance of the high diversity of the MHC is the rare allele advantage hypothesis. Rare alleles should be of advantage in the host-parasite arms race as parasites and pathogens adjust themselves to the most common genotypes in the host population. Thus, individuals possessing rare alleles might have benefits due to a resistance against diseases that

41 41 infect the majority. Nevertheless, beneficial alleles will spread in host populations thereby losing their advantage as parasites will adapt themselves to more common alleles. This coevolution would result in periodic fitness of particular alleles which is a potential mechanism to infinitely sustain the high diversity of MHC alleles (see Penn and Potts, 1999; Sommer, 2005). This frequency-dependent selection was first implied by Hamilton and Zuk (1982). Hill et al. (1991) provided some support for this hypothesis by identifying two MHC alleles (on MHC class I and class II) that - though cannot inhibit an infection - are able to lower the risk of dying of severe Malaria for about 50%. Another study found an association between an MHC allele and spontaneous recovery of hepatitis B virus infection (Thursz et al., 1995). Allele Specific Overdominance Hypothesis Theoretical models indicated that heterozygote advantage alone cannot preserve the high diversity of MHC alleles. Instead, by development of disassortative mating preferences and resulting heterozygosity in offspring, diversity of the MHC could be sustained. In contrast, rare allele advantage could potentially infinitely maintain the high polymorphism of the MHC (see in Penn and Ilmonen, 2005a). In this context, another conceivable approach, called allele specific overdominance, states that heterozygote advantage could result from possessing more different and also more likely rare alleles (see Swaddle and Cuthill, 1995; Sommer, 2005). Hence, both hypotheses may be in accord with each other and are not mutually exclusive. Evidence comes from Carrington et al. (1999) who found an heterozygote advantage at MHC class I loci associated with a slower progression of AIDS and at the same time two MHC class I alleles that were associated with a more rapid development of AIDS.

42 42 MHC-dependent Mate Preferences There are five potential functions of MHC-dependent mate preferences (see Sommer, 2005). First, studies showed that both men and women prefer MHCdissimilar mates (Wedekind et al., 1995; Wedekind and Füri, 1997; Ober et al., 1997; Thornhill et al., 2003) which would result in offspring heterozygosity and hence, is in line with the heterozygosity advantage hypothesis. Second, in contrast to mate preferences based on MHC dissimilarity, a preference for heterozygotes (Thornhill et al., 2003; Roberts et al., 2005) could have been selected for direct benefits to females or through indirect fitness advantages for offspring regarding the allele specific overdominance hypothesis. Third, preferences could have evolved for particular, beneficial MHC alleles in potential mates to guarantee that offspring is resistant against certain parasites and pathogens which would be in line with the rare allele advantage hypothesis. This premise is not easily testable in humans due to the high MHC-polymorphism and the lack of population-wide allele frequencies. Additionally, the existing parasite pressure should be taken into account to test this hypothesis. The preference for rare MHC alleles is another explanation for the failure to find effects of MHC heterozygosity in our study, because rare alleles might cause variation in facial symmetry, skin texture and coloration. However, Thornhill et al. (2003) could not detect any influence of MHC allele rarity on facial symmetry and attractiveness in either sex. This could be due to methodology as they used a sample of wide ethnic variation, which enhanced allelic sample diversity. Moreover, they computed allele frequencies within their sample which may not matched the population-wide frequency. In our study it was not possible to test the rare allele advantage hypothesis, because the sample consisted of thirteen families with on average

43 participating members. Thus, MHC allele distributions would not have correctly represented the regional pattern. Fourth, dissimilar mate preferences could have evolved due to inbreeding avoidance. Individuals that share a great amount of alleles are more likely to be related to each other. Offspring from such matings show less variation in their MHC and hence suffer from lowered fitness and lowered resistance against pathogens and parasites (Brewer et al., 1990). This mechanism is also called kin recognition hypothesis (see Penn and Potts, 1999; Sommer, 2005). Studies have found that humans are able to discriminate the smell of related versus non-related individuals (Gilbert et al., 1986; Jacob et al., 2002; Oberzaucher et al., in prep.). Finally, with regard to inclusive fitness (Hamilton, 1964) and enhanced paternal investment (Trivers, 1972) it could be of advantage in the view of genes (Dawkins, 1976) to share a small amount of genetic material with the partner. However, as extreme homozygosity is linked with health risks (Brewer et al., 1990), there should exist an optimal amount of shared alleles between mates. This theory is called the optimal outbreeding or allele counting hypothesis (see Penn and Potts, 1999; Sommer, 2005). Results that support this hypothesis show that women preferred men with whom they shared a small amount of alleles over zero matches or identical MHC (Jacob et al., 2002; Roberts et al., 2005b). Supertypes A new method for testing these different hypotheses is using HLA-supertypes. Various human MHC alleles belong to only a comparably small number of so called supertypes based on similar specificities in the antigen-binding sides of the HLAs encoded by these alleles (Sidney at al., 1995; Sommer, 2005). Penn and Potts (1999) proposed that MHC heterozygote advantage may have been

44 44 overlooked if functional MHC homozygotes have been misclassified as heterozygotes. This approach was taken up by a study which results showed that HLA-supertypes were highly predictive for the viral load during an HIV infection. The authors found that rare HLA supertypes were of advantage for the progression of AIDS (Trachtenberg et al., 2003). However, latest results of different analyses on binding specificities do not agree about the assignment of single alleles to the so far classified supertypes. Hence, in this study we decided not to work on the functional base, but on the genetical base of MHC. Nevertheless, this seems to be an interesting new method for further studies Mediation of Individual MHC Genotypes The particularly high polymorphism of MHC genes is generally thought to be driven by parasite-mediated selection, but mating preferences may also play a role. There is much evidence, that humans are able to discriminate between individuals with different MHC genotypes (Gilbert et al., 1986; Wedekind et al. 1995; Wedekind and Füri, 1997; Ober et al. 1997; Jacob et al. 2002; Thornhill et al., 2003; Carvalho Santos et al., 2005; Roberts et al., 2005b; Oberzaucher et al., in prep.), though mechanisms for the mediation of individual MHC genotypes are still unclear. Body Scent One assumption is that the human body odor serves as a cue for signaling individual compounds of the MHC. There are three feasible approaches of how MHC genes could influence body odor (review in Wedekind and Penn, 2000). First, HLAs may bind particular peptides whose volatile metabolites such as carboxylic acids could serve as odors. Second, HLAs may determine the microbial flora of the skin. Only strains of bacteria survive that are not

45 45 recognized by HLAs and these resistant strains may metabolize body secretions to volatile substances. Another mechanism constitutes a fusion of the first ones: Microbes may metabolize HLA-bound peptides that thereby get volatile. First evidence for MHC influenced body odor came from Gilbert (1986), who found that humans are able to discriminate odor of mice that differ genetically only at the MHC. Subsequently, Wedekind and his colleagues could detect that humans prefer the body odor of MHC-dissimilar individuals (Wedekind et al., 1995; Wedekind and Füri, 1997). Only recently another study could demonstrate that body odors of siblings differing in their MHC could be discriminated while odors from MHC identical siblings were not distinguished (Oberzaucher et al., in prep.). Bilateral Symmetry Another potential signal for individual MHC is the extent of asymmetry which we tested in this study. The MHC may influence developmental stability during ontogeny that is important for the development of bilateral symmetry. Rikowski and Grammer (1999) found that women preferred the scent of symmetrical men. However, Thornhill et al. (2003) failed to show any correlation between symmetry and MHC heterozygosity or MHC allelic rarity, whereas Roberts et al. (2005a) showed that MHC heterozygosity is associated with facial attractiveness. They found that faces of heterozygous men are judged as more attractive and healthy by women than faces of homozygous men. However, in their study facial symmetry was also not correlated with MHC heterozygosity and they did not look for an effect of rare alleles. Our study is in line with these previous studies that failed to show an association between MHC heterozygosity and facial symmetry. Maybe we should dissociate from this approach and rather look into hormon markers in the face that are developed by means of high levels of immune suppressive sex

46 46 hormones and therefore, might mediate MHC heterozygosity or at least the presence of beneficial MHC alleles. Skin Condition A third conceivable cue for individual MHC could be the condition of skin which we also tested in this study. Roberts et al. (2005a) found health ratings of skin patches from the right cheek of participants to be more healthy in MHC heterozygotes. They proposed to repeat this study with textural and color measurements of skin, but we could not detect any effects of MHC heterozygosity on computed homogeneity or coloration of skin. Thus again, allelic rarity may cause variation in skin condition.

47 47 6. Conclusion So far, there is considerable evidence for human MHC-dependent mate preferences and MHC-dependent mate choice. Although no indication has been found for MHC heterozygosity to be associated with developmental stability or facial symmetry (Thornhill et al., 2003; Roberts et al., 2005a) and only one study yet reported a relation between MHC heterozygosity, perceived facial attractiveness and health judgements of skin patches (Roberts et al., 2005a), heterozygosity on genes of the MHC is associated with disease resistance (Thursz et al., 1997; Carrington et al., 1999). Moreover, body scent seems to signal individual variation of the MHC (Wedekind et al., 1995; Wedekind and Füri, 1997; Oberzaucher et al., in prep.). The influence of the human MHC might be more complex as we think yet. Most likely, there should exist a preference for rare alleles in heterozygotes to provide offspring with parasite resistance as the probability of carrying beneficial, rare alleles is higher in heterozygotes. This hypothesis is difficult to test due to the lack of population-wide frequencies of the highly polymorphic MHC alleles. As these frequencies are dependent from local parasite pressure, primarily one has to define single populations delimited by different strains of pathogens and parasites. Then, allele frequencies for these populations have to be estimated. In a last step, one could compare allele frequencies of research samples with the population-wide frequency to be able to make approximations on mate preferences and mate choice. Nevertheless, Thornhill et al. (2003) examined mate preferences for rare MHC alleles, but could not detect any supporting results. This could be due to their methodology as they computed allele frequencies within their sample which maybe did not match population-wide frequencies. A solution or at least an advancement of this problem is the classification of MHC alleles into HLA supertypes. Nevertheless,

48 48 to test effects of their rarity one also has to know their frequency in a population. The other big missing part of the puzzle is the mechanism through which the quality of the MHC is signaled. Most obvious, the individual composition of the MHC is mediated through the human body scent, but also skin condition and hormone markers should be considered. Since results are rather diverse further research is needed to clarify the role of MHC concerning honest signals in mate choice decisions.

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55 55 Does Facial Attractiveness Convey an Individual s Immunological Constitution?

56 56 8. Appendix. Landmark definitions 1 forehead right corner of the forehead right, located in the hairline 2 forehead left corner of the forehead left, located in the hairline 3 Trichion the local midpoint of the hairline 4 Superciliare laterale right the most lateral point of the eyebrow Upper eyebrow right curve along the upper eyebrow rim with three roughly equidistant points between 4 and 8 8 Superlaterale mediale right the most medial point of the eyebrow Lower eyebrow right curve along the lower eyebrow rim with three roughly equidistant points between 4 and 8 12 Superlaterale mediale left the most medial point of the eyebrow Upper eyebrow left curve along the upper eyebrow rim with three roughly equidistant points between 12 and Superciliare laterale left the most lateral point of the eyebrow Lower eyebrow left curve along the lower eyebrow rim with three roughly equidistant points between 12 and Exocanthion right the outer corner of the eye fissure, where the eyelids meet 21 Top of the iris right highest visible point of the iris 22 Endocanthion right the inner corner of the eye fissure, where the eyelids meet 23 Bottom of the iris right lowest visible point of the iris 24 Lateral side of the iris right the most lateral side of the iris 25 Pupil right midpoint of the pupil 26 Medial side of the iris right the most medial side of the iris 27 Endocanthion left the inner corner of the eye fissure, where the eyelids meet 28 Top of the iris left highest visible point of the iris 29 Exocanthion left the outer corner of the eye fissure, where the eyelids meet 30 Bottom of the iris left lowest visible point of the iris 31 Medial side of the iris left the most medial side of the iris 32 Pupil left midpoint of the pupil

57 57 33 Lateral side of the iris left the most lateral side of the iris 34 Alae origin right the most posterolateral point of the curvature of the base of the nasal alae 35 Alare right the most lateral point on the nasal ala 36 Columella apex right the highest point on the columella crest at the apex of the nostril 37 Subnasale the junction between the lower border of the nasal septum 38 Columella apex right the highest point on the columella crest at the apex of the nostril 39 Alare left the most lateral point on the nasal ala 40 Alae origin left the most posterolateral point of the curvature of the base of the nasal alae 41 Cheilion right the outer corner of the mouth, where the outer edges of the upper and the lower vermilions meet 42 Upper lip point right a point between 41 and 43 at the vermilion border 43 Christa philter right the point on the crest of the philtrum 44 Labrale superius the local philtrum midpoint of the vermilion border of the upper lip 45 Christa philter left the point on the crest of the philtrum 46 Upper lip point left a point between 45 and 47 at the vermilion border 47 Cheilion left the outer corner of the mouth, where the outer edges of the upper and the lower vermilions meet 48 Lower lip point left a point between 47 and 49 at the vermilion border 49 Labrale inferius the local philtrum midpoint of the vermilion border of the lower lip 50 Lower lip point right a point between 41 and 49 at the vermilion border 51 Cleft point right a point between 41 and 52 exactly on the cleft between upper and lower lip 52 Stomion the midpoint of the labial fissure where the lips are closed naturally 53 Cleft point left a point between 47 and 52 exactly on the cleft between upper and lower lip 54 Zygion right the most lateral point on the zygomatic arch Lower face right eight landmarks equidistanly distributed between 54 and 63 along the outline of the lower face 63 Gnathion the most outer point on the outline of the chin Lower face left eight landmarks equidistanly distributed between 63 and 71 along the outline of the lower face 72 Zygion left the most lateral point on the zygomatic arch

58 58 Does Facial Attractiveness Convey an Individual s Immunological Constitution?

59 59 9. Curriculum Vitae Katharina Zimmer phone: Education April 2007 Master Thesis; Ludwig-Boltzmann-Institute for Urban Ethology, Institute for Anthropology, University of Vienna, Austria studies in Anthropology (focusing on Human Ethology); University of Vienna, Austria 2003 Degree in General Biology; University of Vienna, Austria Basic courses in General Biology; University of Vienna, Austria High School (focusing on natural sciences) at the BG&BRG Perchtoldsdorf, Austria; Matura (A-levels) born in Vienna, Austria Occupations Apr. Aug Project Assistance in a funded study about human body scent (Unique Signature Detection Project - USDP) since Jan Administrative Assistant at the Ludwig-Boltzmann- Institute for Urban Ethology, Institute for Anthropology, University of Vienna, Austria since July 2006 Publication for the Konrad Lorenz Institute for Ethology, Austrian Academy of Sciences, Vienna, Austria Status: in review Project Assistance in a funded study about human behavior in semi-public spaces