Contributions to Zoology, 74 (3/4) 213-222 (2005) Morphological variation in two genetically distinct groups of the golden-striped salamander, Chioglossa lusitanica (Amphibia: Urodela) J. Alexandrino1, 2, N. Ferrand1, 2, J.W. Arntzen1, 3 1CIBIO Centro de Investigação em Biodiversidade e Recursos Genéticos, Campus Agrário de Vairão, 4485-661 Vairão, Portugal; 2Departamento de Zoologia e Antropologia, Faculdade de Ciências, Universidade do Porto, Praça Gomes Teixeira, 4099-002 Porto, Portugal; 3Zoology Department, National Museum of Natural His- tory - Naturalis, P.O. Box 9517, 2300 RA Leiden, The Netherlands Corresponding author: João Alexandrino Present address: Departamento de Zoologia, Instituto de Biociências, Universidade Estadual Paulista, 13506-900 Rio Claro, São Paulo, Brazil, e-mail: jalex@rc.unesp.br Key words: Amphibia, Chioglossa lusitanica, clinal variation, colour pattern, golden-striped salamander, morphometrics Abstract Morphometric and colour pattern variation in the endemic Iberian salamander Chioglossa lusitanica is concordant with the genetic differentiation of two groups of populations separated by the Mon- dego river in Portugal. Salamanders from the south have shorter digits than those from the north. Clinal variation with a south to north increase in limb, toe and fi nger length was found superimposed on this dichotomy, resulting in stepped clines for characters describing appendage size. Genetic variability was paralleled by colour pattern variability in the contact zone and in northern populations. To explain the observed parallels we invoke the neutral processes of vicariant isolation, admixture in a secondary contact zone, genetic drift in addition to selection acting along an environmental gradient. Mor- phological constraints imposed by a highly specialized ecological niche may explain why the genetic subdivision of C. lusitanica since the early Pleistocene has remained fairly cryptic. Contents Introduction .................................................................................... 213 Material and methods ................................................................... 215 Results ............................................................................................. 216 Morphometrics ........................................................................ 216 Colour pattern .......................................................................... 217 Testing causal hypotheses ..................................................... 218 Discussion ...................................................................................... 219 Acknowledgements ...................................................................... 220 References ...................................................................................... 220 Introduction The discovery of genetically differentiated groups of populations within species raises the question of how infl uential genetic isolation has been in gener- ating phenotypic diversity. Since morphological traits are products of both genetics and the environment (see e.g. Schluter, 2000) it is relevant, from an evo- lutionary perspective, to determine whether morpho- logical variation refl ects historical isolation or local adaptation despite recurrent gene fl ow. To distinguish between these non-exclusive hypotheses it is impor- tant to study a variety of traits. Differentiation arising from historical isolation of populations (i.e. with the stochastic effects of mutation and drift acting inde- pendently in isolated populations) will likely gener- ate parallel patterns across characters of different nature, e.g. genetics and morphology, whereas adap- tation across environmental selection gradients will not, or not necessarily so. Organismal constraints involving plasticity and autopoiesis may however cause morphological stasis despite considerable levels of genetic and environmental change through evolutionary time, such as in Plethodontid salaman- ders (Wake et al., 1983; see also Larson, 1984). In the case of cryptic closely related lineages one way of testing hypotheses on the extent and nature of phenotypic diversity has been the multivariate sta- tistical analysis of morphometric data (e.g. Good and Wake, 1992; Arntzen and Sket, 1997; Puorto et al., 2001). The golden-striped salamander, Chioglossa lusi- tanica Bocage 1864, is a streamside species inhab- iting low and medium elevation mountainous areas in the northwest of the Iberian Peninsula (Fig. 1). 214 Alexandrino et al. – Morphological variation in golden-striped salamanders 215Contributions to Zoology, 74 (3/4) – 2005 Alexandrino et al. (1997, 2000, 2002) analyzed al- lozyme and mitochondrial DNA variation and un- covered two genetically distinct groups of popula- tions geographically separated by the river Mondego in central Portugal (group 1 south of the river and group 2 north of the river). The two groups represent lineages that separated in the early Pleistocene, probably as a result of climate change in combina- tion with local environmental conditions and formed a secondary contact zone in postglacial times (Se- queira et al., 2005). The northern part of the present range was colonized from a refugium located in between the Mondego and the Douro. Major rivers such as the Douro and, more to the north, the Minho acted as barriers to dispersal, lowering genetic di- versity through sequential bottlenecking (Alexan- drino et al., 2000). Morphological variation in C. lusitanica was fi rst observed by Vences (1990), but with only three populations studied in Portugal, robust spatial patterns could not be discerned. We here describe morpho- logical variation within C. lusitanica to investigate if i) the diversifi cation of C. lusitanica in two popu- lation groups and ii) the decrease of genetic diver- sity in recently colonized areas, have been paralleled by morphological variation. If genetic drift has been underlying morphological evolution we expect con- cordance between genetic and morphological patterns of variation. Conversely, non-concordance between genetics and morphology is expected if directional selection and epistasis have been major sources of morphological variation (see Reed and Frankham, 2001). We specifi cally test morphometric and col- ouration pattern variation across the species’ range against the causal hypotheses of i) vicariance of groups 1 and 2 and ii) isolation by distance. Latitu- dinal increase/decrease in morphological trait diver- sity is examined against causal hypothesis of i) ge- netic heterozygosity ii) hybrid origin of populations and iii) geographic distance between populations. Material and methods A total of 420 adult Chioglossa lusitanica was col- lected from 20 localities (Fig. 1). Sample sizes ranged from 7 to 51. For each individual the dorsal coloura- tion pattern was classified on two accounts i) prominence of colour spots on the dorsum and ii) proportion of colour patches relative to colour stripes, with six ranked (0–5) colour pattern types as follows: type 0, coloured patches absent i.e., melanic; type 1, few coloured patches arranged into incomplete stripes; type 2, two uninterrupted coloured stripes, separated by a dark area of approximately the same width (typical pattern); type 3, stripes wider; type 4, central area with coloured patches diffusely distrib- uted; and type 5, central area with coloured patches densily packed in a mosaic pattern. For a general impression of colour pattern variation in C. lusitanica see Vences (1990). Seven morphometric measure- ments were taken on 275 individuals representing all populations except nos. 14 and 17: snout-vent length (SVL), head (snout-gular) length (HL), head width (HW), forelimb length (FLL), hindlimb length (HLL), third fi nger length (TFL) and fourth toe length (FTL) (Fig. 2). Measurements were taken to the near- est 0.1 mm with digital callipers by a single ob- server (JA) on animals anaesthetized with MS-222. Subsequently, salamanders were released on their localities of origin. Morphometric data were log-transformed to re- duce deviations from normality and distortion ef- fects caused by allometric relationships. Population means were compared by multivariate analysis of variance (MANOVA) with the variable ‘sex’ nested under the variable ‘locality’. All measurements showed signifi cant differences between populations (Wilks’ lambda = 0.098, P< 0.001; univariate results all with P< 0.001) and HL and FLL showed sig- nifi cant differences between sexes (respectively, F = 2.865, P< 0.001 and F = 4.437, P< 0.001). Sub- sequent morphometric analyses were performed for males and females separately by principal compo- nent analysis (PCA) with the software package Fig. 1. The distribution of Chioglossa lusitanica in the north- western Iberian Peninsula (shaded area, from Arntzen, 1999) and the study localities. (1) Muradal, Foz de Giraldo (MU). (2) Lousã, C. Pêra (LCP). (3) Lousã, Fiscal (LFI). (4) Lousã, Vilarinho (LVI). (5) Açor, Margaraça (AC). (6) Várzeas (VA). (7) Buçaco (BU). (8) Saide (SA). (9) Covelo (C). (10) Tarouca (T). (11) Montemuro (M). (12) Valongo, Silveirinha (VSI). (13) Valongo, Águas Fér- reas (VAF). (14) Bom Jesus (B). (15) Cabreira (CA). (16) Gerês (G). (17) Pontevedra (PO). (18) Caaveiro (CAV). (19) Salas (SAL). (20) Cuera (CU). Solid dots denote populations for which genetic data are available (Alexandrino et. al., 2000). Pie diagrams represent colour pattern classes (see text for details): � -0, � � -1, � � -2, �� -3, � � -4, �� -5. 216 Alexandrino et al. – Morphological variation in golden-striped salamanders Statistica/w 4.5 (StatSoft, 1993). Following confi r- mation that size explained most of the observed variability (fi rst axis explained c. 54% of the vari- ance, with variable negative correlations of 0.65– 0.83), the residuals of the regression of log against logSVL were used, hereafter denoted by the symbol # linked to the variable name. Trend surface maps were generated by kriging under default set- tings in Surfer 6.0 (Golden Software, 1996) geosta- tistical software. Morphological differentiation, expressed by pairwise euclidean distances between population means (both of size adjusted variables and scores of size adjusted PCA), was tested against the inde- pendent variables geographic distance (measured on 1:100,000 maps) and group membership by partial Mantel tests (RT 2.0; Manly, 1996), with 10000 randomizations. Similarly, morphometric variability (population distances calculated from the variance on the PCA axes) and colour pattern vari- ability (population distances calculated from the coeffi cient of variation) were tested against genetic heterozygosity and hybridity (Alexandrino et al., 2000). Hybridity distinguished populations located within and outside the putative contact zone (nos. 3, 4, 6 and 7 versus the others). To evaluate the a posteriori classification of individuals in each population to either group 1 or group 2, Discrimi- nant Analysis (DA) was performed on size adjusted morphological measurements with Statistica/w 4.5 (StatSoft, 1993). The hypothesis of association between morphological variation and latitude was tested through linear regression of PCA1 scores with distance along a south to north axis. Results Morphometrics The fi rst and the second PCA axes explained, respec- tively, c. 47% and c. 20% of the variance, for both males and females (Table 1). The fi rst axis had high factor loadings for limb and digit variables (0.76 to 0.89). The second axis had high loadings for head length and head width (0.54 to 0.84). Hence, the variation observed for head shape and the size of the extremities was largely independent. Mean popula- tion factor scores for the fi rst axis increased from south to north, refl ecting an increase in relative size of limbs and digits (Figs. 3 and 6). Mean factor scores for the second axis did not show any readily interpret- able geographic pattern (Fig. 3). Colour pattern Colour pattern type 2 was the most common one in all examined populations, except for populations 10 and 11 in which type 3 was most frequent (Fig. 1). Fig. 2. Morphometric measurements taken in 18 populations of Chioglossa lusitanica: (1) SVL, snout-vent length. (2) HL, head (snout-gular) length. (3) HW, head width. (4) FLL, forelimb length. (5) HLL, hindlimb length. (6) TFL, third fi nger length. (7) FTL, fourth toe length. Table 1. Factor loadings for the fi rst and second Principal Compo- nents axis for six size adjusted (#) morphometric measurements taken in 18 populations of Chioglossa lusitanica. Females Males Variables PCA 1 PCA 2 PCA 1 PCA 2 Head length (HL#) 0.195 0.710 0.483 0.536 Head width (HW#) 0.157 0.800 0.158 0.841 Forelimb length (FLL#) 0.762 -0.193 0.800 -0.248 Hindlimb lenth (HLL#) 0.845 -0.109 0.803 -0.331 Third fi nger length (TFL#) 0.834 0.008 0.772 -0.102 Fourth toe length (FTL#) 0.885 -0.038 0.805 0.007 Variance explained 47.3% 20.2% 46.5% 19.6% 217Contributions to Zoology, 74 (3/4) – 2005 Type 3 was present in all populations south of the Douro River. Other pattern types were present in some populations and not in others. Types other than type 2 were absent north of the Douro, with the ex- ception of type 4 that was present, albeit at low fre- quency, in populations 12, 13 and 20. Testing causal hypotheses Scores on the fi rst PCA axis were signifi cantly as- sociated with geographic distance, group membership (Table 2) and latitudinal distance (Fig. 5). Scores on the second axis were not associated with either of the Fig. 3. First two axes from PCA of six size adjusted morphometric variables in 18 populations of Chioglossa lusitanica. Population centroids and minimum convex polygons enclosing individual factor scores for group 1 and group 2 populations. Open dots and dashed polygon lines represent southern ‘group 1’ populations; solid dots and uninterrupted polygon lines represent northern ‘group 2’ populations. Fig. 4. Means and standard deviations for the a posteriori prob- ability (P) of classifi cation of individuals in each population to northern group 2, from Discriminant Analysis of six size ad- justed morphometric variables in 18 populations of Chioglossa lusitanica. 218 Alexandrino et al. – Morphological variation in golden-striped salamanders independent variables (Table 2). Classifi cation of individuals (both females and males) in group 2, following DA, increased gradually from south to north (Fig. 4). In females, head length and head width were associated with geographic distance and group membership, respectively. Size of the extremities was associated with geographic distance, with the excep- tion of FLL# in males. Digit length and HLL# in males were also associated with group membership. Colour pattern was associated to neither geographic distance or group membership. Morphometric vari- ability was not associated with any of the formulated independent variables (Table 3). Colour pattern variability was associated with hybridity and not with geographic distance or heterozygosity. Additionally, a trend was observed for colour pattern variability to decrease from south to north in group 2 populations (Fig. 1). Discussion Morphometric variation in the endemic Iberian salamander Chioglossa lusitanica was found to be Fig. 5. Regression of mean factor scores of the fi rst Principal Component axis of 18 populations of female Chioglossa lusi- tanica against distance along a south-north axis. Table 2. Partial Mantel test results for association between mor- phological differentiation across 18 populations of Chioglossa lusitanica versus geographic distance and group membership (see text for details). Dependent variables are A) top panel, the fi rst and second PCA-axis and B) lower panel, individual size adjusted (#) morphometric variables and colour pattern. Causal hypothesis (independent variables) Morphological distance Geographic Group (dependent variable) distance membership A) PCA 1 (females) *** ** PCA 1 (males) * *** PCA 2 (females) ns ns PCA 2 (males) ns ns B) HL# (females) * ns HL# (males) ns ns HW# (females) ns ** HW# (males) ns ns FLL# (females) *** ns FLL# (males) ns ns HLL# (females) *** ns HLL# (males) * * TFL# (both sexes) * *** FTL# (both sexes) ** *** Colour pattern (both sexes) ns ns ns - not signifi cant, * - p<0.05, ** - p<0.01, *** - p<0.001. Table 3. Partial Mantel test results for association between morphological variability across 18 populations of Chioglossa lusitanica versus geographic distance, heterozygosity and hybrid versus non-hybrid population status. Causal hypothesis Morphological (independent variables) variability distance Geographic Heterozygosity Hybridity (dependent variable) distance PCA1 (females) ns ns ns PCA1 (males) ns ns ns PCA2 (females) ns ns ns PCA2 (males) ns ns ns Colour pattern (both sexes) ns ns ** ns - not signifi cant, ** - p<0.01 219Contributions to Zoology, 74 (3/4) – 2005 Fig. 6. Trend surface map generated by the kriging of mean fac- tor scores of the fi rst Principal Component axis of 18 populations of female Chioglossa lusitanica. consistent with documented genetic differentiation. Group 1, from south of the Mondego river in Por- tugal, is characterised by shorter digits than group 2, from north of the Mondego. A pattern of south to north clinal variation with increasing limb, toe and fi nger length was found superimposed on this di- chotomy, resulting in stepped clines for each of the characters describing appendage size. We suggest that both historical, vicariant isolation and selection processes account for the observed variation. Short appendages, with a low volume to surface ratio, may represent an adaptation to xeric (i.e. less moist) environments (Nevo, 1972; Lee, 1993). Chioglossa lusitanica is a terrestrial streamside salamander extremely dependent on moist habitats and indeed the level of annual precipitation is the main predic- tor of its range in Portugal (Teixeira et al., 2001; Arntzen and Teixeira, 2006). Given that southern populations appear to occupy a more xeric environ- ment than northern populations (Arntzen and Alex- andrino, 2004) and assuming that rainfall gradients in the past paralleled those of today, selection could have produced the documented (stepped) clines. The general pattern of contemporary restricted gene fl ow between populations of C. lusitanica recently re- vealed by microsatellite data (F. Sequeira et al., unpublished data) would favour local adaptation along selection gradients, originating the observed morphological pattern of latitudinal variation. The action of either selection or stochastic drift, or both, associated with vicariant isolation may have origi- nated the stepped clines at appendage size, across the Mondego. Neither colour pattern nor colour pattern variabil- ity was associated with group membership or with geographic distances between populations. How- ever, colour pattern variability was higher within the group 1-2 contact zone than elsewhere, suggest- ing that the mixing of differentiated gene pools increased phenotypical variability. Two more paral- lels between morphology and genetics were found within group 2 populations. First, a south to north decrease was observed in genetic and colour pattern variability. The processes of sequential bottleneck- ing and drift invoked to explain the decrease in genetic variation (Alexandrino et al., 2000) appear equally applicable to morphological variation. Sec- ondly, the dominance of the otherwise rare colour type 3 in populations 10 and 11, immediately south of the Douro, may refl ect a separate historical refu- gium in this area, as was suggested by the local presence of unique nuclear and mtDNA alleles (Alexandrino et al., 2000, 2002). The genetic subdivision of C. lusitanica is not matched by an equally pronounced morphological differentiation. Selection operating along environ- mental gradients appears to be more important in shaping phenotypic diversity than genetic isolation. Cryptic differentiation and clines are phenomena common to many amphibian species (Larson, 1984; Green et al., 1996), streamside salamanders in par- ticular (Good and Wake, 1992; Carlin, 1997; Tarkhnishvili et. al., 2000). For example, C lusi- tanica and Mertensiella caucasica share ancestry at 14-15 million years before present (Veith et al., 1998) with the further intraspecifi c genetic differentiation (roughly 1-2 and ~10 million years before present, respectively) being accompanied by morphological stasis (Alexandrino et al. 2000, present paper; 220 Alexandrino et al. – Morphological variation in golden-striped salamanders Tarkhnishvili et al., 2000). The two species show strong interdependences in ecological, morpho- physiological, reproductive and developmental fea- tures associated to a streamside life-history (Tarkhnishvili, 1994). In combination with the re- markable evolutionary convergence observed among streamside salamanders, such as C. lusitanica and phylogenetically unrelated plethodontid species (e. g., Eurycea longicauda; Wake and Özeti, 1969), this suggests that constraints are in place that prevent departure from a highly specialized morphotype. Acknowledgements This work was supported by the ‘Instituto da Con- servação da Natureza’ (ICN), the ‘LIFE’ program of the European Community and the ‘Fundação para a Ciência e a Tecnologia’ (PRAXIS XXI / BD / 5917 / 95 PhD grant to JA and FCT project # PRAXIS/P/ BIA/11174/1998). Licenses to collect were provided by ICN for Portugal and by the Galician and Astu- rian ‘Junta de Medio Ambiente’ for Spain. We thank E. Froufe, C. Oliveira, F. Sequeira, D. Tarkhnishvili and J. Teixeira for support and discussions. References Alexandrino J, Ferrand N, Arntzen JW. 1997. Genetic varia- tion in some populations of the golden-striped salamander, Chioglossa lusitanica (Amphibia: Urodela), in Portugal. Bi- ochem. Genet. 35: 371-381. Alexandrino J, Froufe E, Arntzen JW, Ferrand N. 2000. Ge- netic subdivision, glacial refugia and postglacial recoloniza- tion in the golden-striped salamander, Chioglossa lusitanica (Amphibia: Urodela). Mol. Ecol. 9: 771-781. Alexandrino J, Arntzen JW, Ferrand N. 2002. 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For character codes see Table 1, for localities see Fig. 1. Locality Sex N SVL HL HW FLL HLL TFL FTL CP 1 Females 8 43.32 (2.38) 11.41 (0.34) 6.70 (0.34) 11.85 (0.60) 12.67 (0.32) 2.14 (0.19) 3.14 (0.26) 2.27 (0.65) Males 2 42.31 (0.07) 10.98 (0.45) 6.66 (0.02) 11.51 (0.66) 12.55 (0.22) 2.04 (0.05) 2.85 (0.04) 2 Females 28 45.85 (2.13) 11.64 (0.44) 6.91 (0.30) 12.21 (0.37) 14.04 (0.46) 2.50 (0.17) 3.56 (0.22) 2.26 (0.63) Males 23 44.61 (1.57) 11.33 (0.33) 6.90 (0.25) 12.67 (0.30) 13.94 (0.39) 2.37 (0.15) 3.53 (0.28) 3 Females 10 46.76 (2.99) 11.97 (0.61) 7.39 (0.41) 12.01 (0.28) 13.81 (0.55) 2.42 (0.16) 3.35 (0.22) 2.79 (0.94) Males 10 45.73 (0.96) 11.62 (0.31) 7.32 (0.36) 12.26 (0.49) 13.92 (0.32) 2.34 (0.18) 3.46 (0.12) 4 Females 11 46.16 (2.65) 12.09 (0.58) 7.20 (0.41) 11.80 (0.72) 13.62 (0.78) 2.26 (0.22) 3.23 (0.29) 2.21 (0.59) Males 10 46.61 (1.13) 11.84 (0.39) 7.27 (0.23) 12.57 (0.41) 13.88 (0.39) 2.39 (0.12) 3.34 (0.17) 5 Females 12 46.62 (3.54) 11.95 (0.76) 7.13 (0.50) 12.29 (0.80) 14.07 (0.66) 2.40 (0.16) 3.31 (0.32) 2.41 (0.80) Males 12 46.12 (1.30) 11.44 (0.21) 7.12 (0.31) 12.74 (0.46) 13.94 (0.40) 2.36 (0.14) 3.28 (0.22) 6 Females 6 45.61 (3.41) 11.66 (0.74) 7.29 (0.59) 11.64 (0.23) 13.31 (0.36) 2.39 (0.13) 3.33 (0.10) 3.45 (1.43) Males 7 46.56 (1.57) 11.75 (0.33) 7.16 (0.16) 12.58 (0.51) 14.07 (0.37) 2.56 (0.13) 3.68 (0.17) 7 Females 8 49.44 (3.43) 12.50 (0.79) 7.80 (0.31) 12.80 (0.48) 14.52 (0.56) 2.56 (0.38) 3.58 (0.51) 2.95 (1.34) Males 17 48.01 (1.29) 12.11 (0.30) 7.78 (0.30) 13.15 (0.68) 14.69 (0.59) 2.59 (0.33) 3.69 (0.39) 8 Females 9 45.40 (2.49) 11.87 (0.49) 7.15 (0.40) 12.27 (0.42) 13.87 (0.46) 2.50 (0.16) 3.53 (0.20) 2.05 (0.88) Males 17 44.90 (1.02) 11.52 (0.27) 6.97 (0.35) 12.60 (0.43) 13.91 (0.65) 2.46 (0.17) 3.65 (0.29) 9 Females 9 45.94 (2.51) 12.32 (0.68) 7.38 (0.39) 12.21 (0.47) 13.66 (0.54) 2.72 (0.10) 3.78 (0.14) 2.24 (0.89) Males 10 45.23 (1.76) 12.12 (0.41) 7.39 (0.36) 12.97 (0.30) 13.95 (0.26) 2.79 (0.14) 3.92 (0.24) 10 Females 3 47.77 (1.34) 12.07 (0.48) 7.32 (0.32) 13.11 (0.60) 14.88 (0.65) 2.83 (0.12) 3.76 (0.17) 2.82 (0.64) Males 9 45.52 (1.20) 11.88 (0.35) 7.24 (0.23) 14.03 (0.43) 15.05 (0.49) 2.74 (0.31) 4.11 (0.16) 11 Females 19 46.41 (3.34) 11.95 (0.55) 7.27 (0.41) 12.07 (0.82) 14.19 (0.74) 2.67 (0.21) 3.75 (0.30) 2.90 (0.32) Males 7 45.30 (0.93) 11.64 (0.24) 7.40 (0.28) 12.44 (0.25) 14.17 (0.41) 2.68 (0.10) 3.64 (0.14) 12 Females 11 45.01 (1.58) 11.78 (0.34) 7.10 (0.25) 12.24 (0.41) 13.92 (0.43) 2.65 (0.10) 3.75 (0.23) 2.09 (0.42) Males 23 44.02 (0.98) 11.39 (0.25) 7.11 (0.24) 12.49 (0.43) 13.82 (0.46) 2.51 (0.12) 3.68 (0.18) 13 Females 10 45.60 (1.56) 11.82 (0.33) 7.23 (0.25) 12.33 (0.54) 14.06 (0.29) 2.52 (0.10) 3.74 (0.26) 2.10 (0.44) Males 10 44.67 (0.86) 11.36 (0.29) 7.37 (0.23) 12.78 (0.57) 13.99 (0.50) 2.53 (0.25) 3.80 (0.24) 15 Females 10 43.91 (3.92) 11.55 (0.70) 6.95 (0.48) 12.59 (0.67) 14.42 (0.76) 2.87 (0.23) 4.04 (0.35) 2.00 (0.00) Males 6 44.13 (1.36) 11.43 (0.24) 6.87 (0.12) 12.69 (0.50) 14.30 (0.43) 2.70 (0.16) 4.14 (0.37) 16 Females 13 46.01 (1.86) 12.07 (0.44) 7.20 (0.37) 12.83 (0.41) 14.58 (0.54) 2.70 (0.21) 4.09 (0.27) 2.00 (0.00) Males 8 44.05 (1.36) 11.49 (0.50) 7.20 (0.30) 13.11 (0.35) 14.17 (0.31) 2.65 (0.20) 3.99 (0.26) 18 Females 10 47.87 (1.97) 11.91 (0.36) 7.50 (0.58) 12.60 (0.39) 14.48 (0.56) 2.84 (0.10) 4.01 (0.15) 2.00 (0.00) Males 5 47.01 (0.84) 11.75 (0.25) 7.39 (0.23) 13.07 (0.20) 14.68 (0.28) 2.74 (0.18) 4.06 (0.17) 19 Females 6 44.63 (3.57) 12.05 (0.55) 7.20 (0.43) 12.94 (0.79) 14.77 (0.66) 2.81 (0.32) 4.05 (0.30) 2.00 (0.00) Males 9 46.40 (1.38) 12.04 (0.13) 7.25 (0.25) 13.64 (0.74) 14.87 (0.64) 2.89 (0.14) 4.12 (0.16) 20 Females 3 45.72 (3.87) 12.14 (0.90) 7.12 (0.50) 13.26 (0.42) 15.00 (0.54) 2.71 (0.09) 4.04 (0.04) 2.29 (0.76) Males 4 43.73 (2.75) 11.61 (0.80) 6.71 (0.22) 13.18 (0.85) 14.76 (0.42) 2.61 (0.19) 3.92 (0.21) << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /None /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Error /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJDFFile false /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.0000 /ColorConversionStrategy /CMYK /DoThumbnails false /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness true /PreserveHalftoneInfo false /PreserveOPIComments true /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages true /ColorImageMinResolution 300 /ColorImageMinResolutionPolicy /OK /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 1800 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 1800 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1800 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile () /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /Description << /CHS /CHT /DAN /DEU /ESP /FRA /ITA /JPN /KOR /NLD (Gebruik deze instellingen om Adobe PDF-documenten te maken die zijn geoptimaliseerd voor prepress-afdrukken van hoge kwaliteit. De gemaakte PDF-documenten kunnen worden geopend met Acrobat en Adobe Reader 5.0 en hoger.) /NOR /PTB /SUO /SVE /ENU (Use these settings to create Adobe PDF documents best suited for high-quality prepress printing. Created PDF documents can be opened with Acrobat and Adobe Reader 5.0 and later.) >> /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [ << /AsReaderSpreads false /CropImagesToFrames true /ErrorControl /WarnAndContinue /FlattenerIgnoreSpreadOverrides false /IncludeGuidesGrids false /IncludeNonPrinting false /IncludeSlug false /Namespace [ (Adobe) (InDesign) (4.0) ] /OmitPlacedBitmaps false /OmitPlacedEPS false /OmitPlacedPDF false /SimulateOverprint /Legacy >> << /AddBleedMarks false /AddColorBars false /AddCropMarks false /AddPageInfo false /AddRegMarks false /ConvertColors /ConvertToCMYK /DestinationProfileName () /DestinationProfileSelector /DocumentCMYK /Downsample16BitImages true /FlattenerPreset << /PresetSelector /MediumResolution >> /FormElements false /GenerateStructure false /IncludeBookmarks false /IncludeHyperlinks false /IncludeInteractive false /IncludeLayers false /IncludeProfiles false /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe) (CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /DocumentCMYK /PreserveEditing true /UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling /UseDocumentProfile /UseDocumentBleed false >> ] >> setdistillerparams << /HWResolution [2400 2400] /PageSize [612.000 792.000] >> setpagedevice