Orton F , Säfholm M , Jansson E , Carlsson Y , Eriksson A , Fick J , Uren Webster T , McMillan T , Leishman M , Verbruggen B , Economou T , Tyler CR , Berg C .

	Figure 1. Experimental design. Fertilised eggs were exposed to flutamide or linuron until life stage 66 when they were transferred to clean water. Gene expression analysis on a set of target genes was conducted to identify their utility for distinguishing gonadal sex and to investigate the effects of anti-androgens on their expression in developing tadpoles (during sensitive windows of sexual development). Effects of anti-androgens on gonadal development and sex ratio were assessed in metamorphs via histopathology. Frogs were then maintained to adulthood in clean water when they were assessed for reproductive competence and morphology. Breeding studies were conducted to assess the effects of early life exposure to anti-androgens on the gonadal sexual development, secondary sex characters in males (nuptial pads and forearm) and breeding success (fecundity, fertility and time in amplexus). a=sex determination, b=sex differentiation, c=completed sex differentiation. SSC=secondary sexual characteristic, exp.=experimental.
	Figure 2. Sex ratio (a) in stage 51–53 tadpoles (control/linuron low n = 24, linuron high n = 23, flutamide n = 16), stage 55–58 tadpoles (control/linuron high n = 24, linuron low/flutamide n = 16), metamorphs (control n = 37, linuron low n = 24, linuron high n = 33, flutamide n = 11) and adults (control n = 82, linuron low n = 84, linuron high n = 85, flutamide n = 47). Solid bars = males, dotted bars = females. Gonadal differentiation in female (b) control n = 12, linuron low n = 10, linuron high n = 9, flutamide n = 4) and male (c) control n = 23, linuron low n = 10, linuron high n = 22, flutamide n = 5) metamorphs, and female (D: control/linuron low/linuron high n = 9, flutamide n = 7) and male (E: control/linuron low/linuron high/ flutamide: n = 10) adults. *indicates significant difference compared with control group.
	Figure 3. Relationship between cyp19 and amh for distinguishing gonadal sex in tadpole gonado-mesonephros complex (GMC: a,b), gonadal ontogeny of gene expression (dmrt1, cyp17, amh, ar, foxl2, cyp19) in control tadpoles (c,d) and the interrelationships between these genes in control tadpole GMC (e,f). Unsupervised cluster analysis was used to delineate ‘males’ (black) and ‘females’ (red) during sex determination (a) and sex differentiation (b) for all treatment groups collectively (data not shown for dmrt1 v. foxl2/cyp19 or amh v. foxl2 as outcomes of the analysis did not differ). Each letter represents one individual and the asterices indicate the centre of each cluster (C=control, L=linuron low, H=linuron high, F=flutamide). Ontogeny of gene expression in males (normal, filled symbols) and females (italic, open symbols) tadpole GMC (c,d), bold indicates significant change during ontogeny using linear regression analysis *expression is relative to gonad pool standards (see methods for details). Interrelationships between gene expression in male (e) and female (f) GMC analysed using Pearsons’ (or Spearmans’ for non-parametric data), *p < 0.05, **p < 0.01, ***p < 0.001.
	Figure 4. Effects of anti-androgens on expression of target genes during gonad ontogeny (NF stage 51–58). Linear regression of dmrt1 (a), cyp17 (b), amh (c), ar (d), foxl2 (e) and cyp19 (f). Black lines = control males (dmrt1 r2 = 0.59, cyp17 r2 = 0.62, amh r2 = 0.64, foxl2/cyp19 r2 = ns) green lines = linuron low males (dmrt1 r2 = 0.57, cyp17 r2 = 0.67, amh r2 = 0.53, foxl2/cyp19 r2 = ns), blue lines = linuron high males (dmrt1 r2 = 0.79, cyp17 r2 = 0.51, amh r2 = 0.65, foxl2 r2 = 0.58, cyp19 r2 = ns), red lines = flutamide males (dmrt1 r2 = 0.81, cyp17 r2 = 0.83, amh r2 = 0.9, foxl2 r2 = 0.33, cyp19 r2 = ns), grey lines = control females (dmrt1 r2 = 0.41, cyp17 r2 = ns, amh r2 = 0.33, foxl2 r2 = 0.52, cyp19 r2 = ns), light green = linuron low females (dmrt1 r2 = 0.37, cyp17 r2 = ns, amh r2 = 0.79, foxl2 r2 = 0.81, cyp19 r2 = ns), light blue = linuron high females (dmrt1 r2 = 0.42, cyp17 r2 = ns, amh r2 = ns, foxl2 r2 = 0.31, cyp19 r2 = 0.35), pink = flutamide females (dmrt1 r2 = ns, cyp17 r2 = ns, amh r2 = 0.32, foxl2 r2 = 0.50, cyp19 r2 = ns); with matching filled (male) or open (female) symbols. For MANCOVA analysis during sex determination (stage 51–53) or sex differentiation (stage 55–58) effects of flutamide were observed on foxl2 levels in ‘females’ (see Supplementary Table S5 for numerical values/more details). Dashed lines represent significant differences in the linear regression slope compared with controls (ANCOVA, stage as a covariate). *relative gene expression compared to gonad pool standards.
	Figure 5. Effects of antiandrogens on expression of target genes in the brain in both male (circles) and female (diamonds) metamorphs (NF stage 66). Dmrt1 (a), cyp17 (b), amh (c), ar (d), foxl2 (e) and cyp19 (f) expression in controls (filled symbols), linuron low (open symbols), linuron high (dotted symbols) and flutamide (crossed symbols). Control male, n = 22; control female, n = 10; linuron low male, n = 9; linuron low female, n = 7; linuron high male, n = 22; linuron high female, n = 10; flutamide male, n = 5; flutamide female, n = 4. *relative gene expression compared to brain pool standards (see methods for details).

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