Fig. 1. Sex ratio response to temperature in the AB, EKW and NA strains. Percent male for the 28 vs. 36 °C comparison from three independent families each of A) AB strain (AB#1, solid square: n_♂/♀ = 31/26 vs. 33/5; AB#2, solid circle: n_♂/♀ = 9/23 vs. 31/10 and AB#3, solid triangle: n_♂/♀ = 17/15 vs. 34/3 fish). B) EKW strain (EKW#1, solid square: n_♂/♀ = 16/38 vs. 40/13; EKW#2, solid circle: n_♂/♀ = 20/26 vs. 53/1 and EKW#3, solid triangle: n_♂/♀ = 39/16 vs. 47/4 fish. C) Percent male from six independent NA families: three for the 28–34 °C comparison (NAD#4 open square: n_♂/♀ = 45/13 vs. 46/14; NAD#5 solid triangle: n_♂/♀ = 40/16 vs. 59/1 and NAD#6 open triangle: n_♂/♀ = 51/6 vs. 54/4 fish) and three for the 28–36 °C comparison (NAD#1, open circle: n_♂/♀ = 38/16 vs. 48/0; NAD#2, solid square: n_♂/♀ = 48/9 vs. 57/0 and NAD#3, solid circle: n_♂/♀ = 37/17 vs. 48/0 fish). A Fisher's exact test was applied individually for each family comparing phenotypic proportions of males and females between treatments. In all strains, each family was split into three tanks per temperature (technical replicate). Solid and open points are used only for improved clarity due to overlapping symbols. Abbreviations: * = P < 0.05; ** = P < 0.01; *** = P < 0.001; N.S. = not significant.

Fig. 2. Reaction norms of the masculinization rate as a function of genotype and environmental temperature. A) ZZ and ZW genotypes of NA. B) ZZ, ZW₁, ZW₂, W₁W₂, and W₁W₁ genotypes of EKW strain. C) EKW without distinguishing the two W chromosomes. Data shown as mean ± SD of all families tested within each strain.

Fig. 3. Effects of elevated temperature on testis and ovary morphology in adult AB and NA zebrafish exposed to 28°C and 36°C during sex differentiation. A) Percent of different germ cell developmental stages in testes of AB males (n_28°C = 3, n_36°C = 5), NA_ZZ males (n_28°C = 7, n_36°C = 7) and NA_ZW neomales (n_28°C = 7, n_36°C = 8). AStudent's t-test was used to compare the number of each germ cell type between temperature treatments. Abbreviations: type A undifferentiated spermatogonia (A_und), type A differentiated spermatogonia (A_diff), type B spermatogonia (B), spermatocytes (Spc), spermatids (Spd), and spermatozoa (Spz). B) Spermatozoa number per field for AB (n_28°C = 3, n_36°C = 5), NA_ZZ males (n_28°C = 7, n_36°C = 7) and NA_ZW neomales (n_28°C = 7, n_36°C = 8). A Student's t-test was used to compare the number of spermatozoa between different groups. Boxes include values between the lower and upper quartiles, the upper whisker = min (max(x), Q3 + 1.5 *IQR), the lower whisker = max (min(x), Q1 – 1.5 * IQR), where IQR = third quartile (Q3) – first quartile (Q1). The line inside the boxplots indicates the median. C) Percent of different germ cell developmental stages in ovaries of AB (n_28°C = 7, n_36°C = 4), and NA_ZW females (n_28°C = 6). Note: NA_ZW females at 36 °C were not available due to 100% masculinization in all families. A Student's t-test was used to compare the number of each germ cell type between the two temperatures. Abbreviations: primary growth (PG) oocytes, cortical alveolar (CA) oocytes, vitellogenic (Vtg) oocytes, postovulatory follicles (POFs), and atretic (A) oocytes. Data shown as mean ± SEM. * = P < 0.05; ** = P < 0.01; *** = P < 0.001; N.S. = not significant. For sample size in each group see Table S4. For stage descriptions see Supplementary Information and Fig. 4. For detailed information of zebrafish germ cells (Leal et al., 2009).

Fig. 4. Histological sections of adult zebrafish gonads in AB and Nadia strains. Testes of AB males, NA_ZW neomales and NA_ZZ males and ovaries of AB and NA_ZW females exposed to 28 °C (control) and 36 °C (high temperature) during sex differentiation. Abbreviations for males: type A undifferentiated spermatogonia (Aund), differentiated spermatogonia (Adiff), type B spermatogonia (B), spermatocytes (Spc), spermatids (Spd) and spermatozoa (Spz); for females: primary growth oocytes (PG), cortical alveolar oocytes (CA), vitellogenic oocytes (Vtg), postovulatory follicles (Pofs) and atretic oocytes (A). Staining: Hematoxylin and Eosin. Scale bar = 500 μm. Note: high temperature resulted in 100% males in NA, so no NA_ZW females were available for analysis.