Figure 4. Ovum yellowness as a proxy for carotenoid content. Measured as the b* component of the CIE l*a*b* colour mode in Adobe Photoshop CS5. Box plots (median and IQR). Yellowness was measured for all stages combined since a preliminary ANCOVA revealed that developmental stage was not correlated with ovum yellowness. Surface mollies, N = 72; cave mollies, N = 47; sulphur mollies, N = 50. ANCOVA details are presented in the results section; p-values refer to Fisher’s LSD post hoc tests. doi:10.1371/journal.pone.0027377.g004 sulphur molly embryos actually gained body fat during development. Accordingly, we found significant effects of ‘‘ecotype’’ (surface mollies, N = 63; cave mollies, N = 31; sulphur mollies, N = 44; F2,132 = 13.11, P,0.001) and the interaction of ‘‘ecotype by developmental stage’’ (F2,132 = 5.77, P = 0.004) on fat content; however, the effect of ‘‘developmental stage’’ was not significant (F1,132 = 1.75, P = 0.19; Figure 5C). Surface molly embryos began development with the highest percentage of body fat, but at around stage 40 fat content of surface and sulphur mollies converged. Cave mollies, on the other hand, had the lowest fat content of all three mollies throughout development (Figure 5C). 3.4. Embryo development: how to achieve divergent morphologies 3.4.1. Relative head size. In the ANCOVA (surface mollies, N = 31; cave mollies, N = 23; sulphur mollies, N = 20) on head length to SL ratio across embryo developmental stages, both the factor ‘‘ecotype’’ (F2,68 = 4.79, P = 0.011) and the covariate ‘‘developmental stage’’ (F1,68 = 7.66, P = 0.007) had a significant influence, but the interaction of ‘‘ecotype by developmental stage’’ was also significant (F2,68 = 10.19, P,0.001; Figure 6A). Inspection of the raw data indicates that relative head length decreases in surface mollies, but increases in cave and sulphur mollies (Figure 6A). The ratio of ‘head length to SL’ was significantly different between adults of the three ecotypes (N = 10 for all mollies; F2,27 = 72.50, P,0.001) and post-hoc tests revealed that this was due to significant differences between all three mollies (Fisher’s LSD, cave vs. surface and surface vs. sulphur: P,0.001; cave vs. sulphur: P = 0.007; Figure 6B). 3.4.2. Relative eye size. ANCOVA (surface mollies, N = 43; cave mollies, N = 45; sulphur mollies, N = 21) revealed significant effects of the covariate ‘‘developmental stage’’ (F1,103 = 39.16, P,0.001) and ‘‘ecotype by developmental stage’’ (F2,103 = 4.94, P = 0.009) on the ‘eye diameter to SL’ ratio across embryo PLoS ONE | www.plosone.org 6 November 2011 | Volume 6 | Issue 11 | e27377 Embryo Development in Extremophiles Figure 5. How to increase offspring size during development. Scatter plots depicting (A) embryo dry weight, (B) ovum diameter, and (C) embryo fat content in the course of development in molly embryos from one benign and two extreme habitats. Test statistics are presented in the results section. doi:10.1371/journal.pone.0027377.g005 development; however, the effect of ‘‘ecotype’’ was not significant (F2,103 = 0.28, P = 0.75; Figure 7A). Clearly, relative eye size decreases with increasing developmental stage in all populations,