Embryo Development in Extremophiles Figure 2. Comparative embryo development of two ecotypes of P. mexicana and of P. sulphuraria. Stages range from Stage 15 to Stage 30. See Table 1 for details. Scale bar = 1 mm. doi:10.1371/journal.pone.0027377.g002 3.2. Yellowness of the yolk Neither the covariate ‘‘developmental stage’’ (F1,163 = 0.00, P = 0.99) nor the interaction of ‘‘ecotype by developmental stage’’ (F2,163 = 1.11, P = 0.33) had an effect on the yellowness of Poecilia ova, but the model clearly revealed a significant difference between ecotypes (F2,163 = 39.88, P,0.001). Since neither the covariate nor the interaction terms were significant, we conducted post hoc tests from a simplified model that only included the factor ‘‘ecotype’’. This analysis revealed that all three mollies differed significantly from one another (Fisher’s LSD, surface vs. sulphur: P = 0.028; all other comparisons: P,0.001; Figure 4). 3.3. Embryo development: how to increase offspring size 3.3.1. Embryo mass and matrotrophy index (MI). Embryos lost weight during the course of gestation in all three mollies (‘‘developmental stage’’: F1,132 = 44.95, P,0.001); we did not find a significant interaction of ‘‘ecotype by developmental stage’’ though (F2,132 = 0.00, P = 0.99), suggesting no heterogeneity of slopes (surface mollies, N = 63; cave mollies, N = 31; sulphur mollies, N = 44; Figure 5A). However, the three mollies differed drastically from each other in embryo mass (‘‘ecotype’’: F2,132 = 24.38, P,0.001), with cave molly embryos being by far the heaviest, sulphur molly embryos being intermediate in dry mass, and surface molly embryos being the lightest (Figure 5A). This also resulted in different estimated MI values for all three mollies despite similar slopes (surface molly = 0.62; cave molly = Figure 3. Comparative embryo development of two ecotypes of P. mexicana and of P. sulphuraria. Stages range from Stage 35 to the ready-to-be-born embryo (Stage 50). See Table 1 for details. Scale bar = 1 mm. doi:10.1371/journal.pone.0027377.g003 0.77; sulphur molly = 0.69; Figure 5A). In other words: sulphur and especially cave mollies initiated development with heavier eggs, but all employed a similar, mainly lecithotrophic provisioning strategy. 3.3.2. Ovum/embryo diameter. ‘‘Ecotype’’ (F2,141 = 15.48, P,0.001), ‘‘developmental stage’’ (F1,141 = 236.35, P,0.001), and the interaction of ‘‘ecotype by developmental stage’’ (F2,141 = 9.54, P,0.001) all had significant influence on ovum/embryo diameter (surface mollies, N = 69; cave mollies, N = 37; sulphur mollies, N = 41; Figure 5B). While the diameter increased in all three mollies, it did so to a much stronger extent in the two extremophile mollies. Furthermore, surface mollies and sulphur mollies appear to have approximately similar sized ripe ova at the point of fertilization, while cave mollies initiated development with larger unfertilized ova (Figure 5B). (Note the apparent discrepancy to the pattern seen in ovum mass at fertilization, which was higher in sulphur than surface mollies, see above). The ovum diameter at stage 2 was significantly different between ecotypes (surface mollies, N = 9; cave mollies, N = 5; sulphur mollies, N = 4; ANOVA: F2,15 = 6.46, P = 0.009) and post- hoc tests revealed that this was due to significant differences between cave mollies and both other ecotypes (Fisher’s LSD, cave vs. surface: P = 0.015; cave vs. sulphur: P = 0.004; surface vs. sulphur: P = 0.224). 3.3.3. Embryo fat content. Embryos of P. mexicana lost fat during the course of development, albeit at different rates, while PLoS ONE | www.plosone.org 5 November 2011 | Volume 6 | Issue 11 | e27377