Embryo Development in Extremophiles Figure 6. Relative head length in three types of mollies. (A) Scatter plots showing the ‘head length to SL’ ratio vs. stage of development in molly embryos from one benign and two extreme habitats. Stages 0, 2 and 5 are not depicted, because they lack a differentiated head. (B) Box plots (median and IQR) for the head length to SL ratio in adult fish (N = 10 per population). P-values refer to Fisher’s LSD post hoc tests. Test statistics for both graphs are presented in the results section. doi:10.1371/journal.pone.0027377.g006 but closer inspection also suggests that the interaction effect is due to the stronger decrease in relative eye size in cave mollies compared to the other two populations (Figure 7A). Comparison of the absolute eye diameter between mid-stage (stage 35) and late- stage (stage 50) embryos revealed that this strong decrease in relative eye size was due to an arrest of eye growth in cave mollies, in which the median eye diameter at stage 35 measured 0.81 mm (IQR = 0.19 mm) versus 0.72 mm (IQR = 0.16 mm) at stage 50 (Mann-Whitney U-test: U12 = 14.50, P = 0.37). By contrast, eyes still grew larger in surface mollies (U13 = 40.00, P = 0.002); the median eye diameter at stage 35 was 0.74 mm (IQR = 0.11 mm), and 1.09 mm (IQR = 0.18 mm) at stage 50. Due to limited sample size, this comparison was not possible for sulphur mollies. The ratio of ‘eye diameter to SL’ was significantly different between adults of the three different mollies (N = 10 for all mollies; F2,27 = 31.00, P,0.001) and post-hoc tests revealed that this was due to cave mollies having smaller eyes than either surface mollies (Fisher’s LSD: P,0.001) or sulphur mollies (P,0.001; Figure 7B). Figure 7. Relative eye size in three types of mollies. (A) Scatter plots of eye diameter to SL ratio vs. stage of development in molly embryos from one benign and two extreme habitats. Stages 0, 2 and 5 are not depicted, because they lack a differentiated head. (B) Box plots (median and IQR) for the ‘eye diameter to SL’ ratio in adult fish (N = 10 per population). P-values refer to Fisher’s LSD post hoc tests. Test statistics for both graphs are presented in the results section. doi:10.1371/journal.pone.0027377.g007 Discussion General embryo development Pigmentation patterns are thought to play a major role in speciation events because they are important visual cues in species recognition, mate choice, and predator avoidance [74]. Cave organisms, usually living in constant darkness, are often charac- terized by the absence or reduction in melanin body pigmentation [75]. It has long been established that cave mollies, when caught in the Cueva del Azufre, are devoid of most visible pigmentation patterns otherwise characteristic for P. mexicana ([19]; reviewed in [38,50]). Nonetheless, once exposed to light in the laboratory environment, cave mollies partly regain the species-specific colour patterns over a period of several weeks, and a recent analysis revealed that, while their numbers are reduced compared to surface mollies, cave mollies do have melanophores (BL Joachim, I Schlupp, R Riesch, unpublished data). Our results are congruent with this pattern, because cave molly embryos clearly develop melanophores just like other surface-dwelling poeciliids at around PLoS ONE | www.plosone.org 7 November 2011 | Volume 6 | Issue 11 | e27377