ovum at fertilization (e.g., [24,55]). If the eggs were fully provisioned by yolk prior to fertilization (lecithotrophy), then we would expect the embryos to lose 35–40% of their dry mass during development (MI between 0.60 and 0.65; [58,73]). On the other hand, in the case of continuous maternal provisioning even after fertilization (matrotrophy), one would expect the embryos to lose less weight (MI between 0.65 and 1.00) or to even gain weight during development (MI.1.00; e.g., [55]). Thus, maternal provisioning was evaluated by analyzing the relationship between log-transformed embryonic dry mass and stage of development by means of linear regression analysis [55]. For this analysis, data for one of the two species examined, namely P. mexicana (surface and cave mollies), were largely reanalyzed from two previous studies [23,24], but new data were also included. 2.4. Measuring ovum diameter and collection of morphological data Using Spot Imaging Software, version 4.5 (Diagnostic Instru- ments), photos were size calibrated for the specific microscope magnification used to take each picture. Then, using the ‘measurements’ function integrated into Spot Imaging Software, we measured the following traits: ovum diameter [mm] (maximum diameter of ovum and/or embryo), eye diameter [mm], head length [mm] (from tip of embryo mouth to end of head capsule), and body length [mm] (from tip of embryo mouth to caudal peduncle). We then used these traits to calculate two indices, ‘‘relative eye size (eye diameter divided by body length)’’ and ‘‘relative head size (head length divided by body length)’’. These two indices were also calculated for measurements taken from ten randomly selected photographs of adult mollies. 2.5. Statistical analyses Previous studies have demonstrated that the population means for the traits reported here are similar between surface molly populations from southern Mexico (i.e., R´ıo Amatan and Arroyo Bonita) [22–25] and thus both were pooled to increase sample size and statistical power. For the analyses on information extracted directly from the embryonic pictures, for each developmental stage we used offspring from 2.3361.07 (mean6SD) females in surface mollies, 1.6760.65 females in cave mollies, and 1.7560.62 females in sulphur mollies. In other words, some data points for any given stage are from embryos of the same clutch, while other data points are from embryos of different clutches. The analyses on embryo weight and fat, however, utilized data from a much larger data set. Here, each data point is fully independent and represents the mean for all embryos of a single clutch. For embryos of all three types of mollies, we conducted six separate ANCOVAs. For each model ‘‘stage of development’’ served as the covariate and ‘‘ecotype (surface molly vs. cave molly vs. sulphur molly)’’ the independent variable. To test for heterogeneity of slopes, the interaction of ‘‘ecotype by stage of development’’ was also included. The dependent variables in the different ANCOVA models were ‘‘yellowness of the yolk’’, ‘‘embryo dry weight’’, ‘‘ovum diameter’’, ‘‘embryo fat content’’, ‘‘relative eye size’’, and ‘‘relative head size’’, respectively. For adult mollies we conducted two separate one-way-ANOVAs to investigate differences between ecotypes. The first tested for differences in ‘‘relative eye size’’ and the second for differences in ‘‘relative head size’’. In case of significant differences among groups, we then conducted Fisher’s LSD post hoc tests. All statistical analyses were conducted with PASW Statistics 18.0.3 (IBM SPSS). Embryo Development in Extremophiles Results 3.1. General embryo development In agreement with the standardized protocol (Table 1), development of eye pigmentation progressed in a similar fashion starting with the unpigmented optic cups in stage 10 embryos all the way to fully pigmented eyes in stage 20 embryos (Figures 1 and 2). Between stages 20 and 25 the first melanophores appeared in all embryos, including cave mollies (Figure 2). Body pigmentation first appeared above the brain in stage 25 and then appeared along the horizontal dorsal midline (Stages 25 and 30; Figure 2). In general, melanophore pigmentation in cave molly embryos followed similar developmental trajectories as in surface or sulphur molly embryos; however, at stages 40–50 cave molly embryos appeared slightly paler, and individual pigment cells seemed to be smaller than in both other mollies (Figure 3), suggesting a decrease in pigmentation late in development for cave mollies. While 31 out of 45 (69%) P. sulphuraria adults had characteristic lower lip protuberances (Figure S1; please refer to the Supporting Figure Legend), we found no evidence for lip protuberances even in late-stage P. sulphuraria embryos (Figure 3). Figure 1. Comparative embryo development of two ecotypes of P. mexicana and of P. sulphuraria. Stages range from the unfertilised, but ripe oocyte (Stage 0), to the optic cup embryo (Stage 10). See Table 1 for details. Scale bar = 1 mm. Embryos of stage 2 are lateral view. Arrows at stage 5 indicate location of the primitive streak embryo; arrows at stage 10 indicate location of head and optic cups. doi:10.1371/journal.pone.0027377.g001 PLoS ONE | www.plosone.org 4 November 2011 | Volume 6 | Issue 11 | e27377