molly offspring are significantly heavier throughout development. Similarly surprising is the pattern observed for embryo fat content during development. It decreased in both ecotypes of P. mexicana (i.e., surface and cave mollies), albeit to different degrees, but actually increased during development in sulphur mollies. How can the surprising patterns discovered in sulphur mollies be explained? We can only speculate at this point and future research will have to test our hypotheses. Apparently, sulphur mollies are able to create ova with an increased density of nutrients, which would explain how ova could be heavier, yet at the same time be of the same diameter as surface molly ova. Furthermore, we hypothesize that sulphur molly embryos are actually converting some of the nutrients originally stored in the ova into soluble body fats during embryo development. This would explain why sulphur molly embryos are the only ones in which fat content actually increased during development even though overall embryo weight continuously decreased. This pattern is congruent with recent life- history analyses on adult sulphur mollies [27] (R Riesch, M Plath, I Schlupp, RB Langerhans, unpublished data) that revealed sulphur mollies to have surprisingly high levels of body fat that even exceed those of surface mollies from benign habitats. Clearly, at this point in time we can only speculate on the underlying mechanisms for this; however, one has to keep in mind that sulphur and cave mollies are the result of independent coloniza- tions of sulphur habitats. Furthermore, the two habitats are quite different in their physical characteristics. The cave is not completely linear; in other words not all parts of all cave chambers are immediately downstream of the toxic springs, and the innermost cave chamber (XIII) is actually nontoxic [19]. So, cave mollies actually have a better opportunity to escape into microhabitats that are less toxic if the hydrogen sulphide discharge should temporarily increase. Sulphur mollies, in contrast, do not have this option to a similar extent. Almost their entire habitat is immediately downstream of the toxic springs, and if they evade toxicity too much they increase interspecific competition with another sulphur-endemic poeciliid (the widemouth gambusia, Gambusia eurystoma) that inhabits the more peripheral parts of the Ba n˜os del Azufre [27,30]. Given these and (potentially) additional differences (i.e., due to genetic drift), we do not find it unlikely that sulphur mollies evolved a coping mechanism that is lacking in cave mollies. Thus, we hypothesize that at some point during the evolutionary history of the sulphur molly, divergent metabolic pathways must have evolved that enable sulphur mollies to more efficiently create body fat out of available resources (for a similar phenomenon in a variety of cave organisms–but not cave mollies– from resource-limited habitats see [90]). Potentially, these fat reserves could function as an ATP reservoir that can be tapped into during periods of high temporal hydrogen sulphide discharge, and thus, provides sulphur mollies with the energy required to immediately up-regulate physiological detoxification mechanisms. We were able to demonstrate in previous studies that differences in offspring size are not simply a function of differences in female body size between species and ecotypes but rather represent true (evolved) differences [23,27]. Furthermore, the patterns are actually opposite to what would be expected, as cave molly females are on average smaller than surface molly females [23], and sulphur molly females are smaller still [27]; nonetheless, both ecotypes/species have larger offspring than surface molly females. Naturally, some of the differences detected here not only represent the action of evolved (genetic) differences but also environmental factors like resource availability. Since our embryo development data are derived from pregnant females collected in their natural habitat, they represent snap-shots of embryo development in the wild and are therefore also subject to Embryo Development in Extremophiles phenotypic plasticity induced by environmental differences. For example, an important factor in shaping offspring phenotype is variation in parental nutrient provisioning, which, in turn, depends on environmental conditions [59,91]. Still, given that population differences in the traits considered here have been shown to be largely heritable in adults [22,24,38], we argue that most of the differences observed here are unlikely to solely represent the effects of phenotypic plasticity. Embryo development: how to achieve divergent morphologies Not only cave mollies [22,26], but also various other cave organisms, like cave-adapted fishes of the North American family Amblyopsidae or the cave salamander Proteus anguinus show enlarged heads [92,93]. In amblyopsids, in which both surface and several cave forms exist, it was suggested that heads become relatively larger with increasing cave adaptation, and this was interpreted as a neotenic trend that enables sensory adaptations to life in darkness. For example, an enlarged head surface area could facilitate more superficial neuromasts [93]. Our studies on Poecilia spp. do not support this view, as also surface fish from sulphidic surface habitats evolve larger heads [22,26]. As cave ecosystems naturally do not show any photosynthetic oxygen production, hypoxia is common [94,95]. We therefore argue that the necessity to improve oxygen uptake via enlarged gills has selected for enlarged branchial baskets and thus, larger heads not only in extremophile poeciliids, but several other cave organisms as well. All sightless cave-dwelling vertebrates investigated to date exhibit initial eye development followed by secondary degradation during later developmental stages [41,42,68,96,97], because early steps in eye and pigment development are thought to be vital for other essential steps in development [41,42]. In cave-dwelling Astyanax and Rhamdia, early eye development already starts with a smaller eye primordium, and culminates in subsequent eye growth arrest [41,42,68]. Even though cave molly adults have reduced eye size compared to surface and sulphur mollies, this reduction in eye size appears to be mainly the product of growth arrest during the later developmental stages (stage 35 – stage 50) rather than resulting from disproportionally smaller eye primordia at stage 10. Clearly, cave mollies are an evolutionarily young lineage compared to some eyeless Astyanax populations, so growth arrest might be the developmental trait that is easiest to manipulate in the early stages of the evolution of cave organisms; however, we cannot rule out that at a later point during cave evolution, cave mollies will also evolve means to reduce the size of the eye primordium. Conclusions The present study demonstrates that most traits (convergent and unique) under strong selection in extreme habitats diverge early in development in extremophile Poecilia, indicating that both cave and sulphur mollies are in the advanced stages of ecological specialization. Thus, the present study is further evidence for ecological speciation in extremophile poeciliids. Furthermore, certain phenotypic traits (i.e., larger heads) appear to be the result of largely convergent developmental modes among extremophile fish, while other convergent traits (i.e., larger offspring size at birth) seem to be the result of taxon-specific modifications in embryo development and nourishment. Numer- ous studies have investigated to what extent phenotypic divergence to shared selective regimes is convergent or species/population- specific (i.e., unique) among closely related organisms or even different sexes within a species (e.g., [98–101] (R Riesch, M Plath, I Schlupp, RB Langerhans, unpublished data). However, rarely PLoS ONE | www.plosone.org 9 November 2011 | Volume 6 | Issue 11 | e27377