stage 25, but in late-stage cave molly embryos overall strength of pigmentation seems to be reduced compared to surface and sulphur mollies. While the genetic basis and fine-scale develop- mental pathways of pigmentation loss in Astyanax cavefish are well understood (e.g., [76]; reviewed in [41,42]), future studies will have to establish expression patterns of developmental genes in cave mollies. Modified lips with dermal protuberances are deemed a species- specific, apomorphic character of sulphur mollies [34,45]. Clearly these protuberances are traits that are unique to sulphur mollies despite the fact that other poeciliids (including the cave molly) also inhabit similarly toxic and hypoxic habitats; however, as we demonstrated here, only a fraction of adult fish actually exhibit them at any given time. Furthermore, our experience from the field suggests that these are highly plastic traits, because adult fish with prominent lip protuberances caught at the Ba n˜os del Azufre regularly loose them within hours to days of being introduced to non-sulphidic laboratory conditions (M Plath, R Riesch, I Schlupp, personal observation). Thus, they are similar to other fishes from (temporarily) hypoxic habitats, in which lip protuber- ances are also reversible and usually only a certain percentage of fish within a population display the trait at any given time (e.g., [35]). This high degree of phenotypic plasticity also best explains why we found no evidence for lip protuberances in any of the sulphur molly embryos. We propose that this trait may be developed ‘at will’ if the environmental conditions (i.e., sulphide- toxicity and low oxygen levels) require it. Some sulphur mollies are certainly born into less sulfidic microhabitats at the Ba n˜os del Azufre, and these lip protuberances are likely to lead to hydrodynamic disadvantages as a result of increased drag, so not expressing the trait already within the mother’s body cavity may actually be beneficial. Carotenoids serve several important functions in animals, ranging from being Vitamin A precursors and providing the basis for body coloration patterns to providing the precursors for the visual pigment retinal and acting as antioxidants and immune- system enhancers ([77–80]; but see [76]). Animals cannot synthesize carotenoids de novo and consequently, have to acquire them from their diet [67]. In fish, eggs usually obtain their characteristic golden to orange coloration from the presence of carotenoids stored in the oocytes [67], which has been experimentally confirmed by Hubbs and Strawn in their study on darters [65]. The presence of low levels of carotenoids in cave organisms has long been established [63,64] and it is therefore not surprising that cave mollies are able to produce pigmented eyes and (reduced) body pigmentation. Our results demonstrate that although cave molly oocytes are still yellow, the intensity of the yellow colour component is significantly reduced relative to surface and sulphur mollies, suggesting reduced carotenoid content in the yolk of cave molly oocytes. Compared to regular surface habitats that are filled with carotenoid-producing plants, algae and bacteria, carotenoids will be relatively scarce in the natural cave environment. So, where do the carotenoids come from in the Cueva del Azufre? Several mutually non-exclusive scenarios are possible. First, in several parts of the cave, skylights introduce dim light into several cave chambers [19], so it may be possible that carotenoid synthesizing algae and bacteria grow in certain parts of the Cueva del Azufre, in particular since even chemoautotroph sulphur-oxidizing bacteria have been described to produce carotenoids [81]. Other significant sources of carotenoids in caves stem from allochthonous input in the form of fresh plant material that falls through skylights, and detritus carried in by underground rivers [63]. Carotenoids are also found in chironomid eggs and larvae [82], which are numerous in the Cueva del Azufre and are Embryo Development in Extremophiles known to be an important food source for cave mollies [83], even though stable isotope analysis suggests that cave mollies assimilate material obtained from chironomid eggs and larvae only to minor proportions into body tissue [84]. Furthermore, ovum yellowness also differed significantly between surface and sulphur mollies, with sulphur molly ova having slightly reduced yellowness. This is most likely due to the fact that poeciliids in sulphidic surface habitats primarily feed on detritus, arthropods, and bacteria, while surface mollies primarily feed on detritus and algae [83,84]. Even though chemoautotrophic sulphur-oxidizing bacteria have been described to produce carotenoids [81], it is unlikely that they are produced in comparable amounts as in nontoxic surface habitats, where carotenoids are synthesized by algae and other photoautotrophic primary producers. However, we cannot exclude the possibility that some of the variation in ova coloration is due to additional differences in lipid and protein content between ecotypes and species [85]. Clearly, this would also be influenced by the available resources in each habitat and could slightly confound our results. Future experi- ments investigating the actual carotenoid, lipid and protein content of poeciliid ova are needed to clearly resolve this. Finally, in teleost fishes offspring viability correlates positively with oocyte/ovum carotenoid content, with young born from pale ova of low carotenoid content experiencing increased levels of juvenile mortality [86–88]. Our results on ovum yellowness suggest that cave mollies have locally adapted to the reduced carotenoid availability and appear to have evolved a mechanism to offset the cost of reduced carotenoid levels on offspring viability, because cave molly offspring actually have a higher individual fitness compared to surface molly offspring [33]. Hence, the decreased availability of carotenoids likely results in strongly decreased fitness in immigrant fish not showing specific adapta- tions to these conditions and thus helps maintain the strong reproductive isolation detected in this system [20–22,28,89]. Embryo development: how to increase offspring size All three mollies were characterized by lecithotrophy according to the MI analysis. However, a recent comparative analysis of maternal provisioning in surface and cave mollies, based on the detection of transferred radioactively labelled leucine from mothers to embryos, demonstrated that low levels of postfertiliza- tion provisioning are possible even if MI values point towards lecithotrophy [24]. It is therefore highly likely that also sulphur mollies are characterized by dual provisioning rather than pure lecithotrophy (see also [62]). Nonetheless, cave and sulphur mollies clearly show unique developmental patterns to achieve larger embryo size at birth. Cave mollies produced larger ova prior to fertilization and ovum diameter then steadily increased throughout development, mainly driven by the large embryos that, starting around stage 20, led to increasing ovum/embryo diameter. Sulphur mollies, on the other hand, produced ova that at fertilization were not larger than those of surface mollies and only started to diverge at later stages during development (again around stage 20). It is important to keep in mind though, that we only measured ovum diameter on the photos taken of developing embryos rather than on ova that had the developing embryos removed prior to the measurement being taken. This obviously confounded our measurements, because at later stages only small parts of the ovum were visible and the diameter was basically determined as the maximum diameter measurable on ovum or developing embryo and so relatively larger embryos will have confounded our measure of ovum diameter. Nonetheless, this pattern for sulphur mollies is highly surprising, because sulphur PLoS ONE | www.plosone.org 8 November 2011 | Volume 6 | Issue 11 | e27377