surface habitats, but are also found in a subterranean ecosystem, the H2S-rich Cueva del Azufre [19], in which they are under strong selection from two extreme environmental forces: H2S- toxicity and permanent darkness. As a result, P. mexicana display considerable genotypic and phenotypic differentiation between adjacent extreme and benign habitat types [20–25]. Recent studies have demonstrated convergent patterns of morphological [26] and life-history diversification [27] (but see Riesch et al. submitted for non-convergent patterns), and similar mechanisms of selection against migrants from non-sulfidic habitats [28,29] in P. mexicana from the Cueva del Azufre system and livebearing fishes inhabiting the Ba n˜os del Azufre, another sulfidic surface system in Tabasco, southern Mexico. These sulfidic springs are inhabited by two sulphur-endemics, the livebearing fishes Gambusia eurystoma and Poecilia sulphuraria [30]. The sulphur molly, P. sulphuraria, is of particular interest in this context, because phylogenetic analyses suggest that it is the sister-taxon to P. mexicana [26,31] and therefore represents a possible ‘endpoint’ of ecological speciation in H2S-toxic surface habitats. Several targets of divergent selection in extremophile poeciliids have recently been identified. On the life-history level, females from sulphidic, cave, or sulphur-cave habitats produce larger, but fewer offspring [23,27,32]–translating into increased individual offspring survivability [33]–and population differences in those traits have been demonstrated to have a heritable basis [24,32]. Furthermore, the combined selection of H2S and darkness on offspring size in the Cueva del Azufre has resulted in P. mexicana from that cave having the largest offspring [23,27]. Morpholog- ically, extremophile livebearers exhibit convergent evolutionary trends primarily by developing larger heads and thus, larger branchial baskets than fish from non-toxic habitats [22,26]. According to the species description by Alvarez [34], a species- specific trait of sulphur mollies are the dermal lip protuberances not found in any other poeciliid (Figure S1; please refer to the supporting figure legend). These protuberances are thought to enhance the performance of aquatic surface respiration (ASR; [30,35]), a behavioural adaptation to meet oxygen demands in hypoxic habitats (e.g., [36,37]). Cave mollies from the Cueva del Azufre are further characterized by smaller, yet fully functional eyes [22,38–40] and reduced melanin pigmentation [38] (BL Joachim, I Schlupp, R Riesch, unpublished data). 1.2. Developmental patterns of divergent traits Knowledge of development is paramount for understanding the mechanisms of evolutionary change, because changes in develop- ment can result in novel phenotypes [41,42]. More specifically, changes in the timing of various developmental processes (i.e., heterochrony) may explain many of the evolutionary changes observed among closely-related taxa [43,44]. Unfortunately, due to the inherent problem of acquiring embryonic and larval stages, developmental studies are rare in cave biology and research programmes focusing on evolutionary change in other extreme environments. One notable exception is the Mexican tetra, Astyanax mexicanus, which is easy to maintain in the laboratory, reproduces frequently, and therefore, is open to embryological/ developmental investigations at the cellular and molecular levels [41,42]. Albeit a single species, A. mexicanus has a surface-dwelling form that is widespread in the Atlantic slope of Mexico [45] and at least 30 known cave-dwelling forms that differ markedly from their surface-dwelling ancestors [41,42,46]. Typical for a variety of cave organisms, many cave-dwelling populations of A. mexicanus (often also referred to as A. fasciatus) have reduced eye size, while others have completely lost their eyes, and a similar pattern can be found for pigmentation [46–49]. In contrast to most other cave Embryo Development in Extremophiles organisms, the developmental mechanisms underlying these changes in A. mexicanus are quite well understood (reviewed in [41,42]). A major obstacle for these types of developmental studies in extremophile poeciliids has been that they are livebearing, rendering the study of early developmental events difficult. Hence, even though the cave molly has been a model system for evolution of cave organisms for almost four decades now (reviewed in [38,50]), and P. sulphuraria has recently become a model for ecological speciation processes as well (e.g., [18,26,27]), the question of how they achieve their divergent phenotypes during development has so far not been investigated. Convergent phenotypes (e.g., larger heads in extremophile Poecilia) are often, but not always, the result of convergent developmental modes [51]. Similar ecological conditions tend to produce convergent developmental modes, but few cases of convergence in develop- ment have been studied in detail. Wray [51] suggested that convergence in development is probably inversely related to phylogenetic distance with more closely related taxa also being more likely to exhibit convergent developmental modes (but see [52]). Our study system of Poecilia sister taxa that are characterized by several convergent phenotypic traits provides us with the perfect opportunity to test this assumption. Here, we make use of the extensive data available to us from our own life-history analyses that were conducted on wild-caught fish for previous studies [23,24,27]. As part of the traditional life- history sampling protocol (see methods in [23,24,27,53]), devel- oping offspring were removed during the dissection of pregnant, formalin-fixed females and their stage of development determined according to Haynes [54] and Reznick et al. [55] (Table 1), and digital photographs were then taken of representative embryos for each stage of development. This provided us with excellent snap- shots of different stages of embryo development in all three mollies, and allowed us to track some of the most prominent divergent traits through development. In the present study, we examined embryos of surface-dwelling P. mexicana from benign surface habitats (henceforth called ‘‘surface mollies’’), cave- dwelling P. mexicana from the toxic Cueva del Azufre (‘‘cave mollies’’), and the sulphur-endemic P. sulphuraria from a toxic spring (‘‘sulphur mollies’’) to examine at which points during development the local adaptations in life history and morphology would first be detectable and to what extent convergent phenotypes are the product of convergent developmental modes. Our study will provide an important starting point for future investigations into the molecular mechanisms of development, an approach that will also greatly benefit from the recently established protocol for in vitro culture of guppies, Poecilia reticulata [56]. Furthermore, results from transcriptomics studies will likely soon be available for extremophile Poecilia spp., so our present study may provide the basis for future studies linking gene expression patterns to developmental patterns. In this paper, we investigated the following four specific questions concerning the development of some key divergent traits in extremophile Poecilia: (1) When and how do cave and sulphur mollies increase offspring size at birth? Extremophile poeciliids could simply produce larger oocytes (i.e., provide more yolk to individual oocytes prior to fertilization), and embryos might then take roughly the same developmental route as smaller surface molly embryos. Another possibility is that extremophile poeciliids rely more on post-fertilization maternal nutrient transfer to embryos, in which case embryo size could either decrease to a lesser extent or even slowly increase during the course of development. Our first question, therefore, not only concerns PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e27377