Shared and Unique Patterns of Embryo Development in Extremophile Poeciliids Ru¨ diger Riesch1,2*, Ingo Schlupp 2 , R. Brian Langerhans1, Martin Plath3 1 Department of Biology & W. M. Keck Center for Behavioral Biology, North Carolina State University, Raleigh, North Carolina, United States of America, 2 Department of Zoology, University of Oklahoma, Norman, Oklahoma, United States of America, 3 Department of Ecology & Evolution, Goethe–University Frankfurt, Frankfurt, Germany Abstract Background: Closely related lineages of livebearing fishes have independently adapted to two extreme environmental factors: toxic hydrogen sulphide (H2S) and perpetual darkness. Previous work has demonstrated in adult specimens that fish from these extreme habitats convergently evolved drastically increased head and offspring size, while cave fish are further characterized by reduced pigmentation and eye size. Here, we traced the development of these (and other) divergent traits in embryos of Poecilia mexicana from benign surface habitats (‘‘surface mollies’’) and a sulphidic cave (‘‘cave mollies’’), as well as in embryos of the sister taxon, Poecilia sulphuraria from a sulphidic surface spring (‘‘sulphur mollies’’). We asked at which points during development changes in the timing of the involved processes (i.e., heterochrony) would be detectible. Methods and Results: Data were extracted from digital photographs taken of representative embryos for each stage of development and each type of molly. Embryo mass decreased in convergent fashion, but we found patterns of embryonic fat content and ovum/embryo diameter to be divergent among all three types of mollies. The intensity of yellow colouration of the yolk (a proxy for carotenoid content) was significantly lower in cave mollies throughout development. Moreover, while relative head size decreased through development in surface mollies, it increased in both types of extremophile mollies, and eye growth was arrested in mid-stage embryos of cave mollies but not in surface or sulphur mollies. Conclusion: Our results clearly demonstrate that even among sister taxa convergence in phenotypic traits is not always achieved by the same processes during embryo development. Furthermore, teleost development is crucially dependent on sufficient carotenoid stores in the yolk, and so we discuss how the apparent ability of cave mollies to overcome this carotenoid-dependency may represent another potential mechanism explaining the lack of gene flow between surface and cave mollies. Citation: Riesch R, Schlupp I, Langerhans RB, Plath M (2011) Shared and Unique Patterns of Embryo Development in Extremophile Poeciliids. PLoS ONE 6(11): e27377. doi:10.1371/journal.pone.0027377 Editor: Vincent Laudet, Ecole Normale Supe´rieure de Lyon, France Received July 1, 2011; Accepted October 15, 2011; Published November 7, 2011 Copyright: 2011 Riesch et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the National Science Foundation of the United States (NSF: DEB-0743406, DEB-0813783, and DEB 0842364) and the Deutsche Forschungsgemeinschaft (DFG: PL 470/3-1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rwriesch.evolutionarybiology@gmail.com Introduction 1.1. Ecological speciation in extremophile poeciliids During ecological speciation reproductive isolation evolves as a consequence of ecologically based divergent selection [1–3]. Most studies on ecological speciation in animals have focused on divergent selection as a result of biotic interactions, with populations differing, for example, in resource use [4–7], predation pressures [8,9], or parasite exposure [10–12]. On the other hand, the concept of stress and maintenance of homeostasis through adaptation to abiotic habitat parameters is often ignored in the study of speciation in animals and more emphasis is put on its role in population decline and extinction [13], even though stressful environments have long been known to be associated with bouts of strong directional selection [14]. Hydrogen sulphide (H2S) is a widespread natural toxicant that can also originate from anthropogenic pollution, e.g., from pulp mills [15]. Its patchy occurrence often creates environmental gradients in aquatic ecosystems (both horizontally and vertically), e.g., at deep-sea hydrothermal vents, in coastal salt marshes, or in mudflats [16,17]. H2S-toxicity results from its interference with mitochondrial respiration and blood oxygen transport, thereby inhibiting aerobic respiration, and this effect is aggravated by the fact that H2S leads to extreme hypoxia in the water [16,17]. Accordingly, pulses of toxic H2S discharge have been reported to be the source of mass mortalities in aquatic faunas, especially fishes [17]. These adverse effects notwithstanding, a number of livebearing fishes (family Poeciliidae) have been documented to thrive in sulphide-rich habitats (reviewed in [18]). Patterns of trait divergence in these extremophile poeciliids and reproductive isolation towards neighbouring populations inhabiting non-toxic waters are congruent with ecological speciation as a response to adaptation to stressful environmental conditions and have been extensively studied in one particular system in southern Mexico. In this system, Poecilia mexicana inhabit various toxic and non-toxic PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e27377