embryo development per se, but also concerns the field of maternal provisioning, namely resource allocation pre- (vitellogenesis: [57]) and post-fertilization (matrotrophy: [58– 62]). (2) Do cave mollies store fewer carotenoids in their ova? Cave organisms are usually characterized by reduced levels of carotenoids and carotenoid-derived compounds compared to surface-dwelling organisms, due to the scarcity of carotenoid- producing plants and microorganisms in subterranean habitats [63,64]. However, a certain fraction of cave-dwelling organisms of various species are characterized by a distinct yellow body colouration that is thought to be the result of accumulated carotenoids [65–66] and cave mollies are no exception ([38]). In a field sample from January 2009, for example, approximately 26.6% (51 out of 192) of cave mollies from chamber V of the Cueva del Azufre were of the yellow colour variant (R Riesch, unpublished data). Since fish oocytes/ova obtain their characteristic golden to orange colouration from the presence of carotenoids [67,68], measuring their yellowness should provide a reasonable proxy for the amount of carotenoids stored within the oocytes/ova. (3) How do extremophile mollies developmentally obtain their larger heads? This appears pretty straightforward and we therefore hypothesized that, compared to surface mollies, cave and sulphur mollies would boost head growth relative to the growth of other body parts early during development. (4) Finally, we asked how embryonic development leads to reduced eye size in cave mollies. Based on extensive work in Astyanax [41,42] and some other cave fishes like the catfish Rhamdia laticauda [69], we predicted that the eye primordium in cave mollies should be slightly smaller compared to surface and sulphur mollies, and that eye growth in cave mollies should be arrested at a certain point during development [48]. Materials and Methods 2.1. Study populations and sampling protocols Pregnant P. mexicana females were collected in June 2007 and January 2008 in two separate benign surface habitats (surface mollies; R´ıo Amatan and Arroyo Bonita) and in the toxic Cueva Embryo Development in Extremophiles del Azufre (cave mollies) –two sets of populations that are known to be reproductively isolated from one another [21,22]. For more detail on these sample sites, please refer to Tobler et al. [22] and Riesch et al. [23]. Pregnant P. sulphuraria females were collected in August 2008 and January 2009 at the toxic Ba n˜os del Azufre (sulphur mollies; for more details, see [27]). Following well-established life-history protocols [23,27,53], we removed all developing embryos from formaldehyde-preserved, pregnant females. Embryos were counted, their stage of develop- ment determined [53,54], and then dried for 10 days at 40uC and reweighed. To assess embryo condition, embryos were rinsed six times for at least 6 h in petroleum ether to extract soluble nonstructural fats [70], and were then redried and reweighed. This protocol provided us with the following embryonic life history variables: embryonic dry weight [mg], embryonic lean weight [mg], and embryo fat content [%]. Prior to dissections, adult mollies were photographed with an InSight Spot 2 digital camera (Diagnostic Instruments) mounted on an Olympus SX 7 stereomicroscope and pictures were stored in TIFF file format. Immediately following surgical removal from preserved females, representative embryos for each molly and stage were also photographed and stored in TIFF file format. 2.2. Quantification of ovum yellowness Embryo photographs were analyzed using Adobe Photoshop CS5 Extended 12.1 (Adobe Systems Inc., San Jose, CA, USA). TIFF images were opened in 16-bit mode and an L*a*b* colour space was employed [Commission International de l’Eclairage (CIE)]. In this colour space, b* values represent the ‘yellowness’ (balance between blue and yellow) of a photographic object (e.g., [71,72]). 2.3. Quantifying maternal provisioning and the matrotrophy index One potential pathway for extremophile Poecilia to achieve larger offspring size at birth would be to provide more post- fertilization maternal provisioning. To evaluate the mode of maternal provisioning, the matrotrophy index (MI) was calculated (utilizing the slopes and regression coefficients from the regression analysis described below). The MI equals the estimated dry mass of the embryo at birth divided by the estimated dry mass of the Table 1. Embryo development in poeciliid fishes. According to Reznick [103] and compared to the generalized description provided by Haynes [54] (right). Stage Description Stages according to [54] 0 2 5 10 15 20 25 30 35 40 45 50 mature (fully yolked) but unfertilised oocytes, even lipid distribution blastodisc embryos neurula stage: embryonic shield/primitive streak embryos optic cup embryos: head and optic cups are visible optic cups become pigmented (grey-eyed) eyes appear fully pigmented (black) and caudal fin bud visible body pigmentation appears and pectoral fin buds caudal fin rays clearly visible, dorsal and anal fin buds pectoral fin rays clearly visible dorsal and anal fin rays clearly visible operculae conspicuous, scales visible, overall oval shape mature embryos: pericardial cavity almost or completely closed doi:10.1371/journal.pone.0027377.t001 stage 3 stage 4 stage 5 stage 6 stage 7 stage 8 - - - stage 9 stage 10 stage 11 PLoS ONE | www.plosone.org 3 November 2011 | Volume 6 | Issue 11 | e27377