- Top of page
- Materials and methods
- Results and discussion
Caves provide excellent settings to examine evolutionary questions. Subterranean environments are characterized by similar and consistent conditions. Cave-adapted species often share characteristics such as diminished pigmentation, elongated limbs and reduced or absent eyes. Relatively little is known about the evolution and development of troglomorphic traits in invertebrates. In this study, we compare expression of the eye development genes hedgehog, pax6, sine oculis and dachshund in individuals from multiple independently derived cave populations of the amphipod Gammarus minus. hedgehog expression was significantly reduced in cave populations, compared to genetically related surface populations. Interestingly, no differences were found in pax6, sine oculis or dachshund expression. Because hedgehog-related genes are also involved in eye reduced in Astyanax mexicanus, these genes may be consistent targets of evolution during cave adaptation. These results provide support for the hypothesis of genomic ‘hotspots’ of evolution and allow comparison of adaptive mechanisms among diverse animals in subterranean environments.
- Top of page
- Materials and methods
- Results and discussion
In the last several decades, developmental biologists have discovered a set of conserved genes that control the development of similar structures across divergent taxa (Carroll, 1995; Gehring, 1996; Raff, 2000). The realization of this genetic conservation has fostered a novel approach to explore the ways in which phenotypic changes are dependent on modulation of developmental processes that are common to diverse animal lineages. It is clear that distantly related species may converge on similar phenotypes via similar genetic and developmental changes (reviewed by Stern, 2010). However, these studies have mostly focussed within species or genera (e.g. Colosimo et al., 2005; Prud’homme et al., 2006; Shapiro et al., 2006; Sucena et al., 2003; but see also Wlasiuk & Nachman, 2007), and it is unclear to what extent the convergent evolution of more distantly related taxa might also proceed by similar developmental or genetic changes.
Caves are a unique laboratory for tests of convergent and parallel evolution. Geographically isolated cave environments exert strong and consistent selection pressures on animals (Culver et al., 2009). Subterranean habitats are characterized by low nutrient availability, constant temperatures and the absence of light. Subterranean animals of diverse taxa converge on similar characteristics including lack of body pigmentation, reduced or absent eyes, elaborate feeding and sensory structures, and elongated appendages, a suite of adaptations termed troglomorphy by Christiansen (1961). Studies of cave and surface morphs of the Mexican tetra Astyanax mexicanus suggest that among independently derived cave populations parallel changes in development have led to reduced eye size (reviewed by Jeffery, 2005).
The developmental genetic basis of eye reduction has also been examined in A. mexicanus cavefish. Eye reduction in cave populations of this species occurs through expanded midline expression of Hedgehog signalling molecules, encoded by sonic hedgehog (shh) and tiggy-winkle hedgehog (twhh), which are orthologues of the arthropod hedgehog (hh) gene. Expanded Hh activity in cavefish leads to apoptosis in the lens and subsequent degeneration of the developing eyes (Yamamoto et al., 2004). Midline Hh activity also leads to increased numbers of taste buds, which are assumed to be advantageous in a food-poor environment (Jeffery, 2005). It remains unclear whether other subterranean animals might evolve eye reduction by similar or different mechanisms.
Gammarus minus is a freshwater amphipod crustacean prevalent in surface springs and cave streams in the Mid-Atlantic region of the United States. Cave and surface populations of G. minus show heritable, individual variation in classic troglomorphic characters (Fig. 1e,f; Fong, 1989). Hydrological evidence and genetic evidence clearly indicate subterranean populations invaded cave streams from surface springs (Culver et al., 1995); thus, troglomorphy is a suite of derived traits in G. minus. Furthermore, studies of allozyme variation (Culver et al., 1995) and sequence variation at mitochondrial and nuclear loci (Carlini et al., 2009) imply that the multiple troglomorphic populations of G. minus have evolved independently. This species is one of the few eutroglophiles, surface species that are capable of establishing permanent subterranean populations. The only other two well-known examples of eutroglophiles are the isopod Asellus aquaticus and the cavefish A. mexicanus. These attributes make G. minus an excellent species in which to test whether convergent evolution occurs through parallel charges in developmental pathways (Abouheif, 2008; Fong, 2012).
Figure 1. Gene expression in the heads of adult Gammarus minus. (a) hedgehog. (b) pax6. (c) sine oculus. (d) dachshund. Bars indicate the median value for each population, whereas boxes denote the upper and lower quartiles. Whiskers indicate the full range of sampled values. Boxes are shaded grey for cave populations; white for surface populations. (e) An example individual specimen from Ward Spring with normal eye size for surface amphipods. (f) A troglomorphic specimen from Organ Cave illustrates reduced eye size. Insets in (b, c) show detail of the eyes at the same scale.
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One of the most iconic convergent phenotypes among different cave animals is the reduction or absence of eyes. Diverse animals share many of the genes required to initiate eye development (Quiring et al., 1994; Halder et al., 1995; Gehring, 1996; Jensen & Wallace, 1997; Neumann & Nüsslein-Volhard, 2000; Dong & Friedrich, 2010); therefore, vertebrates and invertebrates may present many of the same potential targets to selection during adaptation to caves. Arthropod eye development is best understood in the fruit fly Drosophila melanogaster (reviewed by Pappu & Mardon, 2004). In this insect, eye development initiates late in embryogenesis with expression of the Pax6 homologues eyeless (ey), twin-of-eyeless (toy), eyegone (eyg) and twin-of-eyegone (toe) in the eye-antenna imaginal disc (Quiring et al., 1994; Jun et al., 1998; Czerny et al., 1999; Jang et al., 2003; Yao et al., 2008). Early in the second larval instar, ey expression is sequestered to the posterior of the eye-antenna disc, specifying the eye primordium. Loss of function mutations in these regulatory genes leads to loss of the eyes (Quiring et al., 1994; Halder et al., 1995; Czerny et al., 1999; Jang et al., 2003), and their ectopic expression in other imaginal discs leads to ectopic eye formation (Gehring, 1996; Czerny et al., 1999). Later in the second larval instar, eyes absent (eya), sine oculis (so) and dachshund (dac) expressions are required in the eye primordium (Chen et al., 1997; Pignoni et al., 1997; Curtiss & Mlodzik, 2000). During pupation, the morphogenetic furrow proceeds across the disc as a wave of retinal differentiation. The conserved signalling molecule encoded by hedgehog is secreted from differentiating cells behind the advancing furrow. Hedgehog induces new cells to initiate retinal differentiation (Treisman & Rubin, 1995; Dominguez & Hafen, 1997; Royet & Finkelstein, 1997; Greenwood & Struhl, 1999; Curtiss & Mlodzik, 2000; Pappu et al., 2003). In this way, hh is responsible for progression of the morphogenetic furrow. Interestingly, shh drives a similar wave of retinal differentiation in the developing eyes of zebrafish (Neumann & Nüsslein-Volhard, 2000), chicks (Zhang & Yang, 2001) and mice (Jensen & Wallace, 1997). This finding implies that hh function in eye morphogenesis is conserved in arthropods and vertebrates.
Examining the developmental genetic basis of troglomorphy in G. minus has the potential to illuminate the mechanism of convergent evolution in an invertebrate group. Comparisons to previous studies of cavefish provide insights into cave adaptation at two very different phylogenetic scales (population and superphylum). This short communication reports the expression of several conserved eye development genes in multiple derived cave- and surface spring populations of G. minus. We find evidence of parallel differences in hh expression in cave- and surface sibling populations. Intriguingly, hh is the same morphogen implicated in A. mexicanus eye reduction, suggesting a potentially universal evolutionary target in the eye reduction of cave animals.