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Keywords:

  • adaptation;
  • evo-devo;
  • eye patterning;
  • gene expression variation;
  • troglomorphic traits

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. References

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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. References

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).

image

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.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. References

Animals

Surface and cave specimens of G. minus were collected in the winter of 2010–2011, from sites described in previous studies (Culver et al., 1995), yielding three pairs of cave and surface populations that are hydrologically connected and genetically related within three drainage basins (Carlini et al., 2009). Sites in Greenbrier County, West Virginia included US-219 Spring (US) and The Hole Cave (HC) in the Spring Creek basin, as well as Ward Spring (WS) and Organ Cave (OC) in the Second Creek basin. We also sampled Maiden Spring (MDS) and Fallen Rock Cave (FRC) in the Ward Cove basin of Tazewell County, Virginia. Animals were kept in the laboratory in constant darkness at 10 °C and fed fallen maple (Acer) leaves aged in spring water as described by Fong (1989). Water, containers and container position in the incubator were changed weekly. Individuals were imaged using an Olympus SZX16 dissecting microscope equipped with a Hamamatsu C8484 high-resolution digital camera (Fig. 1e,f).

Cloning of candidate genes

Gammarus minus candidate genes were identified for study based on the literature of eye development in D. melanogaster. We focussed on hh and its upstream regulators including pax6 (orthologous to ey and toy in D. melanogaster), so, and dac. Orthologous arthropod protein sequences were aligned using MUSCLE (Edgar, 2004), and nested degenerate primers were designed using the CODEHOP algorithm (Rose et al., 2003). Total RNA was extracted from a pool of mixed-stage embryos, and first-strand cDNA synthesis utilized a poly-T primer (Promega, Madison, WI, USA). Target gene sequences were amplified from cDNA, and products were ligated into the TOPO4 vector (Life Technologies, Grand Island, NY, USA) and transformed into chemically competent Escherichia coli. Plasmid inserts were then sequenced, and reciprocal BLAST searches were used to determine gene orthology. Gene sequences from G. minus have been submitted to GenBank (Accession numbers: JQ756980-JQ756983).

Gene expression analysis

Gene expression was determined using quantitative real-time RT-PCR (qPCR) amplification of target gene sequences. Total RNA was isolated from the head using the PureLink RNA Mini kit (Life Technologies). The eyes of G. minus exhibit indeterminate growth throughout juvenile development and subsequent adult moults. Ommatidia are added in each moult (Fong, 1989), and morphological divergence between cave and surface populations is more pronounced in older individuals. Therefore, our analysis was restricted to adult G. minus. Sampling was random with respect to timing within the moult cycle. RNA was extracted from between nine and 14 specimens per population and stored at −80 °C for < 1 week. Total RNA concentrations were determined by triplicate measures on a nanoscale spectrophotometer and diluted to 45 ng μL−1 immediately prior to assays. Total RNA was used as a template with three technical replicates in reverse transcription, SYBR Green qPCRs (Quanta BioSciences, Gaithersburg, MD, USA).

For each target gene, specific primers were designed using the Primer3 algorithm (Rozen & Skaletsky, 2000), avoiding conserved functional domains. (dac: forward 5′-ATTGTGTGTAACGTGGATCAAGTTCG-3′, reverse 5′-ATGGAGAGCAACTTACACCTGTTGAC-3′; hh: forward 5′-CAGGAGTAAGGTTACGTGTCAAGGAG-3′, reverse 5′-CTCGTAGTGCAGTGAGTGACTGTTGT; pax6: forward 5′-ATACTTCAAGTTTCCAATGGCTGTGT-3′, reverse 5′-CTTGATGGACCCCGTCTCGTAATATC-3′; so: forward 5′-GCAAAGGCTTTAGTATCGTTCAATC-3′, reverse 5′-TTGTGAGATTCTAAGATCCGGTAGAG-3′). To produce quantitative template standards, clones were linearized and transcribed in vitro from T7 promoters to produce single-stranded RNA. This RNA was treated with DNase I to remove template DNA and purified by precipitation in ammonium acetate and ethanol. Immediately before qPCR assays, the RNA concentration was determined in triplicate (as described above) and the molar quantity was calculated based on the size of the RNA. Dilution series were then prepared fresh for each plate at concentrations of 103, 105 and 107 RNA molecules to serve as a standard curve (Pfaffl, 2004). Dissociation curves for each reaction were used to verify that only single products were amplified.

Differences in gene expression between populations were tested using nested anova (Table 1) and selected t-tests (Table 2). The error term for individual variation is based on technical replicates for each specimen. The coefficient of variation (Table 3) for gene expression was calculated to provide a measure of variation that is corrected for the influence of magnitude. The level of significance for t-tests in Table 2 was adjusted for multiple tests using the Bonferroni method. All statistical tests were conducted using r (R Development Core Team, 2010).

Table 1.   Results of nested anova for log transcript numbers in the heads of Gammarus minus from cave and surface habitats.
 d.f.SSMSSFP-value
  1. Significance: *P < 0.05; **P < 0.01; ***P < 0.001

hedgehog
 Basin  2346.59173.29 517.69< 2.2 × 10−16***
 Basin:habitat  3  9.88  3.29   9.84  3.90 × 10−16***
 Basin:habitat:individual  6  2.45  0.41   1.22  0.2961
 Residuals233 78.00  0.34  
pax6
 Basin  2890.05445.031225.92< 2 × 10−16***
 Basin:habitat  3  2.25  0.75   2.07  0.105
 Basin:habitat:individual  6  5.75  0.96   2.64  0.017*
 Residuals237 86.03  0.36  
dachshund
 Basin  2  8.69  4.35   6.26  0.002**
 Basin:habitat  3  2.15  0.72   1.03  0.38
 Basin:habitat:individual  6  9.72  1.62   2.33  0.033*
 Residuals222154.25  0.69  
sine oculus
 Basin  2161.55 80.78  47.28< 2.2 × 10−16***
 Basin:habitat  3  0.47  0.16   0.09  0.96
 Basin:habitat:individual  6 32.67  5.45   3.19  0.005**
 Residuals216369.01  1.71  
Table 2.   Comparisons of gene expression between sibling populations. Log transcript numbers per ng total RNA were tested for difference using Welch’s two-tailed t-test. The level of significance, corrected for multiple tests using the Bonferroni method, is < 0.05/3 = 0.0167.
  1. Significance: *P < 0.0167; **P < 0.00333; ***P < 0.000333.

  2. Abbreviations of localities: HC, Hole Cave; MDS, Maiden Spring; FRC, Fallen Rock Cave; OC, Organ Cave; US, US-219 Spring; WS, Ward Spring.

 Spring creek US-HCSecond creek WS-OCWard cove MDS-FRC
hh
 d.f.22.64925.43425.563
 t2.8613.93530.7908
 P-value0.0089*0.0006***0.4363
pax6
 d.f.25.99724.59725.834
 t0.2567−1.22941.1144
 P-value0.79940.23050.2754
so
 d.f.24.37225.02716.93
 t−0.30680.474−0.3215
 P-value0.76160.63960.7517
dac
 d.f.18.12425.84125.046
 t2.3622−0.4053−0.5585
 P-value0.029550.68860.5815
Table 3.   Coefficients of variation for log transcript numbers per ng total RNA for genes in different populations. Coefficients of variation are also given for all surface and cave specimens considered together, as well as over all populations.
 hhpax6dacso
  1. Localities abbreviated as in Table 2 and text.

US0.620.980.451.14
HC0.520.620.340.94
WS0.480.651.460.70
OC0.450.662.320.54
MDS0.840.770.974.18
FRC1.150.641.165.56
Surface
 Cave0.700.641.272.35
 Overall0.680.721.122.18

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. References

Significant population-level expression differences in hedgehog

Gene expression in sampled populations of G. minus (Fig. 1) varied significantly among drainage basins (Table 1). hedgehog expression was significantly different in subterranean populations relative to surface populations (Table 1). Expression of hh was also significantly different between two of the three cave- and surface population pairs (Table 2). Each cave population had reduced hh expression relative to its related surface population within the same basin (Fig. 1a). While the expressions of all genes differed significantly between the three drainage basins, expression differences between cave and surface habitats were only significant in nested anova for hh (Table 1).

Drosophila melanogaster mutations that reduce hedgehog expression in the eyes result in adult flies with reduced numbers of ommatidia and smaller eyes (Heberlein et al., 1993). Therefore, reduction in hh expression is a feasible mechanism by which an arthropod might evolve reduced eye size. If so, variation in hh expression within populations might be reduced, by either directional selection or genetic bottleneck. We tested for differences among individuals within populations and found that while significant differences existed among individuals’ expression of other eye development genes, there was no overall difference in the levels of hh expression within populations (Table 1). Furthermore, the coefficient of variation for hh within populations is lower than those of dac and so (Table 3). A genetic bottleneck would be expected to reduce variation for all genes in cave populations relative to surface populations; however, this is not the observed pattern (Table 3). Therefore, findings are consistent with the possibility that reduced hh expression has been involved in eye size reduction in G. minus.

Upstream eye development genes are not expressed differently among G. minus populations

Several regulatory steps precede hh activity in eye development. Reduction in hh expression in subterranean G. minus could be due to mutations in upstream regulatory genes that produce an indirect lowering of hh expression. Therefore, we also examined expression of several other conserved eye development genes, including pax6, so and dac.

In G. minus, no significant differences were detected between cave and surface populations in the expression of upstream regulators of hh, including pax6, so and dac (Fig. 1b–d; Table 1). No correlation between expression of these genes and eye size was observed for individuals or populations (not shown). In contrast, eye size correlates with expression of the two opsin paralogues of G. minus (D.B. Carlini, unpublished). As opsin is strongly expressed in differentiating ommatidia in D. melanogaster (Pollock & Benzer, 1988), it is not surprising that transcript levels correlate strongly with eye size. Differential regulation of opsin and other effectors of eye differentiation must result from differences in expression or function of upstream regulatory genes.

The lack of population-level differences in the expression of pax6, dac or so may be due to at least four factors, which are not mutually exclusive. (i) We cannot eliminate the possibility that while total transcript numbers in the head may not be different, the spatial pattern of gene expression may differ, causing phenotypic consequences (i.e. heterotopy). (ii) Functional analyses of developmental genes have not been conducted in G. minus or other crustaceans, and it is possible that the functions of these genes may differ from their orthologues in insect model species such that they are not involved in eye development. (iii) Differences in pax6, dac and so expression may occur during juvenile and embryonic stages, which could affect adult expression of hh. Adult G. minus continue to moult and add ommatidia as adults (Fong, 1989), suggesting a continuous requirement for eye development genes throughout the life cycle. However, it is possible that pax6, dac and so are necessary at earlier stages (e.g. embryonic eye development) but dispensable for adult eye growth, as in D. melanogaster, hh signalling induces differentiation of new ommatidia. (iv) Alternately, because dac, pax6 and so function in many organs (e.g. appendages, brain and nervous system, respectively), selection may have favoured mutations in genes reducing eye size with fewer pleiotropic consequences. Given the general conservation of eye development in arthropods, we favour the later hypothesis.

Evolutionary implications

Changes in the expression of hh orthologues in A. mexicanus are responsible for troglomorphic eye reduction in that species (Yamamoto et al., 2004) and are also implicated by the present study in G. minus eye reduction. These findings suggest components of the eye development genetic network, close upstream of hh, are involved in the convergent phenotypes shared between A. mexicanus and G. minus. However, the nature of these changes is clearly not identical. shh and twhh are up-regulated in A. mexicanus cavefish, whereas hh is down-regulated in troglomorphic G. minus. These differences in the mode of evolution likely reflect differences in the development of eyes in these different phyla. Nevertheless, hh appears to be a common point of divergence in the regulatory system that permits the evolution of eye reduction. Such genetic hotspots in evolution have been predicted (Stern & Orgogozo, 2008, 2009), and previous studies have found parallel mechanisms for phenotypic evolution of mammalian pigmentation (Hoekstra & Nachman, 2003; Nachman et al., 2003; Hoekstra et al., 2006), fruit fly trichome patterns (McGregor et al., 2007; Sucena et al., 2003) and wing pigmentation (Wittkopp et al., 2002; Gompel et al., 2005; Prud’homme et al., 2006), and skeletal reduction in stickleback fish (Colosimo et al., 2005; Shapiro et al., 2006; Coyle et al., 2007). More work, especially tests of gene function, will be required to compare in detail the mechanisms of eye reduction in amphipods with what is known from cavefish. However, the results presented here suggest an example of convergent evolution via changes at parallel points in development at a phylogenetic scale much greater than any previous study has suggested.

Another similarity exists in the evolution of cave phenotypes in G. minus and A. mexicanus. Cavefish retain the effector genes necessary for eye development, as evidenced by rescue of the eyeless phenotype with transplantation of lens tissue from surface donors (Yamamoto et al., 2004). Thus, despite evolution of a cave phenotype, in which genes for crystallins and opsins would be under relaxed selection, mutations have not completely eliminated effectors of eye development from the cavefish genome. An interesting parallel is seen in G. minus that inhabit karst windows. In these unique habitats, a ceiling collapse generates an exposed area with upstream and downstream cave access. These G. minus populations are most closely related to the hydrologically adjacent troglomorphs, but they have lost troglomorphic adaptations and have enlarged eyes (Fong, 2012). This atavistic evolution implies that like the cavefish, populations of cave-adapted G. minus retain the effector genes for eye differentiation. A recent study of the evolution of antibiotic resistance in E. coli has suggested that such evolutionary trajectories are unlikely, but possible if the required number of mutational steps is low (Tan et al., 2011; Weinreich et al., 2006).

Exploring another cave-adapted crustacean, Protas et al. (2011) recently conducted a QTL analysis using cave and surface populations of the isopod Ascellus aquaticus, for loci affecting eye size and eye loss. Markers were based on SNPs associated with genes conserved in eye development and body pigmentation, including dac, hh, so, eyg, eya and pax6. A single significant eye size QTL was found, which included dac. The presence or absence of eyes was strongly associated with another QTL closely linked to lim1; however, recombination between lim1 and the eye phenotype implied that this developmental gene is not directly responsible for eyelessness. Other marker loci, including those associated with so, pax6 and hh, were not associated with eye size or loss phenotypes (Protas et al., 2011). Studies of eye reduction in G. minus and A. aquaticus have used different methods, and both require additional experiments. As the eyes of subterranean A. aquaticus contain only four ommatidia, potential differences in the genetic cause of eye reduction in these species may be influenced by the initial size of eyes in surface populations. Detailed studies of eye development in each species will help to resolve the issue.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. References

The authors wish to thank Jenny Knauss and Nikita Donti for assistance with real-time PCR. Keya Meyers assisted in imaging and measurement of Gammarus minus. This work was supported by the American University College of Arts and Sciences, through a Mellon Graduate Research Award to ACA, as well as faculty research funds from the College to DRA and funding from Cave Conservancy of the Virginias to DBC and DWF.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. References