What kind of sensory adaptation would you hypothesize the cave fish has to allow

2Department of Molecular and Integrative Physiology, The University of Kansas Medical Center, Kansas City, KS 66160, USA

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1Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110, USA

2Department of Molecular and Integrative Physiology, The University of Kansas Medical Center, Kansas City, KS 66160, USA

e-mail: gro.srewots@orn

One contribution of 17 to a theme issue ‘Evo-devo in the genomics era, and the origins of morphological diversity’.

Accepted 2016 Jun 8.

Copyright © 2016 The Author(s)

Published by the Royal Society. All rights reserved.

Abstract

Animals have colonized the entire world from rather moderate to the harshest environments, some of these so extreme that only few animals are able to survive. Cave environments present such a challenge and obligate cave animals have adapted to perpetual darkness by evolving a multitude of traits. The most common and most studied cave characteristics are the regression of eyes and the overall reduction in pigmentation. Studying these traits can provide important insights into how evolutionary forces drive convergent and regressive adaptation. The blind Mexican cavefish (Astyanax mexicanus) has emerged as a useful model to study cave evolution owing to the availability of genetic and genomic resources, and the amenability of embryonic development as the different populations remain fertile with each other. In this review, we give an overview of our current knowledge underlying the process of regressive and convergent evolution using eye degeneration in cavefish as an example.

This article is part of the themed issue ‘Evo-devo in the genomics era, and the origins of morphological diversity’.

Keywords: Astyanax, cavefish, regressive evolution, eye degeneration, adaptation

1. Introduction

Understanding adaptation has broad implications not only for a basic understanding of evolution but also for developing a better appreciation of the challenges organisms face when confronted with changing environments. Extreme environments represent a unique opportunity to gain insight into adaptation. Some of the solutions animals have evolved in response to living under harsh conditions are simply stunning: desert gazelles that never have to drink, fish surviving high concentrations of sulfuric acid, or alpine marmots hibernating for two-thirds of the year are just a few of many examples of extreme adaptations. A particular widespread and well-studied example of such extraordinary adaptations can be found in cave animals. Organisms that live in caves for their entire life are known as troglobites. These range from snails, worms, beetles, spiders, scorpions and shrimps to vertebrates such as fish and salamanders [1]. To date, no mammal has been found to live entirely in caves (bats do stay inside caves, but venture out for foraging and hence are termed troglophiles). The extreme cave environment has driven the troglobites to adapt and evolve very different morphological, behavioural and physiological traits from their ancestral forms residing outside the caves. The most prominent and widespread troglomorphic traits are the loss of eyes and the reduction in body pigmentation. But many cave animals have also developed a variety of other traits, including many so-called constructive traits, such as elongated sensory organs, extra taste buds, additional neuromasts or metabolic changes.

Cave animals are equally fascinating to scientists and the general public. In Slovenian folklore, for example, a cave salamander is believed to be the larval stage of a dragon [2]. Historically, the first described cavefish were Amblyopsis spelaea [3] and they rapidly gained popularity in the scientific community when Darwin mentioned them in the ‘Origin of Species’. It was the dramatic loss of eyes that caught Darwin's particular interest, urging him to speculate: ‘As it is difficult to imagine that eyes, though useless, could in any way be injurious to animals living in darkness, I attribute their loss solely to disuse’ [4, p. 125]. This short statement launched an ongoing debate about the evolutionary mechanisms responsible for the loss of eyes in cave animals. The neutral hypothesis (the accumulation of hypomorphic mutations in the absence of selection) was favoured until recently when the results of new genetic, developmental and physiological studies supported the adaptive evolution of eye regression in cave animals (see below).

Most studies on eye loss have used the Mexican cavefish Astyanax mexicanus as a model. Accordingly, this review will focus mostly on this species. Towards the end, we will mention work on other cavefish as well, as their diversity is astonishing. Worldwide, more than 150 species of obligate cavefish are described, endemic to all continents except Europe and Antarctica. The Northern cavefish Amblyopsis spelaea [3], the Somali cavefish Phreatichthys andruzzii [5], or the Chinese cavefish Sinocyclocheilus [6] are a few of the better-known species complementing work on Astyanax mexicanus. The cave forms of A. mexicanus are found in the Sierra del Abra of Northeastern Mexico in 29 known caves. The distinguishing advantage of this system is that the ancestral surface forms of A. mexicanus still remain native to the surrounding river systems (figure 1). Although geographically isolated, the fish of the different caves are still completely interfertile with the ancestral surface form (allowing genetic analysis of the inheritance of the cave-specific and surface-specific traits) and with each other (allowing genetic complementation to be assessed). It is estimated that at least five independent events have led to these different cave populations over the past 1–2 Myr [7,8], making it a particularly useful model to study parallel and convergent evolution. Whether the same or different genes regulate repeatedly evolved phenotypes is a long-standing question in evolutionary biology and can be readily addressed in A. mexicanus. Furthermore, functional studies are feasible as the fish can be easily maintained and bred in a laboratory setting. For the last decade, considerable efforts have been devoted into building the tools needed to exploit this unique system. At this point, there are high-resolution genetic maps [9,10], detailed developmental staging tables [11], data from transcriptomic analysis [12], protocols for transgenesis [13] and methods for introducing mutations [14]. Moreover, the genome of A. mexicanus was recently assembled, further developing Astyanax as a tractable model system [15].

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Figure 1.

Surface and cave populations of Astyanax mexicanus. Panel (a) depicts an example of the river habitat of the surface populations of Astyanax mexicanus. Panel (b) shows the entrance to the Tinaja cave. Panels (c,d) depict the obvious morphological differences between surface fish and three independently derived cave populations (Tinaja, Pachón and Molino). While the surface fish are pigmented and have eyes (c), the cave forms have converged on the loss of eyes and strongly reduced their pigmentation (d).

Combined, these advances have enabled the dissection of the mechanism by which the eye develops in surface fish and how the eye regresses in cave fish. In this review, we discuss the developmental and genetic basis of eye loss in cave forms of Astyanax mexicanus and its implications for evolutionary developmental biology.

2. Eye development in fish

Eye development in fish is similar to that in most other vertebrates, with one notable exception. As most fish grow throughout their entire life, so do their eyes. The retina in teleosts, for example, is maintained through life by a continuous influx of new cells from stem cell niches, the so-called ciliary marginal zone and inner nuclear zone of the retina [16,17]. The first sign of vertebrate eye development is bilateral evaginations from a single developmental field in the anterior neural plate giving rise to the optic vesicles (figure 2). As these optic primordia expand, they form the optic stalk and then differentiate into various eye structures, including the neural retina, retinal pigmented epithelium (RPE), ciliary and iris epithelium, and the optic nerves [18,19]. Further in development, the optic cups form a lens vesicle that subsequently accumulate crystallin protein and form a crystalline lens [20]. The patterning of the eye field, however, happens much earlier. At the onset of gastrulation, the future forebrain region is divided into three overlapping domains. The anterior-most region subsequently forms the forebrain and the eye field. This entire process is a coordinated outcome of Wnt, Shh and Bmp signalling pathways [21,22]. Of particular importance is Shh which signals from the ventral midline to upregulate pax2 and vax1 and downregulate pax6. This restricts Pax6 to dorsal regions where it forms the optic cups, neural retina and RPE [23]. Other important transcription factors that are used as early markers and are known to have important roles in patterning the eye field are Rx3, Lhx2/9 and Six3 [18].

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Figure 2.

Eye development in surface and cavefish. The top row illustrates eye development in surface fish. Expression patterns of various key genes are colour-coded. The bottom row shows eye development in cavefish. The level of Pax6 (orange) decreases, while Shh (blue) and Pax2/Vax1 (yellow) levels increase. This results in smaller optic vesicles and longer optic stalks. Lens apoptosis starts at 24 hpf followed by retinal apoptosis ultimately leading to a loss of the eye.

3. Eye degeneration in cavefish

The eyes of surface fish follow the normal pattern of vertebrate eye development leading to fully developed and functional eyes, while the majority of cavefish have small, non-functional or even completely absent eyes as adults [24]. Interestingly, cavefish develop eyes similar to that of surface fish, at least for the first few hours of development. However, soon after these early hours of development, eye growth arrests, and the eyes of cavefish start to degenerate. Various studies have investigated differences in the signalling pathways between surface and cave morphotypes to understand the developmental origin of these distinct eye morphologies in the two populations. An important pathway contributing to eye degeneration in cavefish seems to be mediated by Shh. As mentioned above, in normal eye development, Shh signalling from the anterior midline of the neural plate dictates the levels of Pax6, Pax2 and Vax1. Cavefish show a marked increase in Shh levels along the midline which downregulates pax6 and upregulates vax1 and pax2 [23]. This creates an imbalance between Pax6 levels and Pax2/Vax1 levels that is crucial for determining the gap between the two eyes and the size of the optic cups. This results in increased length of optic stalks and a pair of optic cups reduced in size (figure 2). Other than from these expression analysis experiments, the strongest evidence for a role of Shh in eye regression stems from experiments tinkering with its levels. Overexpression of shh in surface fish results in strikingly similar eye phenotypes as seen in cavefish [23]. The reciprocal experiment of inhibiting Shh in cavefish does not fully restore eye development, but does lead to slightly bigger eyes, underscoring the potentially important role of Shh in eye degeneration [25]. Although these experiments are impressive, it is unlikely that mutations in or nearby shh are directly responsible for the cave phenotypes as none of the eye size quantitative trait loci (QTL) (see below) overlap with the shh locus [10].

Given that Shh is a very powerful morphogen [26], an expansion of shh expression throughout cavefish forebrain development would be expected to have more dire consequences than just losing the eyes. To find out if there are potential compensatory mechanisms in place, Retaux and co-workers [25] studied the timing and expression of potential signalling pathways affected by Shh and found Fgf8 to be a good candidate for such a balancing mechanism protecting the cavefish forebrain from disorganization. While there are no differences in the absolute levels of Fgf8 signalling between cavefish and surface fish [27], the timing of its expression is altered. Using precisely staged embryos [11], Retaux and co-workers [25] found that fgf8 expression was detectable in the cavefish telencephalon as early as 10 hpf, whereas it was apparent only at 12 hpf in surface fish. Using elegant experiments in which the timing of fgf8 expression was altered, they showed that heterochrony between surface and cavefish contributes to the eye degeneration while leaving the organization of the brain undisturbed. Their study further identified another marker of eye field specification, Lhx2, to be differentially expressed. While being expressed at normal levels in surface fish, they could not detect Lhx2 in the medial posterior part of the presumptive cavefish forebrain. Fate mapping of Lhx2-positive cells showed that these cells contribute to the retina in surface fish, but become part of the hypothalamus in cavefish, suggesting a trade-off between retina and hypothalamic fated territories [25]. These observations address an old question in the field: why do cavefish develop an eye in the first place, only to later degenerate it? This appears to be a waste of resources and energy; however, from a neurodevelopmental point of view, it seems to be unavoidable. The cells contributing to the retina and other parts of the forebrain are so highly intertwined that a development of a normal forebrain without an eye may be an impossibility. This presents a remarkable example of a developmental constraint in evolution.

4. The lens as a crucial factor in eye development and regression in cavefish

Apoptosis in the lens marks the first sign of eye degeneration in cavefish [28]. These events precede retinal degeneration, suggesting that apoptosis of the cells in the lens could be responsible for triggering eye degeneration. This observation led to one of the most striking experiments performed in the field of vertebrate evolution (figure 3). The rationale behind the experiment was that if the lens is influencing eye degeneration in cavefish, then transplanting a normal lens from surface fish early in development may be able to rescue the loss. Thus, a 36 hpf surface fish lens was transplanted into the optic cup of an age-matched cavefish embryo [29]. The transplantation was performed only on one side of the fish, while the other eye served as a control. Remarkably, the transplanted eye in the cavefish host developed normally (although to a slightly smaller size). This transplanted eye also grew retina and photoreceptor cells and some of the retinal fibres did project into the optic tectum, but the eye was not able to respond to light [30,31]. In a reciprocal experiment wherein a cavefish lens was transplanted onto a surface fish eye, the eye failed to develop and degenerated as it would have in cavefish [29]. These impressive transplantation experiments demonstrate that the lens serves as a crucial organizer of optic development and in cavefish apoptosis is controlled autonomously within the lens, while the eye and accessory tissues have retained the ability to respond to signals generated by normal surface fish lens.

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Figure 3.

The lens as a crucial factor in eye development and regression in cavefish. Lens from 36 hpf surface fish embryo is transplanted onto the cavefish eye and vice versa. The surface fish lens transplanted onto cavefish is sufficient to grow an eye in cavefish (d). The other side of the fish served as a control (c). In the reciprocal experiment, when a cavefish lens is transplanted onto a surface fish, the eye degenerates (b). (a) The non-transplanted side as a control. Scale bar, 1 mm. The eye images have been reproduced from [24].

5. Genetic basis of eye regression in cavefish

While work on known genes in eye development has led to the identification of some genetic factors that contribute to eye degeneration in cavefish, the study of candidate genes has its limits. It is often impossible to say if an observed change is due to a direct or an indirect effect. Unbiased genetic mapping approaches are needed to distinguish between these possibilities, making Astyanax mexicanus an ideal model. Because eyeless cave forms of Astyanax remain interfertile with the eyed surface form, QTL mapping is feasible and widely used. The eyes of the F1 progeny of a cross between surface and cave form (hybrids) of Astyanax mexicanus are smaller in size but retain their function [32]. In the F2 generation, there is a broad range of eye sizes indicating that multiple genes in cavefish govern eye and lens sizes. The first QTL mapping for troglomorphic traits in cavefish was performed by Borowsky and Wilkens using random amplified polymorphic DNA (RAPD) markers [9]. Using F2 hybrid populations from a cross between a Pachón cavefish and a surface fish, the study revealed three putative QTL for eye size that accounted for 27% of the observed variance. The next significant mapping study was using microsatellite markers which allowed for a more detailed genetic map [10]. This study was again performed in Pachón/surface hybrids and mapped numerous additional traits, such as melanophore number, teeth and taste buds. Among the 48 QTL that were identified, 12 affected eye or lens size. It came as a surprise that none of these QTL were near the earlier studied genes—shh or pax6—indicating that mutations in these genes were not directly responsible for the eye phenotype and that genes upstream were most probably involved; however, in a more recent study, a QTL for eye diameter was found to be near the pax6 locus [33].

Until recently, the identification of candidate genes underlying the regions defined by the QTL studies was only possible by using synteny with the zebrafish genome. While this is a valid approach, Astyanax and Danio diverged 180 Ma [34], making precise predictions about candidate genomic regions challenging. Recently, this hurdle was overcome and the cavefish genome was fully sequenced, which has potentiated the identification of candidate genes within these QTL [15]. The 15 previously identified eye or lens QTL were used to search the assembly and a list of 2408 genes (out of the 23 042 annotated genes in the genome) was compiled. Despite a major step forward, this list has some substantial downsides; because scaffolds often do not span the entire critical region comprising a QTL, candidates could have been missed. On the contrary, the great majority of these genes will have nothing to do with the phenotype and will simply be physically linked to the causal variants. Nevertheless, it was the first time that such an all-encompassing list could be assembled, and with the addition of RNA-Seq data some interesting candidates were identified. Six genes stood out as particularly promising: cryaa (crystallin alpha A—which was identified earlier to be a good candidate [35]), pitx3 (paired-like homeodomain 3), rx3 (retinal homeobox gene 3), olfm2a (olfactomedin 2a), olfml2a (olfactomedin-like 2A) and BCoR (BCL6 corepressor) [15]. Cryaa is an anti-apoptotic chaperon, which—if knocked down in surface fish—induces apoptosis in the lens. It is under the control of sox2, which is also expressed in lower amounts in cavefish compared to surface fish [36]. Studies in zebrafish show that Pitx3 is essential for lens and retinal development [37] and rx3 knock-down leads to an eyeless phenotype [38]. olfm2 encodes a secreted glycoprotein that is involved in nervous system development in zebrafish. Its downregulation adversely affects the development of olfactory pits, eyes and the optic tectum. The downregulation also results in altered expression levels of pax6 [39]. BCoR is a transcriptional corepressor implicated in human diseases that cause small eyes [40]. All these genes showed a marked decrease in expression in the cavefish embryos as compared with surface fish embryos, underscoring their potential role in cavefish eye regression. Future studies will be needed, however, to rule out secondary effects and to identify the responsible genetic loci explaining the expression differences.

The same study provided also some information on the so-called pleiotropic QTL, QTL that were found to affect multiple traits. While it is possible that these traits co-map to similar regions simply by coincidence, one intriguing possibility is that they point towards genes that have multiple functions, so-called ‘super genes’. A classic example in cavefish evolution is shh, hypothesized to be involved in cavefish eye regression. Shh signalling also has positive effects on the development of taste buds, a trait that is largely enhanced in cavefish [41]. In principle, scanning these regions that underlie multiple QTL could reveal additional super genes. An example that emerged from this study is prox1, a gene that affects many traits like lens apoptosis and development of taste buds and neuromasts [42]; shisa2, AIFM1, otx2, crxa and tbx2a are some other interesting candidates that show potential for further study [15].

6. How does selection work: de novo mutations or standing genetic variation

Another important goal in the field of evolutionary genetics is to trace the origins of mutations necessary for adaptation. Do novel mutations originate in the new environment (in this case, the cave) or do they already exist at some low frequency in the founder populations (the surface fish)? The origin of the mutation can have profound effects on the timing of selection, the signature they leave in the genome, as well as the type of alleles that are selected. Changes that occur only after fish have been swept into the caves are termed ‘de novo’ mutations, while mutations that are present in surface fish are titled ‘standing genetic variations’. Standing genetic variations (or simply population polymorphisms) are more common than previously thought and there is an increasing acknowledgement of their importance in evolution and adaptation [43]. Each mode of genetic change has particular advantages and disadvantages when it comes to adaptation. For instance, de novo mutation is generally considered to be a relatively slow process, so less likely to occur in quickly or drastically changing environments [43]. Additionally, de novo mutations come with a bias against recessive alleles (a process called Haldane's sieve [44]) simply because recessive de novo mutations would be less advantageous in the first generation and would be lost sooner from the population. Lastly, the chance that an advantageous variant gets fixed in a population is higher if it is present in many individuals, favouring standing genetic variation, which often is present in the range of several per cent of the population [45,46]. On the other hand, adaptation through standing genetic variation faces a different problem: how is standing genetic variation maintained in the absence of selection since there is no reason to believe that any of these mutations are advantageous in the original environment? There are probably multiple answers to this question; one is that many alleles are usually found in heterozygous condition in the founder populations; another possibility, however, is a mechanism that has recently received a surge in interest: the role of cryptic genetic variation.

Cryptic genetic variation is genetic variation that is ‘invisible’, meaning it has no phenotypic effect under normal conditions (but it can under certain conditions—such as stress or certain genetic backgrounds). The potential role of cryptic genetic variation in evolution was theorized first by Waddington in 1942 in his famous paper on canalization [47] and later in experiments using Drosophila, where he observed that a heat shock during development revealed genetic variation [48]. Canalization has since been defined as a mechanism during development that stabilizes or buffers a phenotype against genetic variation. It took over half a decade before heat shock proteins, especially Hsp90, were identified as a major molecular mechanism underlying Waddington's observations of canalization [49]. Hsp90 keeps unstable signalling proteins poised for activation until they are stabilized by conformational changes associated with signal transduction [50]. It also acts as a buffer against genetic changes that could otherwise have drastic effects on the functionality of the protein (the canalization function). Since the discovery of this dual role (in Drosophila), similar studies have been performed in other model organisms like yeast and Arabidopsis [51,52], but until recently none in a natural system such as the cavefish. Cavefish seemed to be a prime candidate for understanding the role of Hsp90 in evolution. Based on the argument that if such a mechanism contributed to evolution, it would be particularly likely to occur in a setting where animals are suddenly confronted with an extreme change in environment presumably causing substantial physiological stress. Using a chemical inhibitor of Hsp90 and by mimicking an environmental condition of cave environments, surface fish, indeed, revealed cryptic genetic variation affecting eye size in development [53]. Importantly, the variation could be selected for in subsequent generations, providing the basis for heritable evolution. Notably, the same experiments in natural cavefish populations revealed indications of selection for Hsp90-controlled traits, providing the first study in which a potential role of Hsp90 has been documented in morphological evolution in nature [53].

7. Why regress eyes: selection, pleiotropy or drift?

The loss or reduction of ancestral characters is a common event in the evolutionary history of many organisms. Classic examples include the loss of tails in some primate lineages (including ours), reduction of hind limbs in whales, loss of teeth in birds, and the aforementioned loss of eyes and pigmentation in cave animals. While it is relatively easy to imagine that a new trait evolves for a reason, the study of regressive traits has been slowed by the assumption that such characters primarily arise from genetic drift and the unconstrained accumulation of deleterious mutations. Studying these traits, it follows, would not contribute to a broader understanding of adaptation. Even though the evidence for neutral evolution in eye regression remains to be presented, it has still persisted as the main theory for the last century, perhaps, in part, due to Darwin's aforementioned quote about disuse. In the last decade, however, evidence is piling up to support the adaptive significance of eye loss in particular and regressive traits in general. Here, we will discuss the three main theories for eye regression in cavefish and some of the evidence behind them.

• 1) Direct natural selection against eyes to conserve energy or mitigate any other disadvantage of having eyes (entry point for infection, prone to injury, etc.).

• 2) Indirect selection against eyes by selecting for another trait that is negatively linked to eye development.

• 3) No selection at all. Simply due to the accumulation of random mutations after relaxed selection for eyes.

None of these theories have been fully proved, and most probably the final answer will be that all three have contributed to some extent. The strongest genetic argument for the hypothesis that eye loss is adaptive comes from QTL experiments. When the effective size of these QTL was analysed, it was found that for each individual QTL, the cave allele resulted in smaller eyes [54]. This is a strong argument of selection based on the theory of H. Allen Orr [55], as if there were no selection on a trait one would expect that, just by chance, some of the random changes would lead to larger eyes as well (as was seen in the same study for pigmentation traits). Together with the fact that all other known obligate cavefish in the world have reduced eyes [56,57], this makes a selection scenario quite likely. However, it does not allow distinguishing between direct and indirect selection. Evidence for indirect selection comes from several studies both genetically and functionally. Several studies found a clustering of QTL for eye size and neuromast number [33,54], potentially hinting at a shared genetic basis, although the resolution of these genetic studies remains too low to finally prove this [58]. Another study, however, connects the regression of eyes and the enhancement of taste buds and jaws in cavefish by altering expression of shh [59]. Shh has wide-range effects on the development of various organs and it has been identified as a strong candidate for eye regression [23]. Its elevated expression in the oral–pharyngeal region in cavefish taste buds [23,60] and the fact that eye size and number of taste buds are correlated in hybrids [59] makes Shh a promising candidate for functional studies. Conditional overexpression of shh in surface fish, indeed, increased the numbers of taste buds, while the eyes regressed in the same fish [59]. Thus, the opposing effects of Shh are a prime example of negative pleiotropy.

Energy conservation is the major proposed driver of active selection for eye regression. As neural tissue is one of the most energy-intensive tissues to form and maintain [61], the nervous system has to strike a balance between two opposing demands: maintaining good sensory abilities and preserving energy. This becomes even more crucial in energy-deprived environments, such as caves, where procuring food is a major challenge. While the metabolic rate measured as oxygen consumption in cavefish (a proxy for metabolic rate) is lower than in surface/cave hybrids or phylogenetically younger cave populations [62], no systematic study has demonstrated an adaptive reduction in the neural tissues of cavefish. To address this experimentally, a recent study performed a more detailed analysis and measured the oxygen consumption of Astyanax brains and eyes ex vivo [63]. This study calculated significant metabolic costs for the optic tectum and the eyes adding up to 15% of the resting metabolism needs in juvenile fish. Such a high cost of vision, almost reaching the cost of the human brain (at 20–25%), makes an adaptive loss of eyes owing to energy constraints highly probable.

8. Reversal of natural selection in karst windows: Dollo's Law?

A fascinating question in evolution is whether evolution runs along a one-way street or whether it is reversible. Dollo's Law might suggest the former. In 1893, Dollo proposed that ‘An organism is unable to return, even partially, to a previous stage already realized in the ranks of its ancestors’ [64]. While there are obvious exceptions to Dollo's Law (after all whales went back to the sea), it remains an important, even crucial assumption in evolutionary biology. Without Dollo's Law, the construction of phylogenetic trees from fossil data would be a meaningless endeavour. A more updated version of Dollo's Law might read: ‘An organism is unable to return, even partially, to a previous stage already realized in the ranks of its ancestors without leaving obvious signs of this reversal’, meaning that evolution is reversible but not through the exact same mechanisms. Again cave animals are providing a wonderful example to study Dollo's Law through some geological anomalies, the so-called karst windows. These are regions where a cave roof has collapsed and light can again enter the cave and cave animals have regained surface traits. Such a case was initially reported for eyed cave amphipods Gammarus minus [65]. Also Astyanax mexicanus seems to have its own example: the Caballo Moro cave. This cave has a geologically young karst window and carries eyed cavefish populations. Genetic analysis using RAPD markers indicated that these fish are evolutionarily closer to the blind fish of the cave than the eyed surface fish which would make it tempting to speculate that they have indeed regained vision after they came in contact with light [66]. Future studies will be needed to confirm this exciting observation.

9. Work on other cavefish species

While most studies have used Astyanax mexicanus as a model system, research on other cavefish models can provide insight into the question of convergent evolution. Whether independent evolutionary lineages use different genetic and developmental means to achieve similar results, or if there are certain constraints that limit the type of mechanisms is a long-standing question in evolution. There are more than 150 known species of troglobite cavefish in the world, of which China has a particular rich cavefish fauna with over 50 species described to date. The genus Sinocyclocheilus (in the order of the Cypriniformes) contains at least 10 cavefish species. The availability of many closely related surface-dwelling species makes it a good model to study cave adaptation in an evolutionarily distant system from Astyanax mexicanus (which is found in the order of the Characiformes) [6,68]. Both orders share a common ancestor approx. 180 Ma, allowing for predictions over larger evolutionary distances. Unfortunately, these Chinese cavefish do not breed in the laboratory, so no developmental studies are possible. When studying the adult forms of one cavefish species of this genus (S. anophthalmus), it was discovered that they possess regressed eyes similarly buried under the skin as they are in Astyananx, but the overall eye structure remained intact. The authors reported fully formed lenses, cornea, iris and retinal structures, even though the retina was notably thinner than in surface fish [6,69]. As the lens did not show obvious morphological changes in adult Sinocyclocheilus anophthalmus cavefish, this suggests a different route of eye degeneration as in Astyanax, possibly due to decreased proliferation [69]. However, a recent study in another cavefish species of the same genus (S. anshuiensis) found strong lens and retina defects, suggesting that even within a single genus of fish, multiple mechanisms underlying eye regression are feasible. The recently published genome information of three Sinocyclocheilus species (S. grahami, S. rhinoceros and S. anshuiensis) will hopefully help in resolving some of these questions [6].

Another exciting study on a phylogenetically distant cavefish species revealed that there are, indeed, many ways to obtain similar phenotypes [5,70]. The authors of this study used the Somali cavefish Phreatichthys andruzzii, which have recently been introduced as a model for extreme troglomorphism [5]. These cavefish are believed to have been isolated for over 2 Myr, showing not only complete loss of eyes and pigmentation but also a complete loss of scales. While there is no closely related surface form available for this species, the cave form breeds in the laboratory, and developmental studies can be performed. When the authors studied early markers of eye patterning in embryos of these fish, they did not detect any changes. In addition, there were fewer signs of apoptosis in the lens tissues. Both findings were in contrast to the known mechanisms underlying eye degeneration in Astyanax cavefish [70]. On the other hand, they found late retinal differentiation events affected and retinal apoptosis to be the driving force of eye degeneration in these fish. Thus, evolution has targeted different developmental processes in Astyanax and Phreatichthys despite a very similar phenotypic outcome.

10. Conclusion and next steps

Cave forms of Astyanax mexicanus are natural mutants in evolution and have become an excellent model to study the physiological and morphological basis of adaptation to new and extreme environments. Natural populations under strong selective pressure are helpful in deciphering the genetic changes responsible for adaptation, and cavefish present a particular useful model as the polarity of these trait changes is known (from surface to cave). The recent genome assembly has opened up many new promising avenues into the genetic basis of cave adaptation. It is now possible to take QTL studies a crucial step further; so far, QTL studies in Astyanax were mostly used to study the genetic architecture of a trait (e.g. how many loci contribute to a trait, what is their effect size and polarity and to what extent are they linked to other traits?). But as the identified genomic regions in QTL studies often contain many genes, with very few exceptions, this work has never led to the identification of the actual mutation causing the trait under study, the so-called QTN (Quantitative Trait Nucleotide) [71]. Now that intervals can be defined and further narrowed using additional SNPs there is a big opportunity for more QTN to be discovered. The next step after the discovery of QTN is functional testing in vivo. Again new technologies are paving the way in many model organisms, including Astyanax. Genome editing using TALENs has been successful in cavefish [14] and CRISPR/Cas9 will be the obvious next method of choice to alter the genomes of surface and cavefish and ultimately prove causality of identified mutations. With the onset of these new technologies and a growing community of researchers using this and related cavefish systems (www.cavefin.org), cavefish biology awaits exciting times. While the majority of cavefish research has focused so far on the obvious trait of eye loss, there are a plethora of other regressive and constructive traits that await further study. Especially constructive traits are not under the stigma of genetic drift and their adaptive value is more widely accepted. Some of the most striking adaptations are probably the increases in sensory perception, such as the increased sensitivity to vibration and the resulting change in the behaviour and sleep of cavefish [33], or the several thousand-fold increase in olfactory capacities ([72] and S Retaux 2015, personal communication). Another fascinating aspect of cavefish is the change in their metabolism to cope with what otherwise are considered starvation conditions [46]. Studying the genetic basis of metabolic evolution has experienced a surge in interest over the last years and has opened up the new field of ‘Evo-Physio’. Given their extreme metabolic evolution, cavefish provide an excellent model to contribute to this exciting topic in the future.

Most work on eye regression, so far, has focused only on a single or few populations. One of the strengths of Astyanax mexicanus, however, is that many independently derived cave populations exist, which can be harnessed to address the question of repeatability in evolution and adaptation. Again eye regression seems to be a great trait to study this, as previous work is pointing to different genes and mechanisms being involved. For example, crosses between two independently derived populations, Pachón and Los Sabinos revealed bigger eyes in the hybrids than in any of the parents, indicating partial complementation and different genetic loci at play [73]. Last but not least, the phenotypic similarity to some retinal degeneration diseases in humans, such as age-related macular degeneration or Retinitis pigmentosa, has raised the possibility of using this system as an alternative model for evolutionary medicine [74].

Acknowledgements

The authors thank Mark Miller from the Stowers Institute for the help with scientific illustration of the figures and Stacey Williams and Matt Gibson for critical reading of the manuscript.

Authors' contributions

J.K. and N.R. wrote the manuscript.

Competing interests

The authors have no competing interests.

Funding

J.K. and N.R. are supported by the Stowers Institute for Medical Research.

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What kind of sensory variation would you hypothesize if the cavefish?

What is sensory adaptation?