Commonly used evolution assessments often ask about the evolution of blindness in cavefish or salamanders, running speed in cheetahs, and/or the long necks of giraffes. Explaining the loss of function in cave animals, however, is more difficult than explaining evolution involving gains of function resulting from natural selection. In fact, the evolution of cavefish blindness is not yet well understood by scientists. This article presents the three current hypotheses for explaining the evolution of blindness in Mexican tetras (Astyanax mexicanus), related to the Next Generation Science Standards and the Advanced Placement curriculum.
Measures of understanding of evolution often ask students to explain the evolution of blindness in cavefish or salamanders, running speed in cheetahs, and/or the long necks of giraffes (Bishop & Anderson, 1990; Demastes et al., 1995; Project 2061, n.d.; Settlage, Jr., 1994). These items are primarily used to identify student misconceptions, such as inheritance of acquired characteristics and evolution driven by need or “pressure” (Nehm et al., 2010). To the surprise of many, students typically have much more difficulty with the cavefish question than with cheetahs (Nehm & Ha, 2011), but students are not the only ones who have difficulty explaining the evolution of a loss of function (e.g., blindness) vs. a gain of function (increased running speed). Even Darwin (1872) got the explanation of blindness in cavefish wrong, attributing it to disuse: “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” (p. 110).
The evolution of different species with similar structures or functions in spite of their evolutionary ancestors being very dissimilar or unrelated is called “convergent evolution” (Biology Online, 2016). The main question that has confounded biologists for years has been: How do so many different species that inhabit caves end up with very similar phenotypes—how do the phenotypes converge? In particular, how does blindness evolve in cavefish and in essentially every other species of animal whose life is spent in the dark (called “troglodytes” or “troglobionts”)? The surprising answer is that we don't know the full story yet, but the mechanisms underlying the evolution of blindness in the Mexican cavefish have begun to be elucidated.
In the classroom, understanding how evolutionary biology could explain loss of function in the case of troglodytes provides an excellent opportunity not only for identifying student misconceptions, but also for understanding central concepts. These include: Disciplinary Core Ideas such as natural selection, adaptation, and the interplay of genetics and the environment (“evo-devo”) promoted by the Next Generation Science Standards (NGSS Lead States, 2013; Lerner, 2000); as well as more advanced AP “Big Idea 1” (evolution), Science Practices 1 (using models), 3 (scientific questioning), 5 (using scientific explanations and theories), 7 (relating knowledge across scales, concepts and representations); and more specific concepts such as genetic drift, fitness, and homeotic genes (Biology Online, 2008, 2009, 2012). Troglodytes are an excellent case study for students to learn to explain the basic evolutionary question of how species change over time (adapt to shifts in their environment) (Table 1). The existence of vestigial structures such as the nonfunctional eyes of cavefish is also an important component of the evidentiary support for the theory of evolution (Senter et al., 2015; Anonymous, 2011). Also, studying weird creatures that live in a dark world far beneath the surface can be fun because students often find these troglodytes interesting and motivating. To explain troglodyte evolution, first we need some background.
The Lives Of Troglodytes
The underground world of caves is largely unknown even though most of the world's unfrozen water (94%) is stored, not in the oceans, but underground (Culver & Pipan, 2009). There are nearly 50,000 caves in the U.S. alone (Retaux & Casane, 2013). So, what is life like in a cave, and what do we know about life there that might help explain the evolution of cave blindness? Of course, it is dark, and the lack of sunlight means the absence of photosynthesis. Therefore, there are no primary producers to convert light energy into biomass for consumption up the food chain. So how do organisms survive? What do they eat? How do they find mates? What kinds of animals are found there? Why are they all blind? And how did they get into the caves in the first place?
There is a surprising diversity of life in caves. Almost all the major phyla are represented, including fish, beetles, salamanders, shrimp, and spiders, among many others (Protas et al., 2011). There are 86 cavefish species alone (Jeffrey, 2009). The ancestors of most of these species that Darwin called “wrecks of ancient life” (Darwin, 1872, p. 112) were likely washed into the caves by spring flooding. The cave and surface (hypogean and epigean, respectively) populations were then separated when the water receded and connections between the two dried up. This example of the isolation of subpopulations has many parallels with the appearance of Darwin's finches (and other animals and plants) on the Galapagos Islands. Just as with Galapagos organisms, the first members of a species to arrive in a cave were likely few and varied little from their relatives left behind, left behind, but over time cavefish and surface fish subpopulations evolved differently (diverged) so that some sequences in the genomes of cavefish of the same genus are found to be different from their surface relatives (O'Quin et al., 2015). Of course, new spring rains sometimes bring new immigrants into a cave such that the population immediately after a rain actually consists of (sighted and blind) subpopulations.
Probably the most studied of the troglodytes is Astyanax mexicanus (Figure 1) found in certain caves in northern Mexico (Figure 2), a tetra related to the common aquarium fish that students may be familiar with. In the absence of photosynthesis, they mostly survive on the film of bacteria that break down bat and cricket guano (feces), although sometimes flooding brings in additional biomass food (Leighton, 2015; Gross, 2012). Darkness also means that finding mates is more difficult. Mating and sexual selection in most animal species is often based on coloration, but troglodyte species typically have no coloration. Astynanax (as-tie'-a-naks) individuals find mates through their enhanced tactile senses; they have greater ability to sense vibration. In fact, scientists capture these fish simply by putting a net in the water and vibrating it.
The Evolution of Blindness in Astyanax mexicanus
Most cavefish do in fact have tiny eye structures, but these eyes are sunken below the body surface. In many of these species, the initial development of the eye is relatively normal, but the eye structures degenerate (regress) and become nonfunctional as development proceeds.
As Darwin noted, the evolution of blindness by natural selection in cave animals is a conundrum. Natural selection only selects for traits that enhance survival to reproduction, explaining the gain of new structures, traits, and functions. How then can evolutionary theory explain the loss of function in structures that have no value to survival? In darkness, there is no advantage to having functional eyes, therefore there is no natural selection for better functioning eyes. The natural selection pressure is “relaxed,” but there is, likewise, no obvious reason to select for the loss of sight. How can we explain this regressive evolution (the loss of useless characteristics over time) (Jeffrey, 2009)?
Three main hypotheses have been proposed to explain these examples of regressive evolution. According to the first hypothesis, eye loss is indeed caused by direct natural selection because there is an advantage to being eyeless in the dark. Studies have shown that maintaining eye tissue, especially the retina, and the related neural tissue comes at a high metabolic cost (Moran et al., 2015; Protas et al., 2007). Therefore, cavefish without eyes are at an advantage in this environment where energy sources (food) are scarce, because blind fish do not waste energy on these useless structures.
A second hypothesis employs the phenomenon of pleiotropy, that is, cases in which multiple phenotypic effects are caused by the same mutation in a single gene. There is, for example, evidence that one of the genes responsible for eye loss in cavefish also increases the number of taste buds on the ventral surface of the head, which helps cavefish find food more effectively (Gross, 2012). Natural selection for this increase in taste buds would, therefore, also promote blindness.
The third hypothesis is based on neutral mutation and genetic drift. All too often textbooks use the terms “evolution” and “natural selection” interchangeably, ignoring the importance of genetic drift. Genetic drift is “the process of change in the genetic composition of a population due to chance or random events rather than to natural selection, resulting in changes in allele frequencies over time” (Biology Online, 2008). Genetic drift differs from natural selection because observed changes in allele frequency are completely at random, not the result of natural selection for a trait. Genetic drift can have a relatively larger impact on smaller populations such as a typical population of cavefish. According to the neutral mutation and genetic drift hypothesis, therefore, normal mutation processes in a small population of cavefish sometimes produce neutral mutations (mutations that lead to phenotypic changes that natural selection does not act on), and in the absence of natural selection, totally random events can sometimes result in the increased frequency of such mutations over time. Such changes could include eye degeneration.
So, what's the right answer? What genetic evidence is there to support each of these hypotheses? As with so much in science, the answer is probably that these explanations are not mutually exclusive; it is likely that all three partially explain cavefish blindness. To understand that statement, we must have some further background on A. mexicanus genetics.
The Genetics of Astyanax mexicanus
Much is known about the genetics of this cavefish. The genome consists of more than a billion base pairs (NCBI, 2013). Unlike typical Mendelian traits, inheritance of eye structures and eyesight is polygenic, that is, determined not by a single gene but by many (e.g., genes related to eye structure, the lens, the retina, pigments, etc.) This is particularly easy to understand in the case of eye pigments, which are formed by a series of metabolic reactions, each reaction being catalyzed by a different enzyme and coded by a different gene, all of which are required to produce the final active pigment.
Some A. mexicanus genes have an additive effect and thus are called “quantitative” traits. For example, there are at least three genes that determine the extent of ossification (bone development) in the sclera (tough outer covering of the eye) in A. mexicanus (O'Quin et al., 2015). The genes have an additive effect; that is, when two (or three) of these genes are unmutated, more bone development in the sclera occurs than if only one is not mutated. Skin pigmentation in A. mexicanus is also an additive trait (Gross, 2012). In contrast, if the genes were Mendelian (each trait determined by a single gene), the elimination of any one gene product by mutation (in both alleles) would result in no scleral ossification; that is, the effect is qualitative: yes/no, tall/short, and so on.
Most of the genes discussed so far are likely “structural” genes; that is, their DNA sequences code for the sequences of amino acids in the resulting proteins. Cavefish eye development is also determined by “regulatory” genes, genes encoding products that regulate an entire developmental pathway, such as the well-known Antennapedia (antp) gene in Drosophila that produces an entire well-formed pair of legs on the head in the place of antennae (Figure 3).
As mentioned above, some genes involved in A. mexicanus eye development are also pleiotropic, that is, a single gene impacts more than one recognizable trait. For example, the overexpression of the genes sonic hedgehog (shh) and tiggy-winkle hedgehog (twhh)—which are both homeotic genes (genes that encode transcription factors that often control an entire developmental pathway) in A. mexicanus—results in degradation of the lens of the eye, but it also results in an increase in the size of the fish's jaws and in the number of taste buds on the lips (Retaux & Casane, 2013; Protas et al., 2007). In fact, such a gene would also be considered pleiotropic because it has multiple phenotypic effects (on the eye and the mouth). Note that regulatory genetic effects tend to be qualitative, not quantitative, that is, the mutation of a single gene, not many genes, is typically sufficient to determine the effect. Thus, shh is likely to have its effect “upstream” in development, that is, prior to the actions of the structural genes described above.
Evolution of Cave Blindness in A. mexicanus
It is easy to understand how pleiotropic genetic determination of eye development and sensory perception could explain cavefish blindness. Natural selection simply favors mutations that increase the number of taste buds, and the loss of eyesight is a coincidental byproduct—supporting both the natural selection and pleiotrophy hypotheses. Thus, degeneration of the eye is indirectly selected for. On the other hand, studies of the sequences of other genes related to the cavefish eye show high frequencies of substitutions in both coding and noncoding regions, which would support the genetic drift hypothesis (Retaux & Casane, 2013).
In summary, the most current proposed explanation is that the determination of sight in cavefish is a complex process with polygenic determination, involving pleiotropic genes with multiple effects as well as qualitative and quantitative, and structural and regulatory genes. The evolution of blindness in cavefish is best explained by a combination of all three hypotheses described above.
Evolution of Albinism in Troglodytes
It is important to note that not all losses of structure or function are determined in the same way. For example, reduced coloration in cavefish is another loss of function, and the determination and evolution of this trait has similarities to, but also differences from, the evolution of eyesight loss. Although there is not space to discuss this other commonly cited example in detail, pigment regression in Astyanax is also a polygenically determined trait involving both regulatory and structural genes. A prominent structural gene involved is melanocortin 1 receptor (Mc1r), which is involved in determination of the brown component of melanin (a pigment found in the skin). This gene and its functioning were recently discussed at length in this journal (Offner, 2013). Another gene involved is oculocutaneous albinism (oca2), which is known to cause albinism in a variety of vertebrates (Gross, 2012). In the case of pigmentation evolution, current data suggest greater support for the importance of the neutral mutation/genetic drift hypothesis than for the other two hypotheses discussed above (Jeffery, 2009).
The central tenets seem to be these:
Evolutionary science is both an experimental and a historical science; successful evolutionary explanations provide possible explanations (that must be consistent with the data), not “proven” answers. Explanations become increasingly tenable if they are supported by additional research (i.e., supported by the “weight of the evidence”).
Evolution can be used to explain losses of functions or structures over geologic time.
Evolutionary theory includes not only natural selection but also genetic drift.
The exact mechanisms involved are not fully understood, but scientists are learning more and more about the evolution of cave blindness.
Sightedness in A. mexicanus is a polygenic trait, involving structural and regulatory genes, pleiotropic genes, and qualitative and quantitative genes.
Evolution of blindness in A. mexicanus can be explained by a combination of three hypotheses:
The high cost of sight/direct selection hypothesis: Natural selection directly produces blindness in this species because there is advantage to not wasting energy on a useless and costly function and structure.
The pleiotropy/indirect selection hypothesis: Sightedness is determined by individual mutations that code for more than one trait (e.g., increase in sensory perception and loss of eyesight); natural selection for one function necessarily results in increases the frequency of both outcomes. Gains in sensory perception are beneficial and positively selected; loss of eyesight has no effect on selection.
The neutral mutation and genetic drift hypothesis: Many mutations that occur in the genes involved in sightedness are selection-neutral; that is, they are not affected by natural selection. The frequencies of these mutations can increase over time as a result of purely random events (genetic drift). As these mutations accumulate in genes related to eye structure and function over many generations, blindness results.
Assessment, Teacher Talk, Student talk, and Misconceptions
There is, of course, ample reason for using troglodyte blindness in assessing student understanding of evolution: misconceptions about evolution are common, and students often evidence these misconceptions when they try to apply their nascent understanding of evolution by explaining challenging cases such as this. Teachers, textbooks, and even journal articles are, likewise, not immune to these mistakes. In fact, experts who clearly understand the phenomena involved often use misconception language as a shorthand (“but we all understand what we really mean”). Although that may be true, it is also true that many learners do not understand the difference (Ryan, 1985). Table 2 presents some common misconceptions that are easily addressed by instruction about the evolution of A. mexicanus blindness, typical language that demonstrates each misconception, and the preferred language.
How then should teachers explain the evolution of blindness in A. mexicanus? The evolution of loss of function or structure in general? What misconceptions should we be on the lookout for in student explanations of these processes? How might the case of blindness in A. mexicanus be used effectively in introductory and/or AP biology?
Brief Thoughts on Effective Classroom Use
Although the focus of this article is not on explicit recommendations for instruction, a few suggestions may be helpful. Given how difficult it is to explain the evolution of troglodyte blindness, I do not recommend using the cavefish item as a pre-test or advanced organizer where it could be very discouraging to the students. For the same reasons, I do not encourage use of these items for summative assessment.
Questions about loss-of-function evolution could, however, be used in a wide variety of active learning settings, especially after students have a good understanding of the basics of evolution and can adequately explain the evolution of gain-of-function traits such as cheetah running speed. Generating possible evolutionary explanations of cavefish blindness could, for example, be a very demanding challenge used in the Explain and Explore phases of a 5e lesson (Atkin & Karplus, 1962) in which students are asked to work in small groups to propose creative explanations (perhaps without other resources, the internet, etc.). Teams could then present their proposals to other teams or the entire class to receive feedback, then prepare a report or present a poster on their conclusions. Teachers should look for reasoning that gives evidence of the common misconceptions given in Table 1. Likewise, loss-of-function examples could be used in the Extend phase of a 5e lesson in which the earlier stages involved understanding the basics of evolution.
To enhance interest and motivation, teachers might also present a brief Internet video showing blind A. mexicanus (e.g., https://www.youtube.com/watch?v=jOvcB30Yvrg) or invite students to find such videos on their own. Blind A. mexicanus specimens are also often available at local pet stores and are relatively inexpensive. Bringing some of the fish into the classroom for direct observation can be more stimulating for students than looking at pictures in a book or even watching videos. For the most fun, take students on a field trip to a large cave with guided tours if one is available close by. Involving students from other classes such as earth science can also make such field trips more feasible. (For students interested in A. mexicanus conservation or related topics, see Gross, 2012.)
Bonus Activity: Cavefish Blindness and the Nature of Science
Assuming you have addressed the nature of science in a variety of uncontroversial contexts (and that you fostered an atmosphere in which it is “safe” for students to discuss more controversial topics respectfully), the development of blindness in cavefish is an obvious context for further discussion of the nature of science. You might begin your class on the topic with questions such as: Why are most animals that live in caves blind? How do you know why, given that no one was in the cave when they lost their sight? The second question focuses on the difference between evolution and other biological subdisciplines, namely, that evolution is in part a historical science.
Other questions might be fruitful for discussion at the end of the cavefish lesson: What is the goal of science? Does our discussion of cavefish blindness meet that goal? (“Science is an attempt to explain natural phenomena” McComas et al., 1998.) What is the difference between observation and inference? What are some examples of each that we have talked about with cavefish? Why is it important in science to distinguish between observations and inferences? Another option is: “Suppose someone told you that they believe that God just created the cavefish blind in the first place? What would be a scientific response? Because science is empirically based on and/or derived from observations of the natural world” (Lederman, 2007, p. 833), a proper response would be that science is non-theistic (not atheistic), and therefore it takes no position on whether or not there is a supernatural power (God) or the actions thereof.
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