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Darwinism defined the difference between fact and theory.doc
I don't speak of the militant fundamentalists who label themselves with the oxymoron ''scientific
creationists,'' and try to sneak their Genesis literalism into high school classrooms under the guise of
scientific dissent. I'm used to their rhetoric, their dishonest mis- and half-quotations, their constant
repetition of ''useful'' arguments that even they must recognize as nonsense (disproved human
footprints on dinosaur trackways in Texas, risible misinterpretation of thermodynamics to argue that
life's complexity couldn't increase without a divine boost). Our strug- gle with these ideologues is
political, not intellectual. I speak instead of our allies among people committed to reason and
Kristol, who is no fundamentalist, accuses evolutionary biologists of bringing their troubles with
creationists upon themselves by too zealous an insistence upon the truths of Darwin's world. He
writes: ''. . . the debate has become a dogmatic crusade on both sides, and our educators, school
administrators, and textbook publishers find themselves trapped in the middle.'' He places the
primary blame upon a supposedly anti-religious stance in biological textbooks: ''There is no doubt
that most of ur textbooks are still written as participants in the 'warfare' between sci- ence and
religion that is our heritage from the 19th century. And there is also little doubt that it is this
pseudoscientific dogmatism that has provoked the current religious reaction.''
Kristol needs a history lesson if he thinks that current creationism is a product of scientific
intransigence. Creationism, as a political movement against evolution, has been a continually powerful force since the days of the Scopes trial. Rather than using evolution to crusade against religion
in their texts, scientists have been lucky to get anything at all about evolution into books for high
school students ever since Scopes's trial in 1925. My own high school biology text, used in the
liberal constituency of New York City in 1956, didn't even mention the word evolution. The laws
that were used against Scopes and cowed textbook publishers into submission weren't overturned by
the Supreme Court until 1968 (Epperson v. Arkansas).
But what about Kristol's major charge -- anti-religious prejudice and one-dimensional dogmatism
about evolution in modern textbooks? Now we come to the heart of what makes me so sad about
Kristol's charges and others in a similar vein. I don't deny that some texts have simplified, even
distorted, in failing to cover the spectrum of modern debates; this, I fear, is a limitation of the genre
itself (and the reason why I, though more of a writer than most scientists, have never chosen to
compose a text). But what evidence can Kristol or anyone else provide to demonstrate that
evolutionists have been worse than scientists from other fields in glossing over legitimate debate
within their textbooks?
Consider the evidence. Two textbooks of evolution now dominate the field. One has as its senior
author Theodosius Dob zhansky, the greatest evolutionist of our century, and a lifelong Russian
Orthodox; nothing anti-religious could slip past his watchful eye. The second, by Douglas Futuyma,
is a fine book by a kind and generous man who could never be dogmatic about anything except
intolerance. (His book gives a fair hearing to my own heterodoxies, while dissenting from them.)
When we come to popular writing about evolution, I suppose that my own essays are as well read as
any. I don't think that Kristol could include me among Darwinian dogmatists, for most of my essays
focus upon my disagreements with the strict version of natural selection. I also doubt that Kristol
would judge me anti- religious, since I have campaigned long and hard against the same silly
dichotomy of science versus religion that he so rightly ridicules. I have written laudatory essays
about several scientists (Burnet, Cuvier, Buckland, and Gosse, among others) branded as
theological dogmatists during the nineteenth-century reaction; and, while I'm not a conventional
believer, I don't consider myself irreligious.
Kristol's major error lies in his persistent confusion of fact with theory. He accuses us -- without
giving a single concrete example, by the way -- of dogmatism about theory and sustains his charge
by citing our confidence in the fact of transmutation. ''It is reasonable to suppose that if evolution
were taught more cautiously, as a conglomerate idea consisting of conflicting hypothe- ses rather
than as an unchallengeable certainty, it would be far less controversial.''
Well, Mr. Kristol, evolution (as theory) is indeed ''a conglomerate idea consisting of conflicting
hypotheses,'' and I and my colleagues teach it as such. But evolution is also a fact of nature, and so
do we teach it as well, just as our geological colleagues describe the structure of silicate minerals,
and astronomers the elliptical orbits of planets.
Rather than castigate Mr. Kristol any further, I want to discussthe larger issue that underlies both
this incident and the popular perception of evolution in general. If you will accept my premise that
evolution is as well established as any scientific fact (I shall give the reasons in a moment), then
why are we uniquely called upon to justify our chosen profession; and why are we alone subjected
to such unwarranted infamy? To this central question of this essay, I suggest the following answer.
We haven't received our due for two reasons: (1) a general misunderstanding of the different
methods used by all historical sciences (including evolution), for our modes of inference don't
match stereotypes of ''the scientific method''; and (2) a continuing but unjustified fear about the
implication both of evolution itself and of Darwin's theory for its mechanism. With these two issues
resolved, we can understand both the richness of science (in its pluralistic methods of inquiry) and
the absence of any conflict, through lack of common content, between proper science and true
Our confidence in the fact of evolution rests upon copious data that fall, roughly, into three great
classes. First, we have the direct evidence of small-scale changes in controlled laboratory
experiments of the past hundred years (on bacteria, on almost every measurable property of the fruit
fly Drosophila), or observed in nature (color changes in moth wings, development of metal
tolerance in plants growing near industrial waste heaps), or produced during a few thousand years
of human breeding and agriculture. Creationists can scarcely ignore this evidence, so they respond
by arguing that God permits limited modification within created types, but that you can never
change a cat into a dog (who ever said that you could, or that nature did?).
Second, we have direct evidence for large-scale changes, based upon sequences in the fossil record.
The nature of this evidence is often misunderstood by non-professionals who view evolution as a
simple ladder of progress, and therefore expect a linear array of ''missing links.'' But evolution is a
copiously branching bush, not a ladder. Since our fossil record is so imperfect, we can't hope to find
evidence for every tiny twiglet. (Sometimes, in rapidly evolving lineages of abundant organisms
restricted to a small area and entombed in sediments with an excellent fossil record, we do discover
an entire little bush -- but such examples are as rare as they are precious.) In the usual case, we may
recover the remains of side branch number 5 from the bush's early history, then bough number 40 a
bit later, then the full series of branches 156-161 in a well preserved sequence of younger rocks, and
finally surviving twigs 250 and 287.
In other words, we usually find sequences of structural intermediates, not linear arrays of ancestors
and descendants. Such sequences provide superb examples of temporally ordered evo- lutionary
trends. Consider the evidence for human evolution in Africa. What more could you ask from a
record of rare creatures living in terrestrial environments that provide poor opportunity for
fossilization? We have a temporal sequence displaying clear trends in a suite of features, including
threefold increase of brain size and corresponding decrease of jaws and teeth. (We are missing
direct evidence for an earlier transition to upright posture, but wide-ranging and unstudied
sediments of the right age have been found in East Africa, and we have an excellent chance to fill in
this part of our story.) What alternative can we suggest to evolution? Would God -- for some
inscrutable reason, or merely t test our faith -- create five species, one after the other
(Australopithecus afarensis, A. africanus, Homo habilis, H. erectus, and H. sapiens), to mimic a
continuous trend of evolutionary change?
Or, consider another example with evidence of structurally intermediate stages -- the transition from
reptiles to mammals. The lower jaw of mammals contains but a single bone, the dentary. Reptiles
build their lower jaws of several bones. In perhaps the most fascinating of those quirky changes in
function that mark pathways of evolution, the two bones articulating the upper and lower jaws of
reptiles migrate to the middle ear and become the malleus and incus (hammer and anvil) of
Creationists, ignorant of hard evi dence in the fossil record, scoff at this tale. How could jaw bones
become ear bones, they ask. What happened in between? An animal can't work with a jaw half
disarticulated during the stressful time of transition.
The fossil record provides a direct answer. In an excellent series of temporally ordered structural
intermediates, the reptilian dentary gets larger and larger, pushing back as the other bones of a
reptile's lower jaw decrease in size. We've even found a transitional form with an elegant solution to
the problem of remaking jaw bones into ear bones. This creature has a double articulation -- one
between the two bones that become the mammalian hammer and anvil (the old reptilian joint), and a
second between the squamosal and dentary bones (the modern mammalian condi- tion). With this
built-in redundancy, the emerging mammals could abandon one connection by moving two bones
into the ear, while retaining the second linkage, which becomes the sole articulation of modern
Third, and most persuasive in its ubiquity, we have the signs ofhistory preserved within every
organism, every ecosystem, and every pattern of biogeographic distribution, by those pervasive
quirks, oddities, and imperfections that record pathways of historical descent. These evidences are
indirect, since we are viewing modern results, not the processes that caused them, but what else can
we make of the pervasive pattern? Why does our body, from the bones of our back to the
musculature of our belly, display the vestiges of an arrangement better suited for quadrupedal life if
we aren't the descendants of four-footed creatures? Why do the plants and animals of the Galapagos
so closely resemble, but differ slightly from, the creatures of Ecuador, the nearest bit of land 600
miles to the east, especially when cool oceanic currents and volcanic substrate make the Galapagos
such a different environment from Ecuador (thus removing the potential argument that God makes
the best creatures for each place, and small differences only reflect a minimal disparity of
environments)? The similarities can only mean that Ecuadorian creatures colonized the Galapagos
and then diverged by a natural process of evolution.
This method of searching for oddities as vestiges of the past isn't peculiar to evolution, but a
common procedure of all historical science. How, for example, do we know that words have
histories, and haven't been decreed by some all-knowing committee in Mr. Orwell's bureau of Newspeak? Doesn't the bucolic etymology of so many words testify to a different life style among our
ancestors? In this article, I try to ''broadcast'' some ideas (a mode of sowing seed) in order to counter
the most ''egregious'' of creationist sophistries (the animal ex grege, or outside the flock), for which,
given the quid pro quo of business, this fine magazine pays me an ''emolument'' (the fee that millers
once received to grind corn).
I don't want to sound like a shrill dogmatist shouting ''rally round the flag boys,'' but biologists have
reached a consensus, based on these kinds of data, about the fact of evolution. When honest critics
like Irving Kristol misinterpret this agreement, they're either confusing our fruitful consonance
about the fact of evolution with our vibrant dissonance about mechanisms of change, or they've
misinterpreted part of our admittedly arcane technical literature.
One such misinterpretation has gained sufficient notoriety in the last year that we crave resolution
both for its own sake and as an illustration of the frustrating confusion that can arise when scientists
aren't clear and when commentators, as a result of hidden agendas, don't listen. Tom Bethell argued
in Harper's (February 1985) that a group of young taxonomists called pattern cladists have begun to
doubt the existence of evolution itself.
This would be truly astounding news, since cladistics is a powerful method dedicated to reforming
classification by using only the branching order of lineages on evolutionary trees (''propinquity of
descent'' in Darwin's lovely phrase), rather than vague notions of overall similarity in form or
function. (For example, in the cladistic system, a lungfish is more closely related to a horse than to a
salmon because the common ancestor of lungfish and horse is more recent in time than the link
point of the lungfish-horse lineage with the branch leading to modern bony fishes (including
Cladists use only the order of branching to construct their schemes of relationships; it bothers them
not a whit that lungfish and salmon look and work so much alike. Cladism, in other words, is the
purest of all genealogical systems for classification, since it works only with closeness of common
ancestry in time. How preciously ironic then, that this most rigidly evolutionary of all taxonomic
systems should become the subject of such extraordinary misunderstanding -- as devised by Bethell,
and perpetuated by Kristol when he writes: ''. . . many younger biologists (the so- called 'cladists')
are persuaded that the differences among species -- including those that seem to be closely related -are such as to make the very concept of evolution questionable.''
This error arose for the following reason. A small splinter group of cladists (not all of them, as
Kristol claims) -- ''transformed'' or ''pattern'' cladists by their own designation -- have adopted what
is to me an ill-conceived definition of scientific procedure. They've decided, by misreading Karl
Popper's philosophy, that patterns of branching can be established unambiguously as a fact of
nature, but that processes causing events of branching, since they can't be observed directly, can't be
known with certainty. Therefore, they say, we must talk only of pattern and rigidly exclude all
discussion of process (hence ''pattern cladistics'').
This is where Bethell got everything arse-backwards and began the whole confusion. A
philosophical choice to abjure all talk about process isn't the same thing as declaring that no reason
for patterns of branching exists. Pattern cladists don't doubt that evolution is the cause behind
branching; rather, they've decided that our science shouldn't be discussing causes at all.
Now I happen to think that this philosophy is misguided; in unguarded moments I would even deem
it absurd. Science, after all, is fundamentally about process; learning why and how things happen is
the soul of our discipline. You can't abandon the search for cause in favor of a dry documentation of
pattern. You must take risks of uncertainty in order to probe the deeper questions, rather than
stopping with sterile ecurity. You see, now I've blown our cover. We scientists do have our
passionate debates -- and I've just poured forth an example. But as I wrote earlier, this is a debate
about the proper approach to causes, not an argument about whether causes exist, or even whether
the cause of branching is evolution or something else. No cladist denies that branching patterns
arise by evolution.
This incident also raises the troubling issue of how myths become beliefs through adulterated
repetition without proper documentation. Bethell began by misunderstanding pattern cladistics, but
at least he reports the movement as a small splinter, and tries to reproduce their arguments. Then
Kristol picks up the ball and recasts it as a single sentence of supposed fact -- and all cladists have
now become doubters of evolution by proclamation. Thus a movement, by fiat, is turned into its
opposite -- as the purest of all methods for establishing genealogical connections becomes a weapon
for denying the mechanism that all biologists accept as the cause of branching on life's tree:
evolution itself. Our genealogy hasn't been threatened, but my geniality has almost succumbed.
When I ask myself why the evidence for evolution, so clear to all historical scientists, fails to
impress intelligent nonscientists, I must believe that more than simple misinformation lies at the
root of our difficulty with a man like Irving Kristol. I believe that the main problem centers upon a
restrictive stereotype of scientific method accepted by most non-practitioners as the essential
definition of all scientific work.
We learn in high school about the scientific method -- a cut- and-dried procedure of simplification
to essential components, experiment in the controlled situation of a laboratory, prediction and
replication. But the sciences of history -- not just evolution but a suite of fundamental disciplines
ranging from geology, to cosmology, to linguistics -- can't operate by this stereotype. We are
charged with explaining events of extraordinary complexity that occur but once in all their details.
We try to understand the past, but don't pretend to predict the future. We can't see past processes
directly, but learn to infer their operation from preserved results.
Science is a pluralistic enterprise with a rich panoply of methods appropriate for different kinds of
problems. Past events of long duration don't lie outside the realm of science because we cannot
make them happen in a month within our laboratory. Direct vision isn't the only, or even the usual,
method of inference in science. We don't see electrons, or quarks, or chemical bonds, any more than
we see small dinosaurs evolve into birds, or India crash into Asia to raise the Himalayas.
William Whewell, the great English philosopher of science duringthe early nineteenth century,
argued that historical science can reach conclusions, as well confirmed as any derived from
experiment and replication in laboratories, by a method he called ''consilience'' (literally ''jumping
together'') of inductions. Since we can't see the past directly or manipulate its events, we must use
the different tactic of meeting history's richness head on. We must gather its won- drously varied
results and search for a coordinating cause that can make sense of disparate data otherwise isolated
and uncoordinated. We must see if a set of results so diverse that no one had ever considered their
potential coordination might jump together as the varied products of a single process. Thus plate
tectonics can explain magnetic stripes on the sea floor, the rise and later erosion of the
Appalachians, the earthquakes of Lisbon and San Francisco, the eruption of Mount St. Helens, the
presence of large flightless ground birds only on continents once united as Gondwanaland, and the
discovery of fossil coal in Antarctica.
Darwin, who understood the different rigor of historical scienceso well, complained bitterly about
those critics who denied scientific status to evolution because they couldn't see it directly or
reproduce its historical results in a laboratory. He wrote to Hooker in 1861: ''Change of species
cannot be directly proved . . . The doctrine must sink or swim according as it groups and explains
phenomena. It is really curious how few judge it in this way, which is clearly the right way.'' And
later, in 1868: ''This hypothesis may be tested . . . by trying whether it explains several large and
independent classes of facts; such as the geological succession of organic beings, their distribution
in past and present times, and their mutual affinities and homologies.''
If a misunderstanding of the different methods of historical inquiry has impeded the recognition of
evolution as a product of science at its best, then a residual fear for our own estate has continued to
foster resentment of the fact that our physical bodies have ancient roots in ape-like primates,
waddling reptiles, jawless fishes, worm-like invertebrates, and other creatures deemed even lower
or more ignoble. Our ancient hopes for human transcendence have yet to make their peace with
But what challenge can the facts of nature pose to our own decisions about the moral value of our
lives? We are what we are, but we interpret the meaning of our heritage as we choose. Science can
no more answer the questions of how we ought to live than religion can decree the age of the earth.
Honorable and discerning scientists (most of us, I trust) have always understood that the limits to
what science can answer also describe the power of its methods in their proper domain. Darwin
himself exclaimed that science couldn't touch the problem of evil and similar moral conun- drums:
''A dog might as well speculate on the mind of Newton. Let each man hope and believe what he
There is no warfare between science and religion, never was except as a historical vestige of
shifting taxonomic boundaries among disciplines. Theologians haven't been troubled by the fact of
evolution, unless they try to extend their own domain beyond its proper border (hubris and
territorial expansionism aren't the sins of scientists alone, despite Mr. Kristol's fears). The Reverend
Henry Ward Beecher, our greatest orator during Darwin's century, evoked the most quintessential of
American metaphors in dismissing the entire subject of conflict between science and religion with a
single epithet: ''Design by wholesale is grander
than design by retail'' --or, general laws rather than creation of each item by fiat will satisfy our
notion of divinity.
Similarly, most scientists show no hostility to religion. Why should we, since our subject doesn't
intersect the concerns of theology? I strongly dispute Kristol's claim that ''the current teaching of
evolution in our public schools does indeed have an ideological bias against religious belief.''
Unless at least half my colleagues are inconsistent dunces, there can be -- on the most raw and
direct empirical grounds -- no conflict between science and religion. I know hundreds of scientists
who share a conviction about the fact of evolution, and teach it in much the same way. Among these
people I note an entire spectrum of religious attitudes -- from devout daily prayer and worship to
resolute atheism. Either there's no correlation between religious belief and confidence in evolution - or else half these peple are fools.
The common goal of science and religion is our shared struggle for wisdom in all its various guises.
I know no better illustration of this great unity than a final story about Charles Darwin. This scourge
of fundamentalism had a conventional church burial -- in Westminster Abbey no less. J. Frederick
Bridge, Abbey organist and Oxford don, composed a funeral anthem expecially for the occasion. It
may not rank high in the history of music, but it is, as my chorus director opined, a ''sweet piece.''
(I've made what may be the only extant recording of this work, marred only by the voice of yours
truly within the bass section.) Bridge selected for his text the finest biblical description of the
common aim that will forever motivate both the directors of his building and the inhabitants of the
temple of science -- wisdom. ''Her ways are ways of pleasantness and all her paths are peace''
I am only sorry that Dr. Bridge didn't set the very next metaphor about wisdom (Proverbs 3:18), for
it describes, with the proper topology of evolution itself, the greatest dream of those who followed
the God of Abraham, Isaac, and Jacob: ''She is a tree of life to them that lay hold upon her.''
COPYRIGHT 1987 Discover
COPYRIGHT 2004 Gale Group
To See or Not to See: Evolution of Eye Degeneration in Mexican Blind Cavefish1
Jeffery, William R
In 1872 Charles Darwin wrote, "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." This statement
launched more than a hundred years of speculation and debate on the evolutionary mechanisms
responsible for the loss of eyes in cave animals (Culver, 1982). Today this problem is still
unresolved, but prevailing opinions usually support one of two hypotheses.
The neutral mutation hypothesis suggests that eye degeneration is caused by random mutations in
eye forming genes, which gradually accumulate in the absence of selective pressure. In contrast, the
adaptation hypothesis suggests that natural selection causes the loss of eyes due to advantages in
losing eyesight. As exclaimed in Darwin's famous quotation, the actual benefits of blindness are
uncertain. Thus, different versions of the adaptation hypothesis have attributed the loss of eyesight
to energy conservation, citing the high cost of making an eye, or to enhancement of other sensory
organs that are highly beneficial to survival in the cave environment. Through the years, however,
little or no experimental verification has been leveled in support of any version of either hypothesis.
To understand the evolution of eye degeneration, it is necessary to determine the molecular and
cellular mechanisms of the degenerative process, and whether the same or different genes and
mechanisms are involved in loss of vision.
We study the mechanisms of visual degeneration in the Mexican Tetra, Astyanax mexicanus, a
single species consisting of a surface-dwelling form (surface fish) (Fig. IA) and many cave dwelling
(cavefish) forms inhabiting different caves (Fig. IB-E) (Jeffery, 2001). The Mexican tetra is easy to
raise in the laboratory and exhibits many of the attributes that have made zebrafish a popular model
system in developmental biology. These features include external fertilization, frequent and
abundant spawning, transparent embryos, a 4-6 month generation time, and the opportunity for
molecular, developmental, and genetic analysis. The surface and cave forms of A. mexicanus are
interfertile, and successful mating is also possible between different cavefish populations (Sadoglu,
1957; Wilkens, 1971). Because of these attributes Astyanax cavefish represent one of the few cave
animals in which laboratory experiments can be conducted on the mechanisms of eye degeneration
and these mechanisms can be compared in the same species from different caves. Here we review
current progress on the evolution and development of Astyanax cavefish and discuss how these
studies have contributed to understanding the evolutionary basis of eye degeneration.
CAVEFISH EVOLUTIONARY HISTORY
To evaluate differences or similarities in the mechanisms of eye degeneration, it is first necessary to
understand the evolutionary history of cavefish populations. Did all cavefish populations originate
from a common ancestor and lose their eyes only once or did they evolve many times and lose their
eyes independently? Different approaches have been used to determine the evolutionary
relationships of cavefish, including allozyme analysis, biogeography, and phylogenetic
reconstruction using molecular sequences. We will briefly consider the results obtained from the
first two approaches and then describe the phylogenetic studies in more detail.
Figure 2 shows a map of the Sierra de El Abra region in northeastern Mexico illustrating the
locations of known caves harboring Astyanax cavefish populations. The major cavefish region
consists of the Sierra de El Abra, thé Sierra de Guatemala, the Micos region (Fig. 2), and the valleys
lying between these limestone ridges in the states of Tamaulipas and San Luis Potosi, Mexico
(Wilkens and Burns, 1972; Mitchell et al., 1977). An outlying cavefish population has also been
discovered in the state of Guererro in south central Mexico (Espinasa et al., 2001).
In an electrophoretic study showing minimal divergence in 17 allozyme loci, Avise and Selander
(1972) concluded that the Sierra de El Abra cavefish had a common origin. However, a limited
number of cavefish populations (Pachon, Los Sabinos, and Chica; Fig. 2) were sampled in this
study. In contrast, Mitchell et al. (1977), who surveyed 29 different cavefish populations in the
Sierra de El Abra, Sierra de Guatemala, and Micos region, proposed several different origins of
Astyanax cavefish. Mitchell et al. (1977) also estimated the divergence between surface fish and
cavefish to have occurred about 10,000 to 100,000 years ago in the Sierra de El Abra region. The
possibility of multiple cavefish origins is strongly supported by the recently discovered Guerrero
cavefish from a cave located several hundred miles southwest of the main cavefish region (Espinasa
et al, 2001).
The first phylogenetic studies of cavefish populations were done using DNA polymorphisms
amplified by arbitrary primers (RAPDs) (Espinasa and Borowsky, 2001). This analysis supported a
single origin of Sierra de El Abra cavefish and an independent origin of Subterraneo cavefish in the
Micos region (Fig. 2). The limited number of RAPD markers scored in this study, however, left
some uncertainty about the true relationships among the Sierra de El Abra cavefish. Thus far, it has
proved difficult to obtain sufficiently variable sequence information from nuclear genes to construct
robust phylogenetic trees, presumably due to the recent divergence of surface fish and cavefish.
Thus, Dowling et al. (2002) were prompted to use NAD1 dehyrdogenase-2 (ND-2), a rapidly
evolving mitochondrial gene, to infer cavefish relationships (Fig. 3).
Before discussing the resulting ND-2 mitochondrial DNA (mtDNA) phylogeny, it is necessary to
comment on the currently unresolved taxonomy of A. mexicanus and related forms. Some
taxonomists recognize two separate Astyanax species in Mexico: A. mexicanus in northern Mexico
and Astyanax aeneus in southern Mexico (Obregon-Barbosa et al., 1994). Others be lieve that all
Mexican and Central American Astyanax are a single species, Astyanax fasciatus (see Wilkens,
1988). Here, we defer to the first classification, designating the northern Mexican form as A.
mexicanus, the southern Mexican form as A. aeneus, and the Central American form as A.
fasciatus. Our justification is that these taxa are strongly supported by the mtDNA phylogeny (Fig.
The mtDNA phylogeny infers at least two separate origins of cavefish, one before the divergence of
the present day A. mexicanus and A. aeneus, and the other after the bifurcation of these taxa (Fig.
3). Accordingly, two distinct mtDNA lineages are recognized: the A lineage, including A.
mexicanus and A. aeneus surface fish and Pachon and Subterraneo cavefish, and the B lineage,
including Tinaja, Los Sabinos, and Curva cavefish (Dowling et al., 2002). The A lineage exhibits
one of more than 20 different Type A ND-2 haplotypes, which vary from each other in only a few
nucleotide positions and are mostly represented in surface fish. The B lineage exhibits one or two of
only a few Type B ND-2 haplotypes, which differ in 7 or more nucleotide sites from the Type A
haplotypes and are present in cavefish but not in any nearby surface fish populations. Sampling
from Texas to Costa Rica failed to find any surface fish populations with Type B haplotypes
(Dowling et al., 2002), suggesting that the surface fish stock that established the B lineage cavefish
may be extinct.
Although the mtDNA tree has strong bootstrap support, our interpretation of these data must be
treated with caution. First, the tree is based on only a single gene. However, a recent phylogenetic
analysis has confirmed the topology of this tree using a different mitochondrial gene, cytochrome b
(Strecker et al, 2003). second, mtDNA trees could be influenced by hybridization, which is known
to have occurred between some of the cavefish populations and nearby surface fish (Mitchell et al,
1977; Romero, 1983; Langecker et al., 1991). Third, a recent phylogeny using microsatellite loci is
more consistent with a common origin of the Sierra de El Abra cavefish (Strecker et al., 2003),
suggesting replacement of mitochondrial DNA may have occurred by hybridization in Pachon
cavefish. It is clear from the mtDNA data, however, that A and B lineage cavefish are genetically
In summary, separate origins with accompanying episodes of eye degeneration may have occurred
in the Guerrero, Sierra de Guatemala (Molino), Micos (Subterraneo), and Lineage A and B Sierra
de El Abra cavefish populations. Below we will compare the developmental mechanisms of eye
degeneration in some of these cavefish.
THE LENS AS AN ORGANIZER OF EYE DEVELOPMENT
To determine the mechanisms of eye regression, we focused on the nature and timing of
degenerative processes in the embryonic eye primordia. In every cavefish population we have
studied, the eye primordium appears to be smaller than its surface fish counterpart. However, the
cavefish eye seems to develop normally up to about the hatching stage, forming a lens and optic
cup. Subsequently, development gradually arrests, the retina becomes disordered, and the
degenerating eye disappears into the orbit (Cahn, 1958; Langecker et al., 1993). The cavefish lens
does not differentiate arrays of aligned crystallin fibers and the retina, although at first layered
normally, eventually shows disorganization and complete or partial loss of photoreceptor cells. In
many developing systems, an alternative to cell differentiation is apoptosis: programmed cell death
(White, 1996). Therefore, we first investigated whether apoptosis occurred during cavefish eye
If cell death is restricted to a single eye tissue, or begins in one tissue and later spreads to others,
then the tissue that dies first is a strong candidate to initiate the degeneration process. Apoptosis
was compared in surface fish and in Pachon cavefish embryos using the TUNEL assay (Jeffery and
Martasian, 1998), which detects DNA fragmentation. Surface fish embryos showed little or no
programmed cell death in the developing eye (Fig. 4A), except in the isthmus that temporarily
forms between the budding lens and the surface ectoderm, as has been previously described in the
mammalian eye (Silver and Hughes, 1968). Cavefish showed the same apoptotic event in a small
number of isthmus cells as the lens vesicle pinched off from the surface ectoderm. About a day after
the cavefish lens vesicle was formed, however, an additional and more extensive episode of
apoptosis was detected in its central core (Fig. 4B), the region where lens fiber cells would normally
differentiate from lens epithelial cells. No apoptosis was detected at this time in the surface fish lens
(Fig. 4A), and no other cavefish eye tissue died at this stage of development. A few days later, the
retina began to undergo apoptosis. Retinal cell death is restricted to the outer nuclear layer and the
region adjacent to the ciliary marginal zone (CMZ) (A.G.S., unpublished), where most new retinal
cells are produced in the teleost retina (Johns and Easter, 1977; Harris and Perron, 1998). Thus, the
lens is the first tissue to undergo cell death during eye degeneration in Pachon cavefish.
Does the embryonic lens also die in other cavefish populations? Using the TUNEL assay, we
showed that the Los Sabinos cavefish lens also dies before any other tissue in the degenerating eye
(Fig. 4C). The results suggest that lens apoptosis may be responsible for triggering eye degeneration
in both A and B lineage cavefish.
The cessation of retinal growth in cavefish could be caused by the failure of the dying lens to
produce a growth-promoting factor or it could be due to an independent event in the retina. A
reasonable candidate for an independent retinal event would be interference with cell proliferation.
Surface fish have an active CMZ. Proliferating cells can be detected by incorporation of labeled
nucleotides into DNA, the presence of the DNA polymerase cofactor PCNA, and the expression the
homeobox genes RxI and Vsx2 (Fig. 4D, G), throughout the period of eye growth (Strickler et al,
2002; A.O.S., unpublished results). all of these cell proliferation markers were expressed in the
Pachon cavefish CMZ (Fig. 4E, H), although the retina does not markedly increase in size during
this period (Strickler et al., 2002). Presumably, new cells are removed from the retina soon after
they are formed by the apoptotic events that begin a few days after the initiation of lens cell death.
We next asked whether the surprisingly wasteful process in which retinal cells appear to cycle
quickly between birth and death also occurs in other cavefish populations? As shown in Figure F, I,
RxI and Vsx2 are also expressed in the CMZ of Los Sabinos cavefish, despite a comparable lack of
net growth. Thus, we conclude that arrest of cell proliferation is not the major cause of eye
degeneration in A and B lineage cavefish populations.
The results described above focus our attention back to the lens. Does the lens organize the whole
eye and could its removal by apoptosis result in the arrest of eye formation? The central role of the
lens in eye formation has recently been appreciated (Beebe and Coats, 2000; Thut et al., 2001), due
largely to developmental studies with cavefish (Yamamoto and Jeffery, 2000). We developed a lens
transplantation assay to determine the role of the lens in surface fish eye development and in
cavefish eye degeneration (Yamamoto and Jeffery, 2000, 2002).
The embryonic lens was removed from a donor embryo shortly after it pinched off from the surface
ectoderm, about a day before the first detection of largescale apoptosis in the cavefish lens, and it
was transplanted into the optic cup of a host embryo. Lens transplantation was done unilaterally,
with the unoperated eye of the host serving as a control. The first transplantation experiments were
carried out reciprocally between surface fish and Pachon cavefish: a surface fish lens was
transplanted into a cavefish optic cup and vice versa (Yamamoto and Jeffery, 2000). These
experiments also addressed the autonomy of programmed cell death in the cavefish lens: is cell
death determined by the lens itself or is it induced by another tissue, for instance the retina? When a
cavefish lens was transplanted into a surface fish optic cup it died on schedule, just as if it had not
been removed from the donor embryo. Likewise, when a surface fish lens was transplanted into a
cavefish optic cup it continued to grow and differentiated as it would have in the surface fish host.
Together, these results indicate that the Pachon cavefish lens is autonomously fated for apoptosis, at
least by the time of the transplantation (Yamamoto and Jeffery, 2000).
The autonomy of surface fish lens development in the cavefish host is the key part of the
transplantation experiment. After obtaining a surface fish lens, the Pachon cavefish eye reversed its
fate and began to grow and develop (Yamamoto and Jeffery, 2000). Eventually, the cornea and iris
appeared, which are normally missing in cavefish, and the retina enlarged and became more
organized. Further growth resulted in the presence of a highly developed eye containing all of the
expected eye tissues, including the cornea, iris, and photoreceptor cells, in the adult Pachon cavefish
host (Fig. 5B). When the donor lens was labeled with GFP no labeled cells appeared in the restored
tissues of the host (Yamamoto and Jeffery, 2000). Thus, the rescued eye tissues arise from the host
and not the donor. The cornea and iris are derived in part from optic neural crest cells, indicating
that cavefish neural crest cells are present and located in the proper positions to be induced by the
lens. In contrast to the eye with a transplanted lens, the unoperated eye of the cavefish host
degenerated and disappeared into the orbit according to its usual schedule (Fig. 5A). Likewise, after