Evolution and Genetics of Epigean and Cave Astyanax Fasciatus (Characidae, Pisces) : Support For The Neutral Mutation Theory

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Evolution and genetics of epigean and cave Astyanax fasciatus (Characidae,

Pisces): support for the neutral mutation theory
Chapter · January 1988
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From: EVOLUTIONARY BIOLOGY, Vol. 23
Edited by Max K. Hecht and Bruce Wallace
(Plenum Publishing Corporation, 1988)
Evolution and Genetics of

Epigean and Cave Astyanax
fasciatus (Characidae, Pisces)
Support for the Neutral Mutation Theory
HORST WILKENS
INTRODUCTION
Notable progress in the biological sciences is occasionally made pos-

sible by the discovery of a species that exhibits a specialized way of life,
but nevertheless will thrive and breed in the laboratory. In the study of
cavernicolous animals, the Mexican characid fish Astyanaxfasciatus has
come to play a role equivalent to that of the fruit fly, Drosophila, in
genetics.
Not long after the discovery of the first blind, cave-inhabiting deriv-
ative of A. fasciatus (Hubbs and Innes, 1936), Charles M. Breder, Jr.
recognized the unique research opportunities presented by this fish. Be-
tween 1940 and 1958, he and his associates completed a series of exper-
imental studies at the American Museum of Natural History in New York.
The necessity carrying out genetic analyses of the epi- and hypogean
populations of A. fasciatus was first recognized by Curt Kosswig. In
connection with studies he had begun in 1934 on the evolutionary genetics
of complex structures, he stimulated Sadoglu's (1957) work on the ocular
genetics and melanin pigmentation. Subsequently, different hypogean
populations and the ancestral epigean fish have become the objects of
extensive research at the Zoologisches Institut and Zoologisches Museum
in Hamburg.
HORST WILKENS • Zoological Institute and Zoological Museum of the University of

Hamburg, D-2000 Hamburg 13, West Germany.
271
272 H. Wllkens
As is all too often the case, information obtained in the laboratory

can be misinterpreted because of too little knowledge of the conditions
in nature. It was the field work of Robert W. Mitchell and his co-workers
from Texas Tech University, Lubbock, Texas that first revealed to us
the full extent of the complex of caves inhabited by different populations
of Astyanax. A bibliography covering the early history of research on
Mexican blind cave characins was prepared by WHey and Mitchell (1971).
The intent of the present review is to provide an overview of the
comparative studies on epigean and cave-inhabiting Astyanax fasciatus
relevant to an understanding of the troglobitic evolution of these fish. In
addition, some general implications regarding the genetics and evolution
of complex structural and functional features are discussed.
TAXONOMY AND DISTRIBUTION OF EPI- AND HYPOGEAN

ASTYANAX FASCIATUS
The Epigean Fish ..
The epigean fish (Fig. 1) is widely distributed in Mexican waters. Its

northern distributional limit is in Texas. Within the borders of Mexico, '"
five species and subspecies have been described (Eigenmann, 1917). More
recent studies have revealed that the surface Astyanax occurring in Mex-
ico is a variable form, which does not allow further separation at the
species or subspecies level (Schuppa, 1984; Bussing, 1985). The valid
scientific name is Astyanax fasciatus (euvier, 1819) (= A. mexicanus
(Filippi, 1853». Gery (1977) subdivided the subspecies A. fasciatus mex-
icanus, but this process has no nomenclatural status.
The Hypogean Fish
The cave characins with reduced eyes and pigmentation (Fig. 1) are
mainly distributed in the Sierra de El Abra region of San Luis Potosi and
Tamaulipas (Fig. 2). This consists of an area 125 km in length restricted
to a ridge of highly cavernous limestone. Water drainage, physiography,
and geology are described in detail by Mitchell et al. (1977). Twenty-nine
caves are known to be inhabited by cave-adapted Astyanax. The locations
of the caves can be grouped as follows: (1) southern El Abra caves, (2)
Los Sabinos caves, (3) Yerbaniz caves, (4) northern El Abra caves, (5)
Evolution and Genetics of Astyanax fasclatus 273
c
FIG. 1. (a) Epigean, (b) Pachon, and
(c) Micos cave forms of Astyanax fas-
ciatus.
Micos caves, (6) Nicolas Perez caves, (7) Chamal caves, and (8) Gomez
Faria caves (Fig. 2) (Mitchell et al. 1977).
Inhabitants from five of these caves have been subjected to experi-
mental studies that show they can hybridize with epigean A. fasciatus in
the laboratory, as they occasionally do in the natural biotope. Further-
more, analysis of allozymic variation has shown that the coefficients of
genetic similarity do not support the assignment of surface and cave pop-
ulations to different genera or to different species (A vi se and Selander,
1972): The hypogean fish therefore should be characterized as cave-dwell-
ing populations of A. fasciatus (Sadoglu, 1957; Wilkens, 1970a; Mitchell
et al., 1977). Karyotypic studies are incomplete because only specimens
of an unidentified commercial stock were compared with the epigean
ancestor. However, in both, a diploid chromosome number of n = 50
was found (Kirby et al., 1977).
The first cave population discovered was that of La Cueva Chica,
which belongs to the southern El Abra caves. It was originally described
as "Anoptichthys jordani" by Hubbs and Innes (1936). The status of this
variable form has long been a matter of discussion. Allozyme variation
(Avise and Selander, 1972) and eye histology and genetics (Wilkens,
1972a, 1976), as well as ecological studies (Mitchell et al., 1977; Romero,
1983; Wilkens and Hiippop, 1986), have shown that the Chica fish is a
H. Wilkens
274
tN
.blind fish cave
'i
oi 10 15km
FIG. 2. Localities of cave populations of A. fasciatus in the Sierra de El Abra region. (After Mitchell
et al.• 1977.)
Evolution and Genetics of Astysnsx fsSC/stUB 275
hybrid form between the epigean A. fasciatus and a phylogenetically old

cave fish that is now extinct.
The Chica fish, or commercial strains that were undoubtedly derived
from it, have been the subject of a large number of studies concerned
with the problems of troglobitic evolution. Unfortunately, the authors
very often have not realized that the results of their studies must be in-
terpreted in the light of the hybrid character of this form.
The second hypogean population of scientific interest is that of La
Cueva de los Sabinos. It was originally described as "Anoptichthys
hubbsi" by Alvarez (1947). The Sabinos fish is an eye- and pigment-
reduced cave form and is therefore looked upon as phylogenetically old.
The name "A. hubbsi" was erroneously used by Schmatolla (1972), Yew
and Yoshihara (1977), and Voneida and Fish (1984) for the Chica fish.
The population of El Sotano de las Piedras, which was discovered
in 1969 by Mitchell et al. (1977), belongs to the group of Los Sabinos
caves.
The population of El Sotano de Yerbaniz belongs to the Yerbaniz
cave group. Both the Piedras and the Yerbaniz fish are eye- and pigment-
reduced, phylogenetically old forms. They have been studied and kept in
the Zoologisches Institut and Zoologisches Museum Hamburg laborato-
ries.
La Cueva de El Pachon belongs to the northern El Abra caves. The
fish living here was originally described as "Anoptichthys antrobius" by
Alvarez (1946) (Fig. l). Morphological studies have revealed that the Pa-
chon population is the most distinct one in the Sierra de El Abra. This is
probably due to its considerable vertical isolation from the base level
waters. The Pachon population has a somewhat peculiar history. The
material studied by Breder and his associates and originally also main-
tained in the Zoologisches Institut and Zoologisches Museum Hamburg
laboratory (Peters and Peters, 1966; Schemmel, 1967, 1974a; Wilkens
1970a-c, 1971) was heterozygous for an albino gene. In 1972 and 1982 it
was found that the specimens collected in the Pachon cave were homo-
zygous for this feature, possibly due to genetic drift. In 1986 it was found
that part of the Pachon fish population had become hybridized with the
epigean ancestral form (Langecker et al., in preparation).
The population of La Cueva del Rio Subterraneo (= Micos fish) was
not discovered until the 1960s (Wilkens and Burns, 1972) (Fig. 1). It is
one of the few populations to occur outside the Sierra de El Abra (Fig.
2). The population is genotypically and phenotypically variable (Schem-
mel, 1974b; Wilkens, 1976). It occurs associated with specimens of the
epigean fish, that are washed into the cave during the rainy season.
The variable Micos population belongs to the scientifically most in-
276 H. Wilkens
teresting Astyanax cave forms. Its phylogenetically young status has been
questioned several times (Schemmel, 1974b; Mitchell et al., 1977). How-
ever, a series of studies has verified the original interpretation, namely
that the Micos fish is not an Fx hybrid, but is a phylogenetically young
form (Wilkens and Burns, 1972):
First, a comparison of the condition factors exhibited by the epigean
and hypogean Micos specimens that live together in the natural cave bio-
tope has shown that the epigean individuals are starved (see below, p.
317) (Wilkens and Hiippop, 1986).
Second, a genetic analysis of the eye of the Micos fish has revealed
that due to the existence of a genetic equilibrium that is lost in the F2
crosses between epigean and phylogenetically old cave forms, a threshold
effect is able to manifest itself and bring about a discontinuous distribution
of phenotypes (see Fig. 11) (Wilkens, 1976). For the same genetic reasons,
the variability in the size of the pupillary opening is lower in the Micos
fish than in the above-mentioned F2 crosses (see Fig. 17). The retina of
the Micos fish eye is also, on the average, better differentiated than that
of the F2 hybrids (see below, p. 265).
Third, Schemmel (1974b) based his interpretation of the Micos fish
as a hybrid on the great variability shown by its taste buds (see Fig. 39),
but this phenomenon could just as well be attributed to a threshold effect
known to occur in backcrosses between epigean and phylogenetically old
cave fish. Its manifestation in the taste bud system of the Micos fish might
furthermore be proven by the distribution curve of taste buds, which does
not show normality in the F2 crosses between epigean and phylogeneti-
cally old cave forms, but instead a positive skew (see Fig. 39).
Finally, unlike the hybrid Chica fish, the Micos population does not
possess any allele that does not exist in the epigean population (Peters et
al., 1975). All alleles that have been found in the Micos fish exist in the
epigean form at a frequency of 95%. However, the distribution pattern
of alleles does diverge from that of the epigean fish, thus showing that
the hypogean Micos cave specimens share a gene pool of their own (see
below, p. 319).
PREADAPTIVE TRAITS
Lateral Line System
In the epigean A. fasciatus, free neuromasts are distributed over

head, body, and caudal fin, as is typical among teleosteans. With four to
Evolution and Genetics of Astyanax fasciatus 277
FIG. 3. Distribution and density of neu-

romasts of (a) epigean and (b) Pachon b
cave forms (points, neuromasts; dots,
openings of lateral canal). (Schemmel,
1967.)
eight neuromasts on every scale, this fish is among the species best
equipped with these organs (Fig. 3). With respect to number and distri-
bution, no essential differences can be found between epigean and cave
forms (Schemmel, 1967), even though the lateral parts of cave fish heads
possess more neuromasts.
The lateral line canal of the epigean fish can also be characterized
as typically teleostean (Schemmel, 1967). It is well developed and extends
from the operculum to the caudal peduncle, On the head it divides into
the supra- and infraorbital canals and the preopercular, mandibular, and
occipital canals (Fig. 4). Due to fragmentations, considerable inter- and
intraindividual variability of the head canal system may develop (Schem-
mel, 1967),
No differences in the head canal system of epigean and hypogean
forms have been observed (Schemmel, 1967). However, the loss of the
FIG. 4. Head canal systems of

(a) epigean and (b) Sabinos cave
fish (dots, canal neuromasts).
(Schemmel, 1967.) Q b
278 H. Wllkens
relatively large eye and the fragmentation of the suborbital bones into
several separate elements (Alvarez, 1946) seem to have caused some al-
terations of the infraorbital canal in the cave forms. It is no longer char-
acterized by a deep ventral flexure and thus has become shortened. In
many specimens it is interrupted (Fig. 4). As in the epigean fish, inter-
and intraindividual variability are significant.
Very often the lateral line canal displays fragmentations in its caudal
part (Wilkens, 1977). This phenomenon has no genetic basis, but is caused
by the fact that due to a reduction ofthe scales (below, p. 280) the cave
specimens lose these organs much more easily than do epigean fish. The
described fragmentations develop because of irregular regeneration.
In comparison with other species of teleost (Hyphessobryeon, Phox-
inus, Tinea, Lebistes), the lateral line system of the Chica fish is much
more highly developed (Hahn, 1960). There are no recognizable differ-
ences between the lateral line system of the Chica fish and that of the
Pachon and Sabinos populations (Hahn, 1960).
The efficiency ofthe function of the lateral line system in A.fasciatus
has been studied, in particular, in the hybrid Chica population (Breder
and Gresser, 1941a; Liiling, 1953; Grobbel and Hahn, 1958). These fishes
are able to localize stationary objects as well as to discern certain shapes
or the distances between bars (Hahn, 1960; von Campenhausen et al.,
1981; Weissert and von Campenhausen, 1981). The same is true for mov-
ing objects, such as cladocerans (Friihbeiss, 1984). The lateral line system
of the epigean fish appears to be as efficient as that of the Chica fish;
analysis of obstacle avoidance in blinded epigean specimens revealed no
differences when compared to the hypogean derivatives (John, 1957).
The far-reaching morphological, histological, and functional simi-
larity of the lateral line system indicates that constructive improvement
was not necessary for the epigean fish to survive in the cave biotope.
Auditory Capacity
Studies of the auditory capacity in specimens of the epigean and the

Chica population have revealed that both forms can hear a wider range
of sounds than that reported for any other species of fish tested in a
quantitative manner (Popper, 1970). There is no evidence that the hybrid
cave fish is more or less able to detect pressure stimuli than is the epigean
form. This is also true for the phylogenetically old cave populations. Bred-
er's (1943a) assumption of a possible regressive deterioration of the lab-
yrinth could not be confirmed either by morphological analysis (Schem-
mel, 1967) or by the above-mentioned physiological studies.
3 Lamellae
cf'} Epigean
25 C( form
20
..
.- ....
~~.~.~.;:'::~'..-:'
15
· female
10
FIG. 5. Correlation between the number of • male
olfactory folds and the standard length in 5 • juvenile
the Pachon cave form. Regression lines of
these features in the epigean form are o+-~~~,-,-~~~~
drawn for comparison. (Schemmel, 1967.) o 2 3 4 5 6 7 8 9~
Olfactory Organ
The noses of the different hypogean and epigean forms are con-
structed similarly (Breder and Rasquin, 1943; Schemmel, 1967). There
are no histological differences in the olfactory epithelium that might imply
differing efficiencies. Minute improvements in the cave fish could result
from the fact that the olfactory pits are shallower with a larger naris, thus
exposing their olfactory epithelial folds to a greater extent.
The number of olfactory lamellae shows high individual variability,
depending on body size. Male specimens possess more lamellae than do
females (Fig. 5) (Schemmel, 1967).
Comparison between the hypogean Sabinos, Pachon, and Chica pop-
ulations, on one hand, and the epigean fish, on the other, revealed that
the cave fish possess a significantly lower number of lamellae (Fig. 5)
(Breder and Rasquin, 1943; Schemmel, 1967). The numerical differences
are one at 2 cm, two at 4 cm, and four at 6 cm body length in the Pachon
fish. Due to the high morphological variability, Schemmel (1967) argued
that the physiological efficiency of the olfactory organ probably does not
differ between the two forms.
Sexual Behavior
The reproductive behavior of the hypogean populations has been

studied mostly in the Chica fish (Luling, 1954a). More recent analyses
have shown that there are no differences among the epigean, the Chica,
and other hypogean populations (Breder and Rosen, 1966; Wilkens,
1972b).
All forms seem to lack any kind of courtship or display. Females
280 H. Wilkens
about to spawn are characterized by occupation of a relatively small area

in which they slowly swim. This area may be situated in dense plant
material or at one of the vertical walls of an aquarium.
The mating behavior of the males consists of a searching period pre-
ceding the actual spawning. The searching behavior is released imme-
diately after a male comes into direct contact with a spawning female.
Stimulated males try to mate with every other fish in their surroundings.
Ova and sperm are ejected while the mating pair turn their bodies, which
are closely pressed together, upside down. The eggs slowly sink to the
bottom or become attached to plants. This process occurs repeatedly. If
there is more than one male specimen, the mating male usually changes.
In the laboratory, the spawning behavior of the females may be in-
duced by a change of water. The males are stimulated by a substance
secreted by the female, the chemical composition of which is unknown.
Experiments with males whose olfactory nerves had been transsected
indicate olfactory perception of the agent (Wilkens, 1972b).
As has been described previously, the spawning females are char-
acterized by occupation of a relatively small area. This enhances the
chance of a male coming into contact with or finding a spawning female.
This also holds for the epigean males, which, once stimulated, are only
visually enabled to swim directly to other specimens, but are not able to
discriminate spawning females from others. They are not even disturbed
by the different phenotype of the hypogean females. Kauffeld (1954) re-
ports the mating of A. fasciatus with other characin species. In compe-
tition for spawning epigean and hypogean female specimens, the epigean
males do not show preferential mating in light. Parallel experiments in
darkness revealed that competing hypogean and epigean males do not
have different chances of mating.
REGRESSIVE TRAITS
Eye
Epigean Fish
The epigean A. fasciatus is characterized by a typical teleostean eye.

With regard to size and degree of differentiation, it differs sharply from
the eyes of other typical troglophilic species. The retina is well-developed
and possesses a high density of sensory cells. The presence of a consid-
erable quantity of rods makes good optical orientation under twilight
Evolution and Genetic. of Astyanax fasc/atus 281
FIG. 6. Cross section of the eye of the epigean

form of A. 'asciatus: 1, epidermis; 2, epidermal
duct; 3, 4, cornea; 5, ligamentum; 6, anterior
eye chamber; 7, lens capsule; 8, lens core; 9,
lens muscle; 10, vitreous body; 11, falciform
process; 12, retina (a, ganglionic; b, inner plex-
iform; c, Inner nuclear; d, sensory cell layers);
13, pigmentary epithelium; 14, chorioid; 15,
optic nerve; 16, depressions of retina; 17,
sclera. The key also applies for Figs. 7, 18, and
19.
conditions possible (Peters and Peters, 1966) (Fig. 6). The globular lens
is accommodated by a lens muscle. A large falciform process is developed
ventrally. The chorioid possesses an argentea and a large chorioid gland.
Cave Fish
PhylogeneticallyOldPopulations .Theeye rudiment of adult specimens

of cave forms that are presumed to have existed in caves for a long period
of time has a diameter of about 10-20% of that of the epigean fish (Peters
and Peters, 1966; Wilkens, 1970b). Differences in the extent of reduction
and the structural manner of regression such as found by Peters and Peters
(1966) between different cave forms can be attributed to the study of only
a restricted part of the total variability spectrum of eye types.
Lens and visual cells are completely reduced. The degree of differ-
entiation of remaining structures is correlated with eye size. The larger
eyes are also better developed and have a retinal rudiment that consists
of inner nuclear, inner plexiform, and ganglionic layers (Figs. 7 and 8).
In this kind of eye, an optic tract, which mainly consists of enclosing
connective tissues, forms a connection with the brain. The falciform pro-
cess has increased in relative size and almost separates the rudimentary
vitreous body into two chambers. In smaller eyes, nervous tissue and
vitreous body are reduced (Fig. 7).
In eyes of all sizes, the pigmentary epithelium is flattened. The tissue
of the anterior eye chamber has increased, fIlling up the chamber. The
282 H. Wilkens
FIG. 7. Cross sections of the eye of phylogenetically old cave forms of A. fasciatus. (A-C) Different
stages of differentiation. (D) Eye of F, hybrid between two phylogenetically old cave forms (Pachon
x Sabinos). For legend see Fig. 6. (Wilkens, 1970b, 1971.)
argentea has become fibrous. The sclera may consist, in part, of con-
nective, cartilaginous, and bony tissues.
The eye rudiment is situated beneath the body surface. It is connected
with the superficial tissues by an epidermal cord (Wilkens, 1970b; Zilles
et al., 1983).
fJm
eye size o 0
transverse rb o
• 0
00 00
• •• • 0«0
500 • 0 •
••• • t@o 0
• ••
100 FIG.8. Size and degree of the eye of Sabinos cave
fish; (0) retina with inner nuclear and plexiform lay-
500 1000 IJm ers, (e) retina undifferentiated or absent. (Wilkens,
eye size (proximo-distal rb) 1970b.)
Evolution and Genetics of Astyanax faBclatuB 283
Epigean (ave
FIG. 9. Dorsal view of tectum opticum in epigean and Pa- MS

ch on cave forms and their F, hybrid: P, pros-; MS, mes-; MT, MT
metencephalon. (Franck, 1964.)
Correlated with eye reduction is a diminished size of the tectum op-

ticum in the phylogenetically old cave fish (Steffanelli, 1954; Franck, 1964;
Schmatolla, 1972) (Fig. 9). Both retinotectal and retinodiencephalic con-
nections are found (Breder, 1944; Breder and Rasquin, 1947), though these
are greatly reduced as compared to those of the river fish (Voneida and
Fish, 1984). Retinotectal fibers in the Chica fish are entirely contralateral,
penetrating the medial third of the tectum. Application of both light and
electrical stimuli to the optic cyst elicits neither slow wave potentials nor
single unit responses in the cave fish tectum. It appears that if any in-
formation is conveyed to the tectum from the optic cyst, it is so weak as
to be difficult to detect electrophysiologically (Voneida and Fish, 1984).
The Phylogenetically Young Micos Population. The eye size of ran-
domly bred progeny of original Micos individuals is about intermediate
between the epigean ancestor and the extremely reduced cave forms (Fig.
11). Range and standard deviation of eye size surpass those of the epigean
as well as of the extremely reduced forms, but they are lower than the
F2 progeny produced by them. It is possible to influence the eye size by
selective mating. The size is slightly reduced in the offspring of individuals
with relatively small eyes. In addition, the variability is lowered corre-
spondingly. When specimens with larger eyes are mated, the first gen-
eration already inCludes individuals whose eyes are as large as those of
the epigean population (Wilkens, 1976).
Usually the eye sizes are normally distributed. When Micos individ-
uals with intermediate eye sizes are mated, however, the distribution of
the following generation is bimodal. Small-eyed individuals as well as
large-eyed ones turn out to be stable with regard to the eye size of their
offspring (Wilkens, 1976). The histology of the eye of the Micos fish is
described below (p. 263).
Eye Size in Crosses between Epigean and Hypogean Populations
Epigean x Phylogenetically Old Ca ve ,Forms . Comparative studies of

the eye size of crosses between epigean fish and phylogenetically old cave
forms have been carried out, using hybrids between the Pachon and the
284 H. Wilkens
Pachon 81 F2 82 Epigean
M=2.0 M=3.6 M=4.8 M=6.3 M=7.1
32.1% 54.8% 84.0%
pa(Gr.nuI t
00%
L tIm
25%
t
50%
titr ItI 0
75%
t! u1n t 100%
F1
M=5.1
61.8"10
Sabinos 81 Epigean
F2
M=2.1 M=3.6 M=4.6 M=6.1 M=7.1
31.9% 51.1% 80.2%
(Gr. Il .......~----~U-J...-'------'~rlT'1T-r---<>-I.J..L..._---'-'~..........
0.0%
AMean of all breedings

11. Mean of one breeding
FIG. 10. Distribution of mean eyeball size in crosses between epigean and phylogenetically old
Pachon and Sabinos cave forms (standard length 2.5 cm). (Wilkens, 19708.)
Sabinos fish when they were 2.5 and 6.0 cm in standard length. Regardless
of body size, the corresponding crosses do not diverge from each other:
The means are more or less intermediate, slightly shifting toward the
epigean parent (Figs. 10 and 11). In the FI and F2 and the backcrosses
with the cave fish, the distribution curves show normality. In contrast,
in the backcross with the epigean fish, a bimodal, discontinuous distri-
bution is found. With the exception of the F I crosses, the range of the
corresponding crosses includes offspring with eyes the size of the parental
forms.
Crosses Involving the Phylogenetically Young MicosPopulation. In the
crosses with Micos fish, small-eyed and large-eyed Micos parents were
hybridized with epigean and phylogenetically old Pachon and Sabinos
cave fish. Two results are important. (1) The frequency distributions of
the F I generations between small- and large-eyed Micos specimens
crossed with the extremely reduced Pachon cave fish are identical, in
spite of the different eye size of the Micos parent. Furthermore, the F I
of small-eyed Micos crossed with the epigean fish does not deviate from
that of a phylogenetically old cave form crossed with the surface ancestor
(Fig. 12). (2) The F2 and backcross offspring of the Micos fish, crossed
Evolution and Genetics of Astyanax 'asclatus 285
O'~~~~~r-r-~~
20
~ P.Qp.utation
laboratory breeding
10 selected for
intermediate eyes
MiCQS.~t1on
laborutory breeding
for large eyes
o~~~~~~~~
Micos ~lation J1::
i laboratory
2 .
breeling
for small eyes
FIG. 11. Frequency distribution of eyeball size
in the epigean, two phylogenetically old cave
forms and their crosses (-, Pachon; - -, Sabi-
nos), and the phylogenetically young Micos to 2.0 3.0 4D 5D 6D 10 ao
cave fish. (Wilkens, 19708, 1976.) eye size
286 H. Wilkens
Smoll- ey'ed Micas

x epigeon fish x cove fish
0/0
30 F1 F1
20
10
,0
20
10
0
20
10
0
20 F2
10
0
.LM.ge-eyed Micas
30 x epigeon fish x cove fish
20 F1 F1
FIG. 12. Frequency distribution of

eyeball size in crosses of small-eyed
and large-eyed Micos cave fish with
the epigean su rface form and a phy-
logenetically old cave form. (Wilk-
4.0 5.0 60 70 ens, 1976.)
with the epigean or phylogenetically old Pachon and Sabinos cave forms,
are characterized by the skewness or bimodality that develops after the
eyeball has surpassed a minimum size (Fig. 12).
Correlation of Individual Structures of the Eye
Growth of Eyeball, Lens, Pupillary Opening. The growth correlation

between lens and pupillary opening in the epigean fish and in the F 1 of
the cross with phylogenetically old cave fish is isometric. This is also true
in the epigean form for the relation between eye size and lens or pupillary
opening, respectively. In contrast, lens and pupillary opening show pos-
itive allometric growth with respect to eyeball size in the F 1 crosses be-
MU <Hens
o
4.
• y=1.95+x 1.09
r=Q97
n=60
2.
y=1.15+x1.78
r=QS9
n=43
FIG. 13. Growth correlation be-

tween lens and eyeball size in (a) the
epigean fish and (b) its F1 hybrid with <P Eye
a phylogenetically old cave form.
(Wilkens. 1970a.) 5.0 6.0 70 B.O 90 10.011.0 MU
tween epigean and phylogenetically old cave fish (Fig. 13). Thus, Ft hy-
brids are characterized by the fact that, in adult specimens, lenses and
pupillary openings are relatively larger than in juveniles. This kind of
development has also been found in all other crosses. It must be stressed,
however, that in the small-eyed specimens, the process of ontogenetic
reduction negates the growth processes just described. Thus, in the back-
cross with the phylogenetically old cave fish, for example, the mean rela-
tive lens size is larger in younger fish than in adults, which, as a result
of ontogenetic reduction, also possess fewer lenses. Nevertheless, in Ft
hybrids, F2 hybrids (at least all that exhibit larger eyes than do the Ft),
and in all backcross hybrids with the epigean fish the growth of the in-
dividual lens and pupillary opening is positively allometric (Wilkens,
1970a).
Interindividual Size Correlation of the Eye. In addition to the growth

correlations described above, the variable eyes of the crosses between
epigean and cave fish offer the possibility of studying size correlations of
individual structures that are influenced by different genetic backgrounds.
Examination of the eyes of hybrids of the same size (2.5 and 6.0 cm
standard length) has shown that the lens size of both groups shows a
288 H. Wllkens
MU tbLens
10
2.0
y=2.62+x 0.69
r=0.94
n=168
1.0
.
O. 5.. 1_.....,ir-_____..,ir-___. .;.tb. ;.P.; U.:;.;pi pl a; ,;r~Y_o;:;p;,; ,en;.; i;.;jn~ _
0.5 1.0 2.0 3.0 MU
FIG. 14. Correlation between lens and pupillary opening diameter in the F2 of the cross between
epigean and phylogenetically old cave form. (Wilkens, 1970a.)
negatively allometric increase with respect to size of pupillary opening

(Fig. 14). This is also demonstrated by the distribution curves of the size
of the lens and the pupillary opening sizes in the different crosses. In
contrast to the case of eye size, the F2 crosses show bimodality in this
case.
The relationship between eyeball size and lens and· pupillary opening
shows positive allometry. It is characteristic of the F2 crosses that, in
accordance with the bimodal distribution of size of the lens and pupillary
opening, the allometric relation changes, with a shift occurring in the
larger eyes (Fig. 15). A similar correlation can be observed in the Micos
fish (Fig. 16). In this form, the pupillary openings in smaller eyes show
a positively allometric size increase; in larger ones, the correlation with
the eyeball is more or less isometric.
Although correlated, there is a considerable amount of variability in
the different individual structures. Considering all eye sizes, the vari-
ability of the lens diameter observed in pupillary openings of the same
size is highest in smaller eyes. The variability of the pupillary opening
diameter measured in eyeballs of the same size shows parallel curves in
Evolution and Genetics of Astyanax 'asclatus 289
MU
30 Lens
2.0
y= 0OOO2h 2.86
r=O.56
n=151
1.0
FIG. 15. Correlation between lens and eyeball
size in the F2 of the cross between epigean and
phylogenetically old cave forms. (Wilkens, rt>Eye
1970a.) 4.0 5.0 6.0 7.0 MU
rt> Pupillary opening

4.0 MU
y=O 23+x 1.16

r=075
3.0 n= 40
2.
y=0.0000009+x 4.15
r=0.74
n= 48
1.0
FIG. 16. Correlation between pup-

illary opening and eyeball size in the
phylogenetically young Micos cave ~Eye
form. 4.0 5.0 6.0 7.0 aOMU
N
Cl
0
MICOS FISH
%
VARIABILITY OF
~ F1 (MICOS x CAVE)
PUPlLLARY OPENING 6 .or. F1 (MICOS RIVER)
"" X
• • • F2(MICOS X CAVE)
10 .. .. .. F2(MICOS X RIVER)
B (MICOS X CAVE)
------ X
MICOS
............ F2(RIVER X CAVE)
.... ======= 1 F1(RIVER X CAVE)
. ..
\
.r-'+-='~.
." ----2 B (RIVER X CAVE)
X
\V· / \",'" . . ."'--.1

\" ./
RIVER
-,-,-3 B (RIVERx CAVE)
X
CAVE
OI~~--.-~-r~~.--r~~.-~~--.-~~--r-~~~~~-r~~~-r~~M~U
3.0 4.0 5.0 6.0 7.0 EYE SIZE
FIG. 17. Variability of the pupillary opening (= standard deviation in percent of eye size) correlated with eyeball size in crosses
between the epigean and the phylogenetically old cave forms and the phylogenetically young Micos fish (standard length 2.5 cm.).
(Wilkens, 1970a, 1976.)
Evolution and Genetics of AstYBnBx fBsclBtus 291
different crosses between epigean and cave forms (Fig. 17). However,
the degree of variability is greatest in the F2 crosses.
Occasionally, eyes of different sizes develop in the same specimen,
a phenomenon called left-right asymmetry. The individual structures of
such eyes are developed in proper correlation to the respective size. The
study of backcross and F2 hybrids has shown that the size discrepancy
very rarely surpasses 25% of the diameter of the larger eye. In more than
50% of such cases the asymmetry in size of the eyes is less than 4%. The
occurrence of asymmetry is independent of eye size, but its frequency
differs in various crosses (Wilkens, 1970a).
HistologicalAnalysis ofthe Eyes in Different Crosses and in the Micos
Fish. Much histological work has been done on the eyes of hybrids be-
tween epigean and cave forms and in the variable Micos fish (Luling,
1953, 1955; Wilkens, 1972a; Peters et al., 1975; Fack and Wilkens, 1988).
It has been found that the size of the individual structures and their degree
of differentiation are correlated with each other as well as with the total
eye size. The individual structures of the eye show developmental phys-
iological interdependence. In accordance with findings in other verte-
brates, such as chicken and amphibians, by Coulombre (1969) and Kra-
tochwill (1972), within the Astyanax eye two subunits of differentiation
seem to be characteristic (Fack and Wilkens, 1988). On one hand, the
lens plays a dominant role and influences the degree of development of
pupillary opening, cornea, and possibly at least partially the vitreous
body. On the other hand, the retina is closely related to the development
of the pigmentary epithelium, the lens muscle, and probably the vitreous
body (Fack and Wilkens, 1988).
In the following the process of reduction of the individual eye struc-
tures is described.
Lens. The reduction of the lens is primarily characterized by a dimi-
nution in size. The end of this process is almost achieved when a much
larger lens capsule encloses a rudimentary crystalline nucleus (Fig. 18b).
By this stage the lens capsule has lost its globular form and before its
complete reduction even the crystalline has vanished. In small eyes, the
lens capsule is also reduced.
In contrast to the retina, structural differences in lens formation seem
to develop during the early ontogenetic stages (Sadoglu, 1975).
Cornea, pupillary opening, and anterior eye chamber. The devel-
opment of the cornea and the pupillary opening is closely related to that
of the lens. The diminution of lens size not only provokes a reduction in
the size of the pupillary opening (Figs. 18 and 19), but also a thickening
of the cornea propria (Fig. 18). In the anterior eye chamber, the liga-
292 H. Wilken.
FIG. 18. Due to a disharmonious interac-

tion between retina and lens acting as rela-
tively independent inductors of the eye, the
retina is partially pressed into the chorioid
in combination with (a) a very large lens (F2
hybrid Micos x Pachon) and (b) a very small
one (Micos fish). For legend see Fig. 6.
mentum annulare increases in relative size, while the lens becomes

smaller (Fig. 18b). However, as long as a lens rudiment exists, an anterior
eye chamber also remains. In the final state, the ligamentum completely
fIlls up the lumen of the chamber (Fig. 19).
Retina. The histological and ultrastructural phenomena of retinal
regression have not been exhaustively studied. It can be observed that
the visual cells show a negatively allometric correlation with the inner
nuclear, plexiform, and ganglionic layers (Fig. 19). Thus, in small eyes
the retina rudiment only consists of the layers mentioned previously. The
reduction of the visual cells also seems to include a process of diminution
in size. Thus, various stages of differentiation of the outer and inner seg-
ments are developed (Wilkens, 1972a).
Retinal manifestation is characterized by aberrations from the type
of reduction described before. Very often, a well-developed retina par-
tially consists of undifferentiated tissue (Fig. 18). This occurs mainly in
FIG. 19. In small eyes a lens muscle develops, although the

lens is reduced (F2 hybrid Micos x Pachon). For legend see
Fig. 6.
the ventral region. In rare cases, the entire retina is undifferentiated and
develops only as a faint strip of tissue.
The aberration phenomena may be attributed to a disharmonious in-
teraction between retina and lens in their roles as relatively independent
inductors of the size of the vitreous body and the retinal layers of the
eye. This becomes obvious when funnel-shap~d depressions of retinal
tissue develop into the chorioid in the region of the processus falciformis.
This may occur in combination with a small or a large lens (Fig. 18). In
the first case, the small lens is probably not able to induce an adequate
size of the eye to contain a well-developed retina. In the case of a large
lens, the induced eye size is too great for the "genetically smaller" retina.
Both combinations result in the retina being partially pressed into the
chorioid by the vitreous body. This happens in the processus falciformis
region, where the outer eye coats are not tightly grown together.
The aberrations described above seem to develop secondarily during
individual ontogenesis. They are more often formed in F2 crosses and in
hybrid Chica fish than in the phylogenetically young Micos cave fish. It
may therefore be concluded that they are hybrid phenomena (Wilkens,
1972a; Fack and Wilkens, 1988).
Lens muscle. The lens muscle is attached to the tip of the falciform
process. Its development is closely correlated with that of the neural
retina, inasmuch as both are derived from the eye cup. The lens muscle
may develop in eyes whose lenses have completely disappeared (Figs.
18b and 19). This demonstrates the relative independence of lens and
retina as inductors.
Pigmentary epithelium. The pigmentary epithelium is closely cor-
related with the state of differentiation of the visual cells. When the latter
are well-developed, the pigmentary cells assume their characteristic cy-
lindrical form. With the reduction of the outer segments, the pigmentary
epithelium forms only a flattened epithelium. In eyes that show partial
retinal dedifferentiation, the pigmentary epithelium is well-formed in the
294 H. Wllken.
intact retina and flattened in the dedifferentiated portion (Figs. 18 and

19).
Vitreous body, outer eye coats, chorioid, falciform process. All other
individual structures of the eyeball are developed in correlation with its
overall .size. The falciform process is relatively increased in size. De-
pending on the eyeball diameter, the optic rudiment sinks more and more
into the orbital cavity and eventually becomes covered by superficial
tissues. Only a connecting duct keeps contact with the body surface.
Ontogenetic Development of the Eye
The growth of the eye of the epigean fish is characterized by a more

or less isometric correlation with body length. With regard to the eye of
the phylogenetically old cave fish, however, considerable divergences
from this pattern have developed (Wilkens, 1980). During early embryonic
stages its eye shows a growth curve parallel to that of the epigean fish
(Figs. 20 and 49). However, at development between about 4 and 6 mm
body length (2.5-4 days of age) the eye ceases to increase in size, and
the difference in size between cave and epigean fish becomes drastically
enhanced. Subsequently, a strongly negative allometric growth relation-
ship becomes apparent (Wilkens, 1980; Hiippop, 1988b).
It is noteworthy that the unusual variability in the eye size that char-
Eye size(~mm)
a
• Epigean fish
• (ave fish
06
04
•.
. ....:.' :~:.
~= ~ .
...:••~••:".'! ~i-.:::::':.::'•
... :i= :..-1' •• ·:····':11:••. :'
.-.
.~Jitr!.A".~'
~-':'"'II"'.
•::':
"::'" • ''''N'
100%1--;'"""-...b
. :".
FIG. 20. Eyeball growth correlated
with body size in epigean and phylo-
genetically old cave fish. (a) isometric
i "11 "~I I
02 0.3 0.40~06 1.0 2.0 3.0 4.0 ~O 10.0 correlation, (b) occurrence of a lens
Standard length (cm) rudiment. (Wilkens, 1980.)
acterizes the adult cave fish does not appear until a body length of about
1 cm has been achieved. Earlier stages show the same amount of vari-
ability as does the epigean fish.
The comparative histology of the ontogenetic development of the eye
of epigean and cave fish has been analyzed primarily by Cahn (1958).
Recent studies have revealed that her results have to be modified slightly
(Wilkens and Meyer, in press). As recorded by Cahn, the very first anlage
of the eye cup is noticeably smaller in the cave fish than in its epigean
ancestor. However, due to the range of individual variability in the tem-
poral process of organ formation, it is doubtful whether the outpushing
of the optic buds in the cave form as observed by Cabn really is delayed
(Wilkens and Meyer, in preparation; Hiippop, 1988b).
In fact, one of notable features of the early larval development of the
eye is that the formation of the prospective retina does not differ in the
epigean and cave forms. Contrary to Cahn (1958), who in part described
abnormally developed embryos, it can be observed that mitotic activity
is not considerably less evident in the cave fish (Figs. 21a,b). Further-
more, the contact of the optic cup with the outer epidermis is as perfect
and close in the cave fish as it is in the epigean ancestor.
At a length of about 3.9 mm (±60 h) the formation of the retinal
layers has taken place in both the epigean and the cave fish (Fig. 21c).
Although the hypogean eye is already considered smaller, all its retinal
layers are developed. Even horizontal cells can be found. As in the epi-
gean fish at this stage, the outer nuclear layer consists of a single row of
nuclei. The sensory cells differ from the normal ones only in that their
inner and outer segments are of much smaller size. These visual cells will
not fully differentiate in the cave form. Their outer nuclear layer will
continue to consist of but one row of nuclei (Fig. 21d) and is finally re-
duced.
In both cave and epigean specimens, the lens is not formed as a
vesicle, but is developed as a solid bud (Fig. 21a). This seems to proceed
rather simultaneously. Due to the above-mentioned temporal variability
of organ formation, it is difficult to determine a size difference of the
newly formed lenses (Fig. 21a). Immediately after the lens has been
formed, however, remarkable divergences develop. Whereas the lens of
the epigean fish increases in size, that of the cave form remains more or
less fixed (Figs. 21a-c). At a body length of about 3.5 mm (± 34 h),
the differentiation of lens fibers has been completed in the epigean ances-
tor (Fig. 21b). In contrast, lens fibers in the cave fish eye are usually not
developed (Figs. 21b and c); only rarely does a single lens epithelial
cell seem to show the development of rudimentary fibers. In all cave fish
296 H. Wllkens
11
I SOI'Jll I
lBhrs
2.B-2.9mm body length
FIG. 21. Early ontogenetic eye development in epigean fish and the phylogenetically old Pachon
cave form: 1, Eye stalk; 2. lens placode; 3. lens epitheliallfiber cells; 4, retina (undifferentiated);
5, ganglionic layer; 6, inner plexiform layer; 7, inner nuclear layer; 8, outer plexiform layer with
horizontal cells; 9, outer nuclear layer; 10, inner segments of visual cells; 11, pigmentary epithelium;
12, cornea; 13, vitreous body; 14, necrotic centers.
a lens capsule is formed, but the cave fish lens is not able to induce any
thinning of the cornea (Figs. 21b-d).
In summary, it can be concluded that except in size, the development
of the eye tissues in cave and epigean fish is rather similar during early
ontogeny. At about 4 mm in body length (±60 h), however, destructive
phenomena become evident, and necrotic processes take place in the
retina and the lens (Fig. 21c). Remarkably, they occur after the retina has
formed its typical layers. It should also be pointed out that these events
obviously coincide with the cessation of eye growth, between 4 and 6 mm
body length, as previously observed (Fig. 20) (Wilkens 1980).
Evolution and Genetics of Astysnsx 'ssclstus 297
12
I 50 ~m
33hrs
3.5mm body length
(ove fish
FIG. 21. (Continued)
Ultrastructural studies of the hybrid Chic a cave fish retina reveal

considerable signs of degeneration immediately after hatching in the outer
nuclear, inner nuclear, and ganglionic layers. Such Chica fish still possess
visual cells, but the outer segments of these cells may be completely
reduced or may exhibit various degrees of desorientation of the lamellae
(Yew and Yoshihara, 1977; Durand, 1978, 1979; Zilles et al., 1983).
All other individual structures characteristic of the teleostean eye,
such as the vitreous body or the chorioid, are developed in association
with lens and retina. The anterior eye chamber is filled with a specific
tissue, the ligamentum annulare. The eye rudiment gradually sinks into
the orbital cavity (Fig. 21d).
298 H. Wilkens
It is characteristic of the history of the individual structures that

almost no variability is observed in the juvenile cave fish eye; variability
develops only in adult specimens. It appears at about 1-3 cm in body
length and is correlated with the increasing difference in eyeball size. It
is caused by strong reduction processes within the retina and lens, the
result of which is the adult cave fish eye described above.
The Influence of Light and Darkness on the Development of the Eye

In order to elucidate the influence of light and darkness on eye de-
velopment, specimens were bred and kept in darkness. In the epigean
SOlJm
llmm body length
fish, the size of the eyeball does not decrease under these conditions, but
the thickness of the retina is reduced (Fig. 22). On the average, each layer
is reduced by between 20 and 40%. Only the size of the visual cells does
not seem to be affected. As the number of nuclei in the outer nuclear
layer is reduced by about 20%, the reduction of the retina is presumably
primarily caused by a reduction of the number of visual cells.
Peters et al. (1975) tried to show that the eye size of the eye of the
Micos fish can be influenced by different intensities of light. Eyes of
specimens kept in darkness were smaller than those bred in light. This
tendency was confirmed statistically in individuals of the variable Micos
fish as well as of F 1 and F2 crosses between the epigean and the phylo-
genetically old cave forms (Table I).
Earlier suggestions that the eyes of Pachon and Sabinos cave fish
are smaller when these fish are bred in darkness could not be confirmed,
300 H. Wilkens
layers light decreasel'!,)

I
--- I l -
~i
ganglionic 0.0330
---
inner dark
0.0330
plexiform
0.0222
---- -42.7
----
inner
nuclear
0.0285 1\ -453
0.0189
0.1494 mm
-31.1%
0.0156 0.1029mm
outer
jJlexifQ!-m
0.0150 "'-
-30.0
0.0105
outer 0.0174
0.0219 -20h
FIG. 22. Thickness of retinal layers in
nuclear
---- specimens of epigean A. fasciatus
ellipsoids 0.0180 -0.0 0.0183 hatched and kept in darkness. Mean val-
- ues derived from five adult specimens.
however (Wilkens, 1970b). The high degree of variability will probably

rule out the detection of any light-dependent influence on the eyes of the
phylogenetically old cave fish.
Genetic Background
Due to developmental constraints operating within the eye of A. fas-

datus, no individual genes responsible for single structures such as the
lens, visual cells, or pigmentary epithelium have been shown to exist.
Only the effects of genetic factors influencing eyeball size have been
recorded. For such putative genetic unit factors, the term "eye genes"
has been coined (Wilkens, 1970a, 1971, 1984, 1985).
The manifestation of the so-called eye genes is additively polygenic.
This can be concluded from the normal distribution and more or less
intermediate means of eyeball sizes in the different crosses between epi-
gean and phylogenetically old cave populations (Fig. 11). However, the
additively polymeric manifestation characteristic of the equivalent ex-
pressivity of single polygenes is subject to alterations. Backcrosses to the
epigean fish, for example, develop a bimodal distributional curve. Iden-
tical phenomena have been shown in the frequencies of eyes of different
sizes of the Micos fish and in several of its crosses with epigean and
phylogenetically old cave fish (Fig. 12). The skewness and the bimodality
of the distributional curves, as well as the deviations of mean eye sizes
TABLE I. The Influence of Light and Darkness on Eye Size in Different Breedings of
the Phylogenetically Young Micos Cave Fish a
Breeding M s N p Size, cm
Light
Dark
4.75
4.39
0.27
0.19
35
59 } <0.0001 2.5
2 Light
Dark
6.85
6.14
0.36
0.41
60
50 } <0.0001 2.5
3 Light
Dark
5.22
5.07
0.53
0.57
98
90 } <0.08 2.5
4 Light
Dark
6.53
6.43
0.36
0.53
60
75 } >0.1 2.5
5 Light
Dark
4.54
4.54
0.14
0.21
7
39 } 2.5
6 Light
Dark
11.8
10.4
0.8
0.67
35
38 } <0.0001 6.0
7 Light
Dark
9.54
9.52
1.24
1.39
39
15 } >0.8 6.0
8 Light
Dark
7.78
7.14
0.54
0.94
17
5 } >0.5 6.0
a The Mann-Whitney-U-test (two-tailed) was applied because only part of the samples is
normally distributed.
in the direction of the epigean form in the offspring ofthe various crosses,
have the same cause.
These alterations in frequency are the result of the expressivity of
the polygenes, which under certain conditions is discontinuously en-
hanced. This discontinuous jump requires that a minimum number of eye
genes, each with the same amount of expressivity, be recombined. A
further essential requirement seems to be the presence of genetic balance.
This can be concluded from the fact that the above-mentioned alteration
only develops in the F 1 and in the backcross progeny, all of which possess
a complete set of chromosomes of one parental form. It is also charac-
teristic of the Micos fish, which is heterozygous for the eye genes and
thus can develop a discontinuous distribution. In the progeny of the F2
crosses, random recombination of parental genes is the rule. This appar-
ently disturbs the genetic balance, and thus, instead of bimodality, a nor-
mal distribution occurs (Figs. 11 and 12).
The importance of the presence of genetic balance is also reflected
in the variability in the size of the pupillary opening. When correlated
302 H. Wllkens
pm eye size
(transverse t/» 0 0
0 0
1000 8> 0 0
0
"
00
0
0
co 0 0 0
"ococ!>qs>o
Cl' 0 0 0
"'" " "'" + 0 " ooi'
0.66 A6A 00
0 0
o ""~ ~ ~¥8e 0 0 " Sabinos

~oo "'" "<lCI"". cA· 0 o Pachon
o 0 "t8J" 0
o F,-hyl:ri:l
o
o
o " eye size (proximo-distal t/»
500 1000
FIG. 23. Eyeball size of the phylogenetically old Sabinos and Pachon cave fish and their F, hybrids.
Means shown in black. (Wilkens. 1971.)
with eyeballs of the same size, the greatest variability is found in all the
F2 crosses between epigean and Micos fish on one hand and the phylo-
genetically old Sabinos and Pachon cave fish on the other (Fig. 17). In
the Micos fish, which are not hybrids, the level of pupillary variability is
far below that of the F2 crosses.
At least six genetic factors have been calculated to be involved in
eye reduction in the phylogenetically old Pachon and Sabinos fish (below,
p. 307). Crosses between these two cave forms have revealed that the
responsible genes are to some extent nonallelic. In the F 1 progeny, the
eye rudiments are on an average larger, and thus better differentiated,
than in the parental forms (Figs. 7 and 23). Occasionally, single structures
develop, such as the lens capsule, that are never found in adults of the
cave fish (Wilkens, 1971).
Pineal Organ
The pineal organ of A. fasciatus has been studied by several authors

(Grunewald-Lowenstein, 1956; Omura, 1975; Herwig, 1976). In the fol-
lowing, the results of Herwig (1976) are considered, inasmuch as he was
the only investigator to consider ontogenetic regression and the fact that
a hybrid, the Chica cave form, was being studied.
The pineal organ of the epigean population does not reveal essential
Evolution and Genetics of Astyanax (asc/atus 303
morphological and ultrastructural differences in comparison with other

fish species. It is situated below a large frontoparietal fontanelle, which
extends from the olfactory lobes to the caudal part of the cerebellum. The
pineal is divided into a spindle-shaped end vesicle and a slender stalk that
forms a connection to the diencephalic roof. The pineal stalk is almost
completely surrounded by a fold of the underlying dorsal sac.
The pineal organ consists of sensory cells, supporting cells, nerve
cells, and a cell type with phagocytotic characteristics. The sensory cells
possess relatively well-developed, saccularly organized outer segments
on their apical side. On the basal side they have prolongations that make
synaptic contacts with nerve cells. Axons emerge from the nerve cells
and form a pineal tract that runs down the pineal stalk toward the dien-
cephalon.
The outer segments have essentially the same organization as the
cone photoreceptor cells of the lateral eye. They consist of a stack of
about 30-50 parallel saccules. The total thickness of each stack of saccules
does not exceed 2.5 f.1m. The shape of the outer segments may vary con-
siderably. Some are slightly curved and cover the inner segment like a
cap, others have concentric arrangements of saccules, and all interme-
diate stages may be observed as well.
The nucleated part of the sensory cell is connected to the inner seg-
ment by a constricted neck region. The cytoplasm of the inner segment
contains organelles similar to those in the nucleated part of the cell. The
apical part of the inner segment is characterized by a large concentration
of mitochondria, forming the so-called ellipsoid.
Morphologically, the pineal organ of hypogean specimens does not
differ from that of the epigean form. The only differences are in the outer
segments ofthe sensory cells. Injuveniles of the hybrid Chica population,
a number of outer segments are not as well-developed as in the ancestral
form. They possess fewer saccules (10-30), and the stack of saccules has
a maximum thickness of at the most 1 !-lm. There is also a number of
more complicated, distorted outer segments.
The development of the outer segments is subject to a process of
ontogenetic regression. Thus, in adult individuals a variable number of
outer segments lack the saccular arrangement. In phylogenetically old
populations, this situation is presumed to be typical of all specimens.
Color Pattern
Epigean Fish
Besides guanophores, iridophores, and xanthophores, the col or pat-
tern of the epigean fish is primarily formed by melanophores (Rasquin,
304 H. Wllkenl
~A
'\
,glli9mP.Ql1!,!latioo
and F,..!iJmgg!Q!lP..2p'ulations
background black
_-+-+-+background white
backcrosses
5 \Wi th hypogean populations
,
~:,~,
, ,
0,0 ',0 2,0 3,0 4,0 5,0 6,0 7,0 8,0
melanophores lunit
FIG. 24. Frequency distribution of melanophore densities in epigean and phylogenetically old
Sabinos and Pachon cave fish and their crosses. (Wilkens, 1970c.)
1947). These melanophores form a basal pattern of cells that has its great-
est density on the dorsal side. Dark patches of larger melanophores are
developed in the caudal area and on each shoulder (Rasquin, 1947; Wilk-
ens, 1970c).
The melanophores of the epigean fish are subject to physiological
and morphological color change. The number of the cells is about 30%
greater when the fish is living over a dark than over a white background.
Blinded specimens show the same development of color cells typical for
fish kept over a dark substratum (Fig. 24). (Wilkens, 1970c).
Cave Fish
The light body color of the cavernicolous A. fasciatus forms has

several causes. In the phylogenetically old populations, the number of
melanophores is decreased. Specimens of the Sabinos form develop 30%
Evolution and Genetics of Astyansx tssclstus 305
~.,
....
4'
.,. . ...
..
""" .. i'
FIG. 25. Melanophore cells in (a) the epigean and (b) the phy-
logenetically old Sabinos cave form.
and those of the Pachon form about 15% of the number found in epigean
fish. The melanin content of the melanophores is also reduced (Fig. 25).
In the phylogenetically old cave fish, it may be partially reduced, or mel-
anin may be completely lacking (Sadoglu, 1955, 1957; Sadoglu and
McKee, 1969; Wilkens, 1970c). The Pachon and the Yerbaniz forms are
known to belong to completely albinotic populations (Sadoglu, 1957; Wilk-
ens, 1984), while the Piedras and the Sabinos fish exhibit partial reduction
of melanin content.
The melanophores of the cave fish are still able to effect physiological
color changes (Burgers et al., 1962; Wilkens, 1970c). Morphological color
changes, however, are no longer possible. Thus, when maintained in day-
light, specimens of the phylogenetically old populations do not become
darker.
Under normal light conditions, the phylogenetically young Micos fish
show no differences from the epigean form with respect to the melanin
content of their pigment cells or their ability to undergo physiological and
morphological color change (Fig. 26) (Wilkens, 1976). In contrast, Micos
fish in darkness are almost as lightly colored as are the phylogenetically
old cave populations. This is mainly due to a 40% reduction of the number
of melanophores.
Within the phylogenetically old Piedras population, some specimens
show the total reduction of guanin, except for some scales of the lateral
line (Wilkens, in preparation). All phylogenetically old forms seem to
show partial reduction of guanophore pigmentation (Breder and Rasquin,
1947).
306 H. Wilkens
r
CAVE FISH ~ •
MICOS FISH
~
~
...-
I 2s
F1
[MICOSICCAVEl
BACK
[MICOSICCAVEI
IC(AVE
BACK {
• .-
(MICOSIC(AVEI
ICMICOS
F2
[MICOSICCAVE)
{
•I
I
[MIca;F1RIVER!~ $
..
BACK
[MICOS RIVER
xMICOS $ •
BACK
(MICQSICRIVER
ICRIVER ~ •
F2
MICOSICRIVE ~
RIVER FISH
~ I
.. 023
0 1 2 3 4 5 S
MELANOPHORES/UNIT
FIG. 26. Mean, standard error (srn), standard deviation (s), and range (r) of the melanophoral
densities over a white (w, open) or a black (b, solid) background in the phylogenetically young
Micos cave fish and its crosses. (Wilkens, 1976.)
Genetics
The partial reduction of melanin content, as well as albinism, are

based on a monogenic, recessive mode of inheritance. Both mutations
have occurred at allelic loci in the cave populations studied. Albinism
and partial reduction segregate independently (Sadoglu, 1957; Sadoglu
and McKee, 1969; Wilkens, 1970c). The total reduction of guanin in the
Evolution and Genetics of ABtysnsx 'sBclstuB 307
Piedras population also seems to rely on a single recessive gene, which

in this form is heterozygous.
The gene system responsible for the number of melanophores is in-
herited independently from the color genes and is additively polygenic.
This is shown by the normal distribution and intermediate mean in me-
lanophore density of the F2 progeny of crosses between epigean and cave
forms (Fig. 24) (Wilkens, 1970c). However, the additively polygenic mode
of inheritance shows alterations in phenotypic frequencies that are caused
by the ability of certain fish to undergo morphological color change. The
latter does not segregate independently from the gene system that is re-
sponsible for the formation of melano phores. Morphological color change
does not begin to function until a minimal number of melanophore genes
has been recombined, a manifestation of genetic balance.
This genetic balance depends on the presence of at least one complete
parental haplome. Due to a threshold effect, the F 1 and the backcross to
the epigean fish are thus able to show the same pigmentary expression
as the epigean fish. In the backcross to the cave fish this provokes a
skewed frequency (Fig. 24). In the F2 crosses, the genetic balance of which
has been disturbed by the random recombination of genetic factors, the
threshold effect cannot be expressed. Instead, a normal distribution ap-
pears because every polygene shows the same amount of phenotypic
expression.
As described above, the phylogenetically young Micos form does not
deviate from the epigean form with respect to melanophore density and
morphological color change under different light conditions. This also
holds for all crosses between the two forms (Fig. 26) (Wilkens, 1976).
The situation is different, however, in crosses with the phylogenet-
ically old cave fish. The F 1 in this case exhibit an intermediate melan-
ophore density and cannot undergo any morphological color change (Fig.
26). This phenomenon is probably due to the fact that one or more genes
responsible for melanophore formation have mutated in the Micos fish.
Thus, in the above-mentioned F 1 crossing, the number of melanophore
genes is too low in the F 1 progeny to bring about the threshold effect on
which the manifestation of the morphological color change is based (Wilk-
ens, 1976). All the other offspring from crosses between Micos and phy-
logenetically old cave fish show melanophoral densities that develop
under the influence of an additively polygenic mode of inheritance.
Genetic analyses have also shown that the Micos fish as well as the
Pachon and Sabinos fish are homozygous with respect to regressive pig-
mentary loci. A calculation of the minimum effective number of genetic
factors responsible for melanophore formation reveals a difference of at
least one or two loci in the Sabinos and three or four in the Pachon fish.
308 H. Wllkenl
In the Micos fish at least one locus must be involved (Wilkens, 1970c,
1976; Lande, 1981).
Scales
The species of cyprinid cave fish are especially likely to show a partial
or total loss of scales (Bannister and Bunni, 1980; Bannister, 1984). Similar
phenomena can also be observed in the hypogean populations of the char-
acin A. fasciatus (Bohn, 1986).
The body of both the epigean and the hypogean forms is covered
with densely and regularly distributed scales of the cycloid type. They
are largest in the anterior region and smallest in the caudal area and in
the upper and lower horizontal rows. Nevertheless, in comparison with
epigean fish, the phylogenetically old cave forms show a decrease in the
number of scales. The Pachon population, for example, is characterized
by having one or two fewer scales in the lateral line. It should also be
noted that the number of vertebrae is reduced. A tendency to decrease
the number of scales can also be found in the vertical scale rows in the
cave fish (Schuppa, 1984).
Moreover, the craniocaudal scale diameter of the cave fish is short-
ened. In all body regions of cave fish the quotient scale height/scale length
therefore is greater (Fig. 27). Because of this , the foci, which are normally
located at the center of each scale, are located more anteriorly. Due to
the reduction in length, the scale is no longer as deeply embedded in the
skin as it is in the epigean form, and is more easily lost. Nevertheless,
despite their being reduced, the scales of the cave forms of A. fasciatus
still form a complete body covering. In cases where a horizontal row has
been lost, they develop greater height. Their craniocaudal shortening is
compensated by the disjunction of individual scales. As a result, the scales
no longer form the complete double-layered covering that is typical of
the epigean form.
This reduction of scales in the hypogean populations is probably
brought about by a partial loss of biological function. Noting the great
amount of intraspecific aggression shown by the epigean fish, it seems
logical to assume that the scales provide a protection against bites. In the
cave populations, aggressive behavior is lost, and the scales have also
lost a major function and become reduced. This explanation is supported
by the fact that laboratory groups of enucleated epigean individuals that
no longer show aggressive behavioral patterns have a completely intact
covering of scales (see next section).
Evolution and Genetics of Astyanax fasc/atus 309
Cave fish
/ l~~tlllbe'dded part
Epigean fish
~ EmlJedded part
FIG. 27. Comparable epigean and cave fish scales.
Aggressive Behavior
Epigean Population
The aggressive behavior characteristic of the epigean population was

first described as "erratic viciousness" by Breder (1943b). It shows dif-
ferent patterns of expression (Burchard et al., 1985). "Fin-spreading" is
the first reaction to appear. With increasing aggression the head is lowered
and the body takes on an oblique position. In addition, "snake-swim-
ming" and "circling" may be performed. The highest degree of mani-
festation is characterized by "tail-beating," "ramming," and "biting"
(Figs. 28 and 29). Almost every bite results in the loss of scales. Sub-
missive individuals with no possibility of hiding or escaping are eventually
killed.
Aggressive behavior is released visually. Epigean fish will attack their
mirror image as well as specimens separated by a glass pane. Aggressive
behavior also is directed against the phenotypically different hypogean
fish.
Aggression is closely coupled with individual territoriality. Its inten-
310 H. Wilkens
500
400
FIG. 28. Ramming and ramming attempts in

(L) light and (D) darkness by epigean and phy-
logenetically old Piedras cave fish and their F,.
L0 L 0 LD LD LD F2 , and backcross hybrids. Each fish tested
Epigean F, B~ B~ [ove
Epigean Cave against an epigean specimen. (Parzefall, 1985.)
sity depends on the amount of aquarium space available (John, 1964).

Field studies have shown that the epigean A. fasciatus school in rivers.
In pools that dry up during the dry season, however, the fish become
territorial and show the aggressive patterns described above (Parzefall,
1983a, b). The aggression seems to be part of a density-controlling system.
In the laboratory, the fish usually divide smaller tanks into territories
(Burchards et al., 1985), and the occupants direct their aggression pre-
dominantly against fish without territory. No sexually specific differences
have been observed. Dominance is determined mostly by the size of the
fish. Contrary to John (1964), it has been found that juveniles are also
aggressive.
In larger tanks or under the conditions brought about by high pop-
ulation density, territoriality and aggression decrease, and the specimens
start to school. The readiness with which the fish swim in schools in-
120
100
FIG. 29. Frequency of ramming

III (dark bar) and ramming attempts
1:5 20 (open bar) in epigean and phylo-
~ genetically young Micos cave and
!;( phylogenetically o.ld Pachon cave
EPIGEAN MICOS CAVE PACHON CAVE forms. (8uchards et al., 1985.)
creases as the number of fish kept in a given tank increases (John, 1964;
Burchards et al., 1985).
In addition to the available space, state of nutrition also influences
the intensity of aggressive behavior patterns: hungry individuals are more
aggressive than satiated ones.
In darkness, aggressive behavior is practically nonexistent (Fig. 28)
(Burchards et al., 1985; Parzefall, 1985), nor do enucleated specimens
ever show aggression. This is clearly demonstrated by the fact that, con-
trary to sighted specimens, groups ofthem never show any loss of scales.
Hypogean Populations and Crosses
In the phylogenetically old Pachon and Piedras populations, aggres-

sive behavior is reduced (Fig. 29) (Burchards et al., 1985; Parzefall, 1985).
The phylogenetically young Micos population seems to occupy an inter-
mediate position between the epigean and phylogenetically old cave pop-
ulations. In darkness, the aggression of this form is also not manifested.
The progeny of the different crosses between epigean and hypogean forms
also do not show aggressive behavior in the dark (Fig. 28).
Genetics of Aggressive Behavior
The reduction of the aggressive behavior in the cave forms has a

genetic basis (Fig. 28) (Parzefall, 1985). This is demonstrated by the fact
that the mean of the F2 crosses between epigean and cave specimens
exhibits an intermediate amount of aggression. Furthermore, in back-
crosses with the cave form the aggressive behavior disappears. By anal-
ogy with features such as the melanophore system, the particular mani-
festation of aggressive behavior by the F 1 and backcrosses to the epigean
fish can be attributed to epistasis. The studies indicate that aggressive
behavior in A. fasciatus is regulated by a polygenic mode of inheritance
similar to those described for other complex features of A. fasciatus
(below, p. 306).
Causes of Reduction
The reasons for the reduction of aggressive behavior in the cave forms
have not yet been elucidated. Burchards et al. (1985) suggested that, due
to the presumed low population density in caves, the chance meeting of
two specimens is far less probable there than in the epigean biotope.
Because of this "nonusefulness," stabilizing selection no longer was act-
ing. The authors further stress that territorial aggression is only developed
312 H. Wilkens
under conditions ofthe uniform distribution of food. In the Astyanax cave

biotopes, however, the food supply is concentrated below bat roosts.
Thus, territorial aggression would be without function or even a disad-
vantage. In the latter case, it should be reduced by directional selection.
A different, and perhaps more probable, explanation is based on the
fact that in this species aggressive behavior is released almost exclusively
by vision. Although it still exists in epigean individuals in the dark, it
cannot be expressed, and thus it is no longer influenced by stabilizing
selection and becomes genetically reduced.
Schooling Behavior
Schooling behavior has been reported as a common social organi-

zation in epigean fish in their surface habitats (Parzefall, 1983a; Parzefall
and Senkel, 1986). In the natural biotope as well as in the laboratory, it
is abandoned under the conditions of limited space. Under such circum-
stances, individuals become aggressive and territorial (see preceding sec-
tion). In darkness or when blinded, the epigean fish do not school, and
swim in a random spatial distribution without any aggressive behavior
(Fig. 30). Unlike the epigean form, phylogenetically old cave fish do not
school either in light or in darkness.
The loss of schooling behavior has a genetic basis (Parzefall and
Senkel, 1986). This can be quantified in choice experiments, in which a
fish's tendency to follow a school or to leave it is measured. The studies
reveal that visually intact Ft and F2 hybrids between the epigean fish and
the phylogenetically old Piedras cave form show reduced schooling be-
havior (Fig. 30). Especially in the F2 generation, high variability is ex-
hibited, and this shows a distribution between the parental forms.
In the phylogenetically young Micos cave fish, schooling behavior is
also reduced. It shows a stage more or less intermediate between the
epigean ancestral form and the phylogenetically old cave fish (Fig. 30).
The way in which schooling behavior is manifested in the Ft and F2
crosses as well as in the Micos cave fish is more or less identical with
that of the other complex features genetically studied in A. fasciatus
(below, p. 306). It can be concluded that it is polygenically inherited.
Activity Control
Forms of Activity
Activity control in epigean and cave forms has been studied several
times (Thines et al., 1966; Thines and Weyers, 1978). Erckens and Martin
(D)
120 160
T T
,
11=10 0=12
FIG. 30. The tendency to follow a school

(= stay in compartment B) and the num-
ber of changes between chambers A and
B in the epigean fish from the Rio Teapao
and its F1 and F2 crosses with a phylo-
genetically old cave form and the phylo-
genetically young Micos cave fish. (D) Ex-
periment in darkness. (Parzefall and
Senkel, 1986.)
(1982a,b) showed that the activity of single epigean specimens can be

classified as "surface" or "bottom activity" (Fig. 31). Under LD con-
ditions (alternating light and darkness), both can be strongly entrained.
In general, the maximum values of the surface activity correspond to the
dark phases of an LD and the bottom activity follows an inverse course
(Fig. 31). Under DD conditions (continuous darkness) the clear-cut pat-
terns disappear and the oscillations display their maxima against increas-
ing background noise (Fig. 32). It appears obvious that maxima and min-
ima do not mean "activity" and "rest," respectively, but are expressions
of different levels of continuous activity.
In the Pachon cave form, it was necessary to increase the amplitude
of the forcing signal considerably in order to stabilize the forced signal,
due to the fish's lower light sensitivity. However, the same inversion of
314 H. Wilkens
22 11 23 11CET
100.F"~;"=:;,;j '-';;"""~~
B 500 r
JggJ===;"""",/ I=~=-_...d FIG. 31. (Left) Surface and (right) bottom activity of
(A) four epigean specimens during four consecutive
100 F-II:;.,.a.::.I ,..;...,.~~"""" days and (B) one hypogean Pachon specimen during
500 five consecutive days under LD 12:12. Solid horizon-
tal axes, dark phase; open axes, light phase. CET,
100 I=:.='!!!;;;'-'_~ '==~;;;;;";~ Central European Time. (Erckens and Martin,
22 10 22 10CET 1982a,b.)
the two kinds of activity as in the epigean fish was exhibited (Fig. 31).
Furthermore, a greater variability in the activity answer to external light
stimuli could be observed. This mainly means a weaker entrainment, at
least in part, of the time series.
The Passive Control System
The activity of the river fish was entrainable by all the applied LDs.
This leads to the conclusion that a passive system controls the activity.
1~~t~:LJ L...... FIG. 32. Complex demodulation in epigean fish of the time
-'O~~M~M.f.If4"-:D~D~_;:';;'~O.t\~S series of surface, bottom, and total activity under LD 12:12
and following DD. Under LD, surface and bottom activity are
I~~~t~~ J I ~ J
I
entrained and oscillate with a phase-angle difference of 180°.
During 4 days after beginning of DD a section of instability in
-535~~;~ which phase shifts take place occurs in surface activity. There-
after the oscillation stabilizes. Bottom activity becomes ar-
ITotol L 11
o~~~
.
rhythmic in DD. Total activity registers weak entrainment
under LD because of the inversion of both single oscillations.
00- DAYS (Erckens and Martin, 1982a.)
Evolution and Genetics of Astyanax 'asciatus 315
LD 12 ,12
0477:~
5 10 15 20 25 30h
QS34~~
10 15 20 25 30h
FIG. 33. Periodogramm analysis under three different time
series of surface activity in Pachon cave fish. The peaks ex-
tending beyond the shaded areas indicate significant
Q537:~
rhythms. Test level 95%. (Erckens and Martin, 1982b.) 10 15 20 25 3Cl1
After a transition from LD to DD, the entrained oscillations damp out

within one or a few periods under an unchanged frequency (Fig. 32). The
system therefore can be characterized as passive, but not extremely pas-
sive. Furthermore, the system acts like a linear one, because in the tested
range the output frequency (activity oscillation) is always equal to the
input frequency (LD). The system also takes a short time to reach a new
steady state under forcing conditions and can therefore be characterized
as strongly damped in comparison to a circadian time scale.
In the Pachon cave form, activity is under the control of a passive
system, with a nearly unlimited range of entrainment and a linear rela-
tionship between input and output (Fig. 33). The stability of the forced
oscillations is decreased compared with the river fish, but the strong
damping seems to be unchanged. However, the system is unable to os-
cillate after transition from LD to DD, and thus, contrary to that in the
river fish, is extremely passive (Rohler, 1974).
The Nonlinear Self-Sustained System
In addition to the passive system, a self-sustained circadian oscillator

is involved in the activity control ofthe epigean form. If the period length
of an LD is a harmonic of 24 h, the additional circadian rhythm will be
relatively strong and stable. If the period length is not harmonic, the
additional circadian rhythm will be less strong and stable, but still de-
tectable.
The Pachon cave form exhibits an oscillator with a restricted capa-
bility of self-sustainment. Only under special forcing conditions, i.e., in
the range of period lengths of about 24 h, is the oscillator able to act as
a stable system and to remain stable under following constant conditions.
Beyond this range the oscillator seems to remain switched off, and the
activity shows entrained rhythms only under forcing conditions and ar-
316 H. Wllkens
rhythmic patterns only under constant conditions. The oscillator in the

cave fish probably has a rudimentary character and seems to be an ev-
olutionary relic.
Alarm Substance and Fright Reaction
Alarm Substance
The alarm substance has been found to exist in the epigean as well
as in the phylogenetically old Pachon and Piedras cave forms and in the
hybrid Chica population (pfeiffer, 1963, 1966, 1967a; Thines and Legrain,
1973; Fricke, 1988). It is produced in the epidermal club cells. In the
Pachon fish, 2800 club cells have been counted in 1 mm2 of skin (pfeiffer,
1967a). It is unknown whether there is any difference in the number of
club cells or the quantity of alarm substance produced by a single cell in
the epigean and cave fish.
Fright Reaction
The fright reaction is well developed in the epigean fish (pfeiffer,
1963). In tests conducted with single epigean specimens, three stages of
reaction could be differentiated (Fricke, 1988). The most violent reaction
starts 30-60 sec after alarm substance is added to the water in the aquar-
ium. The specimen darts in zigzag movements and hides. It does not leave
its hiding place for several days. In the minimum reaction the specimens
are only slightly startled for a period of time. Almost all fishes react to
the presence of alarm substance by avoiding the surface of the water to
a depth of 5-6 cm, whether the substance has been introduced at the
surface or close to the bottom. These different behavioral elements are
connected by transitional patterns and show a good deal of individual
variability. When kept in schools the fish show the same general behavior,
but they do not hide, schooling more closely instead and remaining mo-
tionless in one place.
The fright reaction has been analyzed quantitatively by determining
the feeding rate at the water surface, inasmuch as the addition of alarm
substance at the water surface causes a significant decrease of feeding
there (Fig. 34) (Fricke, 1988). The feeding rate slowly rises, but it does
not reach its original level for at least 1 week. The introduction of alarm
substance close to the bottom provokes less intense reactions.
In the phylogenetically old Pachon and Piedras cave fish, the fright
reaction no longer includes any zigzag darting. This behavior is only very
Evolution and Genetics of Astysnsx fsscistus 317
• FOOD
50 EPIGEAN FISH ® ALARM SUBSTANCE
~ PTEROPHYLLUM SKIN EXTRACT
!WITHOUT ALARM SUBSTANCEI
A .. DZIBILCHALTUN
~/ TEAPAO n=16
.4#-,#' n=13
10 15 20 DAYS
80
70
CAVE FISlf1
/ : ."...,' ..e,.,
/i
i ~
I I / '.,' ''W'!
I I / \. .I
60
!" ~./
I
~
1;:150 , ,';-
PIEORAS
FIG. 34. Feeding rate at the water surface in a: ../ :.. , n08
1
epigean populations from Rio Teapao and Cen- I
ote Dzibilchaltun, the hybrid Chica fish, and the

phylogenetically old Pachon and Piedras cave
forms after exposure to conspecific or Ptero-
phyl/um sp. (Cichlidae) skin extract. (Fricke,
1988.) 10 15 20005
rarely observed in the hybrid Chica and the phylogenetically young Micos
fish. The fish of all these populations, however, avoid the place where
the alarm substance is introduced and the water surface, including the
space 5-6 cm just beneath (Fricke, 1988). This behavioral element of
avoidance evidently was not recognized by Schutz (1956) and Pfeiffer
(1966). Consequently, both of these investigators reported that the Pachon
and Chica cave fish have lost the fright reaction completely.
As in the epigean fish, the feeding at the surface almost stops after
the alarm substance has been introduced (Fig. 34). The fright reaction
also persists for as long as 6 days before completely vanishes. The intro-
duction of the substance near the bottom also causes a less violent re-
action.
Studies of the ontogeny of the fright reaction have shown that its
manifestation is age-dependent. In F 1 hybrids between epigean and Pa-
chon cave fish, it cannot be observed until they are 46-56 days old and
at least 22 cm long (Pfeiffer, 1966).
This behavioral difference between epigean and cave forms is ge-
netically based. All Fl hybrids show the fright reaction. In the F2 gen-
eration and the backcrosses with the cave fish, some specimens do not
react even when tested several times. Pfeiffer (1966) interprets the in-
heritance as being based on two independently segregating dominant fac-
tors. Unfortunately, this interpretation depends on the erroneous as-
sumption that the cave fish have lost the fright reaction completely.
Additional genetic analyses thus must be performed. Nevertheless, when
318 H. Wilkens
comparing the results described above with the manifestation of the other
complex features found in A. !asciatus, it may be assumed that the fright
reaction is inherited in an additive polygenic fashion.
The partial reduction of the fright reaction in the cave fish can be
explained by the different conditions found in its habitat. Epigean fish
school, and conspecifics can be alarmed visually by rapid zigzag swim-
ming. Such visual alarm signals have lost their biological significance in
darkness and are consequently reduced in cave situations (Fricke, 1988).
On the other hand, the behavioral element of avoidance may still have a
biological function as protection against cannibalism or aerial predation
by bats (Romero, 1985a). It is quite probable that in caves with many
fish, such as the Chica cave, bats (Noctilio leporinus) live at least in part
on cave fish. This could explain why the water surface in particular is
avoided by the cave individuals after the introduction of alarm substance.
However, the possibility cannot be excluded that in caves both features,
the fright reaction and the alarm substance, have no function at all and
are just rudiments.
Light Sensitivity and Phototactic Response
The light reaction of both epigean and cave forms of A. !asciatus has
been the subject of a series of studies (Breder and Gresser, 1941a,b;
Breder, 1944; Breder and Rasquin, 1947; Kuhn and Kfthling, 1954; Liiling,
1954b; Kfthling, 1965; Gertychowa, 1970; Erckens and Martin, 1982b;
Tabata, 1982; Romero, 1985b). Recently Langecker (1988) carried out a
thorough analysis, which may be summarized as follows. In the epigean
form this behavior is highly variable; eyed as well as blinded epigean
individuals cover about the entire range of possible reactions (Fig. 35).
The mean phototactic response under different light conditions never-
theless reveals a clear tendency: In blinded epigean fish an extremely
photonegative reaction is observed under high light intensities, and this
decreases significantly with declining light. In specimens with intact eyes,
the same trend can be observed (Fig. 35). Langecker (1988) states, how-
ever, that in contrast to the blinded fish, the eyed ones cannot be described
as significantly photopositive or photonegative under any light conditions.
This situation is caused by the fact that the reaction to light is greatly
influenced by visual perception. Its dominant role diminishes the expres-
sion of phototactic behavior in epigean fish. This relationship was ne-
glected in previous studies (Breder and Gresser, 1941a; Breder, 1944;
Breder and Rasquin, 1947; Romero, 1984, 1985b).
The prominent requirements of optic orientation prevent epigean
0/0
100 n-6 n=12
-r'" -r
n~8
90
QJ
80 1:
·iii
0
Cl. n=18
70 ~
.c
g n=18
r
60
n~2
~~
5"
~~
40 ~~
§ ~~
ti3
« 0
UJ
er:
.~
t;
0'
QJ
~ .
~2 ()..~
l!l
::J
u..
o 1O·
g
~ .. -
xUJ
FIG. 35. Mean and range of light re- o - - _L-
action of epigean fish under differ- ~ 0

ent light intensities: (0) with eyes; I n III
(e) without eyes. (Langecker, 1988.) 620Lux 50Lux 3Lux
specimens from entering areas without light. Comparison of the occur-

rence of eyed adult fish under different light intensities shows that twilight
is preferred to lower or higher light intensity (Fig. 36) (Langecker, 1988).
The photic behavior of the eyed epigean form is not sufficiently char-
acterized by the term "scotophilic," as used by Romero (1985b), because
it may even turn out to be "scotophobic" under low light (Fig. 35).
Phototactic behavior seems to be subject to ontogenetic change. Ju-
venile epigean fish prefer higher light intensities than do adults (Kiihling,
1961; Thines, 1960; Romero, 1985b).
Blinded epigean fish that have been pinealectomized reveal the im-
portance of the pineal organ for light perception and phototactic behavior
(Kahling, 1961; Langecker, 1988). Under intense light conditions, the ex-
tremely photonegative behavior that is characteristic of blinded individ-
uals decreases in blinded and pinealectomized ones (Fig. 37). Neverthe-
less, they remain photonegative. Besides an altered phototactic response,
pinealectomized blind fish show a strongly reduced reactivity to light; in
contrast to dark-adapted, blinded fish with an intact pineal organ, pine-
320 H. Wllkenl
60 n=18
so
40
~
t::; 30
~
Cl::
~20
g
15 10-
x
~
~ O~---ijL-~--~----~----~r------~~-~I------~~
00 Q2S 0.5 1.0 30 10.0 SOD 100.0 620.0 LUX
LIGHT INTENSrry
FIG. 36. Occurrence of adult epigean fish under different light intensities in the dark (hatched
bars) or lighted chamber (open bars) of a test aquarium. (Langecker, 1988.)
0/0
70- CII
~
.~
60
n=12
SO
i n=6 n=6
n=6
40- ~ .. n:6 ~ ~ t
~
a:
~20 :I:
g ""
.~
t::;30 ....
<t
UJ
~
D
5l'
c:
~~
t
t5 10.
xUJ
~
BUNOED EPliEAN BUNDED YEIIlANIZ PIEmAS PllCIDI
POPU.ATOI PINEAlECTO-
I'tZED
El'lGEAN
POPU.ATlOI
FIG. 37. Mean and range of light reaction in blinded and in blinded and pinealectomized epigean
fish (620 lux) and in three different cave fish (1400 lux). (Langecker, 1988.)
alectomized ones do not react instantly to sudden light exposure (Lan-

gecker, 1988).
In all cave forms studied, the capability of light perception has been
demonstrated. Inasmuch as the light-sensitive cells of the eyes, and prob-
ably also of the pineal organ, in phylogenetically old cave fish are reduced,
their light reaction, and also that of blinded and pinealectomized epigean
ones, must be attributed to extraocular and extrapineal photoreception,
as has also been reported in other fish (Steven, 1963). Studies of specimens
from the phylogenetically old Yerbaniz, Piedras, and Pachon populations
have revealed a photonegative behavior. This is less intense than that of
blinded epigean specimens, but equivalent to that of blinded and pine-
alectomized epigean fish (Fig. 37) (Langecker 1988). The equivalent in-
tensity of the light reaction in blinded and additionally pinealectomized
epigean fish on one hand and in cave fish on the other indicates that the
extraocular and extrapineallight sensitivity has not been subjected to any
obvious regression.
Romero (1985b) has characterized the light reaction of the Pachon
fish as rudimentary. This conclusion, however, relied on inappropriate
testing. Romero used epigean fish with intact eyes and pineal organ. These
cannot be compared with cave fish, because of the above-described dom-
inant role of optic-orientation and the importance of the light sensitivity
of the pineal sensory cells, which influence phototactic behavior (Lan-
gecker, 1988).
From the difference in the phototactic behavior between blinded epi-
gean specimens and blinded and pinealectomized ones (Fig. 37), it can
be concluded that the response is not fully expressed in the latter because
of the loss of the pineal organ. It can therefore not be decided whether
the light reaction in cave fish is really reduced and rudimentary or simply
not expressed due to the regression of the sensory cells of eye and pineal
organ. A regressive development might be postulated, however, inasmuch
as the cave fish are neither threatened nor able to get in contact with the
epigean realm in their natural habitat and thus the biological function of
this behavior has been lost.
CONSTRUCTIVE TRAITS
Gustatory Equipment
Morphology
The gustatory system of the different Astyanax populations is com-
prised of taste buds, which closely resemble those usually found in teleost
322 H. Wllkens
FIG. 38. Distribution of taste buds in (a) the epi-

gean and (b) the phylogenetically old Pachon cave
fish. (Schemmel, 1967.)
fishes. Histological, scanning electron microscopy, and morphological

studies revealed no differences between epigean and cave forms (Schem-
mel, 1967; Zilles et al., 1983).
The taste buds are primarily concentrated on exposed parts within
the mouth as well as outside of it on the head. They are not developed
on the body surface. According to Schemmel (1967), structures reported
as taste buds by Breder and Rasquin (1943) in this area are actually neu-
romasts.
In the epigean form, the taste buds are mostly concentrated on the
lips, whereas their number rapidly decreases in the transition zone to the
head. The density as well as the distributional area have changed in the
hypogean Pachon and Sabinos fish (Breder and Rasquin, 1943; Schemmel,
1967). The number of taste buds on the lips of the Pachon and Sabinos
fish is the same as in the epigean form. However, the distributional area
is considerably extended on the head (Fig. 38) (Schemmel, 1967).
The Pachon fish has the largest number of taste buds (Fig. 39). In
this form they are found primarily on the ventral side of the anterior part
of the head, where their number is three to four times higher than within
the mouth. Taste buds are also found on the dorsal parts of the head.
Originally, the Sabinos fish was supposed to be intermediate between
the epigean and Pachon cave fish (Schemmel, 1967). More recent studies,
however, have revealed that this is not generally true: With respect to
the distribution area of taste buds, the Sabinos fish has not diverged from
the Pachon form. However, the number of these organs is much lower
(Fig. 39) (Schemmel, 1974a). The phylogenetically young Micos fish falls
more or less between the epigean and the Sabinos form (Fig. 39) (Schem-
mel, 1974b).
Genetics
The divergence of the gustatory system in the different hypogean and

epigean forms has a genetic basis, which can be characterized as addi-
tively polygenic. However, certain exceptions have been observed. These
Evolution and Genetics of Astysnsx 'SBC/StUB 323
(ave populations
~
.:.,. 30% A, Pachon:J
':. 20 " '?)ibinos I
,1 l
rJ I '
10
" I
~-1~~~~~~~~~~~~
30
20
10
----O~~~~~~~~~_r~~~~~
~~'~~~~-~-~"~/~'r/~:'~-T~~"~~~~~~~~~
~f~--
-o,A~,., 1 3 5 7 9 12 13 15 17 19 21
i u, re
i'
23 25
i
FIG. 39. Frequency distribution of taste buds on the ventral head region in the epigean, (-) the
phylogenetically old Pachon and (- -) Sabinos cave forms, their hybrids, and the phylogenetically
young Micos fish. Ordinate, VB = square root of taste bud number. (Schemmel, 1967, 1974a,b.)
have been found especially in the backcross, in which mean, range, and
standard deviation surpass expected values (Fig. 39). This is probably due
to a threshold effect (Schemmel, 1967, 1974a).
It has been found in crosses between epigean and cave forms that
the expansion of the area occupied by taste buds proceeds with a constant
density/area correlation: The more of the lower head region that is covered
by these organs, the greater is their average density. This is also true for
the backcross to the cave forms, although these fish are subject to a
threshold effect. Both the density and area occupied of the taste buds are
probably regulated by the same polygene system. According to Schemmel
(1974a), only the Pachon fish are characterized by a different density/
area correlation. This is still an unexplained phenomenon that cannot be
observed in any of the other crosses of this fish (Fig. 39).
Both the Pachon and the Sabinos fish seem to be homozygous for
the gustatory equipment. The phylogenetically young Micos fish is het-
erozygous, as indicated by the different means of the F 1 hybrids between
it and the epigean or the phylogenetically old hypogean populations.
324 H. Wllkens
Food Localization and Feeding Behavior
Food Localization
The epigean A. fasciatus lives mainly in rivers as a facultatively
schooling species. It catches floating prey in the open water by means of
optical fixation (Parzefall, 1983a). In cave fish, schooling behavior is re-
duced and the individual fish swim continuously in the cave biotope. Their
food is located predominantly by means of chemical sense organs.
Studies in the Micos cave have revealed that the epigean specimens
are able to localize the opening of a baited trap directed into the open
water as successfully as do the true cave fish (Wilkens and Hiippop, 1986).
In contrast to this, epigean specimens have great difficulties in localizing
food on the bottom. In competition experiments, 80% of small pieces of
meat on the bottom were found by hypogean specimens of the Pachon
population, but only 20% by epigean fish (Fig. 40) (Hiippop, 1987). Fur-
thermore, the hypogean fish reacted much faster to the presence of food.
Experiments did not improve this kind of food finding in the epigean
individuals.
The superiority of the cave form in feeding in darkness does not
necessarily imply an improved ability to taste, that is, a lowering of tasting
thresholds (Hiippop, 1987). Earlier investigations were not able to dem-
onstrate this (Breder and Rasquin, 1943; Humbach, 1960). Similar im-
provement in the nasal organ can also be ruled out (Schemmel, 1967). It
may be that chemicals bring more and different information to cave fish
than to epigean ones, which find their food predominantly by optical
means and only secondarily by taste or olfaction.
In the cave fish, moving objects can also be located by means of the
lateral line system (Friihbeis, 1984). In particular, certain kinds of water
IriI Epigean fish 0 (ave fish

n=6 n=6
FIG. 40. Food-finding success of epigean and Pachon cave fish in total darkness. (HOppop. 1987.)
turbulences caused by characteristic food searching movements of the

cave fish seem to contain more information for these than for epigean
individuals. Consequently, a cave fish, when stimulated by the charac-
teristic turbulence caused by another individual, searches for food on the
bottom long before an epigean individual starts to do so (Hiippop, 1987).
Epigean specimens can locate food in total darkness, but they need much
more time as compared with cave fish.
Feeding Behavior
Cave fish have developed a specific feeding behavior (Schemmel,

1967, 1980). They search for and pick up food while swimming at a 55°
angle from the bottom. In contrast, the epigean form can only feed on
the bottom while maintaining a steep angle of about 80° (Fig. 41). It then
performs violent and excited movements that make the picking up of food
difficult. The cave fish also have the advantage of being able to very
efficiently apply the extended area on the ventral head side that contains
taste buds due to a smaller angle between body and bottom. This spe-
cialized feeding behavior is characteristic of all the phylogenetically old
cave populations that have been studied. The average angle maintained
between body and ground does not differ between populations (Schem-
mel, 1980). The phylogenetically young Micos fish, however, seems to
show some significant variability.
The behavioral difference between epigean and hypogean forms is
genetically based (Schemmel, 1967, 1980). The manner of inheritance is
additively polygenic and it shows threshold effects (Fig. 42). The number
of genetic factors responsible for the divergence is at least two to four
(below, p. 310). The genetic feeding behavior factors segregate indepen-
FIG. 41. Feeding behavior (angle of attack) in total darkness of (a)

the epigean and (b) a phylogenetically old cave form. (Schemmel,
1980.)
326 H. Wilkens
FIG. 42. The frequency distribution of the angle of

attack (= feeding behavior) in the epigean. a phy-
80 70 60 50 0 logenetically old cave form. and their hybrids.
angle of feeding attack (Schemmel. 1980.)
dently from those responsible for taste bud formation and extension
(Schemmel, 1980).
Metabolic Rates
A comparison of oxygen consumption as a measure of metabolic rate

in A. fasciatus was first performed by Schlagel and Breder (1947). Spec-
imens of the Chica population were compared with epigean fish. The
oxygen consumption rate was found to be greater, although not statisti-
cally significant, in the cave form. However, these results are not truly
representative for the cave populations of A. fasciatus, inasmuch as a
hybrid form was used. Furthermore, nonhomogeneous experimental con-

ditions due to varying group sizes may have complicated the results.
Recent studies have been performed with the epigean form and three
of its cave derivates, the Pachon, the Micos, and the Chica populations
(Hiippop, 1986a). The mean routine metabolic rate [mean postabsorptive
oxygen consumption rate over 24 h, including spontane activity (Fry,
1957)] of the epigean A. fasciatus agrees well with that of the closely
related A. eigenmanniorum from South America [0.44 mg 02g- 1 h- I
(Freyre et al. (1982)] (Table II). In comparison with the cave populations,
the mean routine oxygen consumption rate of the epigean fish was de-
termined to be significantly greater than that of only the Pachon fish (Fig.
43). No statisticaly significant difference exists between the hybrid Chica
fish and the phylogenetically young Micos fish on one hand and the epi-
gean and the phylogenetically old Pachon fish on the other. However, in
accordance with expectation, specimens from the Chic a and the Micos
populations show an intermediate position and a decreasing tendency of
the amount of metabolic rate from the epigean to the Pachon cave fish.
With regard to standard metabolic rate (lowest postabsorptive oxygen
consumption rate measurable during the experiment), the Pachon popu-
lation differs significantly from the epigean and the Micos fish (Fig. 43).
Starvation experiments revealed that the epigean and the Pachon fish
are both able to decrease their metabolism further. After 29 days it is
reduced by about 30% in both forms (Hiippop, 1986a). These experiments
also show, however, that specimens from the Pachon population lose far
less body mass than do epigeans, due to the difference in their metabolic
rates (Fig. 44). Compared to the initial body mass, the epigean fish lost
about 16% during the 29 days of starvation, while the hypogean Pachon
fish lost only 9.5%.
Hiippop (1986a) examined the relationship of body mass to oxygen
consumption by means of regression analyses. No statistically significant
differences between the regression coefficients of epi- and hypogean fish
were found, and all the data could be combined. The correlation of body
mass/metabolic rate was identical in epi- and hypogean populations at all
body sizes. Although the coefficients for standard as well as for routine
oxygen consumption were relatively high, they fit with values generally
characteristic of fish (Heusner, 1984).
However, recent, more sophisticated studies have shown that cave
fish do not have a lower metabolism (Hiippop, 1988b). It was found that
the individuals of the Pachon cave form are able to build up enormous
fat reserves. One-year-old cave fish fed ad libitum had a mean fat content
of 37% of fresh body mass compared to 9% in epigean fish under the same
conditioning. A calculation of the V0 2 (= oxygen consumption rate),
Col
N
CD
TABLE 11. Oxygen Consumption Rates of Seven Groups of Epigean and Hybrid Chica, Phylogenetically Young Micos, and
Phylogenetically Old Pachon Fish a
Teapao-1F Teapao-1S Chica-O Micos-O Pachon-O Pachon-1F Pachon-1S
Mean standard oxygen 0.314 ± 0.081 0.212 ± 0.079 0.284 ± 0.048 0.277 ± 0.063 0.215 ± 0.035 0.243 ± 0.033 0.150 ± 0.025
consumption, mg 02 g -I h - I
Range, mg 0, g-I h- I 0.230-0.481 0.150-0.361 0.231-0.349 0.200-0.444 0.152-0.260 0.202-0.284 0.118-0.185
Mean routine oxygen 0.415 ± 0.071 0.301 ± 0.084 0.356 ± 0.084 0.355 ± 0.080 0.2% ± 0.073 0.319 ± 0.038 0.222 ± 0.076
consumption, mg 02 g -1 h- I
Range, mg 0, g-I h- I 0.328-0.544 0.221-0.435 0.244-0.498 0.251-0.513 0.163-0.405 0.245-0.383 0.150-0.323
Range of wet body mass, g 4.29-9.86 3.22-6.99 3.35-6.82 3.03-11.33 3.17-9.20 4.03-6.66 3.89-8. \1
Number of each sex 2~,50 32,30 5~, 10 6~,60 5~, 30 4~, 50 32,30
Born or caught 1978 1978 1982 1982 1982 1975 1975
a Hiippop (I986a). 0, Fish from natural locality; I, first laboratory generation; F, fed; S, starved.
Evolution and Genetics of Astysnsx 'sscistus 329
035
FIG. 43. Mean and standard deviation (± 1S)

030
015
o Routine
f
Oz- consumption
-Standard D2-consufllllion
!!i
of standard and routine oxygen consumption I I I i
rates of an epigean form (Rio Teapao), the hy- fRlgean (hica Micos Pachon
brid Chica, the phylogenetically young Micos,
and the phylogenetically old Pachon cave fish.
n=7
I
n=6
,
n=12 n=17
(Hiippop, 19868.) (ave fish
based on fat-free body mass, showed no difference in the V0 2 between

cave and surface fish (Table Ill). The similarity in measurements is caused
by the fact that fat tissue has a relatively low maintenance metabolism
compared to other tissues such as muscle, brain, or intestine. Thus, cave
fish, which have a high fat content, only seem to have a low V0 2 because
their total individual oxygen consumption in mg 02/h is related to a high
body mass compared with the surface fish (Table lI).
(ave fish
y=102.33-2.23 Ix
r=0.987
.~
Epigean fish 0
y=103.06-3.34 Ix o
r=0.990
FIG. 44. Loss of body mass in epi- o~
gean (Rio Teapao) and Pachon cave
8
fish during 28 days of starvation. Each
point represents the weight of six
specimens. (Huppop, 19868.)
5 ~ 1~ 17 2; i5 i9
Days of starvation
330 H. Wllkens
TABLE Ill. Median and Range of Fat Content and Oxygen Consumption Rates
Calculated on Fat Body Mass and on Fat-Free Body Mass for Epigean and Cave Fish
of Astyanax fasciatuS'
vo, (+fat)C Vo, (-fat)C
Fat
N contentb N Standard Routine Standard Routine
Swface fish (Rio Teapao) 7 8.9 0.30 0.41 0.33 0.45

(4.3-14.3) (0.23-0.48) (0.33-0.54) (0.25-0.53) (0.36-0.60)
Cave fish (Pachon) 7 34.9 17 0.23 0.32 0.37 0.50
(31.1-48.0) (0.15-0.28) (0.16-0.31) (0.24-0.45) (0.26-0.64)
Significance of Mann- p < 0.001 p < 0.005 p < 0.002 p > 0.25 p > 0.15
Whitney U-test
a From Hiippop (l988b)

b Percent wet body mass.
c Milligrams O2 per g per h.
In summary, these experiments indicate that the Pachon cave fish

probably do not have a lowered V0 2 , such as many other cave animals
do (Hiippop, 1985), but rather exhibit an improved ability to store fat,
presumably for periods of starvation.
In F2 hybrids, it was shown that this metabolic feature could not be
correlated with eye size or the degree of melanin pigmentation (Fig. 45)
(Hiippop, 1988b).
The V0 2 or, more accurately, the fat storage ability is based on a
genetic system of its own. The genetic interpretation of the distribution
curves that appeared in the different crosses was complicated by a high,
environmentally influenced variability.
,..-024
AA~ •
A
'T
'T
~
':"
en
E
0,3
0,2
0,1
i
0
f*
•i
2
-,-a
tI ..• ·
i
3
p) 0.05
4
i
Eye size
N
~
rs=0.11
AA~
'E
[j
B
.. p)0lO
..
~q
•
'C
..-
c: t
FIG. 45. Spearman's rank-correlation test of
~ I
0,1
i
•
•
i i
eye size and degree of pigmentation shows no
correlation with rate of oxygen consumption in
F2 crosses between epigean and phylogeneti-
0 2 3 cally old Pachon cave fish (N = 49). (HOppop,
Pigmentation 1988a.)
x = 0.42 t 0.07
Epigean fish
I i .n. 1J
o ,,
n=7
i , • i , ,n IIH+ 11. ,. n. ,.
.2: , I i
~ 12
10 10
x =0.21t 0.05 x=0.29± 0.10
8
~ 8
~
6 6
F2 F2
4 n =49 n = 49
0.2 03 0.4 os 0.2 0.3 0.4 0.5 0.6

Standard Ve2 [mg' g-1·h-1 ] Routine V0 2 [mg· g-1.h- 1 ]
FIG. 46. Frequency distributions of standard and routine oxygen consumption rates in the epigean
(Rio Teapao) and phylogenetically old Pachon cave forms and their F, and F2 crosses. (HGppop,
1988a.)
Nevertheless, all typical phenomena of inheritance in polygenic sys-

tems of A Jasciatus could be observed. The F 1 and F2 generations showed
a more or less intermediate frequency distribution between the parental
surface and cave form. Most critically, the F2 generation was character-
ized by bimodality (Fig. 46) (HOppop, 1988b).
It can be concluded that this metabolic character is based on a po-
lygenic system, which, in principle at least, is additively polymeric. In
the F2 generation, a threshold effect is developed. This indicates that,
after the recombination of a minimum number of polygenes, gene ex-
pressivity abruptly increases. The number of involved polygenes cannot
be calculated because of high nongenetic variability, the lack of a normal
distribution in the F 2, and the relatively small differences between the
parental forms.
Egg Yolk Content and Early Development

Egg yolk content has been studied in the epigean fish and in five of
its cave derivatives: the phylogenetically old Pachon, Piedras, and Yer-
332 H. Wllkens
Standard length (mm)
fw ~44
f~
3.
f48
2.8
fw
2.6 110
2.4
0.20 Yolk volume (mm 3)
0.18
i"
1 1
0.16 ?w
~50
48
0.14 40
0.12
Ul0 FIG. 47. Mean and standard deviation (15) of yolkvol-
ume and body length in the epigean form (Rio Teapao)
EPIGEAN FISH
.
[..wE FISH
and several cave derivatives 24 h after spawning (n =
number of investigated larvae). (HOppop, 1985b.)
baniz forms, the phylogenetically young Micos fish, and the hybrid Chica
form (Hiippop, 1988b). It has been found that the eggs of all the cave
forms show a significantly greater amount of yolk in comparison with the
epigean ancestor (Fig. 47). Differences among the cave forms studied
cannot be verified statistically, although there is some variability. Within
EPIGEAN FISH
12 hrs
GAG se
~-f±ii9 A
24hrs
FIG. 48. Comparative early embry-
~48hrs
onic and larval development of an epi-
~) gean fish and a specimen of the albi-
notic Pachon cave form: A, anus; ABA,
KlGA
swimbladder;CAG, cemental adhesive
(f~
gland; 10, eye; EM, egg membrane; FIN,
pectoral fin; GA, gill arch; MO, mouth;
72hrs
MY, myotomes; NC, nasal pit; NCH,
FIN
notochord; 0, orbital cavity; PS, peri-
vitelline space; SC, statocyste; TB,
tailbud; YS, yolk sac. (HOppop,
e:z:t£±@ 174hrs 1988b.)
its range, the phylogenetically young Micos fish has already achieved a
stage comparable to that of the phylogenetically old Pachon cave form.
Yolk content and body length are correlated with each other at the
earliest developmental stages (Fig. 48). Cave fish embryos therefore are
longer than epigean ones from the beginning. In accordance with this,
those features whose size depends on body length are also larger. This
phenomenon is made even more apparent by the fact that the epigean
larvae grow more slowly during the first 8 days.
The rate of yolk consumption in epigean and cave larvae does not
differ. Due to the smaller amount of yolk, the epigean fish complete the
process 1 day before the cave larvae (Figs. 48 and 49).
From the beginning, eye volume is smaller in cave fish embryos. In
contrast to this, the growth rate of the eye does not diverge from that of
5.0 standard length (mm)
2.5 , i
0035 eye volume(mm 3)
0030
0020
0010
• • • • 0' J
o
0.20
0.15
0.10
0.05
FIG. 49. Comparative development of yolk con-
tent, eye volume, and body size of epigean and Pa- o
chon cave fish larvae during 4 days after spawning. 4
(HOppop, 1988b.) days
334 H. Wilkens
the epigean form until the age of 2.4 days has been reached (Fig. 49).
During the following period until the fourth day, the cave fish eyes stop
growing (Figs. 20 and 49). The described eye growth stop has no effect
on the development of other features, the body length, or on the yolk
consumption rate (Fig. 49) (Hiippop, 1988b).
Crosses between epigean and cave fish show that the increase in yolk
content has a genetic basis (Hiippop and Wilkens, 1988).
GENETICS AND THE EVOLUTION OF COMPLEX FEATURES
General Principles of Polygene Manifestation in Astyanax fasciatus
The interfertility of epi- and hypogean A. fasciatus has made possible

the genetic analysis of morphological and behavioral differences of an
order of magnitude usually developed only between different species.
Whereas in crosses between separate species, disturbances of phenotypic
expression are very often caused by genetic incompatibility, almost noth-
ing of this nature happens in crosses between epi- and hypogean Astyanax
populations.
The cave fish of A. fasciatus are characterized by both regressive
and constructive features. Thus, the genetic analysis of regressive features
and subsequently improved constructive adaptions is possible. This has
been accomplished, in fact, with analyses of the eye, melanophores, gus-
tatory system, feeding behavior, egg yolk content, and metabolism. The
following modes of inheritance have been found:
1. There are no fundamental differences in the manifestation of re-
gressive and constructive features.
2. Nearly all the complex features that have been analyzed genetically
show polygenic inheritance. The only exceptions are albinism and the
reduced melanin content of melanophores. Each of these depends on a
single recessive gene that shows independent inheritance (Sadoglu, 1955;
Sadoglu and McKee, 1969; Wilkens, 1970c).
3. Except for the above-mentioned color genes, none of the genes
responsible for any specific single structure forming part of a complex
feature has yet been analyzed. This situation arises from the functional
interdependence of the developing individual elements of complex sys-
tems. The above-mentioned polygenes thus appear to have a more quan-
titative than qualitative character. This principle even holds true if, as in
the case of the eye, there are several developmental physiological units
Evolution and Genetics of Astysnsx 'ssclstus 335
that show appreciably less close correlation among one another (see pp.
263,312).
4. Polygenes in principle are responsible for effects of approximately
equal magnitude. This can be concluded from the intermediate means and
the normal distribution of features shown in most crosses. The mode of
inheritance is additively polygenic (Rieger et al., 1968).
5. It is, however, also characteristic of the inheritance of all the com-
plex features studied that alterations in the additive polygenic inheritance
described before develop under the influence of modifying conditions.
Whenever a certain minimum number of polygenes had recombined, gene
expressivity abruptly increases. Usually this change can become manifest
only when the necessary genetic balance has been provided by at least
one set of chromosomes (haplome) of either parental form. This phenom-
enon is due to a threshold effect, and under its influence the increase in
response may be considerable: for example, eye size is enlarged by about
17% (Fig. 50).
6. In all the regressive and constructive features that have been stud-
ied, no phenotypic linkage has been found: for example, eye and melan-
ophore genes show independent inheritance. It is especially noteworthy
that even gustatory system and feeding behavior, which are functionally
correlated to some degree, are not genetically linked (Schemmel, 1967,
1980). The improved ability to store fat is also inherited independently
from eyes and from melanophores (HOppop, 1988b).
Minimum Number of Genetic Factors
The minimum number of genetic factors involved in the manifestation

of the polygenic traits has been calculated by a biometrical method pub-
lished by Lande (1981). In addition, binomial frequency calculations were
used.
Eye
In the extremely regressed Sabinos and Pachon populations, a min-

imum number of six to seven factors responsible for eye reduction has
been calculated (Table IV) (Lande, 1981). The same number has been
determined by means of the binomial calculation based on about 17,000
F2 and backcross specimens (Wilkens, 1970a).
In the variable Micos cave fish, the biometrical calculation revealed
a difference of at least four to five factors between the small-eyed Micos
336 H. Wllkens
-17%
,
r'\r-lxJckcrossing with
~ \ epigean population
8MU
2345678
me!./unit
feeding behavior
Ffgeneration
(hypogeanPachonx
epigean population)
14%
90 80 70 60 50"angle
standard ~02_
Ff generation
(hypogean Pachonx
epigean population)
FIG. 50. The threshold effect. After re-
combination of a minimum number of
polygenes that are responsible for the
formation of a specific structure, gene
expressivity changes. A structure may
be improved by a considerable amount
in one step. (Data from Huppop,
0.1 0.2 0.3 Q.4 1988a,b; Schemmel, 1980; Wilkens,
mg·g-1·h- 1 1970a,c, 1976).
and the extremely regressed Sabinos fish and at least one factor between
the small-eyed Micos and the epigean form (Table IV).
Melanophores
The minimum number of genetic factors involved in the reduction of .

melanophores has been calculated as one to two in the Sabinos and three
TABLE IV. Minimum Estimate of Genetic Factors (nE1, nE2 ) Involved in Regressive
and Constructive Traits in Astyanax fasciatus
N m S2 Genetic factors
Eye
Sabinos P, 55 2.1 0.0361 nE, = 7.47
B, 516 3.63 0.2209 S = ±0.46
F, 183 5.12 0.0576
F2 781 4.63 0.4761 nE2 = 7.24
B2 Bimodal S = ±0.42
P2 30 7.11 0.0256
Pachon P, 48 2.0 0.0324 nE, = 6.88
B, 672 3.64 0.1764 S = ±0.39
F, 177 5.17 0.09
F2 1128 4.80 0.5625 nE2 = 6.46
B2 Bimodal S = ±0.62
P2 30 7.11 0.0256
Micos (selected P, 55 2.1 0.0361 nE, = 5.28
for small eyes); B, 45 3.3 0.16 S = ± 1.33
difference with F, 54 4.05 0.09
Pachon F2 96 4.1 0.25 nE2 = 4.54
B2 105 4.2 0.1225 S = ±0.84
P2 124 4.7 0.04
Micos (selected P, 124 4.7 0.04 nE, = 1.2
for small eyes); B, 86 5.0 0.09 s = ± 0.03
difference with F, 77 5.1 0.04
epigean form F2 141 6.4 0.64 nE2 = 1.2
B2 72 6.7 0.64 s = ±0.04
Pz 30 7.11 0.0256
Melanophores
Sabinos P, 375 1.75 0.0961 nE, = 1.47
B, 610 2.72 1.1025 s = ±O.I
F, 137 5.57 0.2601
Fz 836 3.7 1.6129 nE2 = 1.4
Bz 30 5.9 0.2916 s = ±0.08
Pz 214 5.74 0.16
Pachon P, 311 0.94 0.0784 nE, = 3.35
B, 1071 2.21 0.81 s = ±0.16
F, 7 5.77 0.2916
F2 1250 3.06 0.9801 nE2 = 3.72
B2 30 5.9 0.2916 s = ±0.42
Pz 214 5.74 0.16
Feeding behavior P, 120 55.7 3.61 nE, = 2.1
B, No data s = ±0.39
F, 39 78.7 13.69
Fz 134 54.76 b
Bz Bimodal
Pz 43 81.8 12.96
( continued)
338 H. Wilkens
TABLE IV. (Continued)
N rn S2 Genetic factors
Taste buds ("ViI) PI 97 0.74 1.96 nE I = 12.27

Sabinos BI 237 4.76 2.0164 S = ±2.6
FI 306 7.76 3.0976
F2 405 9.36 5.1984 nE2 = 10.97
B2 250 13.37 6.8121 S = ±2.0
P2 95 15.1 3.24
a N, number of specimens; rn, mean; S2, variance; PI, P2 , parental forms; F I, FI cross; F 2 ,
F2 cross; B I , backcross with cave fish; B2 , backcross with epigean fish.
b The F2 is bimodal. Standard deviation is estimated as twice that of F I.
to four in the Pachon fish (Table IV). Calculation of the divergence be-
tween the Micos and the Pachon fish is disturbed by the fact that the
factors that have already mutated in the Micos form do not manifest in
the latter, but only in the crosses with cave fish that exhibit reduced
pigmentation (Fig. 26).
Taste Buds
In order to calculate the minimum number of factors responsible for

the constructive development of the taste bud system of the Sabinos
fish, the number of taste buds had to be transformed by square roots. At
least 11 or 12 factors are found to be involved in this case (Table IV).
By using the binomial frequency calculation, Schemmel (1974a)
found that at least two to three factors ought be responsible in the Sabinos
and Pachon fish.
Feeding Behavior
The results of F 1, F 2, and backcrosses suggest monogenic inheritance

and nearly complete dominance of the genes responsible for the head-
standing movement during feeding off the substratum (Fig. 42). Thus, the
position of the mean value of F 1 and the segregation ratio of 3: 1 in the
F2 and 1: 1 in the backcross to the cave fish all lead to the assumption of
a relatively simple genetic situation. However, the monogenic mode of
inheritance could not be proven for all crosses and, in fact, polygenes
turned out to be the correct genetic basis. According to Schemmel (1980),
at least three factors are involved. The genetic model developed by
Schemmel (1980) explains the distributional curves of the various pro-
Evolution and Genetics of Astyanax tasc/atus 339
genies as the result of saltatory alterations of expressivity due to threshold

effects.
The estimate of the minimum number of genetic factors obtained by
the method developed by Lande (1981) is also disturbed by the threshold
effect, which causes a bimodal distribution in the F2 generation (Fig. 42).
However, taking into account the estimate of a standard deviation 1.5-
2 times higher in the F2 than in the FJ generation, one could estimate a
minimum of two to four factors (Table IV).
Evolutionary Significance
Threshold Effects
In all the polygenic features studied in A. fasciatus, discontinuous

distributions are commonly seen. They are caused by threshold effects.
These threshold effects develop after a minimum number of polygenes
have recombined. At the threshold, a single additional polygene is able
to bring about a sharp increase in expressivity. In the case of feeding
behavior, for example, it has been shown that monogenic inheritance can
be mimicked by this effect (Schemmel, 1980). Presumably, many other
examples of monofactorial inheritance may turn out to be based on the
inheritance of one polygene that causes a threshold effect, the remaining
polygenes, which are also responsible for the manifestation ofthe feature,
not being analyzable due to too little phenotypic difference between the
parental forms (Wilkens, 1980).
An example for this is probably the sex-determinating system within
the toothcarp genus Xiphophorus (Kosswig and Oktay, 1955; Kosswig,
1964). Here the transition from a strictly polygenic (X. helleri) to a mon-
ogenic (X. maculatus) mechanism is clearly shown. The species X. cortezi
is characterized by an intermediate stage, with both modes existing side
by side (Zander, 1965, 1986). In this form one of the male-determining
polygenes triggers high expressivity and is the basis of an apparently
monogenic mechanism (Wilkens, 1980, 1985).
It is characteristic of the threshold effect in A. fasciatus that it usually
cannot be influenced by the environment. However, the melanophore
system provides an exception to this. Any increase of color cells can be
manifested only over a dark, but not a light, substratum, and it thus is
infuenced by environment. The threshold effect in this feature provides
the genetic basis for a common adaptation in fish, namely morphological
col or change.
This phenomenon is a further example for the validity of the threshold
340 H. Wilkens
effect as a general principle of genetic manifestation. In this case it is

probably not different from what has been described as a switch gene. In
Bonellia viridis (Echiurida), e.g., larvae that attach to a female proboscis
will usually become male. In contrast, those that do not come into contact
will become female. This phenotypic sex-determining system is com-
pletely comparable to the morphological color change of A. fasciatus, in
which the threshold effect can also be suppressed environmentally.
Polygenic Basis of Evolution

The estimates of the minimum number of genetic factors responsible
for the manifestation of these complex features associated with a hypo-
gean existence indicate that the latter are polygenically regulated. This
is another demonstration that evolutionary change is achieved in small
genetic steps. However, in this case especially, the amount of polygenic
expression does not exceed the range of phenotypically induced vari-
ability. Thus, it is difficult for selection to provide a consistent direction.
It is in this connection that threshold effects may play an important role.
The genetic studies on A. fasciatus have shown that certain features can
be discontinuously enlarged by a threshold effect as much as 10-20%
(Fig. 50) (Wilkens, 1985). By this means developmental noise and envi-
ronmental influences may be overridden. Directional selection forces may
then be able to operate effectively.
Developmental and Evolutionary Genetics of Complex Features

Among the polygenic features studied in A. fasciatus, the eye offers
the best opportunities for the analysis of developmental and evolutionary
genetics of complex systems. Histological studies of this organ have re-
vealed that ocular morphogenesis is regulated by developmental con-
straints that are similar to those found in other vertebrates (Coulombre,
1969; Kratochwill, 1972). There seem to be two rather independent sub-
units of differentiation within the eye. On one hand, the lens exerts a
significant inductive influence on the size of the pupillary opening and
the developmental state of the cornea. On the other hand, retinal devel-
opment is closely correlated with that of the pigmentary epithelium and
the lens muscle. In agreement with studies on the chicken eye (Cou-
lombre, 1969), retinal expansion seems to influence eyeball size in A.
fasciatus as well. Although eyeball and lens diameters show a statistically
calculated correlation, the mutual inductive influence between lens and
retina seems to be rather small. This may be concluded from the fact that
rather well-differentiated retinas may be found with tiny lens rudiments
and vice versa (Figs. 18 and 19). In some eyes the lens muscle, which,
like the retina, originates from the eye cup, is developed, while the lens
is reduced (Fig. 18b).
In the chicken, the lens influences the formation of the vitreous body.
In the A. Jasciatus eye this influence seems to be rather weak, inasmuch
as small lenses are found in combination with large vitreous bodies (Fig.
18b). It cannot be excluded that the funnel-shaped depressions that have
been described in certain retinas may be the result of inadequate pressure
of the vitreous body, which is brought about by the disharmonious in-
ductive influence of lens and retina of this organ (Fig. 18).
The nature of the polygenes in A. Jasciatus that are responsible for
the formation of complex structures such as the eye has long been a matter
of speculation. In the case of the eye, Sadoglu (1957, 1975) assumed that
specific lens, retina, eyeball, or other genes should exist. In contrast to
this concept, it has been argued that due to the intimate developmentaV
physiological interrelationships within complex structures, it would be
impossible to segregate and identify such specific genes (Pfeiffer, 1967b;
Peters and Peters, 1968; Wilkens, 1970a).
Accordingly, the existence of eyes of different sizes in various crosses
as well as in the Micos fish can be attributed to a whole series of actions
and reactions: for example, the loss of a lens gene diminishes not only
lens size, but, by induction, also the size of all other individual ocular
structures, and vice versa. As a result, the eyeball as a whole becomes
smaller. For the totality of the genes that determine eyeball size in this
manner, the term "eye gene" was coined (Wilkens, 1979a).
An interpretation of this kind, however, implies that a large number
of genes must be responsible for each specific structure. They would be
required in order to explain the continuous, gradual difference in eye size
that exists in the progeny of crosses between hypogean and epigean pop-
ulations and in the Micos fish. The relatively low number of eye genes
that has been calculated to be responsible for eye development (Table
IV) thus does not give any support to this view of the genetics of complex
structures.
The study of the eye of A. Jasciatus has shown that its development
is characterized by a high degree of self-regulatory ability. This can be
concluded from the fact that eye size and degree of differentiation have
been found to be correlated in hybrid eyes as well as in those of the cave
fish and those undergoing ontogenetic development.
In particular, it seems to be the relative size of a given eye that
determines which individual structures will develop, and vice versa. This
is clearly shown by similar tendencies of allometric correlation that are
developed in eyes whose sizes have quite different causes (Wilkens,
342 H. Wilkens
1970a). In the case of the lens in the crosses between epigean and cave
forms of A. jasciatus, for example, a positive allometric correlation de-
velops during individual ontogenetic growth (Fig. 13) as well as between
the eyes of different individuals whose eye sizes are based on a different
number of "eye genes" (Fig. 15).
Similarly, in cases of left-right asymmetry, the smaller eye shows a
degree of differentiation that is determined by its size and not by the
number of actually available eye genes that formed the larger eye.
Furthermore, it was shown by Cahn (1958) that although the earliest
eye anlage in the cave fish is reduced in size, individual structures such
as the rudimentary lens, which becomes reduced during later ontogeny,
nevertheless are developed. From this, two points can be made: first, the
degree of differentiation attained by the adult eye has to a large extent
already been determined in early embryonic stages and it is therefore very
probable that the so-called "eye genes" are regulatory genes. Second,
certain structural genes such as lens genes have not been lost by mutation.
Their manifestation is simply restricted to early ontogenic development,
and the regulatory nature of the "eye genes" once more is confirmed.
Thus, the so-called eye genes predominantly appear to be develop-
mental genes that operate on early embryonic stages. Inasmuch as there
are two developmental subunits that form the eye (see p. 263), two cate-
gories of relatively independent regulatory eye genes can be postulated.
The regressive evolution of the eye of A. jasciatus proceeds con-
vergently in separate cave populations in a species-specific pattern. It is
a process of diminution of size and it is characterized by certain allometric
correlations. These reflect inductive interactions between different tis-
sues. Thus, suggestions are supported that developmental constraints im-
pose limits on the evolution of complex features (Alberch, 1980, 1982).
That the allometric regressions may change is not contradictory for this
viewpoint. In the epigean form, for example, both lens and pupillary open-
ing, as well as size of eyeball, all show isometric correlations (Fig. 13).
In the progeny of the crosses and in Micos fish, correlation between lens
and eyeball becomes positively allometric, that between lens and pupil
negatively allometric (Figs. 14 and 15). However, this change is not caused
by the mutation of structural genes, but by the loss of regulatory eye
genes. The resulting smaller lens, for example, as a less effective inductor,
secondarily provokes different tissue interaction.
The question of why no structural gene seems to have mutated can
only partially be answered. The total loss of a lens gene would most likely
cause the total failure of eye development during early ontogeny. Ap-
propriate eye development may well be necessary, however, because the
eyeball probably plays an important role as an inductor in head formation.
The genetic analysis of regressive and constructive features in A.

fasciatus has revealed that there are no differences between them as far
as polygenic manifestation is concerned. In the same way as the reduction
of complex features is influenced by diminution in size, constructive ev-
olution is influenced by enlargement (Peters and Peters, 1968). Moreover,
in this context, limits are imposed by tissue interaction of individual struc-
tures. It seems probable that constructive traits in A. fasciatus also
evolved through the interactions of regulatory genes.
THE PHYLOGENETIC AGE OF THE HYPOGEAN POPULATIONS
The epigean A. fasciatus is of South American origin and did not

obtain access to Middle America until the Panamian-Columbian sea bar-
rier closed at or near the end of the Tertiary (Myers, 1966). Thus, the
invasion of the caves by this fish cannot have started before the beginning
of the Pleistocene (Kosswig, 1967; Mitchell et al., 1977; Wilkens, 1982;
Bussing, 1985). Finer dating has been attempted by Avise and Selander
(1972): based on allozymic variation of cave and epigean fish, they suggest
the possibility that the initial entry into caves was even more recent than
the Pleistocene.
As a result of multiple cave colonization by epigean Astyanax (Mitch-
ell et al., 1977), morphological, meristic, and genetic differences have
developed in several cave populations. As has been found in other cave
species (Wilkens, 1982, 1986), some of these features seem to reflect the
length of time since a troglobitic population evolved.
Breder and Rasquin (1947) and Sadoglu (1957) tried to correlate the
degree of eye regression and phototactic behavior with the relative time
of origin of the different cave populations in the Sierra de El Abra. They
suggested that the fishes of the Chica, Sabinos, and Pachon caves formed
a gradient of increasing regression as well as phylogenetic age. This hy-
pothesis, however, ignores the fact that the Chica fish is a hybrid (Avise
and Selander, 1972) and that the eyes of Sabinos and Pachon fishes exhibit
practically no significant differences (Wilkens, 1970b). Furthermore, it is
based on false hydrographic assumptions (M itch ell et al., 1977). Thus, it
can be regarded as obsolete.
Nevertheless, the Pachon fish seems to be the most cave-adapted
among the populations studied so far. It is, for example, characterized
by the greatest number of taste buds (Schemmel, 1974a) and the largest
amount of egg yolk (Hiippop, 1986b). It is very probable that this pop-
ulation belongs to the earliest that gained access to the cave biotope.
344 H. Wllkens
In contrast to this form, the Micos fish can be regarded as phylo-

genetically young. In the constructive features of its taste bud system and
metabolism it shows convergent, intermediate development between the
phylogenetically old Pachon cave and the epigean ancestral form. The
same holds for the regressive eye (Table V, Fig. 53). The Micos fish
undoubtedly colonzied the cave biotope much more recently.
THE SPECIATION PROCESS OF TROGLOBITIC ASTYANAX

FASCIATUS
Speciation and Hybridization
It was pointed out by Hubbs (1938) that the epigean A. fasciatus is

by no means a typical cave-fish predecessor. Neither in phenotype nor
in behavior is this large-eyed fish a typical troglophile. However, the
existence of a large number of rods in its retina indicates good optic
orientation under conditions of reduced light (Peters and Peters, 1966).
This is confirmed by its preference oflight intensities of 1 lux (Langecker,
1988), and the choice of location at which it hovers, presumably seeking
protection against aerial predation (Romero, 1984, 1985a). However, the
epigean fish is neither nocturnally active (Erckens and Martin, 1982a) nor
is it characterized by pronounced scotophilia (Langecker, 1988; Romero,
1985b).
This species has become a cave dweller due to a "fortuitous" com-
bination of certain important adaptations for cave life. Most relevant are
its chemically stimulated spawning behavior, its ability to feed in dark-
ness, and its well-developed lateral-line system for orientation. As has
been demonstrated with blinded specimens and in total darkness, neither
propagation nor orientation is hindered under these conditions (pp. 248,
251) (Schemmel, 1967; Wilkens, 1972b). Moreover, finding sufficient food
presents no problem as long as there is no competition from cave fish
already present (Hiippop, 1987). The epigean fish is able to live in darkness
without transitional stages. No "intermediate" populations (Romero,
1984) are necessary. Controversial findings showing that epigean fish can
neither reproduce nor survive in darkness due to hormonal imbalance
(Rasquin and Rosenbloom, 1954) could not be verified (Franck, 1964;
Wilkens, 1972b).
The epigean forms apparently have not actively colonized cave bio-
topes (Wilkens, 1982). Contrary to earlier deductions (Breder and Gres-
ser, 1941a,b; Breder, 1943a; Breder and Rasquin, 1947), no phototactic
preadaptation for active cave entry by epigean fish exists (p. 290) (Lan-
gecker, 1988). The majority of caves in which hypogean A.fasciatus have
been found are of the type that capture surface streams (Mitchell et al.,
1977). In these, epigean specimens get trapped and are not able to swim
back to the surface again.
This process still occurs today in the range of the hypogean popu-
lations of Astyanax. Epigean individuals get washed into the subterranean
cave system during the rainy season; in several caves, mixed populations
of epi- and hypogean fish have been reported (Wilkens and Bums, 1972;
Mitchell et al., 1977). However, in competition with the cave fish, such
surface forms are not able to start to make any cavemicolous adaptation.
Although interfertile with the cave fish, they are not even able to hybri-
dize. This situation has been observed in the case of the phylogenetically
young Micos population, which forms a mixed population with epigean
specimens (Wilkens and Bums, 1972; Wilkens and Hiippop, 1986). The
epigean fish and the relatively long-term cave individuals can be distin-
guished from each other by the size of their eyeballs, which are smaller
in the hypogean population (Fig. 51). The number of melanophores is also
significantly less in the cave form as long as the fish are kept in darkness
(Wilkens, 1976).
Calculation of condition factors has revealed that the epigean fish in
the Micos cave suffer from malnutrition (Fig. 52). This was also dem-
onstrated by comparing the phylogenetically old Pachon and Pichijumo
cave fishes with the hybrid Chica fish and with epigean specimens caught
in a surface river, the Rio Coy (Wilkens and Bums, 1972; Wilkens and
Hiippop, 1986). Malnutrition of the cave-inhabiting epigean specimens
2. Eye size(mm)
"",0
2.5 n=52 0 0
o ax: o
000
2.0 o~:~B
o 0°£ 0 0
~ 0
1.5
• •• n=38
to ..-.... :
. .......
....:.... ••
FIG. 51. Eye size of (0) epigean and (e) hypogean in- 0.5 2'.4' '3'0' , , '4.0' , , '~O' 5'4
dividuals in the Micos cave. (Wilkens and HOppop. 1986.) Body length (cm)
346 H. Wilkens
24
2.2
FIG. 52. Relation between condition factor and

body length in epigean specimens from the Micos
cave (Me) and the Rio Coy (RC) and in cave fish
from the Micos cave (Mh), the Pachon (Pa) and
Pichijumo (Pi) caves, and the Chica cave (Ch).
(Wilkens and Huppop, 1986.)
develops in competition with the true cave fish and undoubtedly is mainly
the result of their relatively poor food-finding ability (p. 296) (Hiippop,
1987) and limited ability to store fat. These undernourished individuals
do not produce eggs and sperm and thus cannot reproduce.
Strong support for the above interpretation is provided by the Chica
population. A large bat roost in this cave provides a rich food supply,
thus compensating for any deficiencies of the epigean form in the dark
(Mitchell et al., 1977; Parzefall, 1983a). Hybridization has therefore oc-
curred in La Cueva Chica (Avise and Selander, 1972; Wilkens, 1972a;
Romero, 1983). This agrees with laboratory experiments, which show that
only a rich food supply can guarantee adequate nutrition of epigean fish
in competition with the cave form (Hiippop, 1987).
Another feature that makes the establishment of hybrid populations
difficult is the increased yolk content of the eggs of cave fish (p. 303)
(Hiippop, 1986b). The mode of inheritance ofthis feature is critical in this
case. That the egg yolk content of the Fl generation is intermediate (Hiip-
pop and Wilkens, 1988) dictates that even in the unlikely instance that an
epigean individual should succeed in participating in a spawning, any
larvae produced by F 1 hybrids would be overcome in competition with
the significantly larger, homozygous larval cave fish.
It is a notable feature of the Micos fish that they are already repro-
ductively isolated from their epigean neighbors even though they occupy
only an intermediate stage with respect to the constructive adaptations
developed in phylogenetically old cave forms. It is very probable that the
beginning of cavernicolous evolution of every Astyanax cave population
was characterized by the fact that, as in the Micos cave, epigean speci-
mens were washed into the cave biotope each rainy season and there was
no separation even for a short span of time. The speciation process had
to begin while in contact with the epigean ancestral gene pool. It was
probably based on a genetic polymorphism of advantageous features-

such as lowered metabolic rate, change of feeding behavior, or increase
of egg-yolk content-that already existed at low frequencies in the epi-
gean population. According to the genetic studies with A. fasciatus, this
polymorphism could have been increased by genetic threshold effects,
which have proven to be responsible for discontinuously increasing the
manifestation of all features of this species that have been genetically
analyzed.
In contrast to the normal epigean genotypes, carriers of such genes
would have survived in the cave biotope, and thus a rapid accumulation
of these specific genes would have occurred, especially since the popu-
lation density of such young cave forms would have been very low. A
new gene pool would have been founded and further postadaptive traits
now could have been acquired.
Although the connection between the epigean and the hypogean parts
of the given Astyanax fasciatus gene pool was operating only one direc-
tion-from the surface into the cave-the above-described speciation
process has been characterized as sympatric by Wilkens and Hiippop
(1986).
Allozyme Variation
The speciation process also has been analyzed by studying the al-
lozyme variation of several epigean populations, the hypogean popula-
tions that inhabit the Pacnon, Sabinos, and Chic a caves (A vi se and Se-
lander, 1972), and the Micos population (Peters et al., 1975). An analysis
of 17 loci has shown that the epigean populations are among the more
polymorphic vertebrates, with individuals having, on the average, 11.2%
of their loci in the heterozygous state.
In the phylogenetically old cave populations, variability is absent in
the Pachon fish and severely limited in the Sabinos ones. In the hybrid
Chica population variability is less than the average of the epigean pop-
ulations. The phylogenetically young Micos popUlation is monomorphic
for 11 of the 14 loci studied. At three of the 17 loci, the Pachon and the
Sabinos populations are monomorphic or nearly so for different alleles.
At a fourth locus, both are monomorphic for an allele not detected in
epigean populations.
The lower amount of polymorphic loci and the lower mean number
of alleles per locus in the Micos population as well as the almost or com-
plete monomorphic state of the Sabinos and the Pachon populations are
almost exclusively due to the loss of alleles that are rare in the epigean
348 H. Wllkenl
ancestral population. According to Avise and Selander (1972), this is pri-

marily caused by stochastic processes. The nature of these stochastic
processes is uncertain. According to Avise and Selander (1972), small
founding population sizes and actually effective populations of not more
than 50 specimens play an important role. However, the relatively high
allozymic variation of the phylogenetically young Micos in comparison
to the phylogenetically old cave populations indicates that the degree of
variability is correlated with the time of troglobitic evolution. As has been
pointed out previously, it can be assumed that the initial phase of trog-
lobitic evolution in A. fasciatus was a long-lasting process. During this
time there was a continuous influx of epigean fish into the caverniculous
habitats. It is probably erroneous to assume that the founding populations
of the caves inhabited by A. fasciatus were small. It is more likely that
multiple colonization has occurred (Mitchell et al., 1977).
The low level of allozymic variation in the phylogenetically old cave
populations must have developed during troglobitic evolution. It is there-
fore more probable to think of small population sizes and phenomena
such as bottlenecking as being responsible for this situation (Avise and
Selander, 1972). It should be pointed out, however, that at least population
size has been found to be much larger than might have been guessed.
Mitchell et al. (1977) obtained an estimated population size of 8500 fishes
in the Pachon cave in a two-census, mark-recapture experiment. Fur-
thermore, nothing is known about the occurrence of any periodic reduc-
tions in population size (bottlenecking) in caves populated by A. fasciatus.
However, in spite of all these data, it is probable that stochastic
processes played a primary role in the reduction of allozymic variation
in the cave populations. More detailed field studies should help to solve
this problem.
THE NEUTRAL MUTATION THEORY AND ITS GENERAL

IMPORTANCE IN EVOLUTION
General Principles of the Neutral Mutation Theory
Nonselection
It is a common evolutionary phenomenon that features that have lost
their biological function become reduced. If evolution proceeds long
enough, the features may be lost entirely, but during intermediate stages
the process is often associated with vestigial characters.
Evolution and Genetics of Astyanax fasc/atus 349
The causes and the nature of regressive evolution have long been
and still are a subject of controversy. In all, more than 14 Lamarckian,
orthogenetic, and Darwinian theories have been proposed by various au-
thors in an effort to understand this process (Eigenmann, 1909; Barr, 1968;
Kane and Richardson, 1985; Christiansen, 1985).
In accord with the central role that selection plays in the synthetic
theory of evolution, there has been a recent bias toward explanations
based on natural selection (Culver, 1982), especially those involving se-
lection for increased metabolic economy (Poulson, 1964; Mitchell, 1969;
Sket, 1985) or the indirect effects of pleiotropy (Barr, 1968).
Since Darwin, however, efforts have frequently been made to explain
regressive evolution by theories invoking neutral mutation or the accu-
mulation of random mutations (Darwin, 1859; Weismann, 1889; Hubbs,
1938; Kosswig, 1934, 1948, 1960b, 1963; Brace, 1963; Peters and Peters,
1968, 1973; Wilkens, 1971, 1987). Critical reviews of this subject have
been presented recently by Culver (1982) and Poulson (1985).
The central point of the latter theory is that with the loss of biological
function, stabilizing selection, which normally keeps a feature such as
the eye in its appropriate, adapted form by eliminating deleterious mu-
tants, no longer acts: "Organs, originally formed by the aid of natural
selection, when rendered useless, may well be variable, for their variation
can no longer be checked by natural selection" (Darwin, 1899, p. 376).
Now, all deleterious mutations that affect a given feature may be retained
and therefore can accumulate.
Aside from futile semantic disputes (Banister, 1984; Romero, 1985c),
much confusion has arisen in response to the question of whether this
theory can explain all regressive evolutionary processes. It must be
stressed, therefore, that a loss of function does not necessarily imply the
loss of selection. In the cave-inhabiting, live-bearing toothcarp Poecilia
mexicana, for example, aggressive behavior has been reduced by direc-
tional selection, because it disturbs mating in the dark (Parzefall, 1974,
1985). Furthermore, in most troglobites metabolism is lowered-and thus
in a certain sense reduced-by a directional selective force, namely en-
ergy economy in an impoverished environment (Hiippop, 1986a).
Variability
Without selection, the occurrence of individual deleterious mutations

affecting regressive features will lead to genotypic and phenotypic vari-
ability. This is characteristic of regressive evolution. In amblyopsid fish,
variability for constructive traits is less than for regressive ones (Poulson,
350 H. Wilkens
TABLE V. Coefficient of Variation of Regressive (Eye, Pigment) and Constructive

(Taste Buds, Egg Yolk content, Angle of Feeding Attack) Traits in Epi- and
Hypogean A. fasciatus with F2 Crosses for Comparison a
Coefficient of variation (V = (s/m)100)
Population Taste Angle of

or crossing Eye Pigment buds Yolk erection
Epigean 2.25 7.0 No data 7.9 4.4

Pachon 9.0 29.8 11.3 7.1 } 3.4
Sabinos 9.05 17.7 12.0 No data
Micos 12.0 7.9 26.0 8.0 No data
F 2Pa x Ep 15.6 32.4 29.0 11.4 } 11.4
F 2Sa x Ep 14.9 34.0 24.0 No data
a Calculated from data of Hiippop (1988b), Schemmel (1974a,b, 1980), and Wilkens (1970a,
b).
1985). The same can be demonstrated in epigean and cave populations of

A. fasciatus.
Variability of regressive features is most evident during transitional
stages, as shown by the variable eye size of the phylogenetically young
Micos fish. Variability will decrease toward the end of the regressive
evolutionary process, as the deleterious genes slowly become homozy-
gous. This is demonstrated by the eyes of the phylogenetically old cave
fish. Phenotypic variation may also be low at the beginning of the re-
gressive process, because genetic epistasis prevents the expression of
deleterious genes. This has been found to be characteristic of the melan-
ophore system in the phylogenetically young Micos fish (Table V).
Variability requires careful investigation, however. On one hand, de-
leterious genes may not be manifest because of epistasis; on the other,
constructive traits also can show variability. As has been elucidated in
this study, there is no difference in the manifestation of regressive and
constructive polygenic systems, at least in A.fasciatus. Thus, variability
of constructive traits necessarily has to be expected during transitional
evolutionary stages, especially due to threshold effects. A possible ex-
ample is given by the taste buds of the phylogenetically young Micos fish:
The variability in the number of these organs is greater than that in the
phylogenetically old cave forms and is equal to that in the F2 progeny
(Table V).
A critical difference between the above-described variability of re-
gressive and constructive features is probably that the variability of con-
structive traits only exists for a relatively short time span. The influence
Evolution and Genetics of Aslyanax fasclatus 351
Epigeon fish Pachon CCM! fish

0"10 100%
FIG. 53. Means of developmental stage
of two regressive and three constructive
traits in the phylogenetically young Micos
cave fish (arrow) in comparison with the
epigean ancestor and the phylogeneti-
cally old Pachon cave fish.
of directional selection is responsible for this. Its intensity determines

how quickly this variable stage is passed. In the phylogenetically young
Micos fish, the number of taste buds is approximately intermediate be-
tween the epigean and the phylogenetically old cave populations. In con-
trast, the egg yolk content is nearly as great as of that of the phyloge-
netically old cave fish. The variability of this feature is the same in epigean
and hypogean forms, including the Micos fish. From this it can be con-
cluded that directional selection acts much more strongly in the case of
egg yolk than in the case of taste buds (Fig. 53, Table V).
Mutation Rate As a Driving Force
According to the neutral mutation theory, the driving force of re-

gressive evolution is mutation pressure (Kosswig, 1934). The cause of
this is that the probability of deleterious mutations affecting any feature
is quite generally higher than that of those improving it. Actually, at the
beginning of a regressive process even the rarely occurring constructive
mutations may be deleterious, because they alter a structure that had been
kept in its appropriate, adapted form by stabilizing selection.
Human hemophilia provides a good example. In this disease selection
eliminates the mutants immediately, because in the absence of any med-
ical intervention they become lethal. To account for the constant fre-
quency of hemophilia observed in human populations (Haldane, 1935),
new negative mutations are necessary. Actually, one-third of all he mo-
philia fatalities develop in families that never before exhibited the disease.
These casualties therefore must be the result of new mutations.
The question has often been raised of whether the available time span
is long enough to allow for the process of reduction without any selection
(BaIT, 1968; Avise and Selander, 1972; Culver, 1982). In this connection,
one point should be remembered, namely that the random accumulation
of loss mutations is more likely the greater, the number of genes involved
in the development of a structure (Poulson, 1985). In the phylogenetically
young Micos fish, for example, a considerable degree of eye reduction
352 H. Wllkens
has been achieved, while the melanophore system is phenotypically still

unaffected (Fig. 53) (Wilkens, 1976).
Furthermore, all the statistical calculations that have been performed
have been based on the assumption of genetic factors characterized by
equal phenotypic expression (Culver, 1982). However, the studies of po-
lygenic systems, at least in A. fasciatus, have revealed that threshold
effects play an important role. Little or no attention has been paid to the
fact that due to the loss of a single polygene, a structure may be reduced
by >20% in one step.
Nature of Regressive Mutations

The molecular nature of the negative mutations responsible for struc-
tural reduction has yet to be analyzed. Studies of the molecular genetics
of other features, however, have provided important insights. Studies in
the bleeding disorder hemophilia A show it to be caused by defects in
factor VIII, a vital component in the process of blood coagulation. Git-
schier et al. (1985) found that this disease can be caused, independently,
by seven different mutations: four point mutations and three different
partial deletions in the factor VIII gene. The clinically identical Tay-
Sachs-disease of two different human populations also has turned out to
be caused by different mutations (Myerowitz and Hogikyan, 1986).
Recent Criticisms of the Neutral Mutation Theory
Energy Economy
Energy economy is the most commonly invoked selective force in

the regressive evolution of cave animals. Since many cave organisms are
strongly food-limited, a mutation that reduces useless structures such as
an eye should have a selective advantage, provided it saves energy (Cul-
ver, 1982). This hypothesis was proposed again by Mitchell (1969) for
rhadinid cave beetles and by Sket (1985) for eye reduction inA.fasciatus.
Poulson (1985) was the first to calculate savings or extra costs in-
volved in a troglobitic transition on different time scales. His findings do
not clearly support the plausibility of the selective reduction of useless
traits on the basis of energy economy, nor do they falsify it. According
to his studies, selection against useless features would have to be expected
only during or just after embryonic development, when the energy savings
are relatively the greatest (Poulson, 1985).
The Astyanax cave populations are subjected to at least temporary
shortness in food supply (Mitchell et al., 1977; Wilkens and Hiippop,

1986). To help overcome this, the cave fish have not yet been able to
lower their metabolic rate, but they have improved their ability to store
fat (Hiippop, 1988b). In addition, the yolk content of their eggs has in-
creased (Hiippop, 1986b). Genetic analysis has revealed that the genic
systems that regulate fat storage and those responsible for eye reduction
are inherited separately (p. 302) (Hiippop, 1988b). This shows that energy
economy as an indirect pleiotropic effect of eye reduction, an idea pro-
moted by BaIT (1968), has no validity, at least for A. fasciatus.
This situation also holds for larval development. Epigean and hy-
pogean larvae show no difference in growth rate and the amount of yolk
consumption (Fig. 49) (Hiippop, 1986b, 1988b). Thus, the reduced growth
rate of the eye in cave-fish larvae appears to be unimportant for energy
economy. The increase in amount of egg yolk appears to be much more
relevant; it is subjected to a strong directional selection force. The egg
of the phylogenetically young Micos cave fish, for example, possesses
almost as much yolk as does that of the phylogenetically old Pachon cave
fish. In contrast to eye size, which is highly variable due to the lack of
selection, the variability of egg yolk content in the Micos fish is as low
as that in the epigean and the other cave forms (Figs. 47 and 53; Table
V) (Hiippop 1988b).
Further support for the neutral mutation hypothesis is provided by
ethological features such as aggressive and schooling behavior. Both are
optically triggered and thus are not manifested in the epigean A. fasciatus
when kept under cave conditions. Although there was never any possi-
bility for directional selection based on energy economy reducing these
nonmanifested features, they exhibit genetically based reduction (Bur-
chards et al., 1985; Parzefall, 1985). The same is true for the circadian
clock (Erckens and Martin, 1982b; Lamprecht and Weber, 1985) and pos-
sibly even the phototactic behavior (Langecker, 1988), both of which are
reduced in the cave populations of A. fasciatus.
Neoteny
Banister (1984) recently suggested that the characters of cave animals
may be the result of neoteny. This assumption is exclusively based on
studies of the external eye morphology of the cave fish Garra barreimiae
(Cyprinidae) from Oman. According to this, "the eyes in the adult are
identical to those in the juvenile; they have not degenerated, they just
have not developed further." Unfortunately, this observation is not sub-
stantiated by any histological data. As in all the cave fish that have been
studied, histology most likely would have revealed that eye development
354 H. Wilkens
is not, as Banister claims, "truncated in relation to gonadal develop-

ment." In the cave forms of A. fasciatus, the earliest anlage is already
smaller and histologically different from the adult eye; this is caused by
a process of ontogenetic regression (p. 266). As further examples sup-
porting this hypothesis, Banister (1984) calls attention to neotenous cave
amphibians. However, the persistence of their external gills simply may
be a prerequisite oflung breathers for living in an exclusively aquatic cave
biotope; neoteny in cavernicolous salamanders may be some kind of in-
evitable by-product, not a general explanation for regressive evolutionary
processes.
Wilkens (1973, 1986) and Ercolini et al. (1982) tried to correlate the
degree of eye reduction with the time of cavernicolous evolution of cave
fish and crustaceans in Yucatan and Somalia, respectively. This is broadly
questioned by Banister (1984) with the simple argument of "spurious re-
lationships" of some of the fish species that were studied. In the case of
the Mexican synbranchid eels Ophisternon infernale and O. enigmaticum
as used by Parzefall and Wilkens (1972) and Wilkens (1973), this is er-
roneously imputed by Banister (1984) on the basis of a taxonomic revision
of this family by Rosen and Greenwood (1976) (Wilkens, 1982). Moreover,
there is no necessity for any close phylogenetic relationships among the
species studied for this purpose. Studies of the cave faunas of western
Atlantic islands and of the Gulf Coast region (Wilkens, 1986) have shown
this to be the case.
General Evolutionary Significance of Regressive Evolutionary

Principles
Regressive Evolution and the Origin of Species Flocks
The genetic study of phenotypic evolution, as initiated by Kosswig

(1934, 1960a, 1963), made a special point of refuting the overemphasis on
selection. Selection has less a constructive role in evolution than one of
eliminating those mutations that are nonadaptive (stabilizing selection) or
one of simply imparting a certain adaptational direction (directional se-
lection). The basic principles of regressive evolution, as interpreted by
the neutral mutation theory, have general validity for constructive evo-
lution, too. The lack or the lessening of the influence of stabilizing se-
lection may occur not only in biologically functionless features of cave
animals, but also can be observed in other animals undergoing transitional
evolutionary phases (Wilkens et al., 1979; Wilkens, 1985).
Examples of this are provided by the species flocks in geologically
Evolution and Genetics of Astyanax 'asc;atus 355
Mg
100 •
20 40 60 60
Equivalent-%Mg++
.0. Sea
o Lake Geneva
o Cuatro Cienagas
• Lake Victoria
60 ~ • lake Tanganyka
\... + Lake Malawi
60 ~ • Laguna Chichancanab
'0
40~.
FIG. 54. Ionic composition of lakes

100
with fish species flocks in comparison ~~~~~-+--~~~o
with the sea and a normal freshwater 20 40 60 60 100
lake (Lake Geneva). Equivalent-%S0 4
young lakes or on islands. These biota are characterized by depauperate

faunas, which are the result of a geographically isolated position or, in
the case of lakes, by an unusual ionic composition of the water (Ladiges,
1968; Wilkens, 1985). Lakes such as the East African rift lakes, the Laguna
de Chichancanab (Yucatan), or the Cuatro Cienages (Chihuahua), which
are characterized by a high degree of intralacustrine fish speciation, de-
viate from the norm in chemical freshwater composition (Fig. 54). Here,
invading species of fish can find various niches not claimed by other fish.
Thus, stabilizing selection for the relevant adaptations of the specific
niche that the invading species once had acquired is lost or at least lowered
to some extent. By this means, equilibria become punctuated. Variability
arises and may persist because there are no competing species in the
niches occupied by the developing species flock (Fig. 55).
According to Greenwood (1984), variability in body shape in such
flocks arises due to genetically based heterochronic changes in devel-
opment and slight alterations of growth rates of single structures in com-
plex organs. In the haplochromine cichlid species flock of Lake Victoria,
the relative sizes and shapes of many of these fish are functions of simple
allometric relationships with one another, and very often the differences
between species are extrapolations of those found within species (Green-
356 H. Wllkens
Ancestral form
•
FIG. 55. Part of the species flock of Lake Lanao (Philippines). The ancestral form is Puntius
binotatus (Cyprinidae). (Wahl, 1976.)
wood, 1984). In Astyanax, for example, the allometric size correlation

between lens and eyeball changes from isometric in the epigean form to
positively allometric in Ft hybrids due to a mere quantitative reduction
of the number of eye genes (Fig. 19).
Such variability is "permitted," of course, only as long as and to the
extent that the viability of the individual is not affected. Observations on
the above-mentioned cichlid species flock show that, in general, body
form develops little diversity. As empty trophic niches became available,
however, developing cranial and dental differences could persist and these
form the main features of the speciation shown within the flock. It should
be noted, however, that when the skull forms of different species are
drawn to the same scale, they reveal a basic similarity (Greenwood, 1984;
Strauss, 1984).
An essential difference between the colonization of niche-rich lakes
or islands on one hand and the cave biotope on the other is that the latter
offers only one vacant niche, one that in some ways closely resembles
that of the epigean ancestor. Variability is "permitted" to only those few
features that have become functionless. On the contrary, strong direc-
tional selection pressure exists on the features that are constructively
improved, because they are most important for survival under the extreme
environmental conditions characteristic of the cave biotope.
Species flocks only reflect principles and evolutionary processes in

high concentration that in usual evolutionary change are not that con-
spicuous. Nonetheless, occasionally the successive appearance of static
phases followed by rather sudden changes has been observed in the pa-
leontological record and this has brought about speculation questioning
the general validity of Darwinism (Schindewolf, 1936; Gould and Eld-
redge, 1977). These, however, are not necessary to explain evolutionary
progress. In phenotypic evolution, stabilizing selection seems to play a
dominant role and thus provides the impression of stasis. The colonization
of a new environment in which there are empty niches, however, causes
a reduction in the influence of stabilizing selection on some features, and
the phenomenon of punctuated equilibria appears (Wilkens, 1987). Spe-
cies flocks represent such a stage.
Increasing variability in species flocks provides the material for di-
rectional selection, which then brings about the radiation of such species
into free niches. Under the influence of intra- and interspecific compe-
tition, variation will once again be reduced. Genetic threshold effects, as
previously pointed oltt, may play an important role in accelerating this
process. Finally, a new static equilibrium is attained. Besides the spec-
trum of available free niches, the number of developing species is deter-
mined by ethological characteristics of the ancestral species. In cichlids,
homogamy is probably responsible for the overwhelming number of dif-
ferent forms (Kosswig, 1963; Dominey, 1984).
"The Neutral Mutation Theory" and "The Neutral Theory of

Molecular Evolution"
During recent years the neutral mutation theory as advanced in this

chapter has received support from the neutral mutation theory of molec-
ular evolution promoted by Kimura (1987):
1. Because of the variability generated by partial or total loss of
stabilizing selection, great evolutionary importance is attributed to this
event. The molecular as well as the phenotypic "junk" that result from
the regressive mutations-no longer subjected to selection as long as they
do not negatively influence viability and are not eliminated-provides the
raw material for further evolution.
2. Such mutations therefore can be characterized as neutral. When
a feature such as the eye in cave animals has totally lost its biological
function, the responsible, so-called eye genes are subjected to mutational
processes similar to the pseudogenes described by Proudfoot (1980) and
Kimura (1987). The difference is that the loss of their function is not
358 H. Wllkens
caused by a gene duplication, but by the loss of the function of the struc-
ture for which they are responsible.
3. According to Kimura (1987), random drift and fixation of neutral
mutations play an important role in molecular but not in phenotypic ev-
olution. The influence of such processes in phenotypic evolution indeed
seems to be rather restricted. As has been shown, developmental con-
straints also limit the direction and the number of theoretically possible
evolutionary pathways (Kosswig, 1948; Alberch, 1980, 1982). With re-
spect to specialized pharyngeal characters, the molariform morph of Cich-
lasoma minckleyi of the Cuatro Cienagas (Chihuahua), for example, re-
sembles the mollusc-crushing cichlids of East Mrica more closely than
its conspecific papilliform morph living in the same biotope (Liem and
Kaufman, 1984). In separately evolving Astyanax cave fish, the eye ru-
diments do not show phenotypic divergences, although their genetic basis
is different to some extent.
SUMMARY
The epigean characin Astyanax fasciatus (Pisces) has developed a

series of cave-inhabiting populations within a geographically restricted
part of Mexico. The troglobitic forms have convergently evolved differ-
ences in at least four constructive traits (gustatory system, feeding be-
havior, metabolic rate, and egg yolk content) and in at least nine regressive
traits (eye, pineal organ, color pattern, scales, aggressive and schooling
behavior, circadian activity control, fright reaction, and phototactic re-
sponse). The interfertility of epigean and cave fish has provided the unique
opportunity of analyzing these traits genetically.
No differences in the manner of manifestation have been found be-
tween regressive and constructive complex features. Both are based on
polygenic systems. In the eye, for example, at least six to seven genetic
factors have been calculated to be responsible for eye reduction. Each
polygene appears to show the same amount of expressivity. However,
after a minimum number of polygenes have recombined, gene manifes-
tation discontinuously increases. This threshold effect is attributed evo-
lutionary significance.
A study of the process of eye reduction in ontogeny and phylogeny
has revealed processes of general validity in the constructive as well as
in the regressive evolution of complex features. The eye is reduced in a
species-specific pattern, which 'is based on tissue interaction and imposes
limits on the paths of structural reduction. The degree of differentiation
Evolution and Genetics 01 Astyanax fasclatus 359
is correlated with the relative eye size. In the Astyanax cave fish the
regression of the eye and the adaptive development of constructive traits
seem to be exclusively based on the alteration of regulatory gene action.
Further arguments for the reduction of biologically functionless fea-
tures by mutation pressure ("neutral mutation theory") are presented.
The role of energy economy as a driving selection force is furthermore
questioned: e.g., several behavioral features get reduced in A. fasciatus
even though they are not manifested in darkness and thus do not cost
energy.
The general significance of the neutral mutation theory in construc-
tive evolution is also pointed out in the phenomenon of species flocks of
fishes.
ACKNOWLEDGMENTS
This review was made possible by the help of several colleagues. I

especially thank Dave C. Culver (American University, Washington,
D.C.), for critically reading the first version of the manuscript, Kathrin
Huppop (Zoologisches Institut und Zoologisches Museum Hamburg) for
many critical discussions and helpful suggestions, Monika Hanel (Zool-
ogisches Institut und Zoologisches Museum Hamburg) for making all the
drawings, and Renate Dohse (Zoologisches Institut und Zoologisches Mu-
seum Hamburg) for giving steady assistance. Wilhelmine Erckens and
Friedrich Weber (Zoologisches Institut, Universitat Munster, Federal Re-
public of Germany) helped me understand circadian clock problems. I
thank Dr. J. W. Atz and Dr. M. K. Hecht for many editorial comments.
My scientific work on Astyanax fasciatus has been sponsored in the
laboratory and in the field studies by the Deutsche Forschungsgemein-
schaft (Wi 30211-7).
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Evolution and genetics of epigean and cave Astyanax fasciatus (Characidae,

Chapter · January 1988

Book: Evolution in the Dark View project

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Evolution and Genetics of

Notable progress in the biological sciences is occasionally made pos-

HORST WILKENS • Zoological Institute and Zoological Museum of the University of

As is all too often the case, information obtained in the laboratory

TAXONOMY AND DISTRIBUTION OF EPI- AND HYPOGEAN

The Epigean Fish ..

The epigean fish (Fig. 1) is widely distributed in Mexican waters. Its

The Hypogean Fish

.blind fish cave

hybrid form between the epigean A. fasciatus and a phylogenetically old

Lateral Line System

In the epigean A. fasciatus, free neuromasts are distributed over

FIG. 3. Distribution and density of neu-

FIG. 4. Head canal systems of

Studies of the auditory capacity in specimens of the epigean and the

The reproductive behavior of the hypogean populations has been

about to spawn are characterized by occupation of a relatively small area

The epigean A. fasciatus is characterized by a typical teleostean eye.

FIG. 6. Cross section of the eye of the epigean

PhylogeneticallyOldPopulations .Theeye rudiment of adult specimens

FIG. 9. Dorsal view of tectum opticum in epigean and Pa- MS

Correlated with eye reduction is a diminished size of the tectum op-

Eye Size in Crosses between Epigean and Hypogean Populations

Epigean x Phylogenetically Old Ca ve ,Forms . Comparative studies of

AMean of all breedings

Smoll- ey'ed Micas

FIG. 12. Frequency distribution of

Correlation of Individual Structures of the Eye

Growth of Eyeball, Lens, Pupillary Opening. The growth correlation

FIG. 13. Growth correlation be-

Interindividual Size Correlation of the Eye. In addition to the growth

negatively allometric increase with respect to size of pupillary opening

rt> Pupillary opening

y=O 23+x 1.16

FIG. 16. Correlation between pup-

.... ======= 1 F1(RIVER X CAVE)

\V· / \",'" . . ."'--.1

FIG. 18. Due to a disharmonious interac-

mentum annulare increases in relative size, while the lens becomes

FIG. 19. In small eyes a lens muscle develops, although the

intact retina and flattened in the dedifferentiated portion (Figs. 18 and

Ontogenetic Development of the Eye

The growth of the eye of the epigean fish is characterized by a more

FIG. 21. (Continued)

Ultrastructural studies of the hybrid Chic a cave fish retina reveal

FIG. 21. (Continued)

It is characteristic of the history of the individual structures that

The Influence of Light and Darkness on the Development of the Eye

FIG. 21. (Continued)

layers light decreasel'!,)

however (Wilkens, 1970b). The high degree of variability will probably

Due to developmental constraints operating within the eye of A. fas-

o ""~ ~ ~¥8e 0 0 " Sabinos

The pineal organ of A. fasciatus has been studied by several authors

morphological and ultrastructural differences in comparison with other

The light body color of the cavernicolous A. fasciatus forms has

The partial reduction of melanin content, as well as albinism, are

Piedras population also seems to rely on a single recessive gene, which