Download Hormonal Mechanisms as Potential Constraints on Evolution

AMER. ZOOL., 37:482-490 (1997)
Hormonal Mechanisms as Potential Constraints on
Evolution: Examples from the Anura1
TYRONE B. HAYES
2
Laboratory for Integrative Studies in Amphibian Biology Group in Endocrinology,
Museum of Vertebrate Zoology and Department of Integrative Biology,
University of California Berkeley, California 94720-3140
SYNOPSIS. Developmental constraints are limitations on phenotypic variability resulting from developmental mechanisms that produce biases in phenotypic variants
and hence evolution. These constraints ultimately limit the available phenotypes
on which selection can act. Because hormones play important roles in many developmental processes, there is a great potential for hormonal mechanisms to produce (or act as) developmental constraints. In the current study, I present two
examples to show how hormones may produce developmental constraints on evolution in the Anura. One example (a universal constraint in the Anura), examines
evidence that thyroid hormones are required for sex differentiation and reproduction in frogs. The thyroid hormone requirement for these processes may prevent
the evolution of neoteny in anurans. The second example (a local constraint) examines the mechanisms underlying sexual dichromatism in the genus Hyperolius
(Hyperoliidae) and shows how the evolution of sexual dichromatism is limited by
the hormonal mechanisms regulating pigmentation.
amples of developmental constraints in the
Anura, both of which involve hormonal
mechanisms regulating development. The
"Evolutionary constraints are restrictions first example is presented as a universal
or limitations on the course or outcome of constraint, in that it may affect all anurans
evolution" (Arnold, 1992, p. S85). These and prevent a developmental pattern (neoconstraints can be manifested in a variety teny) from emerging in anurans. The secof ways including genetic, selective, phys- ond example is a local constraint, regulating
ical, and developmental. Developmental sexual dimorphism in the hyperoliid genus
constraints are evolutionary constraints pro- Hyperolius. The arguements presented here
duced by the "structure, character, compo- are based on data from several recent and
sition, or dynamics of the developmental ongoing research projects in my laboratory
system" (Maynard Smith et ah, 1985; p. and are intended to present one interpreta265). These restrictions imposed by the de- tion and to put the developmental endocrivelopmental pattern, thus, limit the varia- nology of amphibians into an evolutionary
tion in phenotypes on which selection can framework.
act.
Because many developmental processes Developmental constraints preventing the
are regulated by hormones, the develop- evolution of neoteny in anurans
mental endocrinology of a species may bias
"Neoteny" describes species with the
the direction of evolutionary trajectories by ability to reproduce while maintaining their
imposing developmental constraints. The larval morphology. All members of four
current study examines two potential ex- families of urodeles are obligatory neotenes, and every family of urodeles has
' From the Symposium Developmental Endocrinol- neotenic populations in at least one species
ogy of Non-Mammalian Vertebrates presented at the (Duellman and Trueb; 1986), except RhyAnnual Meeting of the Society for Integrative and
Comparative Biology, 26-30 December 1996, at Al- acotritonidae (which contains only four species). On the other hand, no anurans are
buquerque, New Mexico.
2
E-mail: [email protected]
neotenic: Although a few reports have deHORMONAL MECHANISMS AS POTENTIAL
CONSTRAINTS ON EVOLUTION:
EXAMPLES FROM THE ANURA
482
483
HORMONAL MECHANISMS AND EVOLUTION
scribed non-metamorphosing populations
of anurans (Borkin et al., 1982), none were
reported to reproduce as larvae and were,
thus, not truly neotenic. Among caecilians,
four of the six families of caecelians have
larval life stages, all species of the remaining two families are live-bearing, and none
are known to be neotenic.
In the current study, I suggest that a developmental constraint, in the form of a
hormonal requirement, may have prevented
the evolution of neoteny in the Anura. In
other words, there is no selective disadvantage in neoteny in anurans, but anurans
have inherited a developmental pattern that
does not allow reproduction without metamorphosis; i.e., ". . . there is no reason to
suppose that the developmental mechanisms in question evolved because of the
particular phenotypes that they make readily accessible." (Maynard Smith et al.,
1985, p. 269).
Sexual reproduction requires at least
three processes: Sexual differentiation, sexual maturation, and appropriate reproductive cycling. Sexual differentiation involves
both primary and secondary sex differentiation. In primary sex differentiation, the bipotential gonads differentiate into either
testes or ovaries (in non-hermaphrodites).
Following gonadal differentiation, hormones from the gonads regulate secondary
sex differentiation {e.g., reproductive tract
in males and females, thumb pads and gular
pouch in male anurans, sexual dichromatism). Sexual maturation involves the production of gametes. Once sexually mature,
animals typically enter reproductive cycles,
periods of reproductive activity interspersed
with periods of inactivity. In anurans, all of
these processes are affected and regulated
by sex steroids.
Exogenous sex steroid treatment affects
gonadal differentiation in many species of
anurans, suggesting a role for endogenous
sex steroids. The effects of steroids may
vary greatly between species, however
(Hayes and Licht, 1995). For example, 17[J
estradiol (E2)-treatment in larvae produces
100% females in Xenopus laevis at metamorphosis (Witschi and Allison, 1950; Gallien, 1953, 1955; Chang and Witschi, 1955;
Hayes, unpublished), but testosterone-treat-
X. laevis
THIO
FIG. 1. Effect of [0.37 JJLM] testosterone (T), [0.37
JJLM] 17(3 estradiol (E2), and [3.1 mM] thiourea (Thio)
as compared to the control (Con), on the sex ratio in
the African Clawed Frog (Xenopus laevis; top) and the
African reed frog (Hyperolius viridiflavus; bottom). In
both studies, larvae were treated by direct administration of the compounds to the aquarium water at 28°C
throughout larval development (6-8 weeks). Each
treatment was replicated three times, each replicate
contained 30 animals, and water was changed and new
hormone or thiourea added daily. Sex was determined
after complete tail resorption based on gonadal morphology and histology. Asterisks show treatment
groups in which the sex ratio deviated significantly
from 50:50 based on a G-test statistic (Sokal and
Rohlf, 1981). Figure modified from Hayes et al, 1997.
ment has no effect (Mikamo and Witschi,
1964; Hayes, unpublished., Fig. 1). On the
other hand, E2 does not affect the sex ratio
in the African reed frog {Hyperolius viridiflavus), but testosterone produces 100%
males (Richards, 1982; Hayes et al, 1997;
Fig. 1).
In addition to their effects on primary sex
differentiation, sex steroids regulate a number of secondary sex characteristics. In
males, development of the vocalization apparatus (gular pouch: Hayes et al., 1997;
and larynx: Sassoon et al., 1987) and thumb
pad development are androgen-dependent
(Chang and Witschi, 1955). Androgens also
potentially regulate sexually dimorphic
growth and behaviors in the African bullfrog (Pyxicephalus adspersus, Hayes and
Licht, 1992) and E2 induces female-type
dorsal coloration in a species of sexually
dichromatic reed frog {Hyperolius argus;
Hayes et al., 1997). Also, sex steroids initiate sexual maturation. For example, in fe-
484
TYRONE B. HAYES
males, E2 induces vitellogenin and thus egg
production in females (Clemens, 1974). Finally, steroids also regulate reproductive
cycles in anurans (Licht et al., 1983).
Despite the direct regulation of sexual
differentiation and reproductive processes
by sex steroids, new evidence suggests that
exposure of tissues to thyroid hormones
may be required prior to sex steroid exposure for gonadal differentiation, secondary
sex differentiation, sexual maturation, and
possibly reproductive cycling. These data,
reviewed below, suggest that several components of reproduction (primary sex differentiation, secondary sex differentiation,
and reproductive cycling) require thyroid
hormones (which also induce metamorphosis) in order to occur normally: All tissues
must transform to the adult type (metamorphose) before they can respond to sex steroids.
Several studies have addressed the role
of thyroid hormones in primary sex differentiation, with mixed results (see Hayes,
1997 for review). Administration of the goitrogen, thiourea, to larvae blocked thyroid
hormone production and hence metamorphosis and also resulted in skewed sex ratios (100% females) in Xenopus laevis
(Hayes, 1997; Hayes and Chen, 1997a; Fig.
1). These data suggest that thyroid hormones may be required for testicular development in Xenopus laevis (Hayes, 1997).
Establishing a role for thyroid hormones in
gonadal differentiation in a single species
does not allow generalizations to all of the
Anura, so we (Hayes et al., 1997) examined
sex differentiation in a second species, the
African reed frog Hyperolius viridiflavus.
This species is ideal for comparative studies
for at least two reasons: 1) It is unrelated
to Xenopus laevis (H. viridiflavus is a member of the family Hyperoliidae, and X. laevis a member of the Pipidae) and 2) in H.
viridiflavus, E2 has no effect on primary sex
differentiation, but testosterone-treatment
results in 100% males (the opposite of X.
laevis; Richards, 1982; Hayes et al, 1997;
Fig. 1).
Treating Hyperolius viridiflavus with thiourea resulted in 100% males (Fig. 1).
Thus, in an unrelated species with a completely different sex determining system
(based on the response to steroids), it appears that one sex (male or female, depending on the species) depends on thyroid hormone exposure for differentiation. Although more work is needed to elucidate
fully the mechanisms of hormone action on
gonadal differentiation, the production of
skewed sex ratios in thyrostatic tadpoles of
two unrelated species suggests a general
role for thyroid hormones in anurans: The
production of normal sex ratios cannot occur in the absence of thyroid hormones.
There is also evidence of a role for thyroid hormones in the development of secondary sex characters in anurans. Thyroid
hormones directly induce the testosterone
receptor in the larynx of developing male
Xenopus laevis (Cohen and Kelley, 1996;
Robertson and Kelley, 1996). Subsequent
exposure to androgens from the testes then
induces development of the larynx necessary for vocalizations by males. Thus, in
the absence of thyroid hormones, this important secondary sex character and behavior would not be possible. Furthermore, recent work in my laboratory (Hayes et al.,
1997) showed that estrogens administered
to larvae prematurely induce a change in
the dorsal color pattern in the sexually dichromatic species Hyperolius argus. Normally, both male and female H. argus metamorphose with a bright green dorsum. Females develop a reddish brown dorsal color
with large white spots several months after
metamorphosis. Exogenous E2 induces female coloration in both males and females
at metamorphosis, but does not produce this
effect in larvae if thyroid hormone production is inhibited (thiourea + E2). Likewise,
although exogenous testosterone induced
gular pouch development, this effect does
not occur if animals are concurrently treated
with thiourea. Thus, steroid regulation of
this secondary sex character requires prior
(or possibly concurrent) exposure to thyroid
hormones. Thus, in several examples, steroid regulated secondary sex characters
cannot develop without exposure to thyroid
hormones.
Thyroid hormones may be required for
sexual maturation and subsequent reproductive cycling in anurans, also. Thyroid hormones induce expression of the E2 receptor
485
HORMONAL MECHANISMS AND EVOLUTION
A
c
B
«I
•a
«
E2-trea
H
Con-Fe
«
Con-M
Kidney
M
£
"o
«
I t
a
-fc -is
B
*
FIG. 2. Effect of [0.37 u.M] 17|3 estradiol (E2) on vitellogenin mRNA expression in reproductively mature
Xenopus laevis (A), pre-metamorphic tadpoles (B), and post-metamorphic sexually immature juveniles (C).
Estradiol was administered directly into water in all cases. Expression was analyzed using northern blot hybridization. Total RNA was extracted from tissues using the guanidine thiocyanate method and ultracentrifugation
(Chirgwin et ai, 1979). Samples (15 u.g total RNA) were run on an agarose gel, transferred to a nylon transfer
membrane, and probed with a vitellogenin cDNA (obtained from J. Tata, National Institute for Medical Research,
The Ridgeway, Mill Hill, London, UK) labeled with32 P using an Amersham Megaprime DNA-labeling kit.
Panel A (top; left to right) shows the absence of expression in adult male livers, expression in adult females,
and up-regulation following daily treatment of adult females with E2 for ten days. Expression was tissue-specific
and not produced in the kidney of Ej-treated females (panel A, far right). Panel B shows the absence of
vitellogenin expression in control pre-metamorphic tadpoles and tadpoles treated with E2 for ten days. For
tadpoles, the livers of 30 animals were pooled for RNA extraction. Finally, Panel C shows the absence of
vitellogenin expression in male and female juvenile post-metamorphic animals, but the induction of vitellogenin
following daily treatment with Ej for ten days. Liver tissue from ten post-metamorphic individuals was pooled
for each sex to obtain RNA for controls and E2-treated animals. Expression of Pr28, a non-hormone-inducible
gene coding for a ribosomal protein (Shi and Liang, 1994) was used as a control and revealed that all lanes
were loaded equally (data not shown). Figure modified from Hayes and Chen, 1997ft.
in the liver of adult female Xenopus laevis
(Rabelo and Tata, 1993; Rabelo et al.,
1994). Estrogens from the ovary induce vitellogenin (egg yolk protein) synthesis and
secretion by the liver only after the liver has
been exposed to thyroid hormones (Wangh
and Schneider, 1982). Furthermore, premetamorphic tadpoles are unable to produce
vitellogenin even after exposure to exogenous E2 (May and Knowland, 1980; Hayes
and Chen, 1997b; Fig. 2): Without thyroid
hormones, X. laevis is unable to synthesize
vitellogenin and produce eggs. Thus, even
if sex differentiation for both sexes could
occur in the absence of thyroid hormones
and metamorphosis, reproduction would
not be possible as the secondary sex characters in males, and egg production in females, are thyroid hormone-dependent.
The potential thyroid hormone require-
486
TYRONE B. HAYES
ment for primary and secondary sex differentiation and for sexual maturation may
preclude the evolution of neoteny in anurans. It is not clear if thyroid hormones are
also necessary for reproductive cycling or
if they have a role in spermatogenesis. But,
if thyroid hormones are universally required
for even one of the stages of sexual differentiation or reproduction, the current hypothesis is supported. In fact, it appears that
thyroid hormones may be required for several stages in these processes. The possible
requirement of thyroid hormones for primary and secondary sex differentiation and
sexual maturation couples the capability of
anurans to reproduce with metamorphosis,
because anurans do not show any capability
to undergo "partial" metamorphosis (some
tissues and structures transform but not others) when exposed to thyroid hormones.
This last point is important, because if partial metamorphosis were possible, then one
could imagine that thyroid hormones could
stimulate reproductive tissues and structures
without stimulating other aspects of metamorphosis as a mechanism for producing
neotenic anurans.
To further support the above hypothesis,
however, one has to show that urodeles (in
which neoteny has evolved many times) do
not require thyroid hormones for sex differentiation and reproduction: As stated in
Arnold et al. (1989; p. 411), ". . . it is conceivable that coupling restricts the spectrum
of outcomes and that decoupling or recoupling might sometimes precede the origin
of novelties." Here, I suggest that a decoupling of the hormonal requirements for
metamorphosis and reproduction in urodeles produced the novelty that allowed
neoteny to evolve multiple times.
The fact that at least 40 species of urodeles (at least one species in every family
except Rhyacotritonidae) display neoteny
and reproduce in larval form suggests that
thyroid hormones (and complete metamorphosis) are not required for reproduction in
this order. Like anurans, urodeles do not
show any ability to undergo partial metamorphosis in the presence of thyroid hormones. There are varying degrees of neoteny (Cryptobranchids simply lack eyelids
and some elements of the skull and retain
gill slits, while Sirenids completely lack a
pelvic girdle and hind limbs, retain full
gills, etc.), but this is not accomplished because some tissues (and structures) respond
to thyroid hormones and others do not. At
least two mechanisms result in neoteny in
urodeles. Some neotenic species do not synthesize thyroid hormones (facultative neotenes, such as Ambystomatids) and no tissues transform, while other species lack
thyroid hormone receptors (obligate neotenes, such as Cryptobranchids and Sirenids) and some tissues transform in the absence of thyroid hormones.
The loss of the ability to respond to thyroid hormones in obligate neotenes (Noble,
1924; Gutman, 1926; Kobayashi and Gorbman, 1962) suggests that sex differentiation
and reproduction occur without thyroid hormones. In facultative neotenes, exposure to
exogenous thyroid hormones or stress induces metamorphosis, suggesting that thyroid production is impaired in these species
(Swingle, 1924; Kezer, 1952; Gorbman,
1957; Dundee, 1957, 1961; Dent and Kirby-Smith, 1963; Gabrion and Sentain,
1972; Norris and Platt, 1973, 1974; Brandon, 1976; Galton, 1992). Furthermore, it
appears that thyroid hormone synthesis and
secretion fail as a result of a lack of signal
from the pituitary (Blount, 1950; Dent and
Kirby-Smith, 1963; Norris and Platt, 1973)
or possibly an inability of the pituitary to
respond to stimulation from the hypothalamus (Taurog et al., 1974). In these facultative neotenic species, sex differentiation
and reproduction are carried out normally,
despite the absence of thyroid hormones.
Finally, non-neotenic salamanders arrested
with thiourea or NaCl4 both of which inhibit thyroid hormone production), show
accelerated gonadal development even
though somatic tissues do not change, experimentally producing neotenic larvae
(Wakahara, 1994; Yamaguchi et al, 1996).
Together, the above evidence suggests that
thyroid hormones are not required for sex
differentiation or reproduction in urodeles.
The lack of a requirement for thyroid hormone, thus, does not limit possible developmental patterns and has allowed neoteny
to evolve several times in the urodeles.
These ideas represent a single interpre-
HORMONAL MECHANISMS AND EVOLUTION
tation, but one that is supported by the
available data. More comparative studies
are needed, however. For example, the role
of thyroid hormone in vitellogenesis has
been examined only in a single species (X.
laevis). Likewise, the role of thyroid hormones in the development of secondary sex
characters has been examined in only two
anuran families (Pipidae and Hyperolidae).
Furthermore, the role of thyroid hormones
and molecular mechanisms regulating sex
differentiation and reproduction in urodeles
is even less well studied.
Developmental constraints regulating
sexual dichromatism in African reed frogs
(Hyperoliidae)
The family Hyperoliidae provides another example of how hormonal mechanisms
have potentially imposed developmental
constraints on evolution (in this case, a local constraint within the anuran family, Hyperoliidae). In most anuran species, juveniles at metamorphosis look like small versions of the adults; however, several species
of Hyperolius show ontogenetic changes in
dorsal color patterns. The Hyperoliidae are
also one of as little as four anuran families
that contain species that display permanent
sexual dichromatism in dorsal coloration.
The sexual dichromatism is interesting because anurans are not typically visually oriented with respect to mate choice: Breeding
occurs at night and most communication is
vocal, thus, it is unlikely that sexually dimorphic coloration resulted from sexual selection.
We (Hayes et al., 1997) compared development and the effects of exogenous steroid hormones in two species, Hyperolius
viridiflavus (which is sexually monochromatic) and Hyperolius argus (which is sexually dichromatic). In H. viridiflavus, both
sexes metamorphose with gold dorsal coloration featuring a mid-dorsal and two dorsal lateral brown stripes. Both sexes change
to a bright green with yellow spots (each
with a red dot in the middle) within three
months of metamorphosis. In H. argus,
both sexes metamorphose with a bright
green coloration and females later develop
orange toes and a reddish dorsal color with
large white spots.
487
In Hyperolius argus, E2 treatment induced the female-typical coloration in
males and females at metamorphosis,
whereas females normally do not show this
color change for more than three months
after metamorphosis and normal males never display this color pattern. Although testosterone prematurely induced gular pouch
development, this hormone had no effect on
dorsal coloration, in this species. In H. viridiflavus, however, exposure to either testosterone or E2 induced the adult coloration.
Thus, both androgens and estrogens affected coloration in this sexually monomorphic
species in which both sexes normally undergo an ontogenetic change in color patterns, whereas only estrogens were active
in H. argus in which only females undergo
an ontogenetic change.
At least two mechanisms may explain the
effectiveness of both androgens and estrogens in Hyperolius viridiflavus but only estrogens in H. argus. One possibility is that
H. viridiflavus has androgen receptors in
addition to estrogen receptors in its skin.
Another possibility is that H. viridiflavus
has high levels of aromatase in their skin
and convert the androgen to estrogen. These
possibilities remain to be tested; however,
regardless of the specific mechanism, the
difference between the species produces
sexual monochromatic animals in one species {H. viridiflavus) and sexual dichromatic animals in the other (H. argus).
It is unclear what evolutionary mechanisms would favor sexual dichromatism in
anurans. As discussed earlier, sexual selection is unlikely, because most anurans, including the Hyperoliidae, breed at night and
depend more on vocal signals rather than
visual signals for species and sex recognition. One possibility is niche-partitioning
and differential selection on males and females (Shine 1989). For example, male Hyperolius argus may be more at risk because
they remain in the breeding ponds longer,
vocalizing, and trying to attract females.
Perhaps the green color provides a camouflage coloration in the vegetation around the
breeding ponds that decreases their chances
of being preyed upon. Proper phylogenetic
and/or behavioral ecology data are required
for testing these types of hypotheses further,
488
TYRONE B. HAYES
but clearly the patterns themselves (sexual
monochromatism or dichromatism) are determined by hormonal mechanisms. Even if
the color patterns are maintained by selective pressures, the expression of sexual dichromatism (availability for selection to act
on) is determined by (apparently) heritable
hormonal mechanisms.
CONCLUSIONS
It is unlikely that selection will act on
hormonal mechanisms, only the outcomes
(phenotypes) that these developmental patterns produce. Because hormones coordinate events in development, however, and
because developmental patterns are heritable, taxa may be constrained in terms of the
patterns of development expressed. The
constraints on the developmental pattern
may limit phenotypic possibilities which
themselves may not be maladaptive, but
simply not expressed in some taxa. In addition, the constraints can be universal or
local depending on when they arise, as
shown in the examples discussed.
"The notion of a developmental constraint is important in understanding how
development influences evolution" (Resnik
1995, p. 233): A requirement of thyroid
hormone for sex differentiation and reproduction in anurans may preclude neoteny in
this order just as the presence or absence of
the ability to respond to androgens may allow the expression of sexual dichromatism
or monochromatism in color respectively in
Hyperolius. The pattern of development in
the Anura, i.e., the coupling of sex differentiation and reproduction with metamorphosis reflects an evolutionary history in
which the developmental constraint was acquired, just as the ability to respond or not
respond to androgen in the Hyperoliidae reflects the local evolutionary history of each
species. Recalling that constraints are often
as important in evolution as selection is important in solving evolutionary questions.
ACKNOWLEDGMENTS
I thank Katherine K. Kim for her support.
Marvalee Wake, David Wake, and Dan
Buchholz provided interesting conversations on this topic and have helped shape
my ideas. I also thank Yun Bo Shi who pro-
vided the PR28 cDNA, Jamshed Tata who
provided the vitellogenin cDNA, and Carmen Domingo for technical assistance.
Travel to Kenya (for Hyperolius) was funded in part by a University Research Expeditions Grant. Finally, I thank Robert
Thommes and August Epple who organized
this symposium. This work was supported
by NSF Grants IBN-9513362 and IBN9508996.
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Corresponding Editor: David Norris