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An Integrative Endocrine Model for the Evolution of Developmental Timing and
Life History of Plethodontids and Other Salamanders
Author(s): Ronald M. Bonett
Source: Copeia, 104(1):209-221.
Published By: The American Society of Ichthyologists and Herpetologists
DOI: http://dx.doi.org/10.1643/OT-15-269
URL: http://www.bioone.org/doi/full/10.1643/OT-15-269
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Copeia 104, No. 1, 2016, 209–221
An Integrative Endocrine Model for the Evolution of Developmental Timing
and Life History of Plethodontids and Other Salamanders
Ronald M. Bonett1
In recent years, many molecular endocrine mechanisms that regulate tissue morphogenesis have been detailed in
laboratory amphibian models. However, most of these pathways have not been examined across more closely related
species to understand how deviations in endocrine pathways may have contributed to amphibian diversification. The
timing of metamorphosis and maturation vary extensively across plethodontid salamanders (including direct
developing, biphasic, and paedomorphic species), making them an ideal system for analyzing the evolution of
endocrine mechanisms in a phylogenetic context. Recent phylogenetic-based reconstructions concluded that ancestral
plethodontids were likely direct developers, and free-living larval periods were independently derived multiple times
within this family. Furthermore, within one clade (Spelerpini) there have been multiple independent transitions from
biphasic (metamorphic) to paedomorphic developmental modes. This inspires the question: What endocrine/
developmental mechanisms govern these extreme life history transitions? Prior endocrine models for the evolution
of direct development and paedomorphosis have been largely based on the ontogenetic timing of thyroid hormone
release and/or thyroid hormone responsiveness of target tissues. Here I review endocrine pathways that influence
metamorphosis and maturation in laboratory amphibians (Clawed frog and Axolotl) and other species, and develop a
model that integrates prior thyroid hormone-based patterns with other endocrine axes. This integrated framework
incorporates developmental shifts that result from plasticity or evolution in the timing of hatching, metamorphosis,
and maturation, and can be used to test mechanistic changes that underlie life history variation of plethodontids and
other salamanders.
A
MPHIBIAN metamorphosis involves many concomitant morphogenic changes in different tissue systems. This indicates that common control
mechanisms, such as endocrine signals, may govern these
processes. Amphibians exhibit striking differences in the
timing of developmental events among species (heterochronies), which may ultimately be dictated by evolution and/or
plasticity of endocrine control mechanisms (Dent, 1942,
1968; Rose, 1996; Hanken et al., 1997; Denver, 2013; Elinson,
2013; Johnson and Voss, 2013). A reoccurring conclusion is
that life history variation is correlated with ontogenetic shifts
in the timing of thyroid hormone (TH) release or TH
responsiveness of target tissues. However, many other
endocrine and developmental pathways, particularly stress
response and reproduction, are also known to influence life
history variation of amphibians, but these have not been
previously integrated with TH-based models.
Plethodontid salamanders may prove to be an enlightening system to study the evolution of endocrine mechanisms
given their extensive lineage and developmental diversification (Wake, 1966; Hanken, 1992; Ryan and Bruce, 2000;
Chippindale et al., 2004; Bonett et al., 2014a, 2014b).
Plethodontids exhibit three major developmental (life history) patterns: 1) biphasic: a free-living aquatic larval form that
metamorphosis into a more terrestrial form; 2) paedomorphic:
a permanently aquatic larval form that forgoes metamorphosis; and 3) direct development: a precociously metamorphosing form that lacks a free-living aquatic larval stage.
Despite these categorizations, it may be more informative to
view these ‘‘distinct’’ developmental modes as a continuum
of morphogenic timing, with a primary difference being the
length of time spent in the larval form relative to hatching
and maturation (Ryan and Bruce, 2000; Bonett et al., 2014b).
For example, direct development is the acceleration (speeding-up) of the time to metamorphosis to an age prior to
hatching, whereas paedomorphosis is decelerating (slowingdown) the time to metamorphosis to an age after maturation
1
(see Bonett et al., 2014b for further discussion of this
concept).
Ancestral plethodontids likely exhibited direct development, and therefore the derived biphasic lineages (spelerpines, desmognathines, and Hemidactylium) represent at least
three independent decelerations in the durations of the larval
period (age at metamorphosis; Bonett et al., 2014b; Fig. 1).
The lengths of larval periods within biphasic plethodontids
vary tremendously, from less than a few months to more
than a few years. Some of these differences represent further
derived decelerations leading to very long larval periods (e.g.,
Gyrinophilus porphyriticus or Desmognathus quadramaculatus),
while other taxa likely represent secondarily derived accelerations (e.g., Eurycea quadridigitata). Even though ancestral
spelerpines were likely biphasic, there have been several
independent instances of paedomorphosis across this clade.
In at least one clade (Edwards Plateau Eurycea), there was
likely a very recent reversal from paedomorphosis back to a
biphasic (metamorphic) life cycle (Bonett et al., 2014a,
2014b).
This review describes the general endocrine pathways that
may underlie the development of plethodontids, based on
information about metamorphosis and maturation derived
primarily from studies of laboratory model species and other
amphibians. A model is presented which expands upon the
previously proposed TH-based life history models by integrating endocrine-based plasticity of metamorphic timing, as
well as shifts in hatching and maturation time. This model
can serve as a framework to test how changes in endocrine
mechanisms regulate the plasticity and evolution of life
histories of plethodontids and other salamanders.
GENERAL ENDOCRINE MECHANISMS REGULATING
METAMORPHOSIS AND MATURATION
Several endocrine and environmental factors are known to
regulate the timing of metamorphosis and maturation of
Department of Biological Science, University of Tulsa, Tulsa, Oklahoma 74104; Email: [email protected].
Submitted: 20 March 2015. Accepted: 2 August 2015. Associate Editor: M. E. Gifford.
DOI: 10.1643/OT-15-269 Published online: 31 March, 2016
Ó 2016 by the American Society of Ichthyologists and Herpetologists
210
Copeia 104, No. 1, 2016
Fig. 1. Life history evolution of plethodontid salamanders. The plethodontid phylogeny and reconstruction are based on Bonett et al. (2014b). The
phylogeny was pruned to include relevant major lineages. Branches are colored according to categorical reconstruction of life histories: direct
development (yellow), primarily biphasic (red), and primarily paedomorphic (blue). Lineages with a mix of metamorphic and paedomorphic
populations/species (e.g., Gyrinophilus and Eurycea tynerensis) are shown in purple.
amphibians. The hypothalamus plays a central role in
mediating the influence of environmental factors on endogenous hormone levels, which are important determinants of
how phenotypes and life histories are manifested. Below I
provide an overview of the relevant aspects of three
endocrine systems (thyroid, corticosteroid, and reproductive), which individually and in concert are major drivers of
amphibian development and life histories.
Thyroid hormone and amphibian metamorphosis.—Amphibians have historically been important models for our
understanding of thyroid hormone function, ever since
Gudernatsch (1912) precociously metamorphosed tadpoles
of Rana temporaria by feeding them mammalian thyroid
glands. Subsequently, metamorphosis was successfully induced with thyroid hormone for many amphibian larvae.
These included treatments with both 3,5,3-triiodothyronine
(T3) and the less active form 3,5,3 0 ,5 0 -tetraiodothyronine (T4;
thyroxine). Notable exceptions included some obligately
paedomorphic salamanders (discussed under larval form
paedomorphosis below). Several studies also showed that
plasma levels of T3 and T4 increase during metamorphosis
(e.g., Regard et al., 1978; Suzuki and Suzuki, 1981; Alberch et
al., 1986; Norman et al., 1987), and ablation of the thyroid
gland can prevent metamorphosis (Allen, 1916, 1918;
Hoskins and Hoskins, 1919). Metamorphosis can be recovered in thyroidectomized larvae by treatment with TH (Allen,
1932; Etkin, 1935; Hanaoka, 1966). Taken together, these
studies demonstrate that thyroid hormone is necessary for
amphibian metamorphosis.
The release of T3 and T4 from the thyroid gland is regulated
by the hypothalamic-pituitary-thyroid (HPT) axis (Fig. 2).
Thyrotropin-releasing hormone (TRH) from the hypothalamus causes the release of thyroid-stimulating hormone (TSH)
from the anterior pituitary, which stimulates the thyroid
gland to release T3 and T4 into the circulatory system.
However, TRH appears to only activate the release of TSH in
adult metamorphosed amphibians (Darras and Kühn, 1983;
Denver, 1988; Jacobs et al., 1988). In contrast, hypothalamic
corticotropin-releasing factor (CRF; discussed with corticosteroids below) appears to regulate the release of TSH in larval
amphibians (Denver and Licht, 1989; Denver, 1993, 1997,
2009, 2013; Boorse and Denver, 2002). Plasma TSH levels are
undetectable in early stages of tadpole development, but
elevate prior to metamorphic climax (Dodd and Dodd, 1976).
Treatment of larval amphibians and paedomorphic salamanders with TSH has been shown to stimulate thyroid activity
and induce metamorphosis (Dent and Kirby-Smith, 1963;
Norris et al., 1973; Norman and Norris, 1987). Further
evidence of the central role of this axis comes from
experiments that have inhibited metamorphosis by ablating
the anterior pituitary (Kollros, 1961; Hanaoka, 1966).
At target tissues (e.g., tailfin, external gills, brain), T3 and T4
interact directly with the nuclear transcription factors TRa or
TRb (thyroid hormone receptor alpha or beta, collectively
TRs), which form heterodimers with retinoid X receptor
(RXR) and bind to thyroid hormone response elements
(TREs) in the genome (Ranjan et al., 1994; Buchholz et al.,
2006; Das et al., 2009). Unliganded TRs are bound to corepressor complexes, which inhibit gene expression (Buchholz et al., 2003; Sato et al., 2007). Whereas, when T3 and T4
are bound to TRs they recruit co-activators, which can alter
the expression of genes regulated by thyroid hormone (Paul
et al., 2005). T3 and T4 are known to upregulate and
downregulate hundreds of genes in target tissues of amphibians during metamorphosis (Brown et al., 1996; Denver et al.,
1997; Das et al., 2006, 2009; Page et al., 2008, 2009). Among
these, TRs are autoregulated by T3 and T4 and, at least for
TRb, this upregulation is direct (Machuca and Tata, 1992;
Ranjan et al., 1994; Das et al., 2009). In other words, T3 and
T4 can further accentuate their effects by producing more
Bonett—Endocrinology of salamander metamorphosis
211
Fig. 2. Consensus of general endocrine pathways for amphibian metamorphosis via the HPT and HPI axes. The pathway depicts Corticotropin
Releasing Factor (CRF) from the hypothalamus as the regulator of both Adrenocorticotropic Hormone (ACTH) and Thyroid Stimulating Hormone
(TSH) from the pituitary. These pituitary hormones stimulate the release of corticosteroids from the adrenocortical cells of the interrenal gland, and T3
and T4 from the thyroid gland, respectively. Gene expression is regulated in target tissues (e.g., tailfin) by these hormones through binding to their
respective nuclear receptors: corticoid receptors (CRs, including GRs or MRs) or thyroid hormone receptors (TRs). Sufficient increases in these
hormones can drive the transformation of target tissues and metamorphosis, as depicted here with Eurycea tynerensis (see text for details).
receptors for the hormone to bind. Dominant negative TRb
transgenic tadpoles do not metamorphose (Buchholz et al.,
2004), which further supports the necessity of TH and
functional TRs. The expression patterns of TRs are related to
metamorphic timing and life histories in Spadefoot Toads
(Hollar et al., 2011) and Oklahoma Salamanders (Aran et al.,
2014; discussed more below).
It should also be noted that TH activity can be altered in
target tissue via deiodinases, which are a class of enzymes
that can add or remove iodine to produce more or less active
forms of the hormone (Becker et al., 1997; Brown, 2005; St.
Germain et al., 2009). For example, type-2 deiodinase
converts T4 into the more active T3 with the removal of an
iodine atom. The transcriptional actions of TH and associated
regulatory mechanisms ultimately govern tissue transformation and metamorphosis of larval amphibians.
Corticosteroids and interactions with thyroid hormone.—Corticosteroids are regulated through the hypothalamic-pituitaryinterrenal (HPI) axis and are important mediators of
‘‘environmental stress’’ in vertebrates (Denver, 2009, 2013;
Carr, 2010; Crespi et al., 2013; Fig. 2). CRF from the
hypothalamus regulates adrenocorticotropic hormone
(ACTH) secretion from the anterior pituitary, and ACTH
stimulates the production and release of corticosteroids
(glucocorticoids and mineralocorticoids) from the interrenal
glands. Corticosteroids can regulate transcription in target
tissues by binding to corticoid receptor (CRs, including
glucocorticoid or mineralocorticoid receptor, GRs or MRs,
respectively) homodimers in the cytoplasm, which then
translocate to the nucleus and bind to glucocorticoid or
mineralocorticoid response elements (GREs or MREs) in the
genome (Denver, 2009, 2013). The pathways of corticosteroids and their effects on larval and adult amphibians are
complex and include alterations in activity, behavior,
feeding, body weight, growth, reproduction, shape, and the
timing of metamorphosis (Glennemeier and Denver, 2002;
Coddington and Moore, 2003; Denver, 2009, 2013; Fraker et
al., 2009; Carr, 2010; Bliley and Woodley, 2012; Crespi et al.,
2013; Reedy et al., 2014).
During early developmental stages, tadpoles treated with
corticosteroids or placed in stressful environments can
decrease growth and development but increase tail depth
(Glennemeier and Denver, 2002; Bonett et al., 2010;
212
Middlemis Maher et al., 2013). However, during amphibian
metamorphosis, corticosteroids increase endogenously (Krug
et al., 1983; Carr and Norris, 1988; Denver et al., 1998;
Chambers et al., 2011) and have been shown to alter the
effects of thyroid hormone on morphogenic changes
(Frieden and Naile, 1955; Kobayashi, 1958; Kikuyama et al.,
1983; Kühn et al., 2004; Bonett et al., 2010). Most
importantly, there is significant cross talk between the HPI
and HPT axes at both the neuroendocrine level and in target
tissues. For example, during metamorphosis the anterior
pituitary expresses CRF type 2 receptor, which allows
hypothalamic CRF to directly stimulate the release of TSH
(Manzon and Denver, 2004; Okada et al., 2007; see also
above). In target tissues, corticosterioids can enhance the
effects of thyroid hormone through several avenues. Transcriptional regulatory mechanisms driven by corticosterioids
include the regulation of genes that code for thyroid
hormone converting enzymes (e.g., type-2 deiodinase;
Bonett et al., 2010) or for transcription factors that
upregulate thyroid hormone receptor expression (e.g.,
Krüppel-like factor 9, KLF9, formerly known as BTEB1;
Bagamasbad et al., 2008; Bonett et al., 2009). More complex
interactions between corticosteroids and thyroid hormone
occur at the level of gene regulation, including additive,
synergistic, and inhibitory effects (Bonett et al., 2010;
Kulkarni and Buchholz, 2012). For example, corticosteroids
and thyroid hormone have greater than additive (synergistic)
effects on transcription of thyroid hormone receptors,
deiodinases, and KLF9 (Kühn et al., 2005; Bonett et al.,
2010; Kulkarni and Buchholz, 2012; Bagamasbad et al.,
2015). These regulatory effects may provide a link between
corticosteroids and the rapid morphogenic changes in late
stage larvae, which could expedite their departure from a
‘‘stressful’’ larval environment such as a crowded, drying
pond.
Reproductive hormones.—Understanding the timing of reproductive development is critical for reconstructing the
evolution of maturation with respect to other events (e.g.,
metamorphosis) and also because reproductive hormones
can both stimulate and inhibit other endocrine pathways. It
also is a pivoting point for shifting resource allocation
between growth/morphogenesis and reproduction (Stearns,
1992).
Sex steroids (estrogens and androgens) can function in a
similar manner as corticosteroids, by binding to nuclear
transcription factors (estrogen and androgen receptors) to
regulate gene transcription. There is also significant crosstalk
between the hypothalamic-pituitary-gonadal (HPG) axis and
HPT axes. This topic has been extensively reviewed (Hayes,
1997; Duarte-Guterman et al., 2014), and most research has
focused on the effects of endocrine disruptors on reproductive development and sex ratios. The most relevant aspects
for this review are the influence of thyroid hormone on
gonadal development (Flood et al., 2013) and sex steroids on
the inhibition of metamorphosis. Thyroid-less and thyroidsuppressed (goitrogen treated) tadpoles and larval salamanders can still develop gonadal germ cells (Allen, 1918;
Wakahara, 1994; Yamaguchi et al., 1996; Kanki and Wakahara, 1999; Rot-Nikcevic and Wassersug, 2004), and this may
be mediated through an increase in TSH (Kanki and
Wakahara, 1999). The ability of larval salamanders to develop
gonads without an increase of TH (which could stimulate
metamorphosis) provides the potential for larval form
paedomorphosis. At the same time, both estrogens and
Copeia 104, No. 1, 2016
androgens can inhibit tadpole metamorphosis (Gray and
Janssens, 1990; Hogan et al., 2008), but data for salamanders
are limited. If this mechanism is common among all
amphibians, then the acceleration of maturation and the
production of sex steroids into larval stages could permanently displace metamorphosis (Ryan and Semlitsch, 1998).
ENDOCRINE BASIS OF HETEROCHRONY IN
PLETHODONTIDS AND OTHER SALAMANDERS
Shifts in the timing of developmental events (heterochrony)
such as hatching, metamorphosis, and maturation have
occurred extensively among salamanders and probably most
extremely among plethodontids. These have resulted in three
major developmental-based life histories: direct development, biphasic, and paedomorphic. Studies that examine
the endocrine basis of these developmental patterns in
plethodontids and other amphibians are discussed immediately below.
Direct development.—Direct development is widespread in
plethodontids and has been considered an important
contributor to their diversification, especially in the neotropics (Wake and Hanken, 1996). Direct development is
likely the ancestral condition for the family and has been
maintained in most major lineages except for spelerpines,
one clade of desmognathines, and Hemidactylium (Bonett et
al., 2014b; Fig. 1). The molecular mechanisms that underlie
direct development are compelling, because somatic morphogenesis is accelerated so rapidly, and at such a small body
size, that metamorphosis occurs completely within the egg
(Dent, 1942; Wake and Hanken, 1996; Kerney et al., 2012;
Bonett et al., 2014b). Surprisingly, there have been only a few
molecular endocrine studies on direct developing plethodontids, although there is additional information that can be
derived from the many studies on direct developing frogs.
Dent (1942) proposed, and partially tested, thyroid
hormone-based mechanisms to explain direct development
in plethodontids. He proposed that direct developers either
had precociously developed pituitary and thyroid glands (to
trigger early metamorphosis) or that the tissues of direct
developers had an increased sensitivity to thyroid hormone.
Histological and morphological examination of Plethodon
cinereus showed that the pituitary and thyroid gland develop
at relatively early embryonic stages (Dent, 1942). Furthermore, he observed morphological changes to the thyroid
gland that signified secretory activity, and were coincident
with several morphogenic changes such as gill resorption.
These changes in embryos of P. cinereus (between stages XXII
and XXIII) are similar to those that occur during early
metamorphosis of Eurycea bislineata, which have a multi-year
larval period (Wilder, 1925; Dent, 1942).
The mechanisms of direct development in the frog
Eleutherodactylus coqui have been investigated much more
extensively (reviewed in Elinson, 2013), and this species
shares some important similarities with P. cinereus. For
example, E. coqui also develop their thyroid system embryonically, well before species with free-living tadpoles, and
peaks of thyroid activity are associated with metamorphic
changes (Hanken et al., 1997; Jennings and Hanken, 1998).
CRF is important for metamorphosis in E. coqui and likely
operates by directly stimulating TH release from the thyroid
gland (Kulkarni et al., 2010; Elinson, 2013). However, while
treatment of embryos of E. coqui with the goitrogen
methimazole prevents complete metamorphosis, many frog-
Bonett—Endocrinology of salamander metamorphosis
let features still develop (Callery and Elinson, 2000). This
suggests that either thyroid hormone is provided maternally
in yolk, or other hormones (such as corticosterone) are
driving metamorphosis. The potential influence of corticosteroids on metamorphosis of direct developing amphibians
makes sense from a phylogenetic perspective, because all
direct developing lineages were originally derived from
biphasic ancestors, which likely had plastic larval periods.
This may also explain the variation in metamorphic timing
of biphasic species such D. fuscus that typically have an
aquatic larval period of several months (Danstedt, 1975) but
can complete metamorphosis after less than one month
when raised strictly on wet surfaces without free-standing
water (Noble and Evans, 1932; see also Marks and Collazo,
1998 for further discussion about D. aeneus). This shows that
some biphasic plethodontids can effectively exhibit direct
development under some conditions.
Our experiments on hatchling Eurycea tynerensis, a species
that has minimally a several month larval period (or larval
form paedomorphosis), showed that treatment with exogenous thyroid hormone upregulates TRb in tail tissue and
significantly advances nasal capsule development. Exogenous corticosterone alone has no gross developmental
effects, but in combination with thyroid hormone has strong
synergistic effects on TRb expression and nasal capsule
development (Jackson et al., unpubl.). However, other tissues
that normally transform during metamorphosis of Eurycea,
such as the hyobranchial apparatus, do not seem to be
sensitive to thyroid hormone or corticosterone treatment at
such early stages of development (Jackson et al., unpubl.). As
with direct developing frogs (Elinson, 2013), this indicates
that other hormones may be necessary for the transformation of some tissues, or local differences in tissue sensitivity
determine whether or not metamorphosis can be completed
prior to hatching.
Biphasic.—Biphasic life histories occur in spelerpines, one
clade of desmognathines, and Hemidactylium (Fig. 1). Perhaps
the most interesting aspect of biphasic life cycles of
plethodontids is that they are likely independently derived
from direct developing ancestors (Chippindale et al., 2004;
Bonett et al., 2014b; Fig. 1). This suggests that not only was
there potentially a major endocrine-based acceleration of
larval development in ancestral plethodontids (or earlier) to
achieve direct development, but there may have been
multiple endocrine-based decelerations in the ‘‘re-evolution’’
of larval forms in desmognathines, spelerpines, and Hemidactylium. If these are indeed independent reversals to freeliving aquatic larvae, then their origins may be based on
different mechanisms. These may have involved delays in
the development of the HPT axis or decreasing sensitivity of
tissues to thyroid hormone and/or other endocrine signals.
Larvae of biphasic species/populations of spelerpines have
been transformed using both T3 and T4 (Alberch et al., 1985;
Rose, 1995a, 1995b, 1996; Aran et al., 2014). Hormone
dosage and the age/stage of larvae influence the timing of
transformation of skeletal elements (Rose, 1995a, 1995b).
Treatment of larvae from multiple metamorphic populations
of E. tynerensis with exogenous T3 upregulates TRa and TRb
expression in tailfin tissue (Aran et al., 2014).
Hickerson et al. (2005) found that metamorphic progress of
first year larvae of Desmognathus quadramaculatus was not
influenced by a low dose of T4 (up to 4.8 nM) administered
over approximately two months. We found that similar doses
(1 to 5 nM) of the more active hormone T3 were highly
213
influential on metamorphic changes of larval D. ocoee, D.
santeetlah, and D. brimleyorum over periods of three to five
months (Robison et al., unpubl.).
Alberch et al. (1986) performed radioimmunoassays for T3
and T4 on a series of E. bislineata sampled from across
metamorphosis. They found the common pattern of
increased concentrations of plasma T3 and T4 in metamorphic individuals, but neither hormone was detectable in
some specimens that were clearly in the process of metamorphosis. This suggested that hormone levels may fluctuate
during metamorphosis, or that other hormones are used in
the process.
The lengths of larval periods of biphasic plethodontids
vary extensively among and within species (Petranka, 1998;
AmphibiaWeb, 2015). Several ecological studies have shown
that factors such as temperature, hydroperiod, and stream
order can further influence metamorphic timing (Voss, 1993;
Beachy, 1995; Camp et al., 2000; Freeman and Bruce, 2001;
Bruce, 2005; Hickerson et al., 2005) but food availability does
not (e.g., O’Laughlin and Harris, 2000; Hickerson et al.,
2005). While variation and plasticity indicate that endocrine
processes play a major role in regulating metamorphic timing
(Rose, 2005; Denver, 2009, 2013; Crespi et al., 2013), there
have been relatively few published studies that simultaneously evaluate the effects of environmental and endocrine
factors on biphasic plethodontids (Hickerson et al., 2005).
Temperature is known to influence the rate of larval
growth, development, and the timing of metamorphosis of
plethodontids, with low temperatures delaying metamorphosis (Hickerson et al., 2005). An important endocrine facet
of this effect, which has been generally underexplored in
salamanders, is the potential for low temperatures to inhibit
TH-induced metamorphosis, as seen in studies with larvae of
Hynobius (Moriya, 1983). Integrative studies are needed to
assess how environmental factors and natural stressors
influence TH regulated gene expression programs to control
metamorphic timing of biphasic plethodontids.
Larval form paedomorphosis.—Shifts from biphasic to larval
form paedomorphic developmental modes have occurred
many times in salamanders, and nine of the ten salamander
families include larval form paedomorphic species. In
plethodontids, larval form paedomorphosis is restricted to
several independent instances in the Spelerpini, which are
associated with aquatic subterranean habitats and arid
climate regimes (Bonett et al., 2014a; Fig. 1). These include
isolated instances of paedomorphosis in primarily metamorphic clades (e.g., E. cirrigera; McEntire et al., 2014), clades
with highly variable life histories among populations (e.g., E.
tynerensis, Bonett and Chippindale, 2004, 2006; Emel and
Bonett, 2011; Gyrinophilus, Niemiller et al., 2008), divergent
paedomorphic lineages (e.g., E. subfluvicola; Steffen et al.,
2014), and primarily paedomorphic clades (Edwards Plateau
Eurycea; Chippindale et al., 2000). While most instances of
paedomorphosis are derived, there is strong phylogenetic
support that life history is reversible in spelerpines. In
particular, the Edwards Plateau clade of Eurycea likely
includes a derived instance of metamorphosis after several
million years of paedomorphosis (Bonett et al., 2014a,
2014b).
Obligately paedomorphic salamanders, which never naturally metamorphose and also do not completely transform
with thyroid hormone treatment, include all species in the
families Amphiumidae (Kobayashi and Gorbman, 1962),
Cryptobranchidae (Noble and Farris, 1929), Proteidae (Swin-
214
gle, 1922; Svob et al., 1973), and Sirenidae (Noble, 1924), as
well as the plethodontid species Eurycea rathbuni (Dundee,
1957) and E. wallacei (Dundee, 1962). Analyses of thyroid
hormone sensitivity in the obligately paedomorphic Proteidae showed that their thyroid hormone receptors are
functional and responsive to thyroid hormone (Safi et al.,
2006). Therefore, thyroid hormone control has probably
been decoupled from the process of transformation in
ancestrally metamorphic tissues (e.g., external gills; Safi et
al., 2006; Vlaeminck-Guillem et al., 2006).
Facultatively paedomorphic salamanders, which can naturally metamorphose under some conditions or can be
completely transformed by stimulation of the HPT or HPI
axes, occur at varying frequencies in several families:
Ambystomatidae (Collins, 1981; Semlitsch, 1985), Dicamptodontidae (Nussbaum and Clothier, 1973), Hynobiidae
(Sasaki, 1924), Salamandridae (Denoël et al., 2001), and
Plethodontidae (Dent and Kirby-Smith, 1963; Aran et al.,
2014; Bonett et al., unpubl.). Extensive genetic crosses and
experiments with species from the Ambystoma mexicanum/A.
tigrinum clade have identified multiple allelic variants that
differ in metamorphic timing and thyroxine sensitivity (Voss
and Shaffer, 1997; Voss and Smith, 2005; Voss et al., 2012;
recently reviewed by Johnson and Voss, 2013). These allelic
variants appear to be a primary determinant of metamorphosis in this group.
Approximately half of the populations in the western clade
of Eurycea tynerensis are paedomorphic (Emel and Bonett,
2011). Most populations include only a single life history
mode, and larval form paedomorphosis is associated with
chert streambed substrate (Bonett and Chippindale, 2006;
Emel and Bonett, 2011), which permits access to stable,
subsurface aquatic environments (Treglia et al., unpubl.).
There are a relatively small number of populations of E.
tynerensis that are known to exhibit both paedomorphosis
and metamorphosis. A few of the variable populations are
facultatively paedomorphic, based on the fact that paedomorphic females layed fertilized eggs in the lab, but then
completed metamorphosis in subsequent years. By comparison, other variable populations are composed of sympatric,
but genetically distinct, paedomorphic and metamorphic
populations (Bonett et al., unpubl.). In addition, some
paedomorphic populations of E. tynerensis have been maintained in the lab (at the University of Tulsa) for several years
without spontaneous metamorphosis, whereas larvae from
metamorphic populations raised under the same conditions
always metamorphose eventually. These observations suggest
that variability in metamorphic timing among populations is
not completely driven by environment. Larval E. tynerensis
from metamorphic populations are more sensitive to exogenous T3 treatment than larvae from paedomorphic populations, with respect to both thyroid hormone receptor (TRa or
TRb) upregulation and metamorphosis (Aran et al., 2014).
Furthermore, larvae from paedomorphic populations also
differ in their sensitivity to T3, with extremely reduced
sensitivity in some populations (Aran et al., 2014). This
indicates that both facultative and obligate paedomorphosis
may occur among populations of E. tynerensis occurring
within a very narrow geographic area in the western Ozark
Plateau.
Direct comparisons of circulating hormone levels between
paedomorphic and metamorphic plethodonids have not
been conducted. Analyses of the assimilation of radioiodine
by adult paedomorphic E. tynerensis showed that their
thyroid gland functioned normally, but they exhibited lower
Copeia 104, No. 1, 2016
levels of thyroid activity compared to other vertebrates
(Dundee and Gorbman, 1960). Using similar methods, Dent
and Kirby-Smith (1963) found that paedomorphic Gyrinophilus palleucus exhibited relatively low levels of thyroid activity,
but activity was considerably higher in individuals that
showed signs of spontaneous metamorphosis. These studies
documented general patterns of vertebrate endocrinology,
but it is difficult to evaluate their evolutionary significance
without direct comparisons to related metamorphic species.
INTEGRATIVE ENDOCRINE MODEL FOR SALAMANDER LIFE
HISTORY EVOLUTION
The extensive variation in metamorphic timing among
amphibians and the pervasive influence of thyroid hormone,
as well as changes in the thyroid gland itself, lead several
investigators to suggest that variations in the HPT axis may
be a driver of heterochronic evolution (Lynn, 1936, 1942;
Dent, 1942, 1968; Rose, 1995a, 1995b, 1996; Hanken et al.,
1997; Elinson, 2013; Johnson and Voss, 2013). Most recently,
Johnson and Voss (2013) developed a salamander life history
model (based largely on Ambystoma), which synthesized
commonly identified differences associated with paedomorphosis versus metamorphosis. This model showed that
compared to metamorphosis, paedomorphosis is associated
with greater habitat stability, lower TH responsiveness, and
larger body size (Johnson and Voss, 2013).
Here I provide an extension to previous TH-based life
history models by incorporating additional endocrine and
developmental processes that influence plasticity of metamorphic timing and maturation (Fig. 3). This integrated
model details where the three primary salamander life
history categories (direct development, biphasic, and paedomorphic) occur in this spectrum and shows how plasticity
can result in variation in their expression. It also shows how
similar life histories can be derived from different endocrine
and developmental processes. The model can be used as a
framework for both comparative endocrine analyses and
endocrine manipulation experiments.
Both TH responsiveness and the timing/amount of
endogenous TH release can influence the timing of metamorphosis; therefore, I considered both processes in this
model (Fig. 3A). Timing of metamorphosis is generally
positively correlated with TH release and negatively correlated with TH responsiveness. In other words, early TH release
and high TH responsiveness are related to early metamorphosis (e.g., direct development), and late TH release and low
TH responsiveness are related to late metamorphosis (e.g.,
long larval periods and paedomorphosis). The evolution of
genetic-based differences in ‘‘baseline’’ TH responsiveness/
release could move a lineage along the diagonal between
direct development and paedomorphosis, presumably always
passing through a biphasic life history.
At the same time, many other factors such as environmental stressors, temperature, and reproductive maturity
(described above) can significantly influence the timing of
amphibian metamorphosis for a given genotype (or species).
These factors typically work through molecular endocrine
processes to influence metamorphic timing, but the magnitude and direction of these effects can vary across species, life
histories, and stages (detailed above). Nevertheless, these
effects, individually or in combination, can push metamorphic timing to its theoretical minimum or maximum for a
given genotype. I depict this plasticity along the ‘‘baseline’’
Bonett—Endocrinology of salamander metamorphosis
215
Fig. 3. Endocrine model for salamander life history plasticity and evolution. On all three graphs (A–C), the diagonal depicts the relationship between
metamorphic timing and thyroid hormone (TH) sensitivity and release, as suggested by many authors. The dashed lines depict the degree of plasticity
in metamorphic timing for a given genotype. Horizontal lines indicate hatching (hat) and reproductive maturation (mat), and vertical lines bracket the
major life history categories (direct development, biphasic, paedomorphic) with respect to these events for a given genotype (position along the
diagonal). Facultative and obligate paedomorphosis are also depicted separately, due to the nuances of obligate paedomorphosis (see text). Colors
indicate the most common environment of the individual during the life cycle with respect to hatching and metamorphosis: egg (yellow), aquatic,
(blue), terrestrial (red), and plasticity between aquatic and terrestrial (purple). (A) Arrows between dashed lines indicate plasticity in metamorphosis
for a given genotype. (B) Down arrows in the right margin show the effects of acceleration of maturation and hatching on life history shifts (relative to
A). (C) Up arrows in the right margin show the effects of deceleration of maturation and hatching on life history shifts (relative to A).
TH diagonal of the model, and show how plasticity of a given
genotype could result in a life history shift (Fig. 3A).
The model also shows two primary events that transect the
ontogeny of nearly all salamanders: hatching from an
oviparous egg and maturation. Both of these events likely
have ‘‘baselines’’ for a given genotype (or species), but may
also be influenced by plasticity. Evolutionary or plasticitybased accelerations or decelerations of hatching and maturation could also result in major life history shifts (Fig. 3B, C).
The following describes pathways to transitions between
major life history categories, as well as permanent transitions
to paedomorphosis (obligate paedomorphosis) and the lack
of transitions in some clades.
Transitions between direct development and biphasic.—When
metamorphosis is temporally proximal to hatching, then
genetic changes to baseline TH responsiveness/release or
plasticity effects could shift a lineage from direct development to biphasic or vice versa through accelerations or
decelerations (Fig. 3A) in metamorphic timing. Also, genetic
or plasticity effects that cause hatching time to shift before
(Fig. 3B) or after (Fig. 3C) metamorphosis could also result in
transitions between these life histories.
Transitions between biphasic and paedomorphic.—When
metamorphosis is proximal to maturation, genetic changes
to baseline TH responsiveness/release or plasticity effects
could shift a lineage from biphasic to facultatively paedomorphic, or the reverse. The transitions could occur through
deceleration (neoteny) or acceleration of metamorphic
timing (somatic morphogenesis). These transitions can also
be achieved by acceleration (progenesis; Fig. 3B) or deceleration (Fig. 3C) of maturation relative to metamorphosis
(Alberch et al., 1979). Both mechanisms have been demonstrated in salamanders, sometimes even within a single
species (Denöel and Joly, 2000). The only phylogenetic test of
these alternatives (neoteny vs. progenesis) was performed on
Edwards Plateau Eurycea, where paedomorphosis was likely
derived through neoteny (Bonett et al., 2014b). However,
given the extensive crosstalk between endocrine pathways,
these processes may not be completely independent in all
cases. For example, the loss of thyroid function should cause
a truncation of somatic development (neoteny), but may
simultaneously speed up reproductive development (progenesis) as shown in frogs and salamanders (Gray and Janssens,
1990; Wakahara, 1994; Hogan et al., 2008; see discussion of
reproductive hormones above).
Transitions to obligate paedomorphosis.—Obligate paedomorphs are likely derived from facultatively paedomorphic
ancestors, but are instead infinitely paedomorphic (Fig. 3).
They present an unusual case and may completely deviate
from the model in several ways. Once TH is decoupled from
tissue transformation, the level or timing of TH release does
not need to be low or late to prevent metamorphosis. In
other words, in terms of transformation, the TH responsiveness of the decoupled tissues (e.g., external gills of Necturus;
Safi et al., 2006; Vlaeminck-Guillem et al., 2006) will be
nonexistent, so the level and ontogenetic release time of
circulating TH could fall anywhere on the spectrum compared to other salamander life histories. It should be noted
that some, but not all, tissues remain responsive in some
216
obligate paedomorphs, such as E. rathbuni and E. wallacei
(Dundee, 1957, 1962), so the model should be specified as a
reference to either metamorphic timing of a particular tissue
or a whole animal. Another unique attribute of obligate
paedomorphosis is that since metamorphosis never occurs,
environmental effects (e.g., via ‘‘stress hormones’’) on the
non-responsive tissues should also be nonexistent. Environmental factors could, however, still affect tissues that
metamorphose, such as the external gills of Amphiuma that
resorb just after hatching.
Lack of certain transitions in some clades.—Larval form
paedomorphosis evolved several times among the spelerpines, but is completely unknown for some spelerpine clades,
as well as Desmognathus and Hemidactylium (Fig. 1). This is
surprising given that many of these biphasic clades have
larvae that develop in permanently aquatic habitats. Considering the origins of their biphasic life histories in the
context of the life history model presented here may provide
some clarity. Phylogenetic reconstructions show that biphasic Desmognathus and Hemidactylium (as well as spelerpines)
are independently derived from direct developing ancestors
(Chippindale et al., 2004; Bonett et al., 2014b). This means
that they were derived from ancestors with highly accelerated metamorphic timing, and even though they currently
exhibit free-living larval forms, the ontogenetic timing
between metamorphosis and maturation may be too long
to be bypassed through plasticity of either event. For
example, Hemidactylium and many Desmognathus have larval
periods of less than several months, but take minimally two
years to reach maturation, so plasticity in these events would
have to be collectively shifted by more than a year for
maturation to precede metamorphosis. It is also possible that
a developmental mechanism prevents maturation from
occurring without metamorphosis, such as in frogs. The
Desmognathus quadramaculatus/marmoratus clade is a unique
example that may be an informative test case. These species
have long larval periods (two to three years) and minimally
mature one year later, but always metamorphose even
though D. marmoratus remain permanently aquatic as adults.
Therefore, D. marmoratus accomplishes ‘‘ecological paedomorphosis’’ by retaining their aquatic larval ecology without
a complete truncation of somatic development.
REQUIREMENTS FOR SYNTHESIS AND FUTURE RESEARCH
Much of the data on the timing of hatching, metamorphosis,
and maturation for plethodontids was collected from the
1950s through the 1980s. However, our knowledge of
phylogenetic relationships and species diversity were still
rudimentary at the time, limiting evolutionary interpretations of life history data. Tremendous progress has been
made on plethodontid phylogeny and species diversity over
the last decade, but now much more life history data are
needed to make accurate and comprehensive estimates of the
evolution of developmental timing. An added complication
is the potential effect of plasticity in developmental timing,
which must be separated from genetic-based, geographic
variation. Therefore, measurements of salamanders raised
under common laboratory conditions are required to obtain
baseline estimates of developmental timing.
Several plethodontids have been treated with T3 or T4
(Kezer, 1952; Dundee, 1957, 1962; Dent and Kirby-Smith,
1963; Rose, 1996; Hickerson et al., 2005; Aran et al., 2014),
but more experiments are needed that include a common
Copeia 104, No. 1, 2016
dose and duration to make comparative evaluations of
sensitivity. There have been relatively few analyses of thyroid
gland activity (Dent, 1942; Dent and Lynn, 1958; Dundee
and Gorbman, 1960; Dent and Kirby-Smith, 1963), and no
studies have directly compared related species that exhibit
different life histories. Also, there has been little published
data on transcriptional differences among plethodontid life
histories (Aran et al., 2014; Jackson et al., unpubl.). Such
studies are critical for establishing endocrine correlates for
metamorphic timing (e.g., TH responsiveness and TH
release).
Similarly, several studies have examined a diversity of
environmental effects on metamorphic timing of different
species (Noble and Evans, 1932; Voss, 1993; Beachy, 1995;
Camp et al., 2000; Freeman and Bruce, 2001; Hickerson et al.,
2005), but there is very little data on the effects of stress
hormones on metamorphic timing of plethodontids. Studies
are needed to systematically compare individual factors
among species to establish the degree of the variance around
baseline metamorphic timing. In particular, it will be
informative to test species with baseline metamorphic timing
(or TH sensitivity) that are near the transitions of life history
categories. This will help to establish the degree of plasticity
required for major life history transitions and determine
whether some taxa are unable to make such transitions.
Finally, molecular endocrine experiments that examine the
ontogeny of hormone release and the effects of hormones on
transcription in target tissues are needed to determine
whether patterns of laboratory models are generalizable
across plethodontids. It will also ultimately permit phylogenetic tests of how the evolution of endocrine pathways have
influenced plethodontid life histories.
ACKNOWLEDGMENTS
I would like to thank Matt Gifford for co-organizing the
Plethodontid Conference and co-editing these proceedings. I
would also like to thank C. Beachy, S. Martin, J. Phillips, M.
Steffen, and A. Trujano for their comments on this review
and fruitful discussions of the topic. This study was in part
funded by the National Science Foundation (DEB 1050322
and OK EPSCoR IIA-1301789).
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