Evolution, 56(12), 2002, pp.

2484–2498

THE EVOLUTION OF DEVELOPMENT: TWO PORTRAITS OF SKULL OSSIFICATION IN

PIPOID FROGS
JENNIFER YEH1
Section of Integrative Biology, School of Biological Sciences, The University of Texas at Austin, Austin, TX 78712

Abstract. Development creates morphology, and the study of developmental processes has repeatedly shed light on
patterns of morphological evolution. However, development itself evolves as well, often concomitantly with changes
in life history or in morphology. In this paper, two approaches are used to examine the evolution of skull development
in pipoid frogs. Pipoids have highly unusual morphologies and life histories compared to other frogs, and their
development also proves to be remarkable. First, a phylogenetic examination of skull bone ossification sequences
reveals that jaw ossification occurs significantly earlier in pipoids than in other frogs; this represents a reversal to the
primitive vertebrate condition. Early jaw ossification in pipoids is hypothesized to result from the absence of certain
larval specializations possessed by other frogs, combined with unusual larval feeding behaviors. Second, thin-plate
spline morphometric studies of ontogenetic shape change reveal important differences between pipoid skull devel-
opment and that of other frogs. In the course of frog evolution, there has been a shift away from salamander-like
patterns of ontogenetic shape change. The pipoids represent the culmination of this trend, and their morphologies are
highly derived in numerous respects. This study represents the first detailed examination of the evolution of skull
development in a diverse vertebrate clade within a phylogenetic framework. It is also the first study to examine
ossification sequences across vertebrates, and the first to use thin-plate spline morphometrics to quantitatively describe
ontogenetic trajectories.

Key words. Evolution of development, ossification sequences, pipoid frogs, skull ossification, thin-plate spline mor-
phometrics.

Received January 10, 2002. Accepted August 28, 2002.

Developmental studies have offered numerous insights into
morphological evolution (Alberch and Alberch 1981; Al-
berch 1983; Wake et al. 1983; Hanken 1984; Alberch and
Gale 1985; Trueb and Alberch 1985; Wake 1986; Shubin et
al. 1995; Mabee and Trendler 1996; Smith 1997; Nunn and
Smith 1998). In some cases, dramatic changes in morphology
result from relatively simple developmental mechanisms. The
most familiar of these is heterochrony, a change in the relative
timing of developmental events (Gould 1977; Alberch et al.
1979; McKinney and McNamara 1991).
Significant changes in development often occur concom-
ittantly with the evolution of adult morphology, for example,
see Burke (1989) on the development of the turtle carapace,
a structure homologous to the ribs and vertebrae of other
vertebrates. In other cases, evolution of development is linked
to life history evolution and may not be obviously associated
with morphological change in the adult. The evolution of
direct development in diverse animal taxa, including uro-
chordates (Swalla and Jeffery 1996), echinoderms (Wray and
Raff 1990), mollusks (Duda and Palumbi 1999), salamanders
(Wake and Hanken 1996), and frogs (Elinson 1990, 2001;
Callery et al. 2001), is an obvious example. Distinct life-
history strategies in marsupial and placental mammals also
explain numerous developmental differences between the two
groups (Smith 1997; Nunn and Smith 1998).
Here, I examine the evolution of skull development in
pipoid frogs, a clade exceptional among anurans in both mor-
phology and life history. The skull represents an ideal subject
for development and evolution studies in vertebrates because
it is both osteologically complex and the site of extensive
evolution.
1 Present address: University of California at San Francisco,
Comprehensive Cancer Center, Box 0128, San Francisco, CA
94143-0128; E-mail: jyeh@post.harvard.edu.
2484
q 2002 The Society for the Study of Evolution. All rights reserved.
The Pipoid Frogs
The anuran clade Pipoidea includes two extant families,
Pipidae and Rhinophrynidae. (Fossil pipoid groups, reviewed
in Báez and Trueb [1997], are not considered in this study.)
Pipid frogs remain highly aquatic throughout life, forgoing
the more typical anuran life history of aquatic tadpoles fol-
lowed by terrestrial or semiterrestrial adults. This is related
to many novel features of morphology and life history, in-
cluding depressed shape, laterally placed limbs, dorsally
placed eyes, lack of a tongue (associated with novel food-
capture strategies in an aquatic environment), an unusual
somersaulting courtship routine, and direct development of
eggs into miniature froglets on the female dorsum in cele-
brated species such as Pipa pipa, the Surinam Toad (Duell-
man and Trueb 1986). Rhinophrynidae contains a single ex-
tant species, Rhinophrynus dorsalis, which also occupies an
unusual frog niche in its fossoriality (Duellman and Trueb
1986).
Pipoid frogs make an irresistible study group for several
reasons. First, they possess a derived, unusual skull mor-
phology that is arguably the most deviant among anurans—
the frontoparietal is single rather than paired and overlaps
the robust nasals; the exoccipitals are not medially fused; the
mentomeckelians, palatines, and quadratojugals are absent;
the pars facilias and pars dentalis of the maxilla are reduced
and absent, respectively; the anterior ramus of the pterygoid
invests the maxilla dorsally rather than laterally; the posterior
and medial rami of the pterygoid are expanded to form a
plate that underlies the otic capsule; and the septomaxillae
are enlarged and flat (see Trueb and Cannatella 1986; Can-
natella and Trueb 1988a; Cannatella and de Sá 1993; Trueb
et al. 2000). It is of interest to examine whether attaining
this derived morphology involves significant changes in os-
teological development. Second, phylogenetic relationships
 PIPOID SKULL OSSIFICATION
FIG. 1. Alternative hypotheses for phylogenetic relationships among frog families. Ford and Cannatella (1993) is based on morphological
and life-history characters, Hay et al. (1995) is based on molecular data, and Maglia et al. (2001) is based on adult and larval morphological
characters. Note the differing placement of pipoids.
among pipoids are well studied (Cannatella and Trueb 1988a,
b; de Sá and Hillis 1990; Cannatella and de Sá 1993; Báez
and Trueb 1997), and hypotheses also exist for relationships
among anuran families (Ford and Cannatella 1993; Hay et
al. 1995; Ruvinsky and Maxson 1996; Maglia et al. 2001)
(See Fig. 1). This allows pipoid skull development to be
considered within an evolutionary framework. Finally,
unique attributes of pipoid biology suggest that pipoids may
be less subject to the severe constraints imposed by the speed
and complexity of anuran metamorphosis. Because skull os-
sification occurs largely during metamorphosis and is inti-
mately related to that process, this suggestion is of particular
relevance and will be discussed in detail in the following
section.
Amphibian Metamorphosis and the Exceptional
Nature of Pipoids
A biphasic life history, consisting of larval and adult stages
linked by metamorphosis, is probably primitive in amphib-
ians (Duellman and Trueb 1986). Metamorphosis in sala-
manders is hypothesized to resemble the primitive amphibian
condition—it links larvae and adults with only modest mor-
phological disparity and is gradual and unspectacular (Was-
sersug 1975). In frogs, however, larvae and adults have adapt-
ed to divergent ecological niches, with both stages deviating
sharply from more salamander-like ancestral morphologies.
Whereas adult frogs evolved a short, tailless body in con-
2485
junction with saltatory locomotion, anuran larvae evolved a
variety of morphological specializations related to exploiting
rich, but ephemeral, food resources (Wassersug 1975). An-
uran metamorphosis is a complex, spectacular event involv-
ing the transformation of entire suites of larval specializations
as individuals transition from larval to adult modes of feeding
(tadpoles are usually herbivorous and detritivorous; adult
frogs are predatory insectivores and carnivores) and loco-
motion (tadpoles swim using a well-developed tail; adults
are tailless saltators). Not surprisingly, the dramatic trans-
formation required results in maladapted metamorphics that
experience high mortality (Wassersug 1975). Consequently,
selection has produced a rapid metamorphosis in the group.
Anuran metamorphosis is therefore typically characterized
by large scope and brief duration.
Pipoid metamorphosis differs from that of most typical
frogs in several respects (Cannatella and de Sá 1993; Can-
natella 1999). First, pipoid tadpoles lack certain morpholog-
ical specializations possessed by other anuran larvae. Kera-
tinized beaks and denticles of the mouth are absent, and sev-
eral specialized larval skull cartilages exist only in reduced
conditions (Sokol 1975, 1977; Cannatella 1999; Swart and
de Sá 1999). This decreases the necessary scope of jaw meta-
morphosis in pipoids. Furthermore, larval and adult jaws are
functionally adapted to the same task in pipids. Instead of
tongue flipping like most adult frogs, the tongueless adult
pipids use pumping movements to suck in food and water
2486 JENNIFER YEH

(Duellman and Trueb 1986). These are essentially the same
movements used by tadpoles in feeding. That pipoid meta-
morphosis is less extreme than that of most frog species has
been suggested by other morphological studies; Wassersug
and Hoff (1982) used the degree of rotation of the palato-
quadrate cartilage of the upper jaw to quantify the extremity
of metamorphosis in amphibians. By this measure, pipoids
have a more modest metamorphosis than most other frogs.
In light of the unusual nature of pipoid metamorphosis, a
major question I address is whether the absence of morpho-
logical specializations in pipoid larvae, particularly in the
jaw structures, is associated with unusual patterns of skull
ossification and development.
Strategies for Studying the Evolution of Skull Development
I employ two methods for studying skull development in
pipoids: the evolution of ossification sequences through phy-
logenetic analysis, and the ontogeny of shape change using
thin-plate spline morphometrics.
Ossification sequences describe the order in which bones
begin to ossify. Much information is inevitably lost when
skeletal development is summarized by order of ossification
alone, for example, patterns of bone growth once ossification
has begun. However, ossification-sequence data nonetheless
allow for comparisons of developmentally important events
among diverse species. Ossification sequences may also pro-
vide clues to functional requirements; among fishes, for ex-
ample, the timing of ossification coincides broadly with func-
tional need, with bones related to respiration and feeding
being the first to ossify (Mabee and Trendler 1996; Adriaens
and Verrae 1998; Wagemans and Vandewalle 2001). Differ-
ences in developmental sequences also reveal adaptive dif-
ferences between species or offer insight into critical links
among developmental events (Smith 1997, 2001, 2002).
Thin-plate spline morphometrics (TPS) offers a different
view of the dynamics of skull development. Using landmark
data, TPS considers shape change independent of size, lo-
cation, and orientation (Bookstein 1991). The method also
provides an effective model for visualizing shape change us-
ing deformations reminiscent of D’Arcy Thompson’s classic
transformation grids (Thompson 1917). Most importantly,
TPS describes shape using independent quantitative variables
to which conventional statistical strategies can be applied.
However, the TPS method is constrained by the requirement
that homologous landmarks be identified on all specimens in
the analysis. For studies of osteological development, this
necessitates a trade-off between examining a large number
of bones and including a wide range of developmental stages.
To study a large number of bones, for example, I would be
restricted to examining only ontogenetically advanced spec-
imens that have ossified all these bones. In addition, in this
study, interspecific variation in ossification sequence made
this constraint particularly serious.
Both ossification sequence analysis and thin-plate spline
morphometrics yield results interpretable without reference to
external standards such as age or size, which are problematic
measures of amphibian development. Age, the professed ideal
independent variable of heterochronic analyses (Alberch et al.
1979; McKinney and McNamara 1991), is inappropriate for
amphibians because rates of development are affected by ex-
ternal factors such as temperature and population density
(Duellman and Trueb 1986). Indeed, age seems inappropriate
for developmental studies in many taxa (Hall and Miyake
1995). Size, a common stand-in for age, is also inappropriate
in frogs because snout-vent length changes rather inconsis-
tently during anuran metamorphosis (this study, see electronic
appendices at http://geocities.com/jyevolution/pipoids). De-
velopmental stages (e.g., Gosner [1960]; Nieuwkoop-Faber
[1967]) provide a basis for comparison across frog species and
can be employed as an external reference frame. However,
staging criteria cannot be applied to nonanurans or to direct-
developing anurans, which lack the morphological traits on
which these systems rely.
Although previous studies have used developmental se-
quences (Mabee and Trendler 1996; Velhagen 1997; Smith
1997, 2000, 2001) or TPS (Fink and Zelditch 1995; Reilly
and Altig 1996; Djorovic and Kalezı́c 2000) to examine on-
togeny, this is the first to use both methods to study the
evolution of development in a diverse vertebrate clade within
a phylogenetic framework. It is also the first study to examine
ossification sequences across all vertebrates, as well as the
first to use TPS to quantitatively describe ontogenetic tra-
jectories.
METHODS
Ossification Sequence Evolution
Ossification sequences for anurans and vertebrate out-
groups were obtained from the literature or constructed from
cleared-and-stained developmental series (Fig. 2; specimens
examined listed at http://geocities.com/jyevolution/pipoids).
To obtain ossification sequences from developmental series,
presence or absence of sixteen skull bones was coded for
each specimen: the angulosplenial, dentary, exoccipital, fron-
toparietal, maxilla, mentomeckelian, nasal, parasphenoid,
premaxilla, prootic, pterygoid, quadratojugal, septomaxilla,
sphenethmoid, squamosal, and vomer. (Note that, although
cleared-and-stained series are widely used in ossification

→
FIG. 2. Species included in the ossification sequence study. Phylogenetic relationships illustrated are based on the Ford and Cannatella
(1993) tree for anurans, but analyses were conducted using all alternate phylogenies shown in Figure 1. Ossification sequences are from
the following sources: ANURA: Ascaphus truei (this study); Bombina orientalis (Hanken and Hall 1984; Maglia and Púgener 1998);
Alytes obstetricans (this study); Discoglossus sardus (Púgener and Maglia 1997); Scaphiopus intermontanus (Hall and Larsen 1998); Spea
bombifrons (Wiens 1989); Pelobates fuscus (Smirnov 1992); Rhinophrynus dorsalis, Xenopus borealis, Silurana tropicalis (Trueb 1985);
Xenopus laevis (Trueb and Hanken 1992); Hymenochirus boettgeri (this study); Pipa myersi (this study); Pipa pipa (Trueb et al. 2000);
Ceratophrys cornuta (Wild 1997); Chacophrys pierotti (Wild 1999); Lepidobatrachus laevis (Ruibal and Thomas 1988); Eleutherodactylus
coqui (Hanken et al. 1992); Eleutherodactylus nubicola (Trueb 1985); Bufo boreas (Gaudin 1978); Bufo bufo and Bufo calamita (Dunlap
and Sanchiz 1996); Hyla lanciformis (de Sá 1988); Hyla regilla (Gaudin 1973); Osteopilus septentrionalis, Smilisca baudini, Triprion
 PIPOID SKULL OSSIFICATION 2487

petasatus (Trueb 1985); Pseudacris nigrita (Stokely and List 1954); Uperoleia trachyderma, Uperoleia lithomoda, Uperoleia laevigata,
Crinia signifera, Pseudophryne bibroni (Davies 1989); Pyxicephalus adspersus (Haas 1999; Sheil 1999); Rana aurora, Rana cascadae,
Rana pretiosa (Trueb 1985); Rana pipiens (Kemp and Hoyt 1969); Rana temporaria (Erdmann 1933); and Hamptophryne boliviana (de
Sá and Trueb 1991). CAUDATA: Ambystoma maculatum (Moore 1989); Ambystoma texanum (Bonebrake and Brandon 1971); Triturus
vulgaris (Erdmann 1933); Notophthalmus viridescens (Reilly 1986); and Rhyacotriton variegatus (Worthington and Wake 1971). GYM-
NOPHIONA: Dermophis mexicanus (Wake and Hanken 1982). REPTILIA: Chelydra serpentina (Rieppel 1993a); Alligator mississippiensis
(Rieppel 1993b); Lacerta agilis (Rieppel 1994). MAMMALIA: Monodelphis domestica, Macropus eugenii, Perameles nasuta, Dasyurus
viverrinus, Felis domestica, Tupaia javanica, Mus musculus, Manis javanica, Sus scrofa, Tarsius spectrum (Nunn and Smith 1998); Meriones
unguiculatus (Yukawa et al. 1999). TELEOSTEI: Barbus barbus (Vandewalle et al. 1992); Danio rerio (Mabee et al. 2000); Oryzias
latipes (Langille and Hall 1987); Clarias gariepinus (Adriaens and Verraes 1998); Betta splendens (Mabee and Trendler 1996); Xiphophorus
helleri, Xiphophorus maculatus, Poeciliopsis occidentalis, Poecilia latipinna, Poecilia reticula (Strauss 1990).
2488 JENNIFER YEH
studies, Hanken and Hall [1988] showed that bones may not
stain until some time after they have begun to ossify.) For
each skull element, the total number of specimens exhibiting
ossification of that bone was tallied. Bones were then ranked
from presence in the largest number of specimens (first bone
to ossify) to presence in the fewest (last bone to ossify). This
yields a ‘‘majority-rule’’ consensus ossification sequence,
that is, where intraspecific variation in sequence exists, the
ordering supported by a majority of specimens was employed.
Bones not ossified among available specimens but known to
exist in adults were assigned the final rank. Ossification se-
quences were not always fully resolved.
Identifying homologous bones in vertebrate outgroups pre-
sented a few difficulties. First, frogs and salamanders possess
different complements of mandibular bones, with salaman-
ders lacking a clear homologue to the frog angulosplenial
(Trueb 1993). The salamander prearticular was coded in its
place because of similarity in position and morphology. In
addition, nonanurans are generally characterized by separate
frontals and parietals, in contrast to the fused frontoparietals
of frogs. In these species, the first of the frontal or parietal
to ossify was coded in place of the frontoparietal. Bones
present in anurans but absent in other vertebrates were coded
as absent. Bones present in outgroups but absent in frogs
were not considered.
Changes in ossification sequence
Ossification-sequence evolution was studied using the
method of Smith (1997) and Velhagen (1997). For each spe-
cies, the ossification sequence was used to construct a large
dataset with all possible pairs of skull bones as separate,
although nonindependent, characters. For sixteen skull bones,
there are 120 possible bone pairs. Each pair of skull bones
(Bone A–Bone B) was assigned one of four character states:
(1) Bone A begins to ossify prior to Bone B; (2) Bone B
begins to ossify prior to Bone A; (3) available data do not
permit resolution of ossification order; (4) either Bone A or
Bone B is absent. In my first coding method, States 3 and 4
were treated as missing data. Other coding methods were
employed, for example, State 3 was treated as intermediate
between States 1 and 2, and State 4 was treated as missing
data. However, coding method did not affect substantive con-
clusions, and only results from the first set of assumptions
are discussed. Character evolution was analyzed using alter-
nate anuran phylogenetic hypotheses (Fig. 1) and PAUP*,
version 4.0 (Swofford 2000).
Ossification events by developmental stage
I examined skull bone ossification by Gosner (1960) de-
velopmental stage in anurans, considering all anurans in Fig-
ure 2 for which staging information was available. In light
of the introductory observations on pipoid metamorphosis, I
was interested in whether: (1) the average developmental
stage at which bones ossify is earlier in pipoids than non-
pipoids; (2) ossification occurs more gradually in pipoids,
that is, over a larger number of developmental stages; (3)
pipoids begin skull ossification earlier; or (4) pipoids com-
plete skull ossification earlier than nonpipoids.
Thin-Plate Spline Morphometrics
Thin-plate spline morphometrics uses landmark data to ex-
amine shape change independent of size, location, and ori-
entation (Bookstein 1991). The analysis involves decompos-
ing shape into orthogonal ‘‘principal warps.’’ Each specimen
has a set of principal-warp coefficients that describe its de-
viation from a generalized orthogonal least-square Procrustes
consensus configuration. Although principal warps are math-
ematical constructs that have no necessary biological rele-
vance, the ‘‘relative warps’’ that result from a principal-com-
ponents analysis of the principal-warp coefficients describe
the primary axes along which shape change occurs (Bookstein
1991). Thin-plate spline analyses were conducted on devel-
opmental series of the salamander Ambystoma maculatum and
the frogs Ascaphus truei, Bombina orientalis, Alytes obste-
tricans, Spea bombifrons, Xenopus laevis, Rhinophrynus dor-
salis, Hymenochirus boettgeri, Pipa myersi, Pipa pipa, and
Hyla lanciformis (specimens examined listed at http://
geocities.com/jyevolution/pipoids).
Multiple criteria contributed to landmark choice. First, be-
cause complete sets of homologous landmarks must be avail-
able in all specimens, I tried to include a substantial portion
of the skull while excluding late-ossifying bones that would
limit the range of ontogeny studied. Second, to facilitate in-
terpretation of shape change, most landmarks lie on the
boundaries of skull bones. Midline points (between paired
premaxillae, Meckel’s cartilages, or occipital condyles) were
included as references.
Three separate analyses were conducted. ‘‘Dorsal Skull I’’
included 14 landmarks (Fig. 3a). ‘‘Dorsal Skull II’’ is iden-
tical to ‘‘Dorsal Skull I’’ except that three nasal landmarks
were excluded; because Ascaphus truei and Ambystoma ma-
culatum ossify nasals late in ontogeny, this exclusion per-
mitted less advanced specimens to be considered. ‘‘Lower
Jaw’’ included seven ventral and mandibular landmarks (Fig.
3b). Again, data for the frog angulosplenial were collected
from the salamander prearticular.
Landmark points were drawn using an Olympus (Melville,
NY) SZH microscope with attached camera lucida. Dorsal
landmarks were taken from the left side of the skull, ventral
landmarks from the right. A few specimens exhibited damage
to the preferred side of the skull; for these, data were collected
from the undamaged side and mathematically ‘‘flipped.’’
Drawings were scanned, and landmark coordinates quantified
using NIH Image, version 1.61 (Rasband 1996). Relative-warp
analyses were performed using TPSRELW (Rohlf 1999).
Scatterplots of relative-warp scores were examined to com-
pare ontogenetic trajectories among species. Deformation vi-
sualizations allowed assessment of shape change during de-
velopment. Relative warps were also regressed on Nieuwkoop-
Faber developmental stage (Nieuwkoop and Faber 1967). Note,
however, that Hanken and Hall (1984) report that cranial mor-
phology correlates poorly with the external features of meta-
morphosis used to assign developmental stages to specimens.
RESULTS
Ossification-Sequence Evolution
Changes in ossification sequence
The ossification-sequence study included data from most
major vertebrate groups, including all three amphibian orders,
 PIPOID SKULL OSSIFICATION
FIG. 3. Landmark points for thin-plate spline analysis. (A) ‘‘Dorsal
Skull I’’ landmarks: 1. The point between the anteromedial ends
of paired premaxillae. 2. Anterior tip of maxilla. 3. Posterior tip of
maxilla. 4. Medial tip of nasal. 5. Anterolateral corner of nasal. 6.
Posterolateral corner of nasal. 7. Anterior midline point of fron-
toparietal. 8. Anterolateral corner of frontoparietal. 9. Lateral corner
of frontoparietal. 10. Posterolateral corner of frontoparietal. 11.
Point between paired occipital condyles. 12. Anterior tip of otic
capsule. 13. Lateral tip of otic capsule. 14. Posterior tip of otic
capsule. ‘‘Dorsal Skull II’’ is identical except that landmarks 4, 5,
and 6 were eliminated. (B) ‘‘Lower Jaw’’ landmarks: 1. Anterior
symphysis of Meckel’s cartilages. 2. Anterior tip of dentary (outer
edge). 3. Posterior tip of dentary (outer edge). 4. Anterior tip of
angulosplenial (inner edge). 5. Posterior tip of angulosplenial (inner
edge). 6. Posterior tip of Meckel’s cartilage. 7. Point between paired
occipital condyles.
reptiles, mammals, and bony fishes (Fig. 2; data available in
electronic appendices at http://geocities.com/jyevolution/
pipoids). Previously unpublished amphibian ossification se-
quences, for Ascaphus truei, Alytes obstetricans, Hymeno-
chirus boettgeri, and Pipa myersi, are presented in Table 1.
Vertebrates as a whole show significant variation in ossifi-
cation sequence, although ossification usually begins with
dermatocranial elements; neurocranial and splanchnocranial
elements generally ossify later (see citations in Fig. 2 for the
following descriptions). Among bony fishes, ossification se-
quences reflect functional requirements, with skull elements
related to respiration and feeding ossifying earliest. In most
species, these include the operculum, jaw elements, and par-
asphenoid (the parasphenoid forms the base of the braincase
2489
and protects the brain during deglutition). In species with
unusual feeding strategies, however, there are deviations
from this pattern—in Danio rerio, bones associated with head
lift and hyoid depression ossify earliest (Mabee et al. 2000;
for further examples, see Adriaens and Verraes 1998 and
references therein). Mammals as a group exhibit only slight
variation in skull ossification sequence. Jaw bones—the den-
tary, premaxilla, and maxilla—are invariably the earliest to
ossify, and are followed by other dermal bones such as the
frontal, parietal, jugal, and squamosal. Endochondral ele-
ments typically ossify last, although in a few taxa some en-
dochondral bones precede some dermal bones. The reptiles
examined vary more substantially in ossification sequence.
The lizard Lacerta agilis ossifies the pterygoid, palatine, ju-
gal, prearticular, and surangular first. These are followed by
other jaw elements including the maxilla, premaxilla, and
dentary. Other dermal bones, then neurocranial and splanch-
nocranial bones, follow. A few dermal bones, such as the
lacrimal, ossify very late. The first bone to ossify in the
crocodilian Alligator mississippiensis is the pterygoid. It is
followed by jaw elements such as the maxilla, angular, pre-
maxilla, dentary, and surangular, and then by other derma-
tocranial elements such as the jugal, postorbital, quadrato-
jugal, lacrimal, and frontal. Endochondral elements and a few
dermal elements, including the nasal and parietal, ossify to-
wards the end. The turtle Chelydra serpentina ossifies der-
matocranial elements, including the postorbital, squamosal,
parietal, and pterygoid, first. These are followed by jaw el-
ements such as the prearticular, dentary, premaxilla, and max-
illa, and then by other dermatocranial elements, as well as
neurocranial and splanchnocranial bones. In salamanders,
palatal elements such as the vomer and palatine ossify early
along with jaw elements such as the dentary, prearticular,
coronoid, and premaxilla. Another jaw element, the maxilla,
ossifies significantly later. The nasal and septomaxilla are
among the final bones to ossify. In caecilians, mandibular
elements are among the earliest to ossify. These are followed
by dermal roofing and palatal bones as well as upper jaw
elements. Endochondral elements generally ossify towards
the end. Frogs exhibit considerable variation in ossification
sequence. In general, most species begin ossification with the
parasphenoid, frontoparietal, and exoccipital. These are fol-
lowed by the jaw elements, nasal, septomaxilla, squamosal,
and vomer. The final bones to ossify are usually the men-
tomeckelians, columella, quadratojugal, palatines, and sphen-
ethmoid. However, several species deviate from this general
description.
Evolution in the timing of jaw ossification is one of the
most interesting aspects of ossification sequence evolution
in pipoid frogs and their vertebrate outgroups. My analysis
shows that the primitive condition in vertebrates is for jaw
ossification to occur early, with jaw elements being among
the first cranial bones to ossify. Early jaw ossification is also
primitive in Amphibia. Within Amphibia, early jaw ossifi-
cation is retained in salamanders and the single caecilian
species studied. However, delayed jaw ossification is a syn-
apomorphy of Anura under all phylogenetic hypotheses con-
sidered.
Delayed jaw ossification is subsequently reversed during
the course of pipoid evolution. Under the Ford and Cannatella
2490 JENNIFER YEH

TABLE 1. New anuran ossification sequences. Order of ossification of bracketed elements could not be determined from data available.

Ascaphus Alytes Hymenochirus

truei obstetricans boettgeri Pipa myersi

 frontoparietal 
exocciptal    angulosplenial 
 parasphenoid   exoccipital 
frontoparietal frontoparietal  frontoparietal 
parasphenoid exoccipital  
nasal exoccipital  parasphenoid 
septomaxilla angulosplenial
vomer  premaxilla   
  paraspheoid
 maxilla 
angulosplenial  septomaxilla  maxilla  prootic 
premaxilla
vomer  dentary 
dentary
   nasal 
squamosal  angulosplenial  nasal  
maxilla  dentary   pterygoid   premaxilla 
 maxilla  squamosal  

 pterygoid 
  prootic   premaxilla  

dentary
septomaxilla


 sphenethmoid   squamosal   
 sphenethmoid  pterygoid
 mentomeckelian   pterygoid  septomaxilla squamosal
   
 prootic   quadratojugal  prootic sphenethmoid

nasal  mentomeckelian 
 
quadratojugal  sphenethmoid 

(1993) and Hay et al. (1995) phylogenetic hypotheses, ac-
celerated jaw ossification, involving the angulosplenial, den-
tary, and maxilla, evolved on branches leading to the clades
Pipoidea, Pipidae, and Pipinae, both pipine genera Hymen-
ochirus and Pipa, and Pipa pipa. Under the Maglia et al.
(2001) hypothesis, acceleration characterizes only the Pipinae
and pipine clades (Fig. 4). Some or all pipoids have therefore
partially reverted to the primitive vertebrate condition of ear-
ly jaw ossification, with the direct-developing Pipa pipa rep-
resenting the most extreme instance of this trend. Although
pipoids are almost unique among frogs in this respect, re-
version to early jaw ossification also characterizes the lep-
todactylid genus Eleutherodactylus.
Ossification events by developmental stage
Examination of ossification patterns by Gosner Stage in-
dicated (Table 2): (1) Skull bones ossify at about the same
time in pipoids as in other anurans—pipoid values for mean
stage of ossification fall well within the range of the other
species. (2) One pipid, Hymenochirus boettgeri, ossifies more
gradually than other frogs—the standard deviation for ossi-
fication stage is higher than in other species, and the number
of developmental stages over which ossification occurs is also
high. However, neither Xenopus laevis nor Pipa myersi ex-
hibits a similar trend. (Note, however, that Pipa myersi spec-
imens earlier than Stage 35 were unavailable, and the first
four bones, described as ossifying at Stage 35, might have
begun to ossify earlier.) (3) Skull ossification begins earlier
(Stage 29) in Hymenochirus than in most other species
(though some species of Rana also begin to ossify very early).
(4) Xenopus laevis and Pipa myersi complete ossification ear-
lier (Stage 44) than most other anurans sampled—most an-
urans have some bones which ossify postmetamorphically
(Stage 46). Rana temporaria, which completes ossification at
Stage 29, is a notable exception. In general, these traits are
consistent with hypotheses of accelerated or more gradual
ossification permitted by a relaxation of the constraints on
metamorphosis.

Thin-Plate Spline Morphometrics

FIG. 4. Jaw bone ossification is accelerated relative to other skull
elements during pipoid evolution. Under Ford and Cannatella
(1993) and Hay et al. (1995) phylogenetic hypotheses, all pipoids
are characterized by early jaw ossification. Several clades within
Pipoidea experienced further acceleration. Under Maglia et al.
(2001), only the pipines (Pipa and Hymenochirus) are accelerated.
Phylogenetic relationships from Cannatella and Trueb (1988a, b);
de Sá and Hillis (1990); Cannatella and de Sá (1993); and Báez
and Trueb (1997).
Dorsal Skull I
Relative Warp 1, which explains 42% of the variation in
the total sample, is a general ontogenetic component, similar
to the way principal-components analyses frequently yield a
general size component for Principal Component 1. Onto-
genetic trajectories on the RW 2 versus RW 1 scatterplot go
from smaller to larger RW 1 values for all species (Fig. 5a).
 PIPOID SKULL OSSIFICATION 2491

TABLE 2. Anuran ossification patterns by Gosner Developmental Stage.

Standard
Deviation of Duration of
Mean Stage of Ossification Stage at First Stage at Last Ossification
Species Bone Ossification Stages Ossification Ossification (Stages)
Pipoid Species
Xenopus laevis 40.21 3.40 35 44 9
Hymenochirus boettgeri 40.92 6.69 29 46 17
Pipa myersi 40.09 4.06 35 44 9
Nonpipoid Species
Ascaphus truei 44.08 1.78 40 46 6
Bombina orientalis 42.07 3.17 37 46 9
Discoglossus sardus 40.08 4.17 34 45 11
Alytes abstetricans 39.17 5.11 31 44 13
Pelobates fuscus 40.00 4.90 31 46 15
Scaphiopus intermontanus 40.70 2.84 36 45 9
Hyla lanciformis 41.21 4.71 32 46 14
Ceratophrys cornuta 42.13 2.75 38 46 8
Chacophrys pierotti 42.50 2.58 38 46 8
Hamptophryne boliviana 41.93 4.56 35 46 11
Pyxicephalus adspersus 40.50 4.00 33 46 13
Rana pipiens 39.69 4.27 29 46 17
Rana temporaria 26.83 1.47 25 29 4

Spline deformations (Fig. 5b) further corroborate RW 1 as a
general ontogenetic component, showing: (1) The maxilla
grows in length and shifts from a transverse to longitudinal
orientation. (2) The nasal grows, expanding in area. (3) The
frontoparietal grows rostrally. (4) The otic capsule expands
rostro-caudally.
Relative Warp 2 explains 23% of the variation and dis-
tinguishes pipoids from other frogs. First, pipoids have low-
er RW 2 scores than outgroup species (Fig 5a). Note that
the salamander Ambystoma maculatum clusters with the pi-
poids. Second, pipoid ontogenetic trajectories tend towards
lower RW 2 scores, whereas those of other frogs tend to-
wards higher RW 2 scores. Spline deformations (Fig. 5c)
suggest the following features for pipoid development: (1)
Growth of the frontoparietal is more extensive, both ros-
trally and in breadth. Note that the medial boundary of the
frontoparietal is at the skull midline in pipoids. (2) There
is comparatively less growth of the maxilla. (3) The nasal
is larger and less oblique—the latter is evident from the
comparatively medial position of one corner of the nasal
(landmark 6). (4) The nasal is rostrally translated and close
to the tip of the snout. (5) There is more growth in the otic
capsule. Nonpipoid frogs are characterized by the opposite
ontogenetic trends.
Because ‘‘Dorsal Skull I’’ included only a few, onto-
genetically advanced specimens of critical outgroups As-
caphus truei and Ambystoma maculatum, quantitative com-
parisons of ontogenetic shape change are addressed using
‘‘Dorsal Skull II.’’
Dorsal Skull II
Unexpectedly, exclusion of nasal landmarks did not dramat-
ically affect either ontogenetic trajectories or shape deforma-
tions for RW 1 and RW 2 (Fig. 6). RW 1 and RW 2 explain
40% and 25% of the variation, respectively, similar to ‘‘Dorsal
Skull I.’’ Pipoid ontogenetic trajectories are distinct from those
of nonpipoid species—the salamander and nonpipoid frogs have
ontogenies with positive slopes, whereas pipoid ontogenetic
trajectories have negative slopes (Fig. 6a).
To assess developmental trends more fully, RW 1 and RW
2 scores were regressed on Nieuwkoop-Faber developmental
stage for nine anurans (Fig. 7). (Ambystoma maculatum and
Pipa pipa were excluded because staging criteria could not
be applied to either salamanders or direct developers.) Ear-
lier, RW 1 was described as a general measure of ontogeny;
regressions for all species exhibit positive slopes. Note that
in nonpipoids, earlier stages are not represented; this is not
an artifact of specimen availability but reflects the earlier
stage at which pipoids achieve ossification in all the bones
considered in this analysis. Pipoids also exhibit more gradual
skull ossification, apparent from the generally lower RW 1
regression slopes. RW 2 distinguishes pipoids from other
frogs. Regressions for nonpipoid frogs generally have slopes
that are close to zero or positive; pipoids are characterized
by negative slopes.
Lower Jaw
‘‘Lower Jaw’’ RW 1 encompasses 66% of the variation
and describes general features of ontogeny (Fig. 8): (1)
The dentary grows caudally. (2) The angulosplenial grows
rostrally. Regression of RW 1 on developmental stage (Fig.
9) shows that all species decrease in RW 1 score during
development (with the exception of Hymenochirus, which
is represented by a very small sample size). Pipoids again
ossify more gradually, as evidenced by lower regression
slopes. Species also converge approximately on a final RW
1 value, suggesting that the final lower jaw morphology
of many frog species is fairly similar. However, pipoid
species, approach this end state more gradually; pipoid
regressions are less steep, with development occurring
over a larger number of developmental stages. These fea-
tures are related to early jaw bone ossification in pipoids,
discussed above.
Relative Warp 3 describes 10% of the variation and in-
2492 JENNIFER YEH

FIG. 5. ‘‘Dorsal Skull I’’ results. (A) Scatterplot of RW 2 versus RW 1. All ontogenetic trajectories proceed towards positive values
of RW 1. Pipoids decrease in RW 2 scores during the course of ontogeny, while nonpipoid species increase in RW 2 scores. (B) RW 1
deformations. (C) RW 2 deformations.

creases during ontogeny (Hymenochirus is again anomalous) RW 3 values. Pipoid lower jaw morphology is thus paedo-
(Fig. 9b). Note that all anurans develop roughly along a single morphic relative to other frogs. Morphologically, pipoids
trajectory. RW 3 also distinguishes pipoids from other frogs, have pointed, triangular jaws rather than more square-shaped
with pipoids beginning and completing development at lower jaws (Fig. 8).
 PIPOID SKULL OSSIFICATION 2493

FIG. 6. ‘‘Dorsal Skull II’’ results. (A) Scatterplot of RW 2 versus RW 1. Note the similarity of species’ ontogenetic trajectories to
those from ‘‘Dorsal Skull I’’ (Fig. 5). Each species has a unique ontogenetic trajectory, but pipoid and nonpipoid frogs occupy different
parts of morphospace. (B) RW 1 deformations. (C) RW 2 deformations.

DISCUSSION
Timing of Jaw Ossification in Vertebrates—A Matter
of Function
The most striking features of ossification sequence evo-
lution in this analysis are shifts in the timing of jaw ossifi-
cation. The primitive condition among vertebrates is for jaw
elements to ossify early. Early jaw ossification is functional
in fishes, where feeding and respiration are critical early func-
tions; in addition to the operculum and jaw bones, the par-
asphenoid, which underlies the braincase and allows food to
be swallowed without damaging the brain above, also ossifies
early (Mabee and Trendler 1996; Adriaens and Verrae 1998;
Wagemans and Vandewalle 2001). Delayed jaw ossification
is an anuran synapomorphy. There is a good adaptive reason
for this shift—larval feeding structures are maintained until
2494 JENNIFER YEH
FIG. 7. ‘‘Dorsal Skull II’’ regressions for (A) RW 1 and (B) RW
2 on Nieuwkooop-Faber developmental stage. Significant slopes are
marked with an asterisk.
late in frog metamorphosis, when individuals move to their
adult habitat and transition to adult feeding. Interestingly,
even in overwintering tadpoles of Pelobates fuscus, in which
most skull bones ossify early relative to overall somatic de-
velopment (compared to nonoverwintering tadpoles), jaw and
jaw suspension elements are not accelerated and ossify at the
end of metamorphosis (Smirnov 1992).
Pipoid frogs exhibit a trend towards early jaw ossification,
that is, revert towards the primitive vertebrate condition, al-
though the degree of acceleration varies within the group.
Accelerated jaw ossification is almost certainly related to the
absence of larval morphological specializations in pipoid tad-
poles. Most anuran tadpoles have keratinized beaks and den-
ticles of the mouth which they use to chop food into small
pieces or to scrape food from surfaces. Beaks and denticles
are absent in pipoid larvae, and, consequently, the larval jaw
cartilages associated with beaks—suprarostrals and infraros-
trals for the upper and lower beak, respectively—exist only
in ontogenetically advanced conditions (Sokol 1977; Swart
and de Sá 1999, Trueb et al. 2000). Thus, whereas most
tadpoles reduce and remodel suprarostrals and infrarostrals
during metamorphosis, the cartilaginous jaws of larval pi-
poids are already adultlike, permitting bone deposition to
commence early. Hanken (1992) uses the term ‘‘precocious
metamorphosis’’ to describe the advancement of adult fea-
tures to the larval stage, as occurs in pipoid larval jaws.
However, the term is slightly misleading because species do
not precociously recapitulate the events of metamorphosis,
but skip them altogether.
Accelerated jaw ossification characterizes one other frog
lineage among those studied—Eleutherodactylus. In fact, ac-
celeration is more extreme in Eleutherodactylus than among
the pipoids. Eleutherodactylus frogs are direct developers;
there is no free-living tadpole stage, and eggs hatch as ju-
veniles that resemble miniaturized adults. Direct develop-
ment removes the need for larval specializations and permits
developmental repatterning. Hanken et al. (1992) showed that
some of the most conspicuous instances of repatterning in
Eleutherodactylus pertain to the larval jaw cartilages—su-
prarostrals never form in Eleutherodactylus, and infrarostrals
are minute. The result is an ontogenetically advanced, adult-
like embryonic jaw morphology, similar to that reported by
Sokol (1975, 1977) for pipoid tadpoles. Earlier jaw ossifi-
cation in Eleutherodactylus than pipoids is attributable to the
greater reduction of larval jaw cartilages possible under direct
development. Interestingly, the pipoid species with most pro-
nounced jaw ossification acceleration is Pipa pipa, also a
direct-developer, albeit in its own highly distinctive manner.
In sum, pipoids and Eleutherodactylus converge on a devel-
opmental pattern characterized by (1) unusual life-histories;
(2) loss of larval morphological specializations; (3) onto-
genetically advanced larval jaw morphologies; and (4) ac-
celerated ossification of jaw bones.
A question that remains is why pipoids lack larval mor-
phological specializations of the jaw. One possibility is that
pipoids are basal anurans (Maglia et al. 2001) and merely
retain a primitive character. However, under other phylo-
genetic hypotheses (Ford and Cannatella 1993; Hay et al.
1995), absence of larval jaw specializations is derived in
pipoids. Sokol (1975, 1977) also argues that the traits are
derived and notes that loss of larval specializations may be
easy to achieve if they were merely inserted into develop-
ment. If absence of larval specializations does represent a
pipoid loss, feeding strategies appear to be at the heart of it.
Beaks and denticles function in chopping food into pieces
small enough to ingest, or in scraping food from surfaces.
Unlike other tadpoles, however, free-living pipid larvae are
filter feeders (aside from Hymenochirus, whose predatory tad-
pole clearly sports a derived feeding strategy) (Sokol 1975,
1977). Filter feeding makes beaks and denticles superfluous,
and with the loss of these structures, the suprarostral and
infrarostral cartilages can be reduced.
Microhylid tadpoles are also filter feeders and lack beaks
and denticles. They exhibit some reduction in larval jaw car-
tilages, but less than pipids (Sokol 1975). Among micro-
 PIPOID SKULL OSSIFICATION
FIG. 8. ‘‘Lower Jaw’’ spline deformations for RW 1 and RW 3.
hylids, an ossification sequence exists only for Hamptophryne
boliviana (de Sá and Trueb 1991), and this species does not
exhibit accelerated jaw ossification. Other species which are
known to have lost beaks and denticles, such as Lepidoba-
trachus laevis, retain well-developed suprarostrals and infra-
rostrals, and do not show accelerated jaw ossification (Ruibal
and Thomas 1988). I conclude from these species, as well as
the varying condition of pipoids, that beak and denticle loss
and ontogenetically advanced jaw morphology permit accel-
erated jaw ossification but do not guarantee it.
Function is the crux—reversion to early jaw ossification
occurs only in anurans with ontogenetically advanced larval
jaw morphology if adult-like feeding also commences early.
In Eleutherodactylus, juveniles begin to feed soon after hatch-
ing (Hanken et al. 1992). Among pipoids, Pipa and Hymen-
ochirus species exhibit earliest jaw ossification and are also
the ones whose tadpoles have evolved furthest from microph-
agous feeding towards adult-like feeding. Pipa tadpoles feed
on substantial food items such as large protists and small
planktonic crustaceans (Sokol 1977). Pipa carvalhoi also be-
gins adult-like feeding well before the end of metamorphosis,
when the tail is still half to two-thirds its original length
(Sokol 1977). Hymenochirus is even more extreme—tadpoles
actively hunt large prey, and in fact individuals exhibit
smoothly continuous feeding from larva to adult (Sokol
1977). In contrast, Xenopus retains microphagous feeding
2495
capabilities similar to other anurans (Sokol 1977). Feeding
behavior also affects the timing of jaw ossification in cae-
cilians; Dermophis mexicanus ossifies its jaws earlier than
other caecilians (Wake and Hanken 1982) and is also a vi-
viparous species in which larvae begin to feed extremely
early—in the oviducts prior to birth.
Ontogenetic Shape Change and Morphology
Thin-plate spline morphometrics proved particularly ef-
fective in distilling ontogenetic features which characterize
multiple species—RW 1 described general ontogenetic com-
ponents for the amphibians examined. Higher relative warps
elucidated species-specific aspects of ontogeny, in this study
emphasizing differences between pipoid and nonpipoid frogs
and providing explicit support for the notion (long held by
anuran biologists) that pipoids are morphologically distinct
among frogs.
Unique aspects of pipoid morphology are not merely con-
sequences of hypo- or hypermorphosis, nor do they result
from simple heterochronic transformations. Rather, each spe-
cies was shown to possess a species-specific ontogenetic tra-
jectory from the onset of ossification. Ontogenetically, pi-
poids differ most from salamanders among frogs. This result
contrasts with the ossification-sequence study, which showed
pipoids reverting to a sequence primitive among vertebrates
2496 JENNIFER YEH
FIG. 9. Regressions of ‘‘Lower Jaw’’ relative warps on Nieuw-
koop-Faber developmental stage. (A) RW 1. (B) RW 3. Significant
results are marked with an asterisk.
and similar to that of salamanders. Although pipoid ontogeny
differs hugely from that of salamanders, pipoid morphology
nonetheless shares key features with salamanders—Ambys-
toma clustered with the pipoids in TPS analyses. Evidently,
different ontogenetic paths can result in similar morpholo-
gies. Features of adult skull morphology characterizing both
pipoids and salamanders include broad frontoparietals (sep-
arate frontals/parietals in salamanders) that cover the brain-
case, robust nasal bones, and short maxillae. Thin-plate spline
morphometrics also revealed that pipoid skulls ossify more
gradually than other species, a result of reduced scope of
metamorphosis in these species.
Morphological Effects of One Life-History Stage on Others
In salamanders, it has been argued that larval morphology
constrains adult morphology. For example, larval suction
feeding constrains hyobranchial morphology in such a way
that metamorphosing plethodontids are ultimately less ac-
complished tongue protractors than direct developers as
adults (Deban and Marks 2002). In frogs, some studies ad-
dressing this issue have suggested the opposite—that larval
and adult morphologies do not significantly impact each oth-
er. In Eleutherodactylus, Hanken (1992) and Hanken et al.
(1992) found that, despite dramatic developmental changes
permitted by direct development, adult Eleutherodactylus are
morphologically unremarkable. Hanken (1992) concluded
that morphological evolution at one life-history stage may
occur without correlated effects at other life-history stages.
This is likely explained in anurans by the large-scale re-
modeling that occurs at metamorphosis. Wild’s (1997) com-
parison of the ceratophryine frogs Ceratophrys and Lepidob-
atrachus also supports this hypothesis—high disparity in lar-
val morphology between species is followed by morpholog-
ically more similar adults.
In pipoids, development is characterized in several ways
by a relaxation of constraints imposed by metamorphosis and
the larval stage. A consideration of the developmental stages
over which skull ossification occurs suggested that ossifi-
cation begins earlier, is more gradual, and terminates earlier
in some pipoids than in most other frogs. Thin-plate spline
studies also demonstrated the earlier and more gradual skull
ossification relative to general somatic development in the
group. Early skull ossification may be related to pipoid hy-
perossification of bones such as frontoparietals, nasals, and
pterygoids. Smirnov (1992), for example, showed that early
ossification of skull bones in overwintering tadpoles can re-
sult in hypermorphosis compared to nonoverwintering indi-
viduals. On the other hand, unusual features of pipoid os-
sification sequence, such as early jaw ossification, do not have
an obvious hypermorphic effect on adult jaw morphology.
Portions of the maxilla (the pars facilias and pars dentalis)
are actually reduced in pipoids, and TPS analysis also showed
that the maxilla is shorter in pipoids than in other frog species.
Similarly, TPS results showed that bones of the pipoid lower
jaw, the angulosplenial and dentary, also exist in a paedo-
morphic, rather than hypermorphic, state.
ACKNOWLEDGMENTS
For generous assistance with the loan of specimens, I thank
D. Cannatella (Texas Natural History Collection, University
of Texas, Austin), J. Hanken (Museum of Comparative Zo-
ology, Harvard University), R. de Sá (University of Rich-
mond), R. Heyer (National Museum of Natural History), L.
Trueb (University of Kansas Natural History Museum and
Biodiversity Research Center), and L. Ford (American Mu-
seum of Natural History). Valuable suggestions on the man-
uscript were provided by D. Cannatella, J. Wiens, P. Yeh,
R. Dudley, C. Bell, G. Freeman, and two anonymous re-
viewers. I am grateful to the National Science Foundation
for a Graduate Fellowship and Doctoral Dissertation Im-
provement Grant, as well as to the Zoology Endowment for
Excellence at the University of Texas, Austin for financial
support.
 PIPOID SKULL OSSIFICATION
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Corresponding Editor: J. Wiens