Developmental processes underlying the evolution of a derived foot morphology in salamanders.

Abstract

Interdigital webbing has evolved repeatedly in tropical salamanders (bolitoglossines). This derived foot morphology is only one of many homoplastic traits in this diverse amphibian clade. Indeed, few if any morphological traits sort lineages within this clade. We investigate the processes underlying the homoplastic evolution of morphological characters in these salamanders by analyzing selective and developmental processes that generate interdigital webbing. We show that a pedomorphic developmental change generates the new foot morphology and that pedomorphosis affects a number of morphological traits, thus creating a developmental correlation among them. This correlation among traits is maintained across most species, thus facilitating the repeated evolution of traits. Although we find evidence that the changes in foot morphology are adaptive in one species, the evolution of webbing in all other species does not carry an adaptive signature. The new foot morphology therefore evolves repeatedly, even in the apparent absence of a direct selective advantage.

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Developmental processes underlying the evolution
of a derived foot morphology in salamanders
Martin Jaekel†and David B. Wake‡
Museum of Vertebrate Zoology and Department of Integrative Biology, University of California, Berkeley, CA 94720-3160
Contributed by David B. Wake, October 26, 2007 (sent for review October 3, 2007)
Interdigital webbing has evolved repeatedly in tropical salamanders
(bolitoglossines). This derived foot morphology is only one of many
homoplastic traits in this diverse amphibian clade. Indeed, few if any
morphological traits sort lineages within this clade. We investigate
theprocessesunderlyingthehomoplasticevolutionofmorphological
characters in these salamanders by analyzing selective and develop-
mental processes that generate interdigital webbing. We show that
a pedomorphic developmental change generates the new foot mor-
phology and that pedomorphosis affects a number of morphological
traits, thus creating a developmental correlation among them. This
correlation among traits is maintained across most species, thus
facilitating the repeated evolution of traits. Although we find evi-
dence that the changes in foot morphology are adaptive in one
species, the evolution of webbing in all other species does not carry
an adaptive signature. The new foot morphology therefore evolves
repeatedly, even in the apparent absence of a direct selective
advantage.
geometric morphometrics ? homoplasy ? limb evolution ?
pedomorphosis ? Bolitoglossa
I
extreme case, two morphologically indistinguishable species
have evolved independently from different ancestors (1). The
ubiquity and extent of repeated evolution demand an analysis of
the underlying mechanisms. Traditionally, the repeated evolu-
tion of characters has been interpreted as prima facie evidence
for adaptive processes. Adaptation does lead to similar pheno-
types in similar environments and can thus account for the
repeated evolution of traits. However, developmental processes,
whichbiastherangeofphenotypes,canalsoexplaintherepeated
evolution of morphological traits (2, 3).
We investigate selective and developmental processes under-
lying the repeated evolution of webbed feet in tropical
salamanders. The genus Bolitoglossa is composed of seven
geographically delimited, monophyletic subgenera, four includ-
ing only webbed species and three displaying a range of webbing
(4). Webbing is a derived trait within Plethodontidae and the
clade of tropical salamanders, supergenus Bolitoglossa (5), and
because of its repeated evolution it was thought to be adaptive.
Extensive webbing of hands and feet has been viewed as an
adaptation to a new arboreal lifestyle that evolved in these
tropical salamanders. In particular, webbing was hypothesized to
improve attachment to smooth plant surfaces (6). Webbing in
miniaturized salamander species, however, has been recognized
to be the consequence of truncated limb development rather
than an adaptation for climbing (6, 7). Our morphometrical
analyses now show that all webbed Bolitoglossa species, minia-
turized as well as others, share a juvenile foot morphology. A
pedomorphic developmental change thus gives rise to the
webbed foot morphology.
We show that webbing itself is an adaptation for climbing in
only a single species under the current hypothesis. However,
through pedomorphic changes, webbing is accompanied by the
appearance of other morphological characters, any one of which,
or all, may be under selection. The repeated evolution of these
n tropical salamanders, a wide variety of morphological traits
evolves repeatedly in separate phylogenetic lineages. In the
suites of pedomorphic traits in the genus Bolitoglossa is evidence
that a degree of developmental integration is stable during the
evolutionofthisclade.Thisintegrationmayexplaintherepeated
evolution of similar or identical phenotypes found in nature.
Results
We characterized interdigital webbing in 31 species of salamanders
sampled from four families and seven genera [supporting informa-
tion (SI) Table 2]. To quantify webbing, we devised a measure that
is highly efficient at discriminating foot morphologies of different
species (Fig. 1). This measure uses the sinuosity of the foot to
determine the amount of webbing. The sinuosity is defined as the
length of the outline of the distal part of the foot [from the tip of
digit one (dt1) to digit five (dt5)] divided by the width of the foot.
This dimensionless ratio allows discriminating three morphotypes
with significant statistical support (data not shown). Species dif-
ferences were difficult to capture with previously used measures
(ref. 8 and Fig. 1).
The Bolitoglossa Lineage Has More Webbing than Outgroups. A
comparison of sinuosity among different salamander families,
genera, and species reveals a phylogenetic trend for increased
Author contributions: M.J. and D.B.W. designed research; M.J. performed research; M.J.
analyzed data; and M.J. and D.B.W. wrote the paper.
The authors declare no conflict of interest.
†To whom correspondence may be sent at the present address: Laboratory for Develop-
ment and Evolution, Department of Zoology, University of Cambridge, Cambridge CB2
3EJ, United Kingdom. E-mail: mj279@cam.ac.uk.
‡To whom correspondence may be addressed. E-mail: wakelab@berkeley.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0710216105/DC1.
© 2007 by The National Academy of Sciences of the USA
Fig. 1.
Amountofwebbingmeasuredasaratiooftwodistancesmtandr.mtistaken
from the base of the metatarsal to the tip of digit 3 (dt3) and r from the
metatarsal to the edge of the skin between dt2 and dt3 (8). (b) Amount of
webbing measured as the sinuosity of the foot. The outline of the foot from
the tip of dt1 to dt5 (arrowheads) is measured as linear distance. This distance
is divided by d, the diameter or width of the foot, to yield the sinuosity. The
positionsofthelandmarksusedforfoot-shapeanalysisarelabeledLM1–LM9.
The sinuosity is more efficient at discriminating between different foot mor-
phologies than the previous measure.
Two measures to quantify the amount of interdigital webbing. (a)
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webbing within the tropical genus Bolitoglossa (ref. 9 and Fig. 2).
Even fully terrestrial species of Bolitoglossa have significantly
more webbing than almost all species outside the genus, the
exception being the cave-dwelling Mexican bolitoglossine Chi-
ropterotriton magnipes (ref. 10 and Fig. 2).
Webbed Species Lack Developmental Change in the Amount of Web-
bing. The most webbed species have remarkably similar degrees
of webbing close to ?/2 (sinuosity ?1.56) and little intraspecific
variation in the degree of webbing (Fig. 3a; B. schizodactyla
through B. salvinii). This diminished intraspecific variation has a
telling developmental origin: in webbed species the sinuosity
remains constant over the whole course of development,
whereas in unwebbed species the sinuosity progressively dimin-
ishes as development proceeds (Fig. 3b). In species such as B.
rostrata or B. lincolni, for example, the degree of webbing
decreases (i.e., the sinuosity increases) during growth, whereas
in species like B. salvinii and B. alberchi there is no change in
sinuosity. Consequently, the reduced amount of intraspecific
variation in webbed species is caused by the lack of ontogenetic
change in the degree of webbing.
Ontogenetic Shape Change Occurs in the Distal Part of the Foot Only
in Unwebbed Species. To understand better the changes in foot
shape that occur over the course of development, we used a
morphometrical method that compares juvenile and adult foot
shapes, based on a set of landmarks. Specimens of different
developmental stages were used to trace ontogenetic shape
change in six species: three unwebbed (B. lincolni, B. franklini, B.
rostrata) and three webbed (B. mexicana, B. alberchi, B. occi-
dentalis). The shape change is represented in a grid plot (Fig. 4a
and b). The undistorted grid represents the position of land-
marks in a juvenile salamander. As the salamander matures, the
position of landmarks changes, and the grid becomes deformed.
The grid plot of the unwebbed species B. rostrata (Fig. 4a) is
much more deformed than that of the fully webbed species B.
mexicana (Fig. 4b), indicating more extensive shape change over
the course of development. Regressing the procrustes distance
(representing the total amount of shape change of all landmarks)
onto the centroid size (a size measure of the specimen calculated
as the summed squared distances of all landmarks from the
centroid) shows that the shape change in the analyzed unwebbed
species is significant at the 5% level, whereas no significant
change is found in webbed species. Fully webbed species thus
indeed show less ontogenetic shape change. The grids suggest
that shape change in unwebbed species occurs via outgrowth of
toe tips and perhaps through loss or differential growth of skin
tissue between digits.
A Change in Ossification Mediates the Change in Foot Morphology.
The lack of ontogenetic change in sinuosity and shape suggests
that a webbed foot is the consequence of retention of a juvenile
morphology. A pedomorphic developmental change may thus
give rise to the new foot morphology. A comparison of the foot
skeleton between webbed and unwebbed species demonstrates
this point. We took x-rays of species of Bolitoglossa and other
genera to measure the relative lengths of bony elements in the
third toe. Over the course of salamander development, bony
elements in the toes (phalanges) ossify and lengthen. In webbed
species (B. alberchi, B. platydactyla, and B. dofleini) phalangeal
lengthening does not occur. All phalanges (ph1, ph2, ph3) are
reduced in length and ossification compared with unwebbed
species (Fig. 5 a and b, Inset). The same reduction of distal bony
elements in the foot is found in miniaturized species (B. alta-
mazonica), which also maintain juvenile morphologies into
adulthood (ref. 8 and Fig. 5a). Hence, pedomorphosis mediated
by the reduced ossification of ph2 and ph3 underlies the evolu-
tion of webbed feet (Fig. 5b). In other organisms, connecting
tissue between digits is usually maintained through the down-
regulation of apoptosis (11). The developmental mechanism in
salamanders is therefore different: it adjusts the degree of
ossification and hence does not depend solely on the regulation
of apoptosis.
Growth Trajectories Are Conserved in Bolitoglossa and Have Not
Adapted. Using a mathematical model, we tested whether the
evolution of interdigital webbing is adaptive. The model uses the
Fig. 2.
for a given species. In total, 31 species are analyzed. The white boxes belong to members of Bolitoglossa, the gray boxes to outgroup taxa. All Bolitoglossa,
irrespective of their ecology, have more webbing (i.e., lower sinuosity) than outgroup taxa (with the exception of C. magnipes). The most (Ambystoma,
Dicamptodon, Salamandra, Pseudoeurycea, Hydromantes, Chiropterotriton) webbed species all reach very similar degrees of webbing (sinuosity?1.56).
Degree of webbing in Bolitoglossa and outgroup taxa. Each box plot shows the median, interquartile range, and total range of the degree of webbing
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allometric equation A ? bW?to make specific predictions with
respect to the evolution and scaling of body weight W and foot
surface area A (see Materials and Methods). The allometric
growth trajectory relating A to W, for example, is expected to
evolve if foot webbing constitutes an adaptation to an arboreal
lifestyle. Specifically, given that W in webbed and unwebbed
species is distributed over the same range, the growth trajectory
for webbed species should be closer to isometry (? ? 1) than for
unwebbed species. However, the growth trajectories of webbed
and unwebbed species in the genus Bolitoglossa are identical
(Fig. 6). The residuals from a pooled nonlinear regression
between webbed and unwebbed species are not significantly
different confirming this result (data not shown). These results
show that interdigital webbing does not increase A relative to W
and that webbing thus does not constitute an adaptation accord-
ing to the current hypothesis.
Species-specific estimates of the parameters b and ? were
subsequently obtained for six webbed and four unwebbed Boli-
toglossa species as well as for C. magnipes (Table 1). All
Bolitoglossa species, webbed and unwebbed, share similar pa-
rametervalues(??0.66,asexpectedforanareatovolumeratio;
b ? 0.11). The trajectory of C. magnipes, however, is very distinct
(Fig. 6). The change in C. magnipes is the result of an increase
in parameter b (Table 1). Because C. magnipes is known to climb
on smooth surfaces on the walls of caves, this change may
therefore indeed be adaptive. However, no similar change in
growth trajectories is found in species of Bolitoglossa, webbed or
unwebbed. Accordingly, the parameters ?, b, A, and W have not
been optimized by selection for better climbing performance in
webbed species of Bolitoglossa.
Discussion
Increased foot webbing occurs in bolitoglossine salamanders
irrespective of their lifestyle: terrestrial as well as arboreal
Fig. 3.
depends on their average sinuosity (P ? 0.001). Webbed species vary less in their sinuosity than unwebbed species. The standard deviation is used to measure
the spread in sinuosity in 31 species. This analysis uses the same species as Fig. 2. (b) The ontogenetic change in the degree of webbing is shown for 10 species
ofBolitoglossa.Inwebbedspecies(crosses),thesinuositydoesnotchangeoverthecourseofdevelopment.Itstaysatthesamelevelasintheminiaturizedspecies
B. hartwegi and B. occidentalis (circles). In unwebbed species (diamonds), the sinuosity increases as development proceeds from juvenile to adult. Foot width
increases over the course of development (data not shown) and is taken here as a proxy for age. Nonlinear regressions were used to calculate the trend lines.
Diminished variation in sinuosity in webbed species. (a) A linear least-squares regression shows that the variation in webbing found within a species
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species have more webbing than species outside the genus
Bolitoglossa. We show that a pedomorphic developmental
change leads to this increased degree of webbing, just as in
miniaturized species. Thus, webbing in Bolitoglossa does not
improve attachment (as illustrated by our mathematical model)
and may have evolved alongside another trait affected by pedo-
morphosis. Consequently, the new foot morphology correlates
with other morphological traits: (i) foot surface area scales with
body weight; (ii) foot morphology correlates with skull ossifica-
tion (8, 12); (iii) the developmental correlation between indi-
vidual toe bones is maintained, i.e., phalanges ossify and
lengthen in a concerted fashion over the course of development
in unwebbed species (and fail to do so in webbed species); (iv)
all toes are affected in a correlated way. These developmental
correlations are stable in the genus Bolitoglossa and hence affect
phenotypic evolution in this clade by facilitating the repeated
evolution of similar or identical phenotypes.
There is one interesting exception: C. magnipes not only has
evolved interdigital webbing but also has changed its growth
Fig.4.
visualizeshapechangesoverthecourseofdevelopmentinthewebbedspeciesB.
mexicana (a) and the unwebbed species B. rostrata (b) (landmarks LM1–LM9 are
showninred).Theaveragelandmarkpositionsofthetwosmallestindividualsin
a sample represent the undistorted grid. (a) In B. rostrata, landmarks at the tips
of toes move distally and away from the center (e.g., LM3 and LM7), whereas
landmarks between toes move proximally and toward the center of the palm
(e.g., LM4 and LM6) as development proceeds. This finding accounts for the
observed increase in sinuosity in unwebbed species (Fig. 3b). (b) The grid of B.
mexicana is less deformed than the grid of B. rostrata. Therefore, the shape of
webbed feet changes less than the shape of unwebbed feet over the course of
development. A statistical analysis of shape change supports this visual result
(Pmexicana? 0.1; Prostrata? 0.03; see Results).
Ontogeneticshapechangeinfootmorphology.Griddeformationplots
Fig. 5.
ossifyasmuchasinunwebbedspecies(B.franklini;seeInsetinb).B.altamazonicaisaminiaturizedspeciesthatistruncatedindevelopment(8).Itisindistinguishable
fromlarger,webbedspecies.(b)Thereductionofphalangescausesthewebbedfootmorphology.Thecontributionofph2?ph3tototaltoelengthshrinksfrom40%
in unwebbed to 27% in webbed species, which causes a steep decrease in sinuosity (y ? 7x ? 0.4). (Inset) Bony elements in the third toe.
Pedomorphosis in limb development. (a) In fully webbed species (B. altamazonica, B. platydactyla, B. alberchi, and B. dofleini) phalanges (ph1–ph3) do not
Fig.6.
surfacearea,A,increasesnonlinearlywithbodyweight,W.Webbed(red)and
unwebbed (blue) species of Bolitoglossa share the same trajectory. Data from
sixwebbedandfourunwebbedspecieswerepooledtocalculateleast-squares
nonlinear regressions. Regressions using datasets from individual species
show the same results (Table 1). The foot area relative to body weight is
therefore identical in webbed and unwebbed species. C. magnipes, however,
hasadistinctgrowthtrajectoryclosertoisometry.Themechanismunderlying
limb development in C. magnipes evolves in accordance with our mathemat-
ical model and is different from the mechanism found in Bolitoglossa (see
Results).
Growthtrajectoriesinwebbedandunwebbedsalamanders.Thefoot
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trajectory. As a consequence of these changes, this species has
very large feet compared with its body size, suggesting adapta-
tion to its lifestyle. The increase in parameter b suggests that the
number of cells specified as part of the foot primordium is higher
than in other salamanders. The growth rate (i.e., the rate of
division) of these cells at the tip of the limb bud remains
unaffected because ? remains constant.
Pedomorphic changes initially might have been selected to
allow diversification of body size: both the largest and the
smallest terrestrial salamander species occur in the diverse
tropical clade, supergenus Bolitoglossa. These changes might
then have affected other traits as well (such as skull and foot
ossification). Widespread pedomorphic tendencies in
salamanders suggest a developmental switch that triggers pedo-
morphosis. A major quantitative trait locus triggering pedomor-
phic changes indeed has been identified in Ambystoma (13).
Bolitoglossa development bypasses the larval stage that charac-
terizes Ambystoma development. Pedomorphosis in Bolitoglossa
may therefore employ a different genetic mechanism. However,
evidence that aquatic larvae have reevolved in lineages of
direct-developing plethodontid salamanders (14, 15) raises the
possibility that genes coordinating metamorphosis from larva to
adult may provide such a switch.
Bolitoglossa contains more species than any other salamander
genus. Seven clades have been recognized as subgenera, based
on analyses of mitochondrial DNA (4). A high degree of overall
similarity is evident, and few if any morphological traits sort the
clades. Webbed species are found in all seven clades. Using
out-group comparisons, one must conclude that ancestral Boli-
toglossa arose from unwebbed ancestors. The sister clade of
Bolitoglossa is the Pseudoeurycea (sensu lato; 5) clade, all mem-
bers of which, even miniaturized species, have unwebbed feet.
The sister clade to the entire Bolitoglossa–Pseudoeurycea com-
bined clade is Chiropterotriton, and only the derived C. magnipes
isfullywebbed.However,theparallelisminfootmorphologyand
the increased webbing in all species of Bolitoglossa raise the
possibility that pedomorphosis might have been present in the
common ancestor of Bolitoglossa before it began diversifying. In
this scenario, reduced webbing appears repeatedly in the genus
Bolitoglossa as a derived trait. The underlying developmental
mechanism of such repeated evolution would remain the same
(i.e., ossification of phalangeal elements) but would evolve in an
oppositedirection.AllmembersofBolitoglossadisplaytraitsthat
have been interpreted as pedomorphic: incomplete skull ossifi-
cation with loss of septomaxilla, frequent loss of prefrontals, the
usual presence of a cranial fontanelle, and failure of distal tarsals
to individuate (10).
We believe that these results foster deeper insight into phe-
notypic evolution: phenotypic patterns are used to infer pro-
cesses in evolutionary biology. The repeated evolution of a
morphological character in particular has been used to infer
adaptive processes. Our results show that this inference may not
be robust for individual traits but needs to be considered in a
wider context of morphological characters. We conclude that
without understanding the developmental mechanisms under-
lying character evolution it will remain difficult to infer process
from pattern.
Materials and Methods
Acquisition of Morphological Data. Alcohol-preserved specimens from several
museums were measured for foot surface area (A), snout–vent length, body
weight (W), length of digits, length of individual phalanges and metatarsals,
foot width, degree of webbing, and foot shape. Specimens were photo-
graphed with a digital camera (Nikon Coolpix 995) and x-rayed for 25 s at 25
kV. The digital photographs and digitized x-rays were archived on a Dell PC.
Enlargement of these digitized, high-resolution pictures allowed the precise
measurement of morphological structures with the help of Scion Image
(www.scioncorp.com). Weights were measured on a digital scale. The Inte-
grated Morphometrics Package (IMP) (www2.canisius.edu/?sheets/
morphsoft.html) and tpsdig (http://life.bio.sunysb.edu/ee/rohlf/software.
html) were used for shape analysis. The statistics programs JMP and Prism 4.0
(GraphPad) were used to perform statistical analyses of morphological data.
AlistofspeciesstudiedispresentedinSITable2.Samplesizesdifferedamong
species.Notallspecieswereincludedinallanalysesbecausedifferentanalyses
required different minimal sample sizes. We sampled several species from
each of the seven subgenera of the genus Bolitoglossa (4) wherever possible.
Measuring the Allometric Growth of Foot Surface Area. We measured the
increase in foot surface area A relative to body weight W over the course of
development in 10 species. Preserved specimens that differed with respect to
their developmental stage were measured for A and W. A least-squares
nonlinear regression of A on W was used to estimate the growth trajectory
of A.
Analysis of Foot Shape. We used geometric morphometric techniques to
compare foot shapes over the course of development. The comparison is
basedonachoiceofninelandmarkssetatthetipofeachtoeoftherightfoot
of salamander specimens and at the lowest point between toes (ref. 16 and
Fig. 1b). Differences in shape are defined here as differences that are not
caused by scaling, translation, or rotation of feet (17). The shape difference is
measured as a linear approximation of the procrustes distance, which is the
square root of the summed squared distances between homologous land-
markswhenconfigurationsareinprocrustedsuperimposition(18).Thislinear
approximation is the quantity used by morphometrical software to compare
shapes and perform statistical tests. A thin-plate spline is used to locate shape
differences between two objects. Grid plots visualize this shape change.
Table 1. Growth trajectories in individual species
Species
n
SinuosityAvg. W, g
?
bb*?*100
B. subpalmata
B. dofleini
B. salvinii
B. alberchi
B. lincolni
B. schizodactyla
B. franklini
B. mexicana
B. helmrichi
B. marmorea
C. magnipes
24
26
13
55
24
9
27
56
15
11
33
2.39
1.59
1.55
1.57
2.32
1.60
2.43
1.56
1.80
2.19
1.86
1.6
9.0
7.3
4.3
2.8
2.5
2.0
2.7
1.3
2.5
1.4
0.78
0.68
0.66
0.68
0.71
0.87
0.66
0.66
0.68
0.71
0.73
0.08
0.10
0.11
0.11
0.11
0.09
0.12
0.12
0.12
0.13
0.21
6.2
6.8
7.3
7.5
7.8
7.8
7.9
7.9
8.2
9.2
15.3
Nonlinear least-squares regression was used to estimate ? and b in 10 Bolitoglossa species and in C. magnipes.
?andbaresimilarinallBolitoglossaspecies(seerightmostcolumn).WebbedandunwebbedBolitoglossaspecies
therefore share the same growth trajectory. The increase in the B. schiozodactyla ? value may be influenced by
the small sample size; however, C. magnipes shows an increase in its b value, explaining the increase in relative
foot surface area.
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Mathematical Model. A mechanical model was used to study the adaptive
value of webbing to climbing on smooth surfaces. This model tests whether A
and W have evolved to optimize attachment. Initially, the forces acting on a
salamander hanging upside down on a smooth surface are calculated. Sub-
sequently,givenasetofspecies-specificparameters(A,W,?,andb),theability
of the salamander to attach is assessed. Eqs. 1–3 show that relationships of A
and W resulting in optimal attachment can be predicted (SI Fig. 7).
A ? bW?
[1]
is the allometric equation describing the relationship of A and W as the
organism grows.
??P ? v?A ? gW ? 0
[2]
describes the balance of forces acting on a salamander hanging upside down
on a smooth surface, where ?P is the amount of suction and v the viscosity of
thesecretedmucus.gistheaccelerationofgravity.Bysettingthisequationto
zero, one describes the boundary condition where the salamander reaches its
maximal weight with zero force acting on it in an upward or downward
direction (6).
Eq. 1 ? Eq. 2 ? bW?? gW/??P ? v?.
[3]
Eq. 2 solved for A and then subtracted from Eq. 1 yields Eq. 3. Optimizing Eq.
3 identifies the optimal weight W* for a given species with a given set of
parametersb,?,?P,andv.Alternatively,giventheaverageWofaspecies,Eq.
3 predicts the shape of Eq. 1, resulting in optimal attachment.
ACKNOWLEDGMENTS. We thank D. Adams, N. Shubin, M. Zelditch, C. Spen-
cer, and the Wake laboratory group for helpful discussions and suggestions.
We especially thank Emma Goldberg, who gathered preliminary data and
helped devise the sinuosity measure. This work was supported by National
Science Foundation Amphibian Tree of Life Grant EF-0334939.
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