Articles

DOI: 10.1038/s41559-017-0268-6

Adult frogs and tadpoles have different

macroevolutionary patterns across the Australian
continent
Emma Sherratt   1*, Marta Vidal-García1, Marion Anstis2 and J. Scott Keogh1

Developmental changes through an animal’s life are generally understood to contribute to the resulting adult morphology.
Possible exceptions are species with complex life cycles, where individuals pass through distinct ecological and morphologi-
cal life stages during their ontogeny, ending with metamorphosis to the adult form. Antagonistic selection is expected to
drive low genetic correlations between life stages, theoretically permitting stages to evolve independently. Here we describe,
using Australian frog radiation, the evolutionary consequences on morphological evolution when life stages are under differ-
ent selective pressures. We use morphometrics to characterize body shape of tadpoles and adults across 166 species of frog
and investigate similarities in the two resulting morphological spaces (morphospaces) to test for concerted evolution across
metamorphosis in trait variation during speciation. A clear pattern emerges: Australian frogs and their tadpoles are evolving
independently; their markedly different morphospaces and contrasting estimated evolutionary histories of body shape diver-
sification indicate that different processes are driving morphological diversification at each stage. Tadpole morphospace is
characterized by rampant homoplasy, convergent evolution and high lineage density. By contrast, the adult morphospace shows
greater phylogenetic signal, low lineage density and divergent evolution between the main clades. Our results provide insight
into the macroevolutionary consequences of a biphasic life cycle.

T
he importance of development in shaping the morpho-
logical diversity observed among adult forms has long been
recognized1–3. Perhaps the most pervasive idea in evolution-
ary developmental biology is that phenotypic variation among spe-
cies arises through relative timing of developmental events along a
continuous ontogenetic sequence, that is, from embryo to juvenile
to adult4–6. Indeed, subtle alterations to species-specific ontoge-
netic trajectories among closely related species can result in diver-
gent or convergent phenotypes at the adult stage, for example, refs
7–9
. However, the majority of animal phyla do not develop along a
continuous ontogenetic sequence, but instead have distinct body
plans at different stages of their life history10. This is a phenome-
non known as a complex life cycle (CLC), where individuals pass
through two or more distinct ecological and morphological phases
during their ontogeny, ending with metamorphosis into the adult
form. Distinct life-history stages are a solution to the problem of
different, conflicting selection pressures arising during an animal’s
lifetime. These distinct stages, which are derived from the same
genome, can be so different that larval stages of some taxa have been
misclassified as entirely different organisms than the adult forms
(for example, the leptocephalus larvae of eels, or nauplius larvae of
barnacles). Since the adult phenotype is not simply a change in size
or proportion from previous life stages (as in simple life cycle taxa),
but instead manifests through a complex metamorphosis, it is less
apparent whether earlier life stages contribute to or constrain pat-
terns of adult phenotypic diversity in taxa with CLCs.
Antagonistic selection at each life-history stage has been hypoth-
esized to maintain low genetic correlations across the metamorphic
boundary, that is, between larval and adult traits, known as the
‘adaptive decoupling hypothesis’11,12. Within species, the adaptive
decoupling hypothesis has been examined using a variety of CLC
taxa, for example, refs 13–21, with mixed support. Two approaches
are generally taken to address this hypothesis: either by examining
genetic and/or phenotypic correlations between equivalent traits
in pre- and post-metamorphic stages13,15–18,20, for example, locomo-
tive behaviour in larval and adult stages; or by investigating larval
traits that correlate with different, functionally relevant post-meta-
morphic traits14,21, for example, the relationship between larval life
history and adult morphology. These studies suggest that it is not
an all-or-nothing process, but is trait-dependent. The evolutionary
consequence of adaptive decoupling is that the different life-history
stages are theoretically free to evolve independently of each other,
and would result in markedly different macroevolutionary patterns
at each stage. Such discordance of interspecific phenotypic evolu-
tion in CLCs has been referred to as a form of ‘mosaic evolution’,
the decoupling of traits during evolution22,23. Yet, at the macroevo-
lutionary level, relatively little is known about whether patterns of
phenotypic evolution are congruent between life-history stages. So
far, studies in biphasic taxa have not supported concerted morpho-
logical evolution across metamorphosis23–25. The degree to which
independent evolution between life-history stages of species with
CLCs is important for understanding macroevolutionary diversity,
because it allows us to quantitatively assess the role that develop-
ment, specifically preceding developmental stages, has in shaping
adult diversity.
Anurans, frogs and toads, are a model system for the study of the
phenomena of CLCs. They are a species-rich group with remarkable
levels of morphological, ecological and reproductive diversity, yet
most species have a biphasic lifecycle with ecologically and mor-
phologically distinct life stages: the larval tadpole form is always tied
to an aquatic or very moist environment, for example, streams, per-
manent ponds, ephemeral water bodies, inundated burrows, even

​ ivision of Ecology & Evolution, Research School of Biology, The Australian National University, Canberra, ACT 2601, Australia. 2​School of Environmental
D
1

and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia. *e-mail: emma.sherratt@gmail.com

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Articles
the vocal sacs and stomachs of adults in some species; adult frogs,
on the other hand, occupy a wide range of terrestrial and freshwa-
ter environments, for example, deserts and semi-arid areas, alpine
regions, rainforest, and even permafrost in Alaska. Metamorphosis
between these stages is an extraordinary transformation from an
organism with a composite head and body and a muscular tail (usu-
ally without vertebrae), to an adult with a distinct head and body, no
tail, but with long limbs and fused vertebrae (urostyle).
In anurans, the degree of decoupling between larval and adult
traits across metamorphosis is trait-dependent, and thus support
for the adaptive decoupling hypothesis varies. For example, behav-
ioural traits (for example, activity and exploration) are consistent
and moderately correlated across metamorphosis19. On the other
hand, there are low genetic and phenotypic correlations in morpho-
metric traits between recently metamorphosed juveniles and adult
wood frogs17, and in locomotor performance and morphometric
traits between larval and post-metamorphic frogs15,18. Finally, lar-
val life-history traits show varying degrees of correlation with adult
morphology14. The decoupling of larval and adult stages in anurans
in relation to evolutionary diversification above the species level has
only recently been investigated in Madagascan mantellid frogs26.
Studies examining interspecific phenotypic evolution at a single life
stage show that morphological diversification in tadpoles is pre-
dominantly related to locomotive specializations27–29. There is also
rampant homoplasy30, which is why tadpoles are notoriously dif-
ficult to identify to species. Morphological diversification of adult
frogs often shows convergent evolution in many environments, such
as burrowing, arboreal or terrestrial niches, for example, refs 31–34,
although adult morphology appears to have more phylogenetic
signal than larval morphology35. Thus, the trait of whole body
form at each stage is known to reflect convergent ecomorphologi-
cal adaptations to different locomotive strategies and the niches
they inhabit, but it is unknown whether the patterns are similar
between the stages.
Here we investigate whether there has been a correlated response,
across metamorphosis, to stage-specific processes, whereby larval
and adult evolution has proceeded in tandem along branches of
the phylogenetic tree, or if the tadpole and adult frog life stages are
evolving independently. On the one hand, stage-specific processes
(such as ecological specialization acting on the tadpole stage, or
speciation acting on the adult reproductive stage) may result in cor-
related changes in some traits during the biphasic lifecycle, because
the survival of one stage directly influences the next. However, the
adaptive decoupling hypothesis suggests that low genetic correla-
tions maintained between life stages by antagonistic selection will
result in the two life stages evolving somewhat independently. We
use frogs to examine the relationship between the morphospaces
and inferred histories of morphological diversification of the larval
and adult body plans. We use a comprehensive morphological data-
set of Australian anurans, built from traditional and geometric mor-
phometric data representing adult and tadpole body shape (Fig. 1),
to test the null hypothesis that there is no concordance of interspe-
cific body shape evolution. Furthermore, we place the observed
phenotypic variation in an evolutionary context using phylogenetic
comparative methods to infer the history of morphological diversi-
fication at each stage (evolutionary morphospaces) to characterize
the mode of evolution and whether it differs between the life stages.
Results
Evolution of larval anuran morphospace. Among species, shape
variation is often the result of an evolutionarily association with size
variation, known as evolutionary allometry. In tadpole body shape,
evolutionary allometry is weak and not significant (phylogenetic
generalized least-squares regression (PGLS), R2 =​  0.09, P =​  0.076).
The tadpole morphospace of all 166 species is summarized by prin-
cipal components analysis, and four principal components describe
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NATure EcOlOgy & EvOluTiOn
80.1% of the total shape variation among species (depicted in
Supplementary Fig.  1); the remaining principal components each
describe less that 5% of the shape variation. The main axes explain
changes in shape regarding the whole tadpole (head and body, and
tail). The first axis describes variation in the length of the tail rela-
tive to the head and body region, ranging from a tail that is equal to
the length of the head and body, to a tail that is elongated and twice
as long as the head and body. The second axis describes shape varia-
tion arising from dorsoventral compression of the head and body
region and tail; tadpoles with high-arching tail fins and a deep head
and body are contrasted with shallow tail fins and a flattened head
and body. The phylomorphospace (principal component morpho-
space into which a phylogenetic tree is projected) that is defined
by the first two axes (61.1%) is shown in Fig. 2. There is substan-
tial homoplasy in tadpole shape, as demonstrated by the ‘bird’s
nest’ configuration; there are many crisscrossing branches, and
closely related species are often widely divergent in morphospace.
Consequently, phylogenetic signal is very low in tadpole body shape
(K =​  0.2215, P =​ 0.001). Lineage density of the tadpole morphospace
is high (0.92), indicating that there are many long branches packed
within the morphospace enclosed by the observed species, which
suggests low morphological innovation.
Evolution of adult anuran morphospace. There is significant
evolutionary allometry in the adult shape variables with body
size (snout-to-vent length) (PGLS R2 =​  0.27, P =​ 0.001). The adult
frog morphospace of all 166 species can be summarized by four
principal components describing a total of 82.5% of the variation
(depicted in Supplementary Fig. 2); the remaining principal com-
ponents each describe less than 5% of the shape variation. Overall,
the main axes explain changes to the measurements of the head and
limbs of the adult frogs. The first axis corresponds to the length of
the digits (5th toe, 1st toe, 4th finger and thumb), digits shorten
with an increase in eye size (eye length), such that longer digits
coincide with relatively smaller eyes. The second axis corresponds
to the face length (naris–snout length), changing negatively with
thumb length, such that longer snouts coincide with short thumbs.
The phylomorphospace that is defined by the first two axes (67.2%)
is shown in Fig. 2. The adult morphospace is distinctly divided by
clades: hylids and myobatrachids are divided strongly along princi-
pal component (PC)1, with partial overlap of the myobatrachid sub-
families Limnodynastinae and Myobatrachinae. Phylogenetic signal
is moderate (K =​  0.455, P =​ 0.001), but much higher than in tadpole
body shape. Lineage density of the adult morphospace is very low
(0.18) indicating branches in the morphospace are relatively short
and not densely packed, suggesting higher morphological innova-
tion than in tadpoles.
Dissimilarity in larval and adult anuran evolution. The morpho-
spaces of tadpoles and adults are strikingly different, particularly in
that the clades are well separated in adults and highly overlapping
in tadpoles (Fig. 2). There is little correspondence and low statisti-
cal correlation in the patterns of morphological disparity described
by the two datasets; permuted partial disparity of adults and tad-
pole datasets have a low correlation (all data: Spearman’s rank cor-
relation, ρ =​  0.026, P =​ 0.4053; only PC1–4, ρ =​  0.053, P =​  0.0961;
Supplementary Fig. 3). In terms of dispersion of species in morpho-
space, the correlation between the tadpole and adult datasets is also
low (ρ =​  0.169, P =​ 0.001; only PC1–4, ρ =​  0.176, P =​  0.001). These
results indicate negligible correspondence between larval and adult
evolutionary histories and resultant morphospaces.
Discussion
Ontogeny undoubtedly has an important role in creating phe-
notypic diversity, yet research in this area commonly focusses on
organisms with continuous, simple life cycles, where the adult stages
NATure EcOlOgy & EvOluTiOn Articles
a 24
26 25 23
12 11 10 9 27 22
14 13
15 21
16 7 20
19
17 2
1
46 18
47 48 49 50 51 52 53
3
54 55 8
6 36 37
4 35
38 34
33
39 5 28 31 32
40 45 29 30
41
42 43 44
b
6
5
3 4
7 8
9

2
1

16
17

18 15
23
19 13
14
12

22
21 20

Fig. 1 | Morphometric variables characterizing body shape in tadpoles and adult frogs. a, Landmarks (grey circles) and semilandmarks (blue circles) for
characterizing the body shape of tadpoles (see Supplementary Table 2 for descriptions). b, Linear measurements for characterizing the body shape of adult
frogs (see Supplementary Table 3 for descriptions). Note, measurement numbers in a and b are not analogous.

manifests as a change in size and allometric proportions from previ-
ous life stages. Comparatively less is understood about how much,
if at all, early life-history stages in animals with complex life cycles
influence the diversity we observe in the adult phenotype. Using a
model vertebrate system with a biphasic lifecycle, and taking a mac-
roevolutionary perspective, we investigated whether the outcomes
of stage-specific processes determining body shape diversity in the
larval stage or the reproductive stage (that is, adult) are carried over
to the other stage, or if these stages are evolving independently. Our
results indicate that there is extreme discordance between the body
plan morphospaces of larval and adult Australian anurans, where
morphological variation (disparity) among species in one stage is
not indicative of disparity at the other stage. The inferred evolu-
tionary history of morphological diversification of each life-history
stage is also very different: the tadpole morphospace shows substan-
tial homoplasy, and a clear pattern of convergent evolution, whereas
the adult morphospace has more phylogenetic signal and shows
divergent evolution among the families and subfamilies. These find-
ings suggest the larval and adult life stages are evolving, for the most
part, independently.
Differences in the evolutionary history of larval and adult traits
have often been investigated with respect to their use for cladistic
analyses, and consequently larval forms of many animals are found
to be homoplastic compared to adults, for example, refs 23,25. Larval
anurans are no exception to this pattern, to which anyone who has
attempted to use taxonomic keys to identify tadpoles to species can
attest. Our results from Australian anurans clearly show low phy-
logenetic signal and substantial homoplasy in tadpole body shape,
which is consistent with other studies with broader taxonomic
sampling30,35. A wide-scale study into the phylogenetic signal of
microhabitat use in tadpoles and adult frogs found similar patterns
to ours: there is low phylogenetic signal and extreme plasticity in
tadpoles, and more structure in adults36. Therefore, the substantial
homoplasy in tadpoles may be owing to this apparent ease in which
microhabitat shifts of tadpoles occur during speciation, because
tadpole body shape is strongly linked to ecology37.
The efficiency of morphological innovation (inferred from lin-
eage density in morphospace) was low for tadpoles, and higher for
adult frogs. Although we cannot test it directly, this result may indi-
cate greater disparity in adult morphology compared to tadpoles. If
adult morphology has greater disparity, this may simply be because
a body plan with four limbs with digits has more scope for shape
variation than a ‘finless, fish-like’ body plan of a tadpole. Yet when
the fish body plan is preserved through metamorphosis, a previ-
ous study24 has found greater interspecific variation in body elonga-
tion in adults compared to larvae of bony fishes, this was attributed
to different locomotor demands and physiological constraints (for
example, oxygen uptake through skin). Conversely, some studies of
species with simple life cycles have found a greater disparity among
similarly aged juveniles relative to adult phenotypes, because of
convergent evolution (for example, refs 9,38). In newts, which also
have a biphasic lifecycle, low disparity in body shape was found in
early larval stages as well as in adults, but greater disparity was found
in stages nearer to metamorphosis39. Future research into the phe-
nomenon of differing temporal disparity across ontogeny should
investigate the processes behind greater diversity at one stage or
another, that is, whether it is because of biological constraints or
natural selection.

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Articles NATure EcOlOgy & EvOluTiOn

Cyclorana platycephala Mixophyes fasciolatus Heleioporus eyrei Limnodynastes tasmaniensis Lechriodus fletcheri Neobatrachus sudelli
1 2 3 4 5 6
Tadpole PC1
−0.10 −0.05 0.00 0.05 0.10
Litoria infrafrenata
7 Notaden bennettii
0.10 12

Litoria dahli
8 8 Pseudophryne corroboree
13
0.05 6 1
Litoria nasuta
9 Uperoleia laevigata
2
14
7 10
15 11
Tadpole PC2

9 12
Litoria bicolor 0.00 Spicospina flammocaerulea
10 4 15
5
3 14 13

Litoria xanthomera Crinia signifera

11 16
−0.05

16

Limnodynastinae
−0.10 Hylidae (Litoria) Crinia signifera
Rheobatrachus + Mixophyes
Litoria xanthomera Hylidae (Cyclorana) 16
11 Myobatrachinae

1 6
0.5 12
Litoria bicolor Spicospina flammocaerulea
10 7 15
11 3

8
Adult PC2

15 14
13
Litoria nasuta 0.0 10 4 Uperoleia laevigata
2 5
9 14

16
9
Litoria dahli −0.5 Pseudophryne corroboree
8 13

−0.5 0.0 0.5 1.0

Litoria infrafrenata Notaden bennettii
7 Adult PC1
12

Cyclorana platycephala Mixophyes fasciolatus Heleioporus eyrei Limnodynastes tasmaniensis Lechriodus fletcheri Neobatrachus sudelli
1 2 3 4 5 6

Fig. 2 | Evolutionary phylomorphospaces of tadpoles and adult frogs. The evolutionary morphospaces of tadpoles (top) and adult frogs (bottom), into
which a phylogenetic tree has been projected to visualize the relationships among species (points). Drawings of 16 representative species as both stages
demonstrate the difference in body shape diversity. PC, principal component. Scale bars below each image are 10 mm, for both tadpoles and adult frogs.
Tadpole drawings modified from Anstis52, and adult drawings by E. Walsh. Species of the family Hylidae are denoted by squares (shades correspond to the
two genera), and Myobatrachidae species are denoted by circles (shades correspond to the two subfamilies, and Rheobatrachus with Mixophyes).

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NATure EcOlOgy & EvOluTiOn
Our study focussed on whole body shape in tadpoles and adults,
because it is a complex trait that strongly relates to functional per-
formance and niche specialization27–29,31–34. In this way, comparing
body shape in larval and adult stages is analogous to comparing tad-
pole swimming and adult frog jumping performance, for example,
refs 15,18. These traits have also been shown to have little correlation
across the metamorphosis boundary. Animal morphology is only
one aspect of the animal, however, and previous studies that have
tested adaptive decoupling in animals with CLCs, have examined
a great variety of traits, that is, behavioural traits, morphological
traits, performance, life history and even colouration, with vary-
ing support for the adaptive decoupling hypothesis13,15–21. Traits that
are genetically determined and directly passed over through meta-
morphosis represent the ideal test for decoupling and consequently
independent evolution. However, the nature of metamorphosis and
its complete overhaul of the body plan makes this a notable chal-
lenge. The suit of developmentally homologous traits at each stage
is limited, and this is perhaps why the adaptive decoupling is often
inferred from correlated responses in different, but functionally
related traits, for example, refs 14,21, or investigated in non-morpho-
logical traits, for example, refs 15,16,18. If morphology is the focus, a
character-based approach to describing trait variation (as in ref. 23,
see below) permits a greater range of traits to be captured, partic-
ularly presence or absence of a trait, in a way that morphometric
methods usually cannot (although character and morphometric
datasets do often provide congruent results40–42). Regardless of the
trait, this study adds to a growing body of evidence that larval and
adult life stages of CLC species are distinct and have the propensity
to evolve along their own macroevolutionary trajectories.
Early literature discussing the discordance of interspecific phe-
notypic evolution in CLCs co-opted the term ‘mosaic evolution’22,23,
which generally refers to the decoupling of traits during develop-
ment and evolution2 (also known as modularity43), but in this case
refers to larval and adult morphology evolving independently
within a lifecycle. Few other studies have considered the evolution-
ary consequences of maintaining a CLC, and the potential adaptive
decoupling of life stages, by taking a macroevolutionary approach
to explicitly test whether patterns of interspecific phenotypic evolu-
tion are congruent between life-history stages. A previous study23
has carried out a broad cladistic analysis of patterns of morphologi-
cal evolution in echinoids (reanalysing data from ref. 22) and found
that there was little evidence for concerted morphological evolution
between larval and adult life-history stages. Recently, a transcrip-
tomic approach was used for analysis of the Madagascan mantellid
frog radiation26 and it was demonstrated that genes associated with
morphological structure formation are expressed in a life stage-spe-
cific pattern, thus arriving at the same conclusion of the decoupling
of phenotypic evolution across metamorphosis. Although these are
not organisms with a CLC, others38 have examined shell shape mor-
phospace occupation of juvenile and adult ammonites, and found
substantially higher morphological disparity in adults, but they
did not explicitly examine the similarity of dispersion patterns or
inferred evolutionary diversification as was carried out here. CLC
is the most common life-history strategy in the animal kingdom,
yet only few studies have considered evolution in these taxa at mul-
tiple life stages. The association of larval and adult morphospaces
in CLC taxa, which has been referred to as developmental and non-
developmental morphospaces44, is important for the understanding
of the genotype–phenotype map and addressing the fundamental
question of how ontogeny relates to phylogeny. Further investiga-
tions using the plethora of CLC taxa, both invertebrate and verte-
brate, are encouraged.
Research on the evolution of taxa with CLCs has predominantly
focused on why distinct life stages are maintained11,12, and the evolution-
ary consequences of losing the free-living larvae and instead directly
developing into the adult form45–47. In amphibians, evolutionary
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Articles
shifts from a biphasic lifestyle to direct development are numer-
ous48. The loss of a free-living larval stage in plethodontid salaman-
ders has been implicated in the high morphological and ecological
diversity and species richness of this clade, which forms the basis
for a hypothesis that a biphasic lifestyle could represent an ontoge-
netic constraint on the adult phenotype46,49. In marine invertebrates,
where developmental strategy is extremely evolutionarily labile,
the degree to which a CLC affects adult phenotype, compared to
direct developing species, appears to vary from clade to clade22,47.
Whether the presence of a free-living larval stage provides an onto-
genetic constraint on adult phenotypic diversity has yet to be explic-
itly examined in most CLC taxa. We propose that Australia’s frogs
are a potentially good system to examine this hypothesis, given that
12% of the continent’s native anuran taxa are direct developers. The
results of this study suggest that the ontogenetic constraint hypoth-
esis may not apply to Australian frogs, or to Anura generally. Future
research is encouraged along this avenue.
Methods
Samples and morphometric body shape data. Australia’s indigenous anuran
biodiversity comprises three Neobatrachian families. We only sampled species
that have a free-living larval stage from Hylidae (68 species) and Myobatrachidae
(99 species), for a total of 166 species (70% of the total amphibian diversity
in Australia; Supplementary Table 1). We excluded all microhylid species and
Arenophryne, Metacrinia and Myobatrachus (Myobatrachidae), because these
species do not have a free-living larval stage (they are direct developers). We
also excluded the single Ranidae species Papurana daemeli (formerly Hylarana),
because it is a relatively more recent migrant into Australia from New Guinea50.
All the following procedures and analyses were performed in the R statistical
environment v.3.3.1 (ref. 51) unless otherwise stated.
Tadpoles were sampled from the comprehensive work of Anstis52 and
comprised animals that were at mean Gosner stage 35.4 (±​3.09; hindlimb bud
started to grow, toes gradually developing on hindlimbs). For each species,
Anstis52 produced a detailed and accurate drawing of a tadpole in lateral view
from multiple specimens where the position was standardized. The difference in
morphology between photographs and the high-quality drawings is negligible
relative to among species variation (Supplementary Fig. 4). To circumvent
phenotypic plasticity to environmental conditions, tadpole drawings from ref. 52
had been, where possible, based on specimens reared in captivity under consistent
conditions and therefore represent the phenotype without ecological influences.
We used two-dimensional (2D) landmarks and semilandmarks to characterize
tadpole body shape (Fig. 1a and Supplementary Table 2). Landmarks were digitized
using tpsDig2 v.2.26 (ref. 53) on published lateral view photographs and drawings
of tadpoles52, and semilandmarks were automatically digitized by routines written
in R and ImageJ54. Details of the digitizing process are given in the Supplementary
Information. The landmark and semilandmark coordinates were aligned using a
generalized Procrustes superimposition55 implemented in the R package geomorph
v.3.0.2 (ref. 56), where the semilandmarks were permitted to slide along their
tangent directions to minimize bending energy57. The resulting Procrustes shape
coordinates were used as tadpole body shape variables in the subsequent analyses.
To characterize adult frog body shape we used published species-average
morphometric data (five female specimens per species) comprising 23 external
linear measurements collected using digital calipers on alcohol-preserved
museum specimens33 (Fig. 1b and Supplementary Table 3). The 22 variables
(excluding snout-to-vent length) were transformed using the log-shape ratios
method58, where the species data are divided by species size (as geometric mean)
and ln-transformed, producing shape variables that were used in the subsequent
analyses. This approach is analogous to the size correction in the Procrustes
superimposition method for landmark data, and both approaches correct for size,
while retaining the allometric shape variation59. It is important to note that the
juvenile and adult body shape variables are on a different scale.
Statistical analyses. In order to do phylogenetically informed statistical analyses
we generated a Bayesian molecular phylogeny, with branch lengths, for all 166
species of Australian hylids and myobatrachids used in this study. The phylogeny
was consistent with previously published molecular phylogenies that have been
used for macreoevolutionary analyses in these groups50,60 (phylogeny available on
Dryad (http://dx.doi.org/10.5061/dryad.23j6t)).
We investigated allometry in both the tadpole and adult datasets using
multivariate regressions based on a PGLS model for multivariate data61,
implemented in geomorph. Size in the tadpole dataset was centroid size (calculated
from the landmark configurations), and snout-to-vent length was used in the adult
dataset. Statistical significance was assessed by permutation (1,000 iterations).
To visualize the tadpole and adult body shape morphospaces, we performed
principal component analysis of the Procrustes shape coordinates (hereafter
1389
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tadpole shape variables) and the regression residuals of log-shape ratios (hereafter
adult shape variables). The shape variation described by each principal component
axis of the tadpole morphospace was visualized using thin-plate spline warp grids
of the principal component loadings, implemented in geomorph, which plots the
eigenvectors of each landmark coordinate simultaneously to represent the overall
shape change from the mean shape of the sample to the minima, and maxima, of
the axes. For the adult morphospace, the importance of the variable loadings on
the first four axes were interpreted from biplots. We then used a complementary
suite of analyses to compare these two morphospaces.
To estimate the overall strength of correlation between the two morphospaces
and estimate the similarity of species dispersion, we performed a Mantel test
between adult and tadpole matrices of the pairwise Euclidean dissimilarities
(distances) between species implemented in vegan v.2.3-5 (ref. 62). Scale differences
between the datasets are irrelevant for this approach. The null hypothesis of a
Mantel test is the absence of relationship between the dissimilarities in the two
matrices, and statistical significance was assessed using a permutation approach
(1,000 iterations) on the Spearman’s rank correlation coefficient.
To examine whether the two datasets describe similar morphological disparity,
we applied a disparity permutation procedure designed to compare different
datasets of the same taxa sensu40. Disparity was measured as variance, calculated
as the trace of the covariance matrix divided by the number of observations8 as
implemented in geomorph. We randomly subsampled sets of 84 species (half the
total sample) and calculated disparity for each dataset, this was repeated 1,000
times. Correlation between the subsampled disparities of the two datasets was
calculated using the Spearman’s rank correlation coefficient. Spearman’s rank
correlation is the most appropriate, because it evaluates the monotonic relationship
between the two matrices by using the ranked values for each, and, as such,
does not assume a linear relationship. To account for the difference in the
number of variables (110 in tadpole shape dataset, 22 in adult), we repeated this
disparity permutation procedure using only the first four principal component
axes of each dataset.
To estimate the evolutionary history of body shape diversification in these
morphospaces, we used a phylomorphospace approach63. The phylogeny was
projected into the morphospaces by calculating ancestral states of the internal
nodes through maximum likelihood, as implemented in geomorph. This
approach allows inference of the magnitude and direction of shape change
along any branch. From the phylomorphospaces, we investigated the ‘efficiency
in morphological innovation’ during evolution, which is the degree to which
taxa explored more novel regions of morphospace and expanded out from their
inferred ancestral positions, or remained relatively clumped within morphospace.
This is done by calculating the lineage density63, which describes the degree of
packing by the branches of the phylogeny in the morphospace, and is calculated
as the sum of morphometric branch lengths divided by the volume occupied
in the morphospace. Lineage density is a ratio and can therefore be compared
between datasets irrespective of scale. Higher lineage-density values indicate
that the morphospace is filled with long branches that are more densely folded
to pack into the total occupied space, suggesting low morphological innovation,
whereas lower lineage-density values indicate shorter branches, less packing, and
this suggests higher morphological innovation. The volume was calculated as a
minimum bounding hyper-ellipsoid of the space defined by the first four principal
component axes of each morphospace, implemented in cluster v.2.0.4 (ref. 64).
The absolute magnitude of change per branch (to estimate whether there is more
morphological variation at one life stage versus another) was not possible to
estimate, because of the difference in scale of the two datasets.
Finally, we measured phylogenetic signal, the degree to which related species
resemble each other phenotypically, using the multivariate extension of the
generalized K statistic65 implemented in geomorph. A K value greater than 1
implies that closely related species are more similar than expected under a
null model of Brownian motion (more phylogenetic signal), whereas a value
that is less than 1 suggests that relatives resemble each other less than expected
(less phylogenetic signal). Rather than absolute values of K on this scale,
we focus on how K differs between tadpoles and adults. Statistical significance
was assessed by permutation (1,000 iterations).
Data availability. The digitizing protocol (including R computer code) is provided
in the Supplementary Methods, and R computer code for analyses, all data and
phylogenetic tree are available on Dryad (http://dx.doi.org/10.5061/dryad.23j6t).
Received: 4 December 2016; Accepted: 3 July 2017;
Published online: 14 August 2017
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Acknowledgements
We thank T. J. Sanger for comments on the manuscript, and E. Walsh (http://www.
negaleg.com/) for the beautiful adult frog drawings she produced for us and help
with figure preparation. Funding came from the Australian Research Council
DP150102403 to J.S.K.
Author contributions
E.S. and J.S.K. conceived the study. E.S., M.A. and M.V.-G. collected the data.
E.S. performed the analyses. E.S., J.S.K. and M.V.-G. wrote the paper. All authors read
and approved the final manuscript.
Competing interests
The authors declare no competing financial interests.
Additional information
Reprints and permissions information is available at www.nature.com/reprints.
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