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Phylogenetic relationships of the Cretaceous frog Beelzebufofrom Madagascar and the placement of fossil constraints basedon temporal and phylogenetic evidence
S. RUANE*� , R. A. PYRON� & F. T. BURBRINK*�*Department of Biology, The College of Staten Island, The City University of New York, New York, NY, USA
�Department of Biology, The Graduate School and University Center, The City University of New York, New York, NY, USA
�Department of Ecology and Evolution, Stony Brook University, Stony Brook, NY, USA
Estimating the ages of clades by integrating fossil data
and molecular phylogenies has become commonplace
and expands the range of evolutionary hypotheses that
can be tested with phylogenetic data. The placement of
fossil calibrations on molecular phylogenies is ideally
based on explicit phylogenetic analysis of extinct species,
for which only morphological data is typically available
(e.g. Donoghue et al., 1989; Shaffer et al., 1997; Manos
et al., 2007; Lee et al., 2009). Recent work has also
suggested that combined analysis of morphological and
molecular data may improve the estimation for the
support and placement of both extinct and extant taxa
(Wiens, 2009; Wiens et al., 2010), and the placement of
fossil calibrations (e.g. Shaffer et al., 1997; Gatesy et al.,
2003; Sauquet et al., 2009; Magallon, 2010). However,
uncertainty regarding the placement of extinct taxa
represents a continuing source of error, which should
be incorporated into estimates of divergence dates (Ho &
Phillips, 2009; Lee et al., 2009).
Additional information regarding the placement of
fossil calibrations can be derived from other well-
supported constraints placed throughout the tree. Using
other calibrations, one can predict the age of the targeted
node for the uncertain fossil. Several methods for
incorporating this information into divergence time
analyses have been developed (e.g. Near & Sanderson,
2004; Near et al., 2005; Rutschmann et al., 2007; Pyron,
2010; see Marshall, 2008). However, these methods vary
in their effectiveness in highlighting and combating
improper calibrations and choosing the optimal set of
constraints (Rutschmann et al., 2007; Marshall, 2008).
To better understand the impact that the placement of
a fossil on a particular node has on date estimates
throughout a tree, we expand on the protocols suggested
by Lee et al. (2009) and Rutschmann et al. (2007). In
particular, we assess the impact that the phylogenetic
placement of the Late Cretaceous fossil frog Beelzebufo
ampinga from Madagascar has on estimates of divergence
dates on Anura. This massive fossil frog has been
hypothesized to be a crown-group member of the
New World (NW) hyloid subfamily Ceratophryinae
(Evans et al., 2008), comprised of eight species in the
extant genera Ceratophrys (known commonly as the pac-
man frogs), Chacophrys and Lepidobatrachus (as defined
by Fabrezi, 2006). This assignment would offer support
for a remnant connection between South America,
Correspondence: Sara Ruane, Department of Biology, The College of Staten
Island, The City University of New York, 2800 Victory Blvd, Staten Island,
NY 10314, USA. Tel.: 718 982 3850; fax: 718 982 3852;
e-mail: [email protected]
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274 J O U R N A L O F E V O L U T I O N A R Y B I O L O G Y ª 2 0 1 0 E U R O P E A N S O C I E T Y F O R E V O L U T I O N A R Y B I O L O G Y
Keywords:
Beelzebufo;
Ceratophrys;
dating error;
divergence time estimation;
fossil calibration;
Gondwanaland;
Madagascar.
Abstract
The placement of fossil calibrations is ideally based on the phylogenetic
analysis of extinct taxa. Another source of information is the temporal
variance for a given clade implied by a particular constraint when combined
with other, well-supported calibrations. For example, the frog Beelzebufo
ampinga from the Cretaceous of Madagascar has been hypothesized to be a
crown-group member of the New World subfamily Ceratophryinae, which
would support a Late Cretaceous connection with South America. However,
phylogenetic analyses and molecular divergence time estimates based on other
fossils do not support this placement. We derive a metric, Dt, to quantify
temporal divergence among chronograms and find that errors resulting from
mis-specified calibrations are localized when additional nodes throughout the
tree are properly calibrated. The use of temporal information from molecular
data can further assist in testing phylogenetic hypotheses regarding the
placement of extinct taxa.
doi: 10.1111/j.1420-9101.2010.02164.x
Madagascar and India via Antarctica that may have
existed well into the Late Cretaceous (Hay et al., 1999)
and imply a much older age for the hyloids than
previously thought (e.g. � 50–60 Ma, as estimated by
Roelants et al., 2007; Wiens, 2007). We use both mor-
phological and combined molecular + morphological data
to assess the phylogenetic affinity of Beelzebufo. We also
use molecular divergence time estimates derived from
other, well-supported anuran calibrations to assess the
likelihood of alternative placements of the taxon in
the combined-data phylogeny, and as a calibration in the
molecular divergence time analyses.
Placement of Beelzebufo within the crown-group of
Ceratophryinae is supported by several aspects of cranial
morphology (Evans et al., 2008) and potentially offers
support for a Late Cretaceous connection between SA
and Madagascar (Hay et al., 1999). However, in the
phylogenetic analysis presented by Evans et al. (2008),
the sister relationship between Beelzebufo and Ceratophrys
is supported by only a single character (out of 81) in a
maximum parsimony analyses (Evans et al., 2008).
Unfortunately, this relationship cannot be tested using
molecular data alone. Therefore, we integrate molecular
and morphological data to assess the phylogenetic place-
ment of Beelzebufo and use divergence time estimation to
test relationships in a temporal context (e.g. van Tuinen
& Hedges, 2004; Waggoner & Collins, 2004). In doing so,
we examine two major aspects of molecular divergence
time estimation: (i) how molecular divergence time
estimates can be used to assess hypotheses concerning
phylogenetic relationships, and (ii) how a misplaced
fossil calibration can influence age estimates across the
phylogeny with and without other constraints.
First, we re-analyse the data presented by Evans et al.
(2008) alone and in combination with molecular data for
extant species using statistical phylogenetic methods to
assess the hypothesized crown-ceratophryine affinity of
Beelzebufo. We use molecular divergence time estimates
from a larger anuran data set (Roelants et al., 2007) to
determine the temporal likelihood of the placement of
Beelzebufo within Ceratophryinae. We would not reject a
sister relationship between Beelzebufo and Ceratophrys if
the estimated dates for Beelzebufo fall within the range of
the ages estimated for Ceratophryinae by the other
calibrations. However, if the estimates for the crown-
group Ceratophryinae are younger than Beelzebufo, this
would challenge the hypothesis of a sister relationship
between Beelzebufo and Ceratophrys. We also test whether
Beelzebufo is temporally compatible as a stem-group
ceratophryine.
Second, we derive a simple metric to assess the impact
of a fossil on date estimates for a tree and determine
whether these effects are consistent across the tree. This
test allows us to examine whether using Beelzebufo as a
calibration significantly alters dates across the tree or
induces only localized errors in the vicinity of the
ceratophryine crown group. Although it is widely known
that misplaced or improperly dated fossils on a tree will
result in poor estimates of divergence dates (Graur &
Martin, 2004), it is unclear how these incorrectly
assigned fossil calibrations impact divergence time esti-
mates on nodes nearest to the calibration point (proxi-
mal) relative to nodes farther away in the tree (distal).
We use this statistic to test the impact that a poorly placed
fossil calibration has on node ages distributed across a
phylogeny.
Materials and methods
Molecular data and tree inference
We used Bayesian inference (BI) methods to construct an
anuran phylogeny and assess placement of fossils for
divergence dating. The molecular data set of Roelants
et al. (2007), consisting of four nuclear genes (CXCR1,
NCX1, RAG1 and SLC8A3) and one mitochondrial gene
(16S), was used for all molecular analyses. This data set
includes 120 anurans, and we included three salaman-
ders and one caecilian as outgroups. We simultaneously
estimated trees and support using BI in the program
MrBayes v3.1.2 (Ronquist & Huelsenbeck, 2003) to
determine the correct fossil placement for our divergence
time analyses. We used the Bayesian Information Crite-
rion (BIC) in jModelTest, (Posada, 2008) with a maxi-
mum-likelihood–optimized base tree to determine the
substitution model for each gene; molecular data were
partitioned by gene and codon position. Each analysis
(two runs of four chains each) was run for 40 million
generations and sampled every 1000 generations. Con-
vergence was assessed using Gelman & Rubin’s r statistic
(Gelman et al., 1995). The analysis was considered
complete when the standard deviation of split frequen-
cies between the chains in MrBayes was < 0.01 and r
approached 1 for all parameters.
Morphological data and tree inference
To test the strength of the hypothesized sister relation-
ship between Beelzebufo and Ceratophrys using BI, we
analysed the 81 character morphological data set used by
Evans et al. (2008; 66 taxa) and a combined data set of
molecular and morphological data, using only the 35
taxa that had both molecular and morphological data
available, and the four extinct taxa represented by fossils
which were only scored from morphological variables. To
assess topology and estimate posterior probability (Pp)
support, we used the standard discrete (morphology)
model (Lewis, 2001) with the default settings in MrBayes
v3.1.2 (Ronquist & Huelsenbeck, 2003) using the 81
morphological characters. This analysis was run for 40
million generations, sampled every 1000 generations,
and the first 10 million samples were discarded as burnin.
For the mixed molecular ⁄ morphological analysis of
35 taxa, we used the models determined by BIC in
Phylogenetic placement of Beelzebufo 275
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jModelTest (Posada, 2008) for the molecular data. We
also performed a parsimony analysis using only the
morphological data used by Evans et al. (2008; 66 taxa) in
PAUP* (Swofford, 2003), with 10 000 nonparametric
bootstrap replicates to estimate node support (the mul-
tistate characters were unordered and unweighted in the
analyses, as per Fabrezi, 2006 and Evans et al., 2008). In
addition, we ran the parsimony analysis a second time
ordering the appropriate multistate characters in the data
matrix (Wiens, 2001; D. Marjanovic, pers. comm.).
Divergence time estimation
We used molecular divergence time estimation to address
two categories of hypotheses that consider (i) the effect of
using Beelzebufo as a calibration date for the extant
ceratophryines on divergence time estimates for other
anuran clades and (ii) the temporal likelihood of a sister-
group relationship between Beelzebufo and Ceratophrys.
First, we ask what effect Beelzebufo has on date estimates
for all nodes throughout the tree. Second, we determine
whether divergence times in the absence of Beelzebufo
support a timeframe consistent with a sister-group
relationship between Beelzebufo and Ceratophrys. Diver-
gence time estimation was performed using the program
BEAST v1.5.4 (Drummond & Rambaut, 2007). The tree
in all BEAST analyses was fixed to the topology gener-
ated by the initial MrBayes analysis of the molecular
data. We applied an uncorrelated lognormal tree prior, a
Yule process speciation prior and lognormal fossil priors
for divergence date estimations. Sequences were parti-
tioned by gene and codon position, and each analysis was
run for a minimum of 40 million generations to generate
a high effective sample size (ESS > 200; Drummond
et al., 2006) and allow the Markov chain Monte Carlo
(MCMC) chains to achieve stationarity.
Four calibration references were used to calibrate
internal nodes for divergence time estimation:
(C1) 249–275 million years (Ma) as the divergence time
between Caudata and Anura, with a lower bound
provided by the stem-anuran Triadobatrachus and
an upper bound based on high number of temno-
and lepospondyls, but a lack of lissamphibians in
the Artinskian fossil record (Marjanovic & Laurin,
2007); lognormal mean (LNM) = 5.569, standard
deviation (LNSD) = 0.0255.
(C2) 170–185 Ma as the divergence time between
Discoglossoidea and Pipanura, based on a lower
bound provided by the earliest discoglossoid
Eodiscoglossus and an upper bound provided by
the nonanuran salientians Vieraella and Prosalirus
(Marjanovic & Laurin, 2007); LNM = 5.178,
LNSD = 0.0216.
(C3) 155–175 Ma as the divergence time between
Xenoanura and Neobatrachia + Pelobatoidea
based on a lower bound provided by the rhi-
nophrynid xenoanuran Rhadinosteus and an upper
bound bracketed by the divergence of Discoglos-
soidea and Pipanura (Marjanovic & Laurin, 2007);
LNM = 5.104, LNSD = 0.0309.
(C4) 65–70 Ma as the divergence time between Lepido-
batrachus and Ceratophrys based on Beelzebufo
ampinga as a crown-group ceratophryine, putative
closest relative to the genus Ceratophrys, (Evans
et al., 2008); LNM = 4.2114, LNSD = 0.0189.
(C4a) 65–70 Ma as the divergence time for the node
preceding the divergence of Lepidobatrachus and
Ceratophrys (the divergence between Ceratophryi-
nae and a clade containing the genera Acris,
Trachycephalus and Hyla), based on Beelzebufo
ampinga as a stem-group ceratophryine, (Evans
et al., 2008); LNM = 4.2114, LNSD = 0.0189.
To test whether the use of Beelzebufo as a calibration
point within Ceratophryinae yields credible dates for four
major lissamphibian clades, Batrachia, Hyloidea sensu
stricto (i.e. Nobleobatrachia, Frost et al., 2006; referred to
as Hyloidea throughout the text), Ranoidea, and Cerat-
ophryinae, and what, if any, effects Beelzebufo has on
dates when other fossil constraints are included, we ran
analyses using the following calibration sets:
1. C4 alone and C1, C2, C3 together to determine
whether Beelzebufo alone produces similar dates to the
other three calibration points.
2. C1, C2, C3, C4 together to determine what influence
Beelzebufo has when used with multiple calibration
points.
3. C1, C4 together and C1 alone, to determine whether
Beelzebufo affects dates when used with only a deep
node calibration.
4. C3, C4 together and C3 alone, to determine whether
Beelzebufo affects dates when used with a calibration
nearer to the tips.
We also tested the possibility that Beelzebufo may be a
stem-group ceratophryine (C4a) using this placement
alone, as well with the other three calibration points
(C1–C3).
All BEAST analyses that used only a single fossil
calibration (C1, C3, and C4 ⁄ C4a) were constrained at the
root by a maximum upper bound of 4.57 Ga, the
estimate for the maximum age of the earth (Allegre
et al., 1995), essentially allowing dates to increase unfet-
tered. We examined the temporal likelihood that Beelze-
bufo is the sister taxon to Ceratophrys by determining
whether the estimated date for the most recent common
ancestor (MRCA) of the crown group of Ceratophryinae,
when using Beelzebufo (C4) as the only calibration, is
included in the 95% highest posterior density (HPD) for
Ceratophryinae (Lepidobatrachus + Ceratophrys) when
Beelzebufo is excluded as a calibration. This allowed us
to determine whether the date for the MRCA of
276 S. RUANE ET AL.
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Ceratophryinae given by Beelzebufo is compatible with the
dates given by the other constraints we tested. We used
the Wilcoxon signed rank test in STATISTICA v.6
(StatSoft, Inc., Tulsa, Oklahoma) to calculate if the dates
for all nodes were significantly different when using the
calibration set that included Beelzebufo (C1–C4) compared
to the calibration set that did not include this fossil (C1–
C3). We also compared the mean dates for all nodes that
resulted when using C1–C3 with those from the analyses
using C4a alone and using C1–C4a using a Kruskal-
Wallace ANOVAANOVA-by-Ranks test followed by multiple
comparisons among the means (STATISTICA v.6;
StatSoft, Inc., 2001).
Quantifying temporal discordance
We introduce a simple metric, Dt, to quantify the
temporal discordance between two dated chronograms
(f1 and f2) which differ in the age of a single fossil
constraint. Given a rooted phylogenetic tree containing a
single node (n), which has two alternative potential fossil
calibration placements, the difference in the mean ages of
the rest of the nodes on the tree form a set DX of n ) 2
deviations D�Xi ¼ �Xijf1 � �Xijf2 , where �Xijf1 is the mean date
estimate at a node for chronogram one and �Xijf2 is the
mean date estimate at a node for chronogram two. Thus,
D�X2i +1 gives a non-negative estimate of the differences
between the two node ages, corrected for continuity. The
natural logarithm of this quantity yields Dti, a log-scaled
estimate of the per-node temporal deviation between the
two trees. This value is equal to zero when there is no
difference in inferred times. Thus,
Dti ¼ ln ð�Xijf1 � �Xijf2 Þ2
� �þ 1
� �
The mean of this quantity, D�t; gives an estimate of the
absolute temporal deviation between two dated chron-
ograms relative to the fossil constraint. Regressing Dtiagainst the patristic distance (branch length measured in
expected substitutions per site) from the original phylo-
genetic tree assesses the relationship between tree length,
node distance, and the temporal deviation induced by a
poorly assigned fossil. These calculations were performed
using a script developed for this research implemented in
the statistical package R (R Development Core Team,
2010). The script is available from http://www.colubroid.
org.
To quantify how Beelzebufo affects date estimates at
proximal vs. distal nodes on the tree, we calculated the
difference in the mean dates (D�t) for all nodes of
Batrachia using the calibration set that includes C1, C2,
C3, C4 and the same calibration set excluding C4.
Additionally, we calibrate the tree using a hypothesized
date for the node E4 (the same node as C4, the clade
containing Ceratophrys and Lepidobatrachus) based on the
mean divergence date estimated from the BEAST anal-
ysis using only C1–C3. This hypothesized calibration
point E4 (mean age = 12 Ma, 95% HPD 7.6–17.1 Ma,
LNM = 2.514, LNSD = 0.261, when dated using C1–C3)
permits us to assess whether or not the presence of
simply having a calibration point at the MRCA of
Ceratophryinae (rather than the Beelzebufo calibration
specifically) causes a significant change in date estima-
tion across the tree. We used linear regression to
determine whether there was a significant relationship
between patristic distance and temporal deviation (D�t)when using Beelzebufo and when using the hypothesized
calibration E4. This was also calculated using the script
developed here in the statistical package R (R Develop-
ment Core Team, 2010). We then performed the same
procedure to determine what effect using the alternate
placement of Beelzebufo (C1–C4a) as a stem ceratophryine
had on mean date estimates across the tree.
Results
Molecular phylogeny
Using jModeltest (Posada, 2008), a HKY+G+I model was
determined to be the best fit for the CXCR4, NCX1 and
RAG1 genes according to the BIC, whereas GTR+G+I was
the best fit model for SLC8 and 16S. The topology of the
molecular tree using BI agreed with several other recent
anuran phylogenies regarding the content and placement
of major clades such as Batrachia, Discoglossoidea,
Pipoidea, Pelobatoidea, Myobatrachidae, Neobatrachia,
Hyloidea and Ranoidea, as well as Ceratophryinae
(Roelants & Bossuyt, 2005; Marjanovic & Laurin, 2007;
Wiens, 2007; San Mauro, 2010). Our placement of the
Ceratophryinae as sister taxon to the Hylidae is consistent
with previous analyses (Biju & Bossuyt, 2003; Roelants
et al., 2007), although this is poorly supported and differs
from some other phylogenetic analyses that include
anurans (e.g. Frost et al., 2006; Wiens, 2007). Support for
most nodes (73.7%) in this tree was high (‡ 95%). The
BI tree from MrBayes is identical in topology with respect
to the BI tree from BEAST, so the BEAST chronogram is
shown with the Pp from the MrBayes tree (Fig. 1).
Morphological analyses
Poor node support for a sister relationship between
Beelzebufo and Ceratophrys was found in all morphological
analyses. Bayesian inference, using only the morpholog-
ical data set, produced a tree consisting primarily of
polytomies (Fig. 2). The maximum parsimony analyses
(using both ordered and unordered characters) for the
70% majority rule trees as well strict consensuses of the
most parsimonious trees were all topologically similar
and recovered the same relationships between the
crown-group ceratophryines, Beelzebufo, Wawelia and
Baurubatrachus (results similar to the 70% majority
rule tree presented by Evans et al., 2008). Although
both Bayesian and parsimony trees do suggest a sister
relationship between Ceratophrys and Beelzebufo, this is
Phylogenetic placement of Beelzebufo 277
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Fig. 1 Chronogram of 120 extant anurans using Bayesian inference (BI) in BEAST v1.5.4 (Drummond & Rambaut, 2007) applying three
calibration points excluding Beelzebufo (C4). Calibration points used in the study are as follows: C1, divergence of the Batrachia; C2, divergence
between Bombinanura and Pipanura; C3, divergence between Xenoanura and Neobatrachia + Pelobatoidea; C4, divergence within the
Ceratophryinae; C4a, alternate placement of Beelzebufo-based calibration as a stem ceratophryine. Clades of interest are indicated. As the BEAST
topology was fixed to that from the MrBayes analysis, posterior probabilities ‡ 95% using BI in MrBayes v3.1.2 (Ronquist & Huelsenbeck,
2003) are indicated by filled circles.
278 S. RUANE ET AL.
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only weakly supported by the posterior probability
(68%) and bootstrap proportions (53% unordered data
set ⁄ 52% ordered data set). In the Bayesian analysis, the
remaining ceratophryines (Chacophrys and Lepidobatra-
chus) form a polytomy with the Ceratophrys and Beelzebufo
clade (85% Pp). The South American taxa Baurubatrachus
and Wawelia are also allied with the ceratophryines (85%
Pp), as has been compiled by other authors (see Marja-
novic & Laurin, 2007).
Better resolution was obtained using the combined
molecular and morphological data set, but weaker
support was found for the sister relationship between
Beelzebufo and Ceratophrys (58% Pp; Fig. 3) than in the
morphological analysis alone (68% Pp; Fig. 2). In this
analysis, Baurubatrachus and Wawelia form the sister
group to crown group of Ceratophryinae, including
Beelzebufo (90% Pp; Fig. 3). The enigmatic frog Thaumas-
tosaurus from the Eocene of Europe is allied with Ranidae
(the resulting polytomy makes it unclear whether this
taxon is a stem or crown-ranid; Fig. 3); though, this
relationship is weakly supported (51% Pp for the poly-
tomy including Thaumostosaurus; Fig. 3). This taxon was
also thought to be allied with the ceratophryines, which
would represent a European affinity of some Meso-
zoic ⁄ Cenozoic taxa (Holman & Harrison, 2002; Rage &
Rocek, 2007), in addition to the putative connection
between Madagascar and South America possibly implied
by Beelzebufo (e.g. Evans et al., 2008).
Divergence time estimation
When using any combination of calibration points, ESS
values were > 200 for most clades of interest, indicating
that convergence was likely achieved (lower ESS values
indicate poor mixing of the Markov chain; Drummond
et al., 2006). Convergence was also assessed by visually
checking the trace plots for each run. However, when
using only a single point (e.g. Beelzebufo) as a calibra-
Fig. 2 Morphological analysis of the phylogenetic relationships of 66 anurans using Bayesian inference in MrBayes v3.1.2 (Ronquist &
Huelsenbeck, 2003). Clades with posterior probability distribution of ‡ 50% (Pp), ‡ 95% (filled circles) and extinct taxa are indicated (�).
Phylogenetic placement of Beelzebufo 279
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tion, the ESS for most parameters did not reach 200.
This may be attributed to under-parameterization when
using only a single calibration point located close to the
tip of the tree, subsequently producing a very flat
likelihood surface for other nodes (Drummond et al.,
2006). Although the ESS remained low (< 100), we
include the estimates from the analysis of Beelzebufo as
the sole calibration (C4 and C4a) to help illustrate one
of the problems associated with incorrect fossil calibra-
tions. Using the three calibration references to the
exclusion of Beelzebufo resulted in mean divergence time
estimates that correspond to previous studies (Fig. 1;
Table 1; Marjanovic & Laurin, 2007; Wiens, 2007). The
inclusion of Beelzebufo as a fossil calibration on the
crown-ceratophryine node in BEAST analyses always
resulted in an older mean date for the MRCA of
Ceratophryinae and for all other clades of interest
(Batrachia, Hyloidea, and Ranoidea) than when it was
excluded (Table 1; Fig. 4). Also, the use of Beelzebufo as
a single calibration reference for the crown-group
Ceratophryinae always yielded the oldest dates for a
given clade when compared to any other fossil calibra-
tion sets (Table 1; Fig. 4). However, when combined
with other calibrations, the impact of Beelzebufo was
lessened, especially when estimating the age of nodes
distant from the ceratophryines (e.g. the divergence
time of Ranoidea vs. Hyloidea; Table 1).
A significant localized effect of Beelzebufo as a crown-
group ceratophryine on divergence time estimates was
found when using Beelzebufo plus the other three
calibration references (C1–C4), with the nodes nearest
C4 having the greatest difference in mean divergence
times when compared to the calibration set excluding C4;
this difference decreased in magnitude as nodes increased
in distance from C4 (t = )7.5, d.f. = 121, P < 0.001, r =
)0.559; Fig. 5a). When compared to a chronogram that
Fig. 3 Combined morphological and molecular analysis of the phylogenetic relationships of 35 anurans using Bayesian inference
in MrBayes v3.1.2 (Ronquist & Huelsenbeck, 2003). Clades with posterior probability distribution of ‡ 50% (Pp), ‡ 95% (filled circles)
and extinct taxa are indicated (�).
280 S. RUANE ET AL.
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was calibrated using only C1–C3, we found the closer a
node was (as measured by patristic distance) to the
MRCA of the crown group of Ceratophryinae the larger
its mean temporal deviation (Dt) when dates were
estimated using all fossils including Beelzebufo (C1–C4);
conversely, the farther a node was from the Beelzebufo
calibration, the lower its Dt score (Fig. 5a). Using Beelze-
bufo as a stem-group constraint on Ceratophryinae gave
similar results (C1–C4a; t = )4.874, d.f. = 121, P < 0.001,
r = )0.405; Fig. 5b). However, no significant effect was
found when using C1–C3 with the hypothesized cerat-
ophryine calibration (E4, calibrated using C1–C3;
t = 0.083, d.f. = 121, P = 0.934, r = 0.089; Fig. 5c).
Temporal and phylogenetic position of Beelzebufo
The resulting date estimates from the analyses which did
not include Beelzebufo were similar to those from other
studies of Anura (Marjanovic & Laurin, 2007; Wiens,
2007; Table 1; Fig. 4). Additionally, these dates were
highly consistent across all the non-Beelzebufo calibration
combinations in our analyses, despite C1 being less well-
constrained than C2 or C3 and the fact that the
calibrations are based on estimates using different lines
of evidence (Marjanovic & Laurin, 2007; Table 1; Fig. 4).
In assessing the likelihood that Beelzebufo is the sister
taxon to Ceratophrys, we found the mean date estimated
for the MRCA of Ceratophryinae (� 67 Ma; Table 1)
when Beelzebufo was used as the sole calibration point
was not included in the 95% HPD for the MRCA of
Ceratophryinae for any calibration set that excluded
Beelzebufo (Fig. 6). The age of the Lepidobatrachus +
Ceratophrys node was estimated to be � 12 Ma when
Beelzebufo was not included as a calibration, � 55 Ma
younger than the age of the Beelzebufo fossil (Fig. 1;
Table 1). Node age estimates were also significantly older
Table 1 Mean divergence date estimates (Ma) for the most recent common ancestor of Batrachia, Hyloidea, Ranoidea, and the crown
group of Ceratophryinae using different sets of calibrations in BEAST v.1.5.4 (Drummond & Rambaut, 2007) for 40 million generations; 95%
highest posterior density shown in parentheses. C4a indicates the Beelzebufo-based calibration placed on the stem group for Ceratophryinae.
Calibrations Batrachia Hyloidea Ranoidea Ceratophryinae
C1, C2, C3, C4 266.6 (255.1–275.1) 80.1 (74.5–85.2) 106.6 (94.3–124.9) 64.0 (61.7–66.3)
C4 1141.9 (872.5–1411.5) 247.6 (194.5–298.5) 400.8 (301.1–485.5) 67.3 (64.8–69.9)
C1, C2, C3, C4a 265.4 (253.5–277.0) 71.4 (67.2–75.5) 101.6 (94.3–109.4) 13.6 (8.9–19.2)
C4a 354.1 (306.9–406.9) 77.7 (71.8–84.4) 125.2 (112.1–138.8) 16.3 (10.5–23.3)
C1, C2, C3 262.0 (250.2–273.9) 58.1 (52.2–64.5) 93.5 (85.9–101.7) 12.0 (7.6–17.1)
C1, C4 282.2 (267.8–293.9) 85.3 (78.9–92.1) 119.6 (109.9–129.1) 64.9 (62.4–66.9)
C1 260.9 (247.9–273.9) 56.1 (48.8–63.6) 90.3 (78.8–101.5) 12.0 (7.3–16.7)
C3, C4 308.8 (267.7–352.8) 82.2 (76.5–89.2) 112.6 (104.3–121.3) 64.2 (61.9–66.6)
C3 251.2 (224.5–281.7) 59.6 (48.5–62.1) 88.2 (79.7–96.8) 11.6 (7.3–16.4)
Fig. 4 Trace of the posterior probability distribution from BEAST analyses over time for five million generations of the individual
divergence time analyses using the indicated calibration constraints. Traces represent the estimates for the most recent common ancestor of the
Batrachia, Hyloidea, Ranoidea and Ceratophryinae for several calibration combinations. The approximate dates for oldest known fossils for
the Porifera (Li et al., 1998), Craniata (Myllokunmingia fengjiaoa; Shu et al., 1999), Tetrapodomorpha (Kenichthys campbelli; Muller & Reisz,
2005), Batrachia (Triadobatrachus massinoti; Rage & Rocek, 1989) and Beelzebufo ampinga (Evans et al., 2008) are indicated on the right Y-axis.
Phylogenetic placement of Beelzebufo 281
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when Beelzebufo was included in the calibration (C1–C4)
than when it was excluded (C1–C3; Wilcoxon signed
rank test; Z = 9.585, d.f. = 121, P < 0.001).
When using the Beelzebufo-based calibration point as a
stem ceratophryine (C1–C4a, C4a), the mean divergence
time estimates for Ceratophryinae were similar to both
previous (� 15–20 Ma; Marjanovic & Laurin, 2007;
Wiens, 2007) and our own estimates using calibration
sets sans Beelzebufo; in our analyses, these divergence
time estimates for Ceratophryinae were included in the
95% HPD of all the calibration sets we analysed
(excluding those that placed Beelzebufo as a crown-group
ceratophryine; Table 1). However, we found that using
Beelzebufo alone as a stem-group constraint (C4a) resulted
in overall significantly different mean divergence time
estimates when compared to the two calibration sets that
used the three other constraints alone and in combina-
tion with Beelzebufo (C1–C3, C1–C4a; Kruskal–Wallis
ANOVAANOVA-by-Ranks test; H = 13.476, d.f. = 2, P = 0.001).
When using the stem-group placement of Beelzebufo,
dates estimated using C1–C4a resulted in divergence time
estimates that were older, but not significantly different
from the results using the three non-Beelzebufo calibra-
tions (C1–C3; P = 0.347). However, when C4a was used
alone, the mean date estimates across the tree were
significantly older than those estimated using C1–C3
(P < 0.001) and the 95% HPDs of the Batrachia, Hyloidea
and Ranoidea were not included in the 95% HPDs of
dates estimated using C1–C3 (Table 1).
Discussion
Phylogenetic analysis and fossil calibrations
One of the major critiques of molecular divergence time
estimation is the uncertainty associated with using fossil
calibrations from extinct organisms, which may be
attributed to improperly dating the matrix from which
the fossil is derived, poor sampling of fossils, or incorrect
phylogenetic placement of fossils (Conroy & van Tuinen,
2003; Graur & Martin, 2004; Donoghue & Benton, 2007;
Pyron, 2010). Although explicit phylogenetic analysis of
extinct taxa can improve their placement as fossil
calibrations, residual phylogenetic uncertainty can still
impact estimates of molecular divergence times (Lee
et al., 2009; Sauquet et al., 2009). Our results also suggest
that the effects of an improperly placed fossil are
amplified when additional calibrations are not included
in the analyses. Incorrect fossil placement can have
significant effects on divergence dates (e.g. Graur &
Martin, 2004; van Tuinen & Hedges, 2004; Lee et al.,
2009) and can ultimately impact tests that rely on
accurate temporal information (e.g. Hugall & Lee, 2004;
Fig. 5 Graphs of linear regressions between Dt and patristic distance
(PD), where (a) used calibrations C1–C4, (b) C1–C4a and (c) C1 )C3 + E4 (hypothesized calibration). The Dt statistic was calculated
for (a), (b) and (c) using the previously specified calibration sets and
the mean dates from a chronogram using only calibrations C1–C3.
For (a) and (c), PD was taken from the basal node of the crown-
group of Ceratophryinae, and for (b) PD was taken from the stem-
node of Ceratophryinae; adjusted R2 and P-values are shown. Graphs
(a) and (b) show the diminishing effects of date estimates on nodes
distal to the Beelzebufo calibration.
Fig. 6 Marginal densities from BEAST analyses for the most recent
common ancestor of the crown group of Ceratophryinae shown
using the following calibrations: C1, divergence of the Batrachia; C2,
divergence between Bombinanura and Pipanura; C3, divergence
between Xenoanura and Neobatrachia + Pelobatoidea; C4, diver-
gence within the Ceratophryinae. For clarity, only four of the
calibration sets are shown.
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Burbrink & Pyron, 2008). The inclusion of additional
well-constrained fossils (e.g. Muller & Reisz, 2005)
reduces but does not eliminate global error, particularly
in the vicinity of the erroneous constraint. Although a
poorly placed calibration is likely to be mitigated by other
constraints, it may still affect dates across the tree. Thus,
understanding how inaccurate calibrations actually affect
the estimation of dates across trees is crucial.
Here, we have shown that considering the fossil
Beelzebufo as a crown-group ceratophryine results in date
estimates for numerous anuran clades that are much
older than those estimated using other well-supported
calibrations. Using the mean estimate for the origin of the
crown group of Ceratophryinae (E4) calculated from the
other calibrations alone (C1–C3) as a hypothetical cali-
bration point did not result in a significant relationship
between patristic distance and Dt (Fig. 5c). These results
suggest that the increases in divergence time estimates
were not caused by simply placing calibrations on this
node, but rather by using Beelzebufo as a calibration
(Fig. 5). The inclusion of additional constraints (C1–C3)
appears to mitigate the global overestimation of diver-
gence times caused by the incorrectly placed fossil,
whereas nodes nearest to the misplaced constraint
are particularly susceptible to local overestimation
(Fig. 5a, b). For example, Batrachia (Dt126 = 3.26), at a
distance of 0.429 substitutions ⁄ site from the Beelzebufo
calibration (i.e. Ceratophryinae), is estimated to be
262.0 Ma using calibrations C1–C3 and increases in
mean age by only 1.8% when using C1–C4 (Table 1).
In contrast, Hyloidea (Dt197 = 6.18), at a patristic distance
of 0.05 substitutions ⁄ site from the Beelzebufo calibration,
is estimated to be 58.1 Ma when using C1–C3 and increases
in mean age by 27.5% when using C1–C4 (Table 1).
Despite the more localized effects of the misplaced
calibration, most nodes were still estimated to be older
when Beelzebufo was included in the analyses at both the
stem- and crown-group placement. Although this study
demonstrates that the impacts of a poorly placed fossil
may be reduced across the tree when using several well-
placed fossils, it does not negate the importance of
identifying and removing an improperly placed calibra-
tion point. In combination with other methods intro-
duced by Waggoner & Collins (2004), Near & Sanderson
(2004), Near et al. (2005), Rutschmann et al. (2007),
Marshall (2008), and Pyron (2010), the Dt metric can be
used to identify incorrectly placed calibration points and
specify their effects across the tree.
Phylogenetic hypotheses and biogeography
The molecular phylogenies dated without the use of
Beelzebufo yield significantly younger ages for the MRCA
of Ceratophryinae than those trees dated with the
inclusion of Beelzebufo (� 12 Ma without Beelzebufo vs.
� 67 Ma with Beelzebufo; Table 1; Figs 1, 4 and 6). Using
the Beelzebufo fossil alone produced date estimates for
crown group of Ceratophryinae that are outside the 95%
HPD for any other fossil combination tested (Fig. 6).
Additionally, phylogenetic analyses using either mor-
phological or mixed morphological and molecular data
did not strongly support a Beelzebufo + Ceratophrys sister
relationship (Figs 2 and 3). Based on the temporal
evidence in addition to the molecular and morphological
phylogenetic estimates, Beelzebufo seems unlikely to be a
crown ceratophryine.
As such, a hypothesized relationship between Beelze-
bufo and Ceratophrys does not provide strong evidence for
a Late Cretaceous connection between South America
and Madagascar via the Kerguelen Plateau connecting
India ⁄ Sri Lanka to Antarctica. This is especially relevant
given the possibility of overwater dispersal of even
potentially salt-intolerant organisms such as lissamphib-
ians (Duellman & Trueb, 1994; Vences et al., 2003a,
2003b; de Queiroz, 2005; Laurin & Soler-Gijon, 2010).
The sister relationship of Baurubatrachus and Wawelia to
the extant ceratophryines supports a South American
origin of the group, as do several other extinct species
from South America not included in this analysis
(Marjanovic & Laurin, 2007). Additionally, the extinct
Thaumastosaurus is allied with the ranoids rather than the
hyloids (Fig. 3). Although this is not strongly supported,
it seems unlikely that it is a ceratophryine. An alternative
scenario for the proposed ceratophryine affinities of
enigmatic taxa such as Beelzebufo and Thaumastosaurus is
convergence on a similar set of cranial characters during
the late Mesozoic or early Cenozoic, resulting in a
‘pac-man’ morphotype.
Finally, the hypothesis that Beelzebufo represents a
stem-group ceratophryine, or occupies another position
in the crown-group Hyloidea, cannot be ruled out. The
mean date estimate for the MRCA of the crown group of
Ceratophryinae using the stem-group placement of Beel-
zebufo is compatible with those found using the other
calibration sets in our analyses, both using C4a alone or in
conjunction with C1–C3 (Table 1). Although not as
pronounced as the results where Beelzebufo was used to
calibrate the crown-group ceratophryines, we found that
using Beelzebufo alone as a stem calibration produced dates
that were significantly older than those estimated using
C1–C3 (Table 1); these dates (e.g. a mean date estimate
for origin of the Batrachia of � 354 Ma) also disagree
with recent publications on the origin of anurans based
on molecular divergence time estimation (Marjanovic &
Laurin, 2007; Wiens, 2007; San Mauro, 2010) as well as
stratigraphic evidence (Marjanovic & Laurin, 2008).
Additionally, the use of Beelzebufo to calibrate the stem
of Ceratophryinae results in mean dates that are not
included in the 95% HPDs of Batrachia, Hyloidea, or
Ranoidea when estimated using the other fossil calibra-
tions (Table 1). Therefore, it is likely that Beelzebufo does
not represent a stem or crown-group ceratophryine fossil,
but may occupy a position deeper in the hyloid crown
group, possibly to the exclusion of all NW hyloids.
Phylogenetic placement of Beelzebufo 283
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Conclusions
The extinct frog Beelzebufo ampinga from the Cretaceous
of Madagascar is unlikely to represent a crown-group
ceratophryine and does not provide strong support for a
late Cretaceous connection between Madagascar and
South America. As we have demonstrated with Beelze-
bufo, it is crucial that the phylogenetic position of newly
discovered fossils be tested rigorously before being
applied as calibration points. However, even with explicit
phylogenetic analyses, topological uncertainty can still
affect divergence times. Here, the use of molecular date
estimates as a tool for testing phylogenetic hypotheses is
utilized and provides additional means for assessing the
temporal likelihood of evolutionary relationships in
extinct taxa. Protocols such as that suggested by Lee
et al. (2009), Pyron (2010) and the Dt approach devel-
oped here can provide a preliminary avenue for the
integration of such information into the placement of
fossil calibrations and the estimation of divergence times.
Acknowledgments
Thanks to K. Roelants for providing the molecular data
set used in this work, T. Guiher for providing assistance
in BEAST analyses, and to M. Laurin, D. Marjanovic,
M. Lee, S. Edwards, M. McPeek, S. Heard and D. Shepard
for their comments on an earlier version of this manu-
script. This work was supported in part by a National
Science Foundation grant (DBI-0905765) issued to
R. A. Pyron and by the Graduate School and University
Center and the College of Staten Island, both of the City
University of New York.
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