phylogenetic relationships of the cretaceous frog

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]

ª 2 0 1 0 T H E A U T H O R S . J . E V O L . B I O L . 2 4 ( 2 0 1 1 ) 2 7 4 – 2 8 5

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


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

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.



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




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.



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 (�).




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.



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.




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.

282 S. RUANE ET AL.



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.




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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Received 26 August 2010; revised 22 September 2010; accepted 27

September 2010




phylogenetic relationships of the cretaceous frog

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