doi: 10.1111/j.1420-9101.2010.02164.x Phylogenetic relationships of the Cretaceous frog Beelzebufo from Madagascar and the placement of fossil constraints based on temporal and phylogenetic evidence S. RUANE* ,R.A.PYRONà & 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 àDepartment of Ecology and Evolution, Stony Brook University, Stony Brook, NY, USA Keywords: Abstract Beelzebufo; The placement of fossil calibrations is ideally based on the phylogenetic Ceratophrys; analysis of extinct taxa. Another source of information is the temporal dating error; variance for a given clade implied by a particular constraint when combined divergence time estimation; with other, well-supported calibrations. For example, the frog Beelzebufo fossil calibration; ampinga from the Cretaceous of Madagascar has been hypothesized to be a Gondwanaland; crown-group member of the New World subfamily Ceratophryinae, which Madagascar. 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. Estimating the ages of clades by integrating fossil data supported constraints placed throughout the tree. Using and molecular phylogenies has become commonplace other calibrations, one can predict the age of the targeted and expands the range of evolutionary hypotheses that node for the uncertain fossil. Several methods for can be tested with phylogenetic data. The placement of incorporating this information into divergence time fossil calibrations on molecular phylogenies is ideally analyses have been developed (e.g. Near & Sanderson, based on explicit phylogenetic analysis of extinct species, 2004; Near et al., 2005; Rutschmann et al., 2007; Pyron, for which only morphological data is typically available 2010; see Marshall, 2008). However, these methods vary (e.g. Donoghue et al., 1989; Shaffer et al., 1997; Manos in their effectiveness in highlighting and combating et al., 2007; Lee et al., 2009). Recent work has also improper calibrations and choosing the optimal set of suggested that combined analysis of morphological and constraints (Rutschmann et al., 2007; Marshall, 2008). molecular data may improve the estimation for the To better understand the impact that the placement of support and placement of both extinct and extant taxa a fossil on a particular node has on date estimates (Wiens, 2009; Wiens et al., 2010), and the placement of throughout a tree, we expand on the protocols suggested fossil calibrations (e.g. Shaffer et al., 1997; Gatesy et al., by Lee et al. (2009) and Rutschmann et al. (2007). In 2003; Sauquet et al., 2009; Magallon, 2010). However, particular, we assess the impact that the phylogenetic uncertainty regarding the placement of extinct taxa placement of the Late Cretaceous fossil frog Beelzebufo represents a continuing source of error, which should ampinga from Madagascar has on estimates of divergence be incorporated into estimates of divergence dates (Ho & dates on Anura. This massive fossil frog has been Phillips, 2009; Lee et al., 2009). hypothesized to be a crown-group member of the Additional information regarding the placement of New World (NW) hyloid subfamily Ceratophryinae fossil calibrations can be derived from other well- (Evans et al., 2008), comprised of eight species in the extant genera Ceratophrys (known commonly as the pac- Correspondence: Sara Ruane, Department of Biology, The College of Staten Island, The City University of New York, 2800 Victory Blvd, Staten Island, man frogs), Chacophrys and Lepidobatrachus (as defined NY 10314, USA. Tel.: 718 982 3850; fax: 718 982 3852; by Fabrezi, 2006). This assignment would offer support e-mail: [email protected] for a remnant connection between South America, ª 2010 THE AUTHORS. J. EVOL. BIOL. 24 (2011) 274–285 274 JOURNAL OF EVOLUTIONARY BIOLOGY ª 2010 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY Phylogenetic placement of Beelzebufo 275 Madagascar and India via Antarctica that may have that misplaced or improperly dated fossils on a tree will existed well into the Late Cretaceous (Hay et al., 1999) result in poor estimates of divergence dates (Graur & and imply a much older age for the hyloids than Martin, 2004), it is unclear how these incorrectly previously thought (e.g. 50–60 Ma, as estimated by assigned fossil calibrations impact divergence time esti- Roelants et al., 2007; Wiens, 2007). We use both mor- mates on nodes nearest to the calibration point (proxi- phological and combined molecular + morphological data mal) relative to nodes farther away in the tree (distal). to assess the phylogenetic affinity of Beelzebufo. We also We use this statistic to test the impact that a poorly placed use molecular divergence time estimates derived from fossil calibration has on node ages distributed across a other, well-supported anuran calibrations to assess the phylogeny. likelihood of alternative placements of the taxon in the combined-data phylogeny, and as a calibration in the Materials and methods molecular divergence time analyses. Placement of Beelzebufo within the crown-group of Molecular data and tree inference Ceratophryinae is supported by several aspects of cranial morphology (Evans et al., 2008) and potentially offers We used Bayesian inference (BI) methods to construct an support for a Late Cretaceous connection between SA anuran phylogeny and assess placement of fossils for and Madagascar (Hay et al., 1999). However, in the divergence dating. The molecular data set of Roelants phylogenetic analysis presented by Evans et al. (2008), et al. (2007), consisting of four nuclear genes (CXCR1, the sister relationship between Beelzebufo and Ceratophrys NCX1, RAG1 and SLC8A3) and one mitochondrial gene is supported by only a single character (out of 81) in a (16S), was used for all molecular analyses. This data set maximum parsimony analyses (Evans et al., 2008). includes 120 anurans, and we included three salaman- Unfortunately, this relationship cannot be tested using ders and one caecilian as outgroups. We simultaneously molecular data alone. Therefore, we integrate molecular estimated trees and support using BI in the program and morphological data to assess the phylogenetic place- MrBayes v3.1.2 (Ronquist & Huelsenbeck, 2003) to ment of Beelzebufo and use divergence time estimation to determine the correct fossil placement for our divergence test relationships in a temporal context (e.g. van Tuinen time analyses. We used the Bayesian Information Crite- & Hedges, 2004; Waggoner & Collins, 2004). In doing so, rion (BIC) in jModelTest, (Posada, 2008) with a maxi- we examine two major aspects of molecular divergence mum-likelihood–optimized base tree to determine the time estimation: (i) how molecular divergence time substitution model for each gene; molecular data were estimates can be used to assess hypotheses concerning partitioned by gene and codon position. Each analysis phylogenetic relationships, and (ii) how a misplaced (two runs of four chains each) was run for 40 million fossil calibration can influence age estimates across the generations and sampled every 1000 generations. Con- phylogeny with and without other constraints. vergence was assessed using Gelman & Rubin’s r statistic First, we re-analyse the data presented by Evans et al. (Gelman et al., 1995). The analysis was considered (2008) alone and in combination with molecular data for complete when the standard deviation of split frequen- extant species using statistical phylogenetic methods to cies between the chains in MrBayes was < 0.01 and r assess the hypothesized crown-ceratophryine affinity of approached 1 for all parameters. Beelzebufo. We use molecular divergence time estimates from a larger anuran data set (Roelants et al., 2007) to Morphological data and tree inference determine the temporal likelihood of the placement of Beelzebufo within Ceratophryinae. We would not reject a To test the strength of the hypothesized sister relation- sister relationship between Beelzebufo and Ceratophrys if ship between Beelzebufo and Ceratophrys using BI, we the estimated dates for Beelzebufo fall within the range of analysed the 81 character morphological data set used by the ages estimated for Ceratophryinae by the other Evans et al. (2008; 66 taxa) and a combined data set of calibrations. However, if the estimates for the crown- molecular and morphological data, using only the 35 group Ceratophryinae are younger than Beelzebufo, this taxa that had both molecular and morphological data would challenge the hypothesis of a sister relationship available, and the four extinct taxa represented by fossils between Beelzebufo and Ceratophrys. We also test whether which were only scored from morphological variables. To Beelzebufo is temporally compatible as a stem-group assess topology and estimate posterior probability (Pp) ceratophryine. support, we used the standard discrete (morphology) Second, we derive a simple metric to assess the impact model