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Appendix: Materials and Methods

Time-calibrated models support congruency between Cretaceous continental rifting and titanosaurian evolutionary history

Eric Gorscak1,2*, and Patrick M. OConnor2,3.

1Department of Biological Sciences, 107 Irvine Hall, Ohio University, Athens, Ohio 45701 U.S.A., eg377304@ohio.edu;

2Ohio Center for Ecology and Evolutionary Studies, Irvine Hall, Athens, Ohio 45701 U.S.A.;

3Department of Biomedical Sciences, 228 Irvine Hall, Ohio University Heritage College of Osteopathic Medicine, Athens, Ohio 45701 U.S.A., oconnorp@ohio.edu

Titanosaurian Data

Autapomorphies

Phylogenetic Analyses

Paleobiogeographic Analyses

Phylogenetic Trees

Paleobiogeographic Reconstructions

Model Scores

Morphological Characters

References

A. Titanosaurian Data.

Morphological character data were collected from several previous titanosaurian or titanosauriform phylogenetic analyses and several characters came from literature of anatomical descriptions (see Morphological Characters below). Many titanosaurians in our study have been included in at least one previous phylogenetic analysis and their character scorings were recorded and transferred into the nexus file utilized in this study. Character scorings were further re-evaluated based on the respective descriptive literature or any subsequent published re-interpretations. The same applies for age estimates for the deposits from which specimens were recoved (i.e., these were taken from the respective original descriptive literature or subsequent updated publications). Although certain ambiguity exists in the demarcation of the stratigraphic ranges, such as the words early or middle, in order to assign explicit numeric dates their descriptions were taken literally into dividing the range. The reported stages were converted into the approximate dates based on the information provided by Gradstein and Ogg (2004). After assigning the numerical age ranges, a uniform distribution prior sampled the age range of each respective taxa in order to account for the uncertainty of tip age based on the stratigraphic range. E.Gorscak scored the characters for Rukwatitan bisepultus, Malawisaurus dixeyi, Alamosaurus sanjuanensis (NMNH 15560; National Museum of Natural History), and Dreadnoughtus schrani based on personal observations.

Sauropod Taxa in Dataset

Aeolosaurus maximus (Santucci and de ArrudaCampos, 2011)

Alamosaurus sanjuanensis (Gilmore 1922, 1946; DEmic, 2012; E.G. pers. obvs., 2014)

Andesaurus delgadoi (Calvo and Bonaparte, 1991; Mannion and Calvo, 2011)

Angolatitan adamastor (Mateus et al., 2011)

Ampelosaurus atacis (Le Loeuff, 1995; 2005)

Argentinosaurus huinculensis (Bonaparte and Coria, 1993)

Argyrosaurus superbus (Lydekker, 1893; Mannion and Otero, 2012)

Baurutitan britoi (Kellner et al., 2005)

Bonitasaura salgadoi (Apestegua, 2004; Gallina and Apestegua, 2011, 2015)

Brachiosaurus altithorax (Riggs, 1903; Wilson, 2002)

Camarasaurus (Cope, 1877; Wilson, 2002)

Chubutisaurus insignis (del Corro, 1975; Carballido et al., 2011)

Diamantinasaurus matildae (Hocknull et al., 2009; Poropat et al., 2014a)

Dreadnoughtus schrani (Lacovara et al., 2014; E.G. pers. obvs., 2014)

Epachthosaurus sciuttoi (Powell, 1990; Martinez et al., 2004)

Euhelopus zdanskyi (Wiman, 1929; Wilson and Upchurch, 2009; DEmic, 2012)

Futalognkosaurus dukei (Calvo et al., 2007b, c)

Gondwanatitan faustoi (Kellner and de Azevedo, 1999)

Isisaurus colberti (Jain & Bandyopadhyay, 1997; Wilson and Upchurch, 2003)

Ligabuesaurus leanzai (Bonaparte et al., 2006; DEmic, 2012)

Lirainosaurus asitbiae (Sanz et al., 1999; Dez Daz et al., 2011, 2012, 2013a, b)

Malarguesaurus florenciae (Gonzlez Riga et al., 2009)

Malawisaurus dixeyi (Haughton, 1928; Jacobs et al., 1993; Gomani, 2005; DEmic, 2012; E.G. pers. obvs., 2014, 2015)

Maxakalisaurus topai (Kellner et al., 2006)

Mendozasaurus neguyelap (Gonzlez Riga, 2003, 2005)

Muylenesaurus pecheni (Calvo et al., 2007a)

Nemegtosaurus mongoliensis (Nowinski, 1971; Wilson, 2005)

Neuquensaurus australis (Powell, 1986; Otero, 2010; DEmic and Wilson, 2011)

Normanniasaurus genceyi (Le Loeuff et al., 2013)

Opisthocoelicaudia skarzynskii (BorsukBialynicka, 1977; DEmic, 2012)

Overosaurus paradasorum (Coria et al., 2013)

Paludititan nalatzensis (Csiki et al., 2010)

Panamericansaurus schroederi (Porfiri and Calvo 2010)

Paralititan stromeri (Smith et al. 2001)

Pellegrinisaurus powelli (Salgado, 1996)

Phuwiangosaurus sirindhornae (Martin et al., 1994; Suteethorn et al., 2009, 2010; DEmic, 2012)

Rapetosaurus krausei (Curry Rogers & Forster, 2001; Rogers, 2004, 2009)

Rinconsaurus caudamirus (Calvo and Gonzalez Riga, 2003)

Rukwatitan bisepultus (Gorscak et al., 2014)

Saltasaurus loricatus (Bonaparte and Powell, 1980)

Tangvayosaurus hoffeti (Allain et al., 1999; DEmic, 2012)

Tapuiasaurus macedoi (Zaher et al., 2011)

Tastavinsaurus sanzi (Canudo et al., 2008; Royo-Torres et al., 2009)

Trigonosaurus pricei (Campos et al., 2005)

Wintonotitan wattsi (Hocknull et al., 2009; Poropat et al., 2014b)

Clade Definitions

Titanosauria: Andesaurus delgadoi, Saltasaurus loricatus, their most recent common ancestor and all descendants (Bonaparte and Coria, 1993).

Lithostrotia: Malawisaurus dixeyi, Saltasaurus loricatus, their most recent common ancestor and all descendants (Upchurch et al., 2014).

Saltasauridae: Opisthocoelicaudia skarzynskii, Saltasaurus loricatus, their most recent common ancestor and all descendants (Bonaparte and Powell, 1980).

Andesauroidea: Most inclusive clade that includes Andesaurus delgadoi but not Saltasaurus loricatus (Salgado, 2003).

Euhelopodidae: The most inclusive clade that includes Euhelopus zdanskyi but not Neuquensaurus australis (Romer, 1959).

Saltasaur-lineage: The most inclusive clade that includes Saltasaurus loricatus but not Aeolosaurus maximus (Informal clade, this study).

Aeolosaur-lineage: The most inclusive clade that includes Aeolosaurus maximus but not Saltasaurus loricatus (Informal clade, this study).

Stratigraphic Information

Taxon

Stratigraphic Range

Age Range (Ma)

Mean Age (Ma)

Aeolosaurus

CampanianMaastrichtian

83.565.5

74.5

Alamosaurus

Late CampanianMaastrichtian

77.165.5

71.3

Ampelosaurus

Late CampanianEarly Maastrichtian

77.168.1

72.6

Andesaurus

AlbianCenomanian

11297.5

104.8

Angolatitan

Late Turonian

91.489.3

90.4

Argentinosaurus

AlbianCenomanian

11293.5

102.8

Argyrosaurus

CampanianMaastrichtian

83.565.5

74.5

Baurutitan

Maastrichtian

70.665.5

68.1

Bonitasaura

SantonianEarly Campanian

85.877.1

81.4

Brachiosaurus

KimmeridgianTithonian

155.7145.5

150.6

Camarasaurus

KimmeridgianTithonian

155.7145.5

150.6

Chubutisaurus

Cenomanian

99.693.5

96.6

Diamantinasaurus

Cenomanian

99.693.5

96.6

Dreadnoughtus

CampanianMaastricthian

83.565.5

74.5

Epachthosaurus

Late CenomanianEarly Turonian

96.691.4

94.0

Euhelopus

BarremianAptian

130112

121.0

Futalognkosaurus

TuronianConiacian

93.585.8

89.7

Gondwanatitan

TuronianSantonian

93.583.5

88.5

Isisaurus

Maastrichtian

70.665.5

68.1

Ligabuesaurus

Late AptianAlbian

118.599.6

109.1

Lirainosaurus

Late CampanianEarly Maastrichtian

77.168.1

71.3

Malarguesaurus

Late TuronianEarly Coniacian

91.487.6

89.5

Malawisaurus

Aptian

125112.0

118.5

Maxakalisaurus

TuronianSantonian

93.583.5

88.5

Mendozasaurus

Late TuronianLate Coniacian

91.487.6

89.5

Muyelensaurus

Late TuronianEarly Coniacian

91.487.6

89.5

Nemegtosaurus

Middle Maastrichtian

69.167.1

68.4

Neuquensaurus

Early Campanian

83.577.1

80.3

Normanniasaurus

EarlyMiddle Albian

112107.9

110

Opisthocoelicaudia

Early Maastrichtian

70.668.1

69.4

Overosaurus

Campanian

83.570.6

77.1

Paludititan

Early Maastrichtian

70.668.1

69.4

Panamericansaurus

Late CampanianEarly Maastrichtian

77.168.1

72.6

Paralititan

Cenomanian

99.693.5

96.6

Pellegrinisaurus

CampanianEarly Cretaceous

83.568.1

75.8

Phuwiangosaurus

BarremianAptian

130112

121.0

Rapetosaurus

Maastrichtian

70.665.5

68.1

Rinconsaurus

Late TuronianConiacian

91.485.8

88.6

Rukwatitan

AptianAlbian

110100

105.0

Saltasaurus

Late CampanianEarly Maastrichtian

77.168.1

71.3

Tangvayosaurus

AptianAlbian

12599.6

112.3

Tapuiasaurus

Aptian

125.0112.0

118.5

Tastavinsaurus

Late Aptian

125.0118.5

121.8

Trigonosaurus

Maastrichtian

70.665.5

68.1

Wintonotitan

Cenomanian

99.693.5

96.6

B. Autapomorphies

Of the total 492 characters, 230 characters are autapomorphic characters and 262 characters are variable. The inclusion of autapomorphic characters is vital for the likelihood-based analyses as these characters explicitly aid in estimating the length of terminal branches and for calculating the overall model likelihood (Lewis, 2001; Mller and Reisz, 2006; Prieto-Mrquez, 2010). In this sense, autapomorphic characters essentially estimates the amount of morphological evolution that occurs along the terminal branches. Autapomorphies were obtained from either the original diagnosis of the respective descriptive paper of the taxon or from more recent revised studies. Typically, morphological studies focus on variably-scored characters and not parsimony-uninformative characters such as autapomorphies. The exclusion of of such parsimony-uninformative characters may interfere with the appropriate probability calculations over the character sites (ascertainment bias; Lewis, 2001). Nonetheless, the data matrix developed herein consists of the different character types. It is important to note that previous studies that incorporated different character inclusion criteria (e.g., running analyses with and without autapomorphic characters) did not substantially differ in terms of general topologies and support metrics (Mller and Reisz, 2006; Prieto-Mrquez, 2010; Gorscak et al., 2014); however, these studies also did not use time-calibration methods. Nevertheless, we advise the inclusion of autapomorphies in future morphological, model-based time-calibrated phylogenetic approaches as they provide estimation on the morphological evolution that occurred along the terminal branches. The list of autapomorphies are listed below following the other morphological characters used in this study.

C. Phylogenetic Analyses

Following previous studies (Pyron, 2011; Ronquist et al., 2012a; Wood et al. 2013), we treated terminal taxa as non-contemporaneous tips in the analyses herein. We constructed the XML files using the R package BEASTmasteR (Matzke, 2014) and ran the subsequent analyses in BEAST v2.1.3 (Bouckaert et al., 2014). In order to run the following time-calibrated BEAST analyses, the following packages need to be installed within BEAUTi v2.1.3: BEASTlabs, BDSKY, SA, phylodynamics, and CA. The Mk model for morphological character evolution (Lewis, 2001) was used and we tested both equal (Er) and variable rates (Vr; drawn from a gamma-distribution) of character evolution in separate analyses. All discrete characters were assumed to be unordered and all characters assumed to be independent. We utilized a relaxed (i.e. uncorrelated) clock with rates drawn from a lognormal distribution in order to allow rates to vary rather than confined by a strict clock. We used the birth-death-skyline-serial-sampling (BDSKY) tree model with serial samples to account for the non-contemporaneous tips and allow for birth (speciation) and death (extinction) rates to vary through time (Stadler et al., 2013). The BDSKY tree model is based off of the earlier birth-death serial-sampling tree model (BDSS: Stadler 2010; Stadler et al., 2011) that has been used previously for viral and bacterial strain evolution in order to estimate mutation rates as these types of datasets utilize samples collected from different times. Moreover, this approach can be used in an analogous manner to model species-level phylogenies that may include both extinct and extant taxon (Stadler, 2010; Stadler and Yang, 2013). We allowed the Markov Chain Monte Carlo (MCMC) to run for 20 million generations with sampling occurring every 1,000 generations. Convergence, effective sample sizes (>200) of pertinent parameters, and model likelihood scores (harmonic mean) were conducted and visualized in Tracer v.1.6 (Drummond et al. 2006). The burn-in was set to the first 25% of samples to eliminate the initial climbing phase of the MCMC and prevent over-sampling. Model comparison was conducted by both Bayes Factor (Kass and Raftery, 1995) and model posterior probability based on relative log likelihoods (see Model Scores below).

Similar analyses were conducted using MrBayes 3.2 (Ronquist et al., 2012b) in order to account for program variability as the two programs process clock models somewhat differently and MrBayes has been commonly used in previous divergence dating studies (e.g., Ronquist et al., 2012a; Lee et al., 2014a). Here, we follow the methodological protocols from Lee et al (2014a). Clock models differ slightly, as MrBayes 3.2 is not capable of using a birth-death clock model with non-contemporaneous tips, unlike in BEAST. Instead, we used the uniform clock model for our prior to draw the branch lengths. The only parameter is the total tree height which contrasts the additional speciation and extinction rate parameters in the birth-death models. Clock variance was relaxed (i.e., uncorrelated) over the branches using the independent gamma rate (IGR) option and drawn from an exponential distribution (similar to the uncorrelated clock model in the BEAST analyses). Additionally, the MrBayes models tested for rates of character evolution that were assumed to be equal or variable (drawn from a gamma distribution). The models were developed and processed with a similar approach to that of the BEAST runs: 20 million generations with sampling every 1,000 generations, a 25% burn-in, and model parameters checked via Tracer v1.6.

Finally, parsimony analyses were conducted for additional comparisons across methodologies. These analyses were conducted in PAUP v4.0b10 (Swofford, 2003) using tree-bisection-reconnection, random addition within the stepwise heuristic search. The maximum number of trees retained was reached at 10,000 with the resultant tree score equaling 740 steps. The majority-rule tree is reported below.

D. Paleobiogeographic Analyses

Paleobiogeographic analyses were conducted using the R package BioGeoBEARS (BioGeography with Bayesian Evolutionary Analysis in R Scripts; Matzke 2013; http://cran.r-project.org/web/packages/BioGeoBEARS/ index.html). BioGeoBEARS is able to perform DEC (Dispersal-Extinction-Cladogenesis; Ree and Smith, 2008) and likelihood-interpretations for both DIVA (DIVALIKE; Dispersal-Vicariance Analysis; Ronquist, 1997) and BayArea (BAYAREALIKE; Landis et al., 2013) by means of parameterizing the underlying processes these models assume (see Matzke, 2013 for further details). Likewise, the package can perform counterpart models with an additional parameter, +j, to model a situation similar to the founder effect or an approximation of long-distance dispersal such that a daughter lineage is able to disperse to non-contiguous areas from the ancestral range. The +j parameter alleviates the need of range expansion followed by range contraction in order for a daughter lineage to disperse to a new area (e.g., A to AB to B as opposed to A to B). The DEC and BAYAREALIKE models may not be appropriate because these models tend to over-estimate how widespread the ancestral range is at the ancestral nodes as well as frequency of vicariance events to break apart the range into a narrow range if all the terminal taxa are from a single area, a bias previously noted in these models (Matzke, 2014). Given that this study is on the continent-level scale, it is not too unreasonable that the taxa, more or less, were limited in their distribution on their respective continent (or sub-continent). Generally, the resultant reconstructions did not differ substantially from one another with respect to the without j and the +j models (see below). The main differences at each reconstructed node between the without j and +j models tend to either support for multiple areas (AB) or a single area (A), respectively, which is what the j parameter tends to resolve for the case of long-distance dispersal. The best-fit time-calibrated phylogenetic model was used to run these paleobiogeographical analyses, in this case the BEAST variable character rates model. Model comparisons between the without j and +j models used the AIC scores and likelihood ratio test (see Model Scores below). Eight general area states were used with a maximum ancestral range of eight states: North America, South America, Africa, Europe, Asia, India, Madagascar, and Australia.

E. Phylogenetic Trees

The following are the resultant topologies from the BEAST and MrBayes analyses. Nodal ages are to the right of the respective node and posterior probabilities are in italics along the branches leading up to their respective node. 95% highest posterior density of the nodal age is represented by a purple bar. The time scale is in millions of years. For the parsimony tree, the number at each node represents the percentage that clade appears within the majority rule consensus tree of the maximum 10,000 tree limit. Though the many differences, as subtle or as overt as they are, could be attributed to a number of factors including the varying evolutionary models (e.g., clock models, tree models, morphological character rates), the amount of missing data, morphological character selection, and/or the stochastic nature of the MCMC process as it explores tree space.

BEAST ModelsSubtle differences are present between both BEAST models with varying values for the estimated nodal ages, posterior probabilities, 95% highest posterior density (HPD), and orderings within subclades. Tastavinsaurus is placed as the sister lineage outside of titanosauria within the the Er model whereas this taxon is placed outside of the more inclusive group that includes the Euhelopodidae and Titanosauria in the Vr model. Additionally, within the Er model, Andesauroidea is a more inclusive clade compared to the Vr model: Wintonotitan, Chubutisaurus, Angolatitan, and Malarguesaurus are recovered within Andesauroidea in the Er model but form a monophyletic non-titanosaurian clade in the Vr model. The Australian Diamantinasaurus is recovered as a non-lithostrotian titanosaurian in the Vr model but is an early-branching saltasaur-lineage lithostrotian in the Er model. Between the two BEAST models certain taxa vary in relative position around the basal node of the aeolosaur-line clade that implicates Tapuiasaurus, Normanniasaurus, Rapetosaurus and the clade consisting of Bonitasaura, Rinconsaurus, and Muyelensaurus (Rinconsauria of Calvo et al., 2007a). Considering the rest of the aeolosaur-lineage, the ordering differs in placement of Gondwanatitan as the earliest branching taxon (Vr model) or sister lineage to Overosaurus (Er model). Concerning the saltasaur-lineage part of the topology, differences in orderings within the subclades occur but the overall pattern of relationships amongst the saltasaur-lineage subclades remain consistent between the two BEAST models. In the Vr model, Paralititan is recovered as the sister lineage with Epachthosaurus whereas Paralititan is the sister lineage to Maxakalisaurus in the Er model. Futalognkosaurus and Mendozasaurus either form a clade (Lognkosauria; Calvo et al. 2007b) in the Vr model or recovered as a paraphyletic clade outside of the biconvex first caudal vertebra clade (Pellegrinisaurus, Deadnoughtus, Alamosaurus, Baurutitan) and the Laurasian clade (Opisthocoelicaudia, Nemegtosaurus, Lirainosaurus, Ampelosaurus, Paludititan) in the Er model. Finally, the relative orderings within the biconvex first caudal vertebra clade and Laurasian clade differ slightly between the two models.

MrBayes ModelsSimilar to the BEAST models, subtle differences occur within the estimated topology parameters concerning nodal dates and their respective 95% HPD, branch lengths, and posterior probabilities. Within both MrBayes models, Malarguesaurus, Tastavinsaurus, Angolatitan, and Chubutisaurus were all recovered outside of Titanosauria. The Australian Wintonotitan is recovered within the titanosaurian clade of Andesauroidea in the MrBayes Vr model but outside of titanosauria with Chubutisaurus and Angolatitan in the Er model. Andesauroidea is consistent between both MrBayes analyses and includes the South American titanosaurians of Andesaurus, Argentinosaurus, and Ligabuesaurus. The other Australian taxon, Diamantinasaurus, is recovered outside of Lithostrotia in the MrBayes Vr model but recovered as an early-branching saltasaur-lineage lithostrotian in the Er model. Similar to the BEAST analyses, the basal node of the aeolosaur-lineage part of the topology differs in ordering of Normanniasaurus, Tapuiasaurus, Rinconsauria (Bonitasaura, Rinconsaurus, and Muyelensaurus), and the rest of the aeolosaur-lineage branch. The relative ordering within the rest of the aeolosaur-lineage also differ in relative placement of taxa but with Rapetosaurus being an early-branching aeolosaurid in both models. As in the BEAST analyses the relative orderings and membership of the subclades within the saltasaur-lineage part of the topologies are consistent between the two MrBayes model. The main difference between both MrBayes models is the placement of Epachthosaurus and Maxakalisaurus as either both early-branching saltasaur-lineage lithostrotian (Vr model) or within the subclade composed of Paralititan, Saltasaurus, and Neuquensaurus (Er model).

BEAST and MrBayes ModelsDifferences between BEAST and MrBayes models (e.g., BEAST Vr and MrBayes Vr) include placement of certain taxa, relative ordering within consistent subclades, and, to a more noticeable extent, certain topological parameters. It appears that the MrBayes topologies tend to favor lengthening the terminal branches and shortening the internodal branches when compared to the BEAST analyses. The varying branch internodal branch lengths is noticeable along the base of the tree to the titanosaurian node before resembling each topology from the two different programs. This may be due to the different tree models for each program. The BEAST analyses have the virtue of estimating varying rates of speciation and extinction with the BDSKY model whereas the MrBayes analyses only rely on the tree height prior. Overall, the topologies are similar to each other in terms of relative placement of subgroups and their relationships to one another. Consistently present among the different models are various subclades (though some of the memberships vary slightly concerning select taxa): Euhelopodidae, Andesauroidea, Lithostrotia, aeolosaur-lineage, saltasaur-lineage, and Saltasauridae.

Parsimony and Bayesian TopologiesThe topologies between the Bayesian-based analyses and the more traditional parsimony-based approach differ significantly with one another in several regards. In the parsimony analysis, the euhelopodids are recovered within Titanosauria rather than excluded from this clade. Additionally, Argentinosaurus and Ligabuesaurus are not recovered as part of the early branching Andesauroidea, rather located in a relatively more nested position than the Bayesian-based counterpart analyses. Furthermore, Argyrosaurus is recovered outside of Lithostrotia rather than part of the aeolosaur-lineage of the topology as in the Bayesian-based analyses. In the parsimony analysis, Rukwatitan is recovered outside of Lithostrotia as an early-branching titanosaurian. The relative ordering within the saltasaur-lineage significantly differs from the Bayesian-based analyses, placing Saltasaurus and Neuquensaurus subclade into a more nested position as the sister lineage to, a less-inclusive, Laurasian titanosaurian subclade consisting of Opisthocoelicaudia and Lirainosaurus. The monophyletic grouping of Alamosaurus, Pellegrinisaurus, Dreadnoughtus, and Baurutitan (united by exhibiting a biconvex first caudal vertebra) from the Bayesian-based analyses is broken up in the parsimony analysis and variably placed within the saltasaur-lineage part of the topology. The European clade of Paludititan and Ampelosaurus is recovered as an early-branching lineage within the aeolosaur-lineage from the parsimony analysis rather than within the Laurasian clade of Opisthocoelicaudia and Lirainosaurus from the Bayesian-based analyses. The aeolosaur-lineage region of the parsimony topology is divided into two clades: those leading to a more traditional aeolosaurid clade (e.g., Santucci and de Campos-Arruda, 2011) and a clade that preserves cranial material. Maxakalisaurus, recovered within the saltasaur-lineage of the Bayesian-based analyses, is recovered within the aeolosaur-lineage. Malawisaurus, Nemegtosaurus, Rapetosaurus, Tapuiasaurus, Muyelensaurus, and Bonitasaura all preserve significant portions of the skull and are all clustered together as a monophyletic group. This may be due to the tendencies of the parsimony approach which may group these taxa based on virtue of preserving cranial elements. For titanosaurians within the current data matrix, only Lirainosaurus and Saltasaurus bear significant cranial material but also exhibit significant portions of post-cranial material to aid in differentiating their phylogenetic placement. The occurrence of a titanosaurian clade known from cranial material (e.g., Nemegtosauridae [Upchurch, 1995]) has been recovered and/or noted before in Wilson (2002), Curry Rogers (2005), Gallina and Apesteguia (2011), and Zaher et al. (2011).

Past AnalysesSeveral older studies that have initially sought out to, at least attempt, resolve titanosaurian phylogenetic relationships more broadly are limited in scope by the available morphological characters and number of taxa at the time they were published: 6 taxa: Upchurch, (1995); 10 taxa, Salgado et al. (1997); 7 taxa, Sanz et al. (1999); 7 taxa, Wilson (2002); and 8 taxa, Upchurch et al. (2004). Though it should be noted several of these studies looked at the interrelationships of sauropods more broadly (Upchurch, 1995; Wilson, 2002; Upchurch et al., 2004). Rogers (2005) conducted a significantly larger effort in resolving titanosaurian interrelationships including analyses varying between 26 and 14 taxa. Other later studies are comparable in scope such as Calvo et al. (2007b) with 16 taxa, Santucci et al. (2011) with 17 taxa, or Gallina and Apesteguia (2011) with 19 taxa. Though indirectly, Mannion et al. (2013) looked at titanosauriforms more broadly and depending on the data set and methodology, the number of titanosaurians based on the formal definition (Andesaurus, Saltasaurus, most recent common ancestor and all descendants [Bonaparte and Coria, 1993]) ranged from 11 (Lusotitan standard discrete matrix) to 38 (Lusotitan continuous and discrete matrix). In comparison with the analyses presented here, the BEAST Vr analysis includes 35 titanosaurian taxa, the BEAST Er analysis includes 39 titanosaurian taxa, and the parsimony analysis includes 43 titanosaurian taxa of the total 45 taxa used.

Topologically, the resultant phylogeny of the BEAST Vr model broadly agrees with some recent titanosaurian phylogenetic studies where there is a significant split within lithotrotian titanosaurians that includes a saltasaur-lineage and an aeolosaur-lineage (Gallina and Apestegua, 2011; Santucci and de Campos-Arruda, 2011). However, the Gallina and Apestegua (2011) topology differs from our analyses in several regards: Lirainosaurus is placed outside of the aeolosaur- and saltasaur-lineage split; Lognkosauria is nested within Rinconsauria; Epacthosaurus is more closely related to Aeolosaurus than to Saltasaurus; Nemegtosaurus and Rapetosaurus are grouped together within Nemegtosauridae; and Malawisaurus is recovered as a sister lineage to Andesaurus. Compared to Santucci and de Campos-Arruda (2011), a similar aeolosaur- and saltasaur-lineage split is present. Furthermore, differences between the Santucci and de Campos-Arruda (2011) topology and the topology presented in this study includes: the presence of Nemegtosauridae (Rapetosaurus and Nemegtosaurus); Muyelensaurus placed outside of the aeolosaur- and saltasaur-lineage split; Maxakalisaurus more closely related to Aeolosaurus rather than Saltasaurus; and Baurutitan as an early-branching member of the saltasaur-lineage. Though some sort of broad consensus in titanosaurian phylogenetics is starting to emerge, many more titanosaurian taxa have yet to be included into a more comprehensive analysis.

BEAST Equal-rates Relaxed Clock Model

BEAST Variable-rates Relaxed Clock Model

MrBayes Equal-rates Relaxed Clock Model

MrBayes Variable-rates Relaxed Clock Model

Parsimony Majority Rule Consensus Tree

Parsimony Compared to Bayesian (BEAST VrRc)

F. Paleobiogeographic Reconstructions

The following are the resultant paleobiogeographic ancestral area reconstructions from the BioGeoBEARS analysis. The BEAST variable-rates model was utilized. The pie chart at each node indicates relative likelihood for each possible estimated ancestral area. Reconstructed areas that include multiple ranges are represented by the blending of the colors representing the areas, e.g. an ancestor for both a European lineage (light blue) and a South America lineage (red) would be a shade of purple to represent a Europe+South America ancestral range. North America is represented by light green; Asia is represented by magenta; South America is represented by red; Africa is represented by orange; Europe is represented by light blue; Madagascar is represented by purple; India is represented by dark blue; and Australia is represented by dark green.

A consistent difference between the without j and the +j+ models is the noticeable lack of combined or widespread ancestral ranges in the latter models. For example, in the DIVALIKE model, the ancestral range for Lithostrotia (and surrounding nodes) is likely to have been South America and Africa whereas in the DIVALIKE+j model, the ancestral range is likely to have been South America whereas Africa is the second likeliest area of origin. This pattern is also true in the DEC -/+ j and BAYAREALIKE -/+j models. Though this may make sense for the without j models, as the two continents were conjoined in some way for the early half of the Cretaceous, the fact remains that none of the titanosaurians in our data setor even outside of our data setare recovered from multiple areas on the continental scale. Though this may be telling that we would expect the range of South American titanosaurians extended within continental Africa (or vice versa) during the early half of the Cretaceous, the current fossil record of Africa, the data in our analyses, and the models utilized herein do not necessarily support this notion of widespread titanosaurian species ranges. Overall, the reconstructions amongst the DEC, DIVALIKE, and BAYAREALIKE are broadly similar to one another and the same can be said for the +j models with differences in relative proportions of the likelihood for each reconstructed area at each node.

DIVALIKE (likeliest ancestral range)

DIVALIKE (proportion of likely ancestral areas)

DIVALIKE+j (likeliest ancestral areas)

DIVALIKE+j (proportion of likely ancestral areas)

DEC (likeliest ancestral areas)

DEC (proportion of likely ancestral areas)

DEC+j (likeliest ancestral areas)

DEC+j (proportion of likely ancestral areas)

BAYAREALIKE (likeliest ancestral areas)

BAYAREALIKE (proportion of likely ancestral areas)

BAYAREALIKE+j (likeliest ancestral areas)

BAYAREALIKE+j (proportion of likely ancestral areas)

G. Model Scores

The phylogenetic analyses were compared by using the Bayes Factor (twice the difference of the model likelihoods: Kass and Raftery 1995) and relative log-likelihood scores represented as posterior probabilities. Model scores were computed using Tracer v1.6 to obtain the harmonic mean likelihood. The paleobiogeographical analysis results are summarized by both model log-likelihood and AIC scores. Given the separate set of assumptions and variables used, each model (DEC, DIVALIKE, BAYAREALIKE) was tested against their +j counterpart models. The AIC score differences are also reported to assess relative probability and the p-values of the likelihood ratio test are shown to assess whether or not to reject the null model.

Bayes Factor (Kass and Raftery, 1995)

B10

Evidence against H0

13

No distinction

320

Positive

20150

Strongly posititve

>150

Very strongly positive

Phylogenetic analyses

Program

Character Model

Tree Model

Clock Model

lnl

Bayes Factor

Posterior Probability

BEAST 2.1.3

Gamma

Skyline BDSS

Relaxed; Lognormal

-3781.12

>0.99

MrBayes 3.2

Gamma

Height Prior

Relaxed; IGR

-3837.666

113.092