Supporting Information Sauquet et al. 10.1073/pnas.0805607106 SI Materials and Methods most fossils, fossil taxa were analyzed one by one in PAUP* by Dataset Assembly. New DNA sequences from the nuclear ribo- using heuristic searches of 100 random addition replicates with somal internal transcribed spacers (ITS) and plastid matK (plus TBR branch swapping. The resulting most parsimonious trees 3Ј end of the trnK intron), rbcL, trnL intron, and trnL-trnF were then examined one by one to identify the set of alternative intergenic spacer were produced following standard protocols branching positions on the phylogeny. To assess confidence in (1–3). These sequences were combined with available GenBank these assignments, the analyses were repeated keeping all trees data for these regions as well as the plastid atpB, atpB-rbcL that are 1 or 2 steps longer than the most parsimonious trees. In intergenic spacer, and rpl16 intron. Taxon sampling was designed addition, weighted analyses were conducted in which characters to include at least one representative of each genus of Pro- were reweighted by maximum value of their rescaled consistency teaceae while maximizing the number of characters sampled for index, an inverse measure of homoplasy. Unweighted and each terminal taxon, combining sequence data from different weighted analyses gave, in general, very similar results (Fig. S2). species of the same genus where necessary (Table S6). Previous Fossil taxa that matched Ͻ10 most parsimonious positions in studies have shown that Dryandra is nested in Banksia (4, 5), and weighted analyses were considered suitable for calibration. The all species of Dryandra have recently been transferred to Banksia oldest stratigraphic record of a fossil was used as minimum age (6). For practical reasons, we do not follow this nomenclatural evidence for the most recent common ancestor of all of the most change here but we have included several species of Banksia as parsimonious positions of this fossil. In a few cases, Ͼ1 fossil terminal taxa in our dataset and only consider Banksiinae (the constrained the same node, and the fossil with the oldest record monophyletic group of all species of Banksia and Dryandra)as was used to constrain this node. Stratigraphic ages were con- a terminal taxon in all diversification rate analyses. Represen- verted into absolute ages by using the geological timescale of tative species of the nearest outgroups of Proteaceae (Platanus Gradstein et al. (13). The upper (youngest) bound of the oldest and Nelumbo) and a selection of other early-diverging eudicot stage in which a fossil has been confirmed was used as a lineages (Buxales, Sabiaceae, and Trochodendrales) were in- minimum age constraint. cluded as outgroups. All sequences were aligned by using This approach produced a set of 5 fossil pollen age constraints BioEdit v. 7.0.9 (7) and ambiguous regions were removed before (Table S1; Fig. S2). By using the most recent common ancestor combining all datasets into a NEXUS matrix of 97 taxa and 9,914 of all alternative positions of a fossil, we may have obtained fewer characters. and less informative calibration points than are potentially available from the fossil record. However, we argue that this is Phylogenetic Analyses. Parsimony analyses were conducted in the safest approach to follow, given the uncertainty in fossil PAUP* v. 4.0b10 (8) by using heuristic searches of 1,000 random placement revealed by our analyses. Another possible, but less addition replicates and TBR branch swapping. Bootstrap sup- conservative, approach would have been to try all of the port was evaluated with 1,000 replicates each of one random combinations of most parsimonious positions for all of the fossils addition replicate and a maximum of 100 trees saved per considered suitable for calibration (e.g., ref. 27). However, the replicate. Bayesian analyses were conducted in MrBayes v. 3.1.2 huge number of possible combinations (Ͼ850,000) of fossil (9, 10) by using 2 parallel runs each of 4 chains and 106 positions for this study made this approach prohibitively time- generations, sampling every 100th generation. The best model consuming. for each partition of the combined dataset was determined by In addition, a well-preserved fossil inflorescence with a com- using the Akaike Information Criterion in MrModeltest v. 2.2 bination of apomorphic features only found in a small clade of (11). Because of missing data in some of the datasets, partitioned 2 extant genera (subtribe Musgraveinae) (26) was used as a analyses in MrBayes did not converge within a reasonable minimum age constraint for the stem node of this clade. Finally, number of generations so we present results of unpartitioned the well-documented origin of tricolpate pollen in the fossil analyses with a GTR ϩ⌫ϩI model. However, inspection of the record near the Late Barremian/Early Aptian boundary (28–30) results revealed an almost identical topology and similar poste- was used as a maximum age constraint on the root node of our rior probabilities from both analyses. This was also verified by phylogeny, which is probably only slightly younger than crown conducting both unpartitioned and partitioned analyses of a eudicots as a whole (31). smaller, 5-locus dataset with much less missing data. In the final To infer the phylogenetic relationships of fossil taxa, an analysis, the first 25% of the sample were discarded as burn-in. alternative approach to the use of a backbone constraint derived Parsimony and Bayesian analyses yielded highly congruent trees from the molecular analysis of extant taxa is to combine the and similar branch support (Fig. S1), although bootstrap values morphological and molecular datasets in a single maximum were generally lower than posterior probabilities (12). parsimony search. We experimented with this approach after reducing the molecular dataset to represent each extant genus Fossil Calibration. Twenty-five fossil palynomorph species were with a single exemplar species (thereby eliminating multiple selected and scored with the same characters as an extensive representatives of Platanus, Banksia, and Protea). The resulting database of pollen variation across all extant genera of Pro- combined dataset contained 113 taxa and 9,936 characters and teaceae (recorded at the species and reference level). A mor- was analyzed by using heuristic searches as above. Without the phological matrix of 22 pollen characters and 113 taxa (Dataset fossils, this ‘‘total evidence’’ approach produced 1,152 most S1) was compiled and submitted to parsimony analysis, using the parsimonious trees, of which the strict consensus was congruent molecular tree of extant taxa as a backbone constraint, in which with the maximum parsimony and Bayesian analyses of the all of the branches recognized in the majority-rule Bayesian tree molecular dataset (Fig. S1; except for branches with low boot- (Fig. S1) were used as a constraint. By doing so, we aimed at strap or posterior probability, most of which collapsed in the evaluating more comprehensively the uncertainties in fossil total evidence consensus tree). When fossil morphological char- placement, regardless of uncertainties in relationships among acters were used in the unconstrained total evidence searches, extant taxa. Because of many alternative branching points for the resulting trees were congruent with those found by using a Sauquet et al. www.pnas.org/cgi/content/short/0805607106 1of53 molecular backbone constraint. The major difference is that a groups or, as in our approach to fossil calibration, an entire given much higher number of trees were recovered in the uncon- topology. However, the maximum clade credibility tree we strained total evidence searches, because of the uncertainty in obtained from the BEAST analyses was remarkably similar to relationships among extant taxa. For example, the sister group the majority-rule consensus tree obtained with MrBayes, both in relationship of Granodiporites nebulosus and Embothrium was topology and branch support (Fig. S1). The main difference lies supported in all of the 1,152 most parsimonious trees obtained in the shift of Carnarvonia to the base of Grevilleoideae, but in the total evidence analysis of this fossil taxon. These results are neither alternative is well supported. congruent with those of Manos et al. (32), who also compared different approaches (i.e., backbone constraint vs. total evi- Diversification Rates. The relative extinction rate (␬) was esti- dence) to analyze the relationships of 3 fossil taxa of Juglan- mated by nonlinear least-squares regression of the log- daceae by using morphological and molecular data. We concur transformed version of Eq. 3 of Ricklefs (41) using the nls with these authors that using a molecular backbone constraint is function of R v. 2.7.1 (42). The regression was performed on 2 a more practical and straightforward method in studies integrat- samples of independent clades, first the sample of all genera of ing numerous fossils such as the present one. Proteaceae (terminal taxa of Fig. 1), and then the sample of all independent clades present 20 mya, excluding monospecific ␭ Dating Analyses. Penalized likelihood (PL) dating (33) was im- clades in both cases (Fig. S3 h and i). Speciation rates ( ) for a ␬ 4 plemented in r8s v. 1.71 (34) by using the majority-rule consensus given value of were calculated for each clade by using Eq. of Ricklefs (41). Nonparametric rank-order Kruskal–Wallis tests phylogram from the Bayesian analysis as input tree. The cross- were performed in R on both speciation and net diversification validation procedure determined an optimal value of 10 for the rates for a range of values of ␬, on both samples above, either smoothing parameter. Because the input tree for this PL analysis including or excluding monospecific clades (Table S4). had 2 polytomies, we ran an additional PL analysis by using a Ricklefs (43, 44) proposed an alternative method to estimate fully resolved tree, chosen as the maximum clade credibility tree global rates of speciation and extinction using lineage-through- with mean branch lengths computed by using TreeAnnotator v.