A NGIOSPERM DIVERSIFICATION THROUGH TIME 1

Transcription

1 American Journal of Botany 96(1): A NGIOSPERM DIVERSIFICATION THROUGH TIME 1 Susana Magallón2 and Amanda Castillo Departamento de Bot á nica, Instituto de Biolog í a, Universidad Nacional Aut ó noma de M é xico, 3er Circuito de Ciudad Universitaria, Del. Coyoac á n, M é xico D.F Mexico The extraordinary diversity of angiosperms is the ultimate outcome of the interplay of speciation and extinction, which determine the net diversification of different lineages. We document the temporal trends of angiosperm diversification rates during their early history. Absolute diversification rates were estimated for order-level clades using ages derived from relaxed molecular clock analyses that included or excluded a maximal constraint to angiosperm age. Diversification rates for angiosperms as a whole ranged from to net speciation events per million years, with dates from the constrained analysis. Diversification through time plots show an inverse relationship between clade age and rate, where the younger clades tend to have the highest rates. Angiosperm diversity is found to have mixed origins: slightly less than half of the living species belong to lineages with low to moderate diversification rates, which appeared between 130 and 102 Mya (Barremian-uppermost Albian; Lower Cretaceous). Slightly over half of the living species belong to lineages with moderate to high diversification rates, which appeared between 102 and 77 Mya (Cenomanian-mid Campanian; Upper Cretaceous). Terminal lineages leading to living angiosperm species, however, may have originated soon or long after the phylogenetic differentiation of the clade to which they belong. Key words: crown group; diversification; extinction; fossils; molecular clock; penalized likelihood; stem group. Through the interplay of evolutionary and ecological factors, flowering plants (angiosperms) have accumulated an extraordinary species diversity that encompasses a vast morphological, functional, and ecological versatility and constitutes the structural and energetic basis of the great majority of present-day terrestrial ecosystems. The outstanding diversity and abundance of angiosperms has allowed the development of rich and complex interactions within and among trophic levels and fostered the diversification of other biological lineages (e.g., Schneider et al., 2004 ). Substantial progress in our understanding of the origin and evolution of angiosperms has been accomplished, including, for example, knowledge of relationships at all levels (e.g., Jansen et al., 2007 ; Moore et al., 2007 ), the likely identification of the most ancestral living members of the group (e.g., Mathews and Donoghue, 1999 ), possible morphological and ecological attributes of early representatives (e.g., Endress, 2001 ; Feild et al., 2004 ), and the nature and effect of genomic mutations, and duplications and interactions in determining angiosperm form and structure (e.g., Kramer et al., 1998 ) among many others. Some fundamental questions have nevertheless defied unequivocal resolution ever since Charles Darwin referred to angiosperm rapid diversification as the abominable mystery ( Darwin and Seward, 1903 ). For example, when did angiosperms initiate the diversification that lead to their present-day species diversity? Has their rate of diversification been constant through time? 1 Manuscript received 15 February 2008; revision accepted 13 October The authors are grateful to the editors of the Darwin s Abominable Mystery issue for inviting them to contribute a manuscript. They thank L. Segovia for setting parallel-processing and providing computing resources, I. Cacho and P. Vinuesa for useful Bayesian suggestions, T. Hern á ndez for help in compiling fossil first appearances, J. C. Aguilar for mathematical advice, and B. Moore for comments regarding the capabilities of SymeTREE. They also thank the editors and two anonymous reviewers for their thorough and careful editing and for helpful suggestions. This research was partially funded by the Consejo Nacional de Ciencia y Tecnolog í a, México (CONACYT-2004-C ). The Coordinación de la Investigación Científica, UNAM, provided postdoctoral funding to A.C. 2 Author for correspondence ( s.magallon@ibiologia.unam.mx) doi: /ajb At the most fundamental level, angiosperm species richness must be accounted for in terms of the interplay of origination and extinction, which determines the rate of species diversification. Previous works have shown that species diversity is unequally distributed among angiosperm lineages (e.g., Sanderson and Donoghue, 1994 ; Barraclough and Savolainen, 2001 ; Magall ó n and Sanderson, 2001 ; Sims and McConway, 2003 ); however, little is known about the relative role of speciation and extinction, their long-time trends, and their causal factors in angiosperm diversification. Long-term temporal trends of species accumulation in the fossil record have informed us about the dynamics of angiosperm diversification. By constructing plots of cumulative species diversity through time for vascular plant lineages, Niklas et al. (1985) found a gradual increase in angiosperm species and a decline of other plant lineages, that led to an overall increase in net species diversity in the Cretaceous. The dynamics of early angiosperm diversification and concomitant changes in the composition of the land flora were documented through independent but complementary quantitative analyses of leaf macrofloras ( Lidgard and Crane, 1988, 1990 ) and palynofloras ( Crane and Lidgard, 1989 ). These studies documented the rapid increase in angiosperm diversity and the sharp decline of several land plant lineages, including cycadophytes and free-sporing plants, through the Cretaceous. Angiosperms first became prominent at lower paleolatitudes and subsequently increased their diversity at higher latitudes ( Crane and Lidgard, 1989 ). Lupia et al. (1999) elaborated the quantitative analysis of biotic replacement by using both diversity and abundance data obtained through a stringent selection of paleopalynological samples from the Cretaceous of North America. Absolute species diversity showed no strong trends toward increasing or decreasing within-flora diversity through the Cretaceous. Nevertheless, a pronounced increase of angiosperm diversity and a marked decrease of free-sporing plants were documented. These pronounced trends support the competitive exclusion of plant lineages that were dominant in pre-cretaceous floras by angiosperms ( Lidgard and Crane, 1988, 1990 ; Crane and Lidgard, 1989, 1990 ; Lupia et al., 1999 ).

2 350 American Journal of Botany [Vol. 96 The rate of angiosperm diversification has been explicitly measured in some studies. Niklas et al. (1985) estimated the rate of diversification of angiosperms using counts of speciation and extinction events per geological interval. Rates of angiosperm diversification were calculated as 0.37 net speciation events per million years (Myr 1 ) for monocots, and 0.47 Myr 1 for dicots. Eriksson and Bremer (1992) calculated the average diversification rate of angiosperm families as 0.12 Myr 1 using a maximum likelihood (ML) estimator ( Stanley, 1979 ) that considered the standing diversity and earliest fossil occurrence of 109 angiosperm families. Magall ó n and Sanderson (2001) introduced estimators of diversification rate that consider stem group or crown group age and are contingent on the survival of the lineage to the present. Using ages derived from the fossil record, the rate of diversification of angiosperms as a whole was calculated as Myr 1 assuming a high relative extinction rate and as Myr 1 assuming no extinction. Bokma (2003) introduced ML estimators for speciation and extinction probabilities that account for extinction based on the observed distribution of species among taxa and on their age. The rate of speciation ( λ ) of angiosperms was calculated as 1.0 Myr 1 and the rate of extinction ( μ ) as Myr 1. Several studies have tested for significant shifts in diversification rates in angiosperms. Sanderson and Donoghue (1994) used a ML model-testing approach to evaluate the hypothesis that angiosperm-level key innovations triggered high diversification rates. Models in which a rate shift occurred within angiosperms better explained the distribution of species numbers among the two earliest branches within angiosperms and their sister taxon (Gnetales) and thus provided no support for the hypothesis that angiosperm apomorphies confer high diversification rates. Magall ó n and Sanderson (2001) found that unexpectedly species-rich clades belong to different major angiosperm lineages, suggesting that traits that confer high rates of diversification are different and independently evolved among angiosperm lineages. Sims and McConway (2003) established a ML context to test the hypothesis that the asymmetry in species diversity between angiosperm family or genus sister pairs is within the expectation of a branching model with constant origination and extinction rates. The distribution of species diversity among angiosperm sister pairs was found to be inconsistent with constant net diversification rates. Bokma (2003) used the distribution of species diversity among angiosperm clades and ML-estimated rates of speciation and extinction (described earlier) to test if speciation and extinction probabilities differ among angiosperm higher taxa. The results showed that equal rates of speciation and extinction were unlikely to yield the observed distribution of species diversity among angiosperm clades. Correlation between intrinsic or extrinsic traits and diversification rate or species diversity in angiosperm groups has been abundantly evaluated (e.g., Farrell et al., 1991 ; Sanderson and Donoghue, 1994 ). A dominant approach has attempted to document positive or negative correlation between traits and diversification (e.g., Heilbuth, 2000 ; Sargent, 2004 ). These studies consider absolute species diversity or estimated diversification rate (e.g., Eriksson and Bremer, 1992 ; Ricklefs and Renner, 1994 ; Verd ú, 2002), or rely on relative comparisons of species richness (e.g., Ricklefs and Renner, 1994 ; Heilbuth, 2000; Sargent, 2004 ) or diversification rate (e.g., Farrell et al., 1991 ; Eriksson and Bremer, 1992 ) between sister groups (e.g., Barraclough et al., 1996 ; Dodd et al., 1999 ; Davies et al., 2004 ). A great variety of different traits, alone or in combination, have been advocated as drivers of diversification or as correlates of species-richness. Biotic pollination and a herbaceous life form have been found to be positively correlated with species richness or high diversification rate (e.g., Burger, 1981 ; Stebbins, 1981 ; Eriksson and Bremer, 1992 ; Ricklefs and Renner, 1994, 2000 ; Dodd et al., 1999 ; Silvertown et al., 2000 ). Other positively correlated traits include the rate of chromosomal evolution ( Levin and Wilson, 1976 ), the rate of molecular substitution ( Barraclough et al., 1996 ; Barraclough and Savolainen, 2001, but see Davies et al., 2004 ), defense against predators (Ehrlich and Raven, 1964; Farrell et al., 1991 ); floral zygomorphy ( Sargent, 2004 ), environmental energy ( Davies et al., 2004 ) and the capacity of species to adopt new life history attributes ( Ricklefs and Renner, 1994, but see Dodd et al., 1999 ; Ricklefs and Renner, 2000 ; Silvertown et al., 2000). Other traits such as dioecy ( Heilbuth, 2000 ) and age at maturity among woody species (Verd ú, 2002) have been found to be negatively correlated with angiosperm species-richness/diversification. Burger (1981) considered that a rapid reproductive cycle, flexible breeding modes, improved dispersal capacity and protection for seeds, an extraordinary variety in growth form and anatomy, and a varied array of chemical defenses are some of the intrinsic traits that have allowed angiosperms to outcompete and outnumber free-sporing plants and gymnosperms. He emphasized that biotic pollination probably acts by guarding small populations against extinction in species-rich and specialized biotas, whereas the driving forces promoting speciation are most likely those that divide large populations into small, genetically isolated species ( Burger, 1981 ). Empirical observations and quantitative evaluations ( Sanderson and Donoghue, 1994 ; Magall ó n and Sanderson, 2001 ; Sims and McConway, 2003 ) of the vastly different number of species in phylogenetically defined angiosperm taxa strongly indicate a substantial variation in the rates of diversification among angiosperm lineages. In species-rich clades, diversity is also heterogeneously distributed: there are some branches that have diversified profusely, while others have given rise to only a few species. Angiosperm species diversity results from the combination of lineages that have achieved an extraordinarily high species diversity and lineages that contain small or moderate numbers of species. This study provides estimates of the net diversification rate of angiosperms as a whole and of a comprehensive set of angiosperm order-level clades. Based on the estimated rates, general trends of the process of diversification are documented through the initial part of angiosperm history. By estimating absolute rates of diversification for angiosperm orders and for nonnested clades appearing every 10 Myr and by assessing the general trend of the magnitude of diversification rates through time, we attempt to answer (1) whether rates of diversification of major angiosperm lineages have remained constant through time, (2) whether present-day angiosperm species diversity preponderantly belongs to ancient or to young lineages, and (3) whether there are particular times during angiosperm history when lineages with high diversification rate appeared. Absolute diversification rates were calculated with estimators that distinguish between the age of differentiation (stem group age) and the age of diversification (crown group age) of a clade and are conditional on the survival of the lineage to the present. Previously, these estimators were used to calculate diversification rates for a set of angiosperm clades with ages obtained from fossil first appearances ( Magall ó n and Sanderson,

3 January 2009]Magallón and Castillo Angiosperm diversification ). Problems associated with the direct use of fossil first appearances as ages of clades have been elaborated on more specifically by Sims and McConway (2003) and McConway and Sims (2004) in the context of diversification rate estimation. Fossil first appearances can only provide minimal ages of clades, which are separated by a time lapse of unknown magnitude from the true age of the clade ( Magall ó n, 2004 ). If the true age is substantially older than the first fossil appearance, diversification rates derived from fossil ages will be overestimated. The study here presented is strongly based on the ideas and methods of Magall ó n and Sanderson (2001), but it differs in two important aspects. First, methodologically, rates of diversification are here based on clade ages derived from a relaxed molecular clock analysis, which in turn relied on abundant fossil information to guide molecular-based age estimations. Second, whereas the previous study focused on differential diversification rates among lineages, in this study, we concentrate on differential diversification rates through time. MATERIALS AND METHODS Phylogenetic relationships Phylogenetic trees used to estimate diversification rates were obtained from a data set including 265 genera belonging to 52 angiosperm orders (and unplaced isolated families), according to the Angiosperm Phylogeny Website (APW; Stevens, 2001 ). The APW is strongly based on the APG system ( APG, 1998 ; APG II, 2003) and updates its contents with numerous recent publications. The orders sampled in this study represent over 94% of angiosperm living species. The gymnosperms Ginkgo biloba (ginkgophytes) and Gnetum gnemon (gnetophytes) were also included, and Ginkgo biloba was specified as the outgroup (Appendix S1, see Supplemental Data with the online version of this article). The data are the concatenated nucleotide sequences of three plastid genes ( atpb, rbcl, and matk ) and two nuclear markers (18S nuclear ribosomal DNA [nrdna] and 26S nrdna). Sequences were obtained through a bioinformatic search in GenBank, which aimed to balance a comprehensive representation of angiosperm orders with the largest possible number of molecular markers for all included taxa. The matrix was complete in that all genes were available for all taxa, although in several cases, a single genus was represented by different species. Sequence alignment for each marker was achieved with the program MUSCLE ( Edgar, 2004 ), followed by manual refinements with the program BioEdit v5.0.6 ( Hall, 1999 ). The sequences of each marker were subsequently concatenated in a single data set ( treebase.org, TreeBASE accession SN4019 ). An examination of parameter values of best fitting models for individual molecular markers and codon position partitions (for atpb, rbcl and matk ), obtained with the Akaike information criterion (AIC) implemented in the program Modeltest ( Posada and Crandall, 1998 ; Posada and Buckley, 2004 ), indicated that the data could be appropriately divided into four partitions: (1) first and second positions of atpb and rbcl, (2) third positions of atpb and rbcl, (3) matk, and (4) 18S and 26S. Phylogenetic relationships among the 265 angiosperm genera were estimated with Bayesian analysis using MrBayes v3.1.2p ( Huelsenbeck and Ronquist, 2001 ). Independent models with unlinked parameters were applied to the four data partitions, implementing variable rate priors. Two independent Metropolis coupled-markov chain Monte Carlo (MC 3 ) runs of generations, each consisting of four incrementally heated chains (temp = 0.2), were conducted, sampling one tree every 200 generations. After examination of generation vs. likelihood plots, trees corresponding to the initial generations (10%) were discarded as burn-in. Post burn-in sampled topologies were summarized as a 50% majority rule tree to obtain the posterior probability (PP) credibility interval of each clade. Age estimation Ages of clades were estimated with penalized likelihood (PL; Sanderson, 2002, 2004 ), a molecular-based semiparametric method that incorporates among-lineage rate heterogeneity and can use fossil information as auxiliary in divergence time estimation. We relied on two considerations to select a phylogenetic tree to estimate dates. First, avoid an incompletely resolved tree (e.g., the 50% majority rule consensus), to circumvent possible complications in the cross validation procedure (described later), probably the most computationally difficult step in PL dating ( Sanderson, 2004, pp ). Second, use a phylogram (i.e., topology with branch lengths) in which branch lengths were obtained through the optimization of best-fitting model parameters for each data partition. Hence, we selected the topology with highest PP found in the two Markov chains as a phylogenetic working hypothesis and used all the phylograms that have a topology identical to it to estimate ages. The topology with highest PP was identified with MrBayes, and topologically identical phylograms were found and extracted with the program PAUP* version 4.0b10 ( Swofford, 2002 ). Because of technical conventions for optimizing branch lengths around the root node of a tree (e.g., Sanderson, 2004, pp ), it became necessary to remove Ginkgo biloba, the outgroup used during phylogeny estimation, from dating analyses. The divergence between Gnetum gnemon and angiosperms became the new root node. Phylograms were temporally calibrated by fixing the age of the root node, which represents the crown group of seed plants, at 350 Myr. This age was selected because it is younger than the Famennian (Upper Devonian; Rothwell et al., 1989 ) age of the oldest known fossil seeds ( Elkinsia polymorpha ), and older that the Namurian (Lower-Upper Carboniferous; Taylor and Taylor, 1993 ) age of the oldest presumed crown group seed plants (Cordaitales). It also corresponds approximately to the mean age for the crown group of seed plants obtained in different relaxed molecular clock analyses for a representation of vascular plant lineages (S. Magall ó n, unpublished results). Forty-nine nodes within angiosperms were constrained with minimal ages (minage) derived from a critical examination of the angiosperm fossil record ( Fig. 1, Appendix S2, see Supplemental Data with the online version of this article). In addition to the minage constraints, two maximal age (maxage) constraints were alternatively applied. In the first case, here referred to as relaxed dating, a maxage of 125 Myr was applied to the eudicot crown node. The appearance of tricolpate pollen grains in late Barremian-early Aptian (ca. 125 Myr) sediments ( Hughes and McDougall, 1990 ; Doyle, 1992 ; Hughes, 1994 ; Friis et al., 2006 ), has been regarded among the best indications in the fossil record of the origin of a biological lineage. There is an exact correspondence between the presence of a distinctive morphological attribute, which was abundantly produced and became easily fossilized (i.e., tricolpate pollen), and membership to a monophyletic group (i.e., eudicots, or tricolpate angiosperms). The age of the eudicot crown node appears to be constrained narrowly around 125 Myr, given the Barremian-Aptian age of the oldest tricolpate pollen, presumably produced by eudicot stem lineage representatives, and the Barremian-Aptian report of putative eudicot grown group members ( Leng and Friis, 2003 ; Friis et al., 2006 ). Previous studies (e.g., Soltis et al., 2002 ; Anderson et al., 2005 ) have used the earliest record of tricolpate pollen as a definitive temporal landmark for the origin of eudicots. In the second case, referred to as constrained dating, a maxage of 130 Myr was applied to the angiosperm crown node. This maxage is based on the oldest report of fossil angiosperm pollen, typically characterized by a thin and granular endexine ( Friis et al., 2006 ), from Valanginian-Hauterivian (ca Myr) sediments ( Hughes and McDougall, 1987 ; Hughes et al., 1991 ; Hughes, 1994 ; Brenner, 1996 ). Assuming that these early pollen grains were produced by angiosperm stem lineage representatives, we speculate that the origin of the angiosperm crown group may have occurred shortly afterwards, by the Hauterivian-Barremian boundary (130 Myr). The age of the angiosperm crown group is limited by a lower (younger) bound of approximately 125 Myr, corresponding to the late Barremian-early Aptian age of unequivocal angiosperm crown group members (Chloranthaceae, Friis et al., 2006; Winteraceae, Doyle et al., 1990; Doyle, 2000 ; eudicots, described earlier). The stratigraphic position of fossils used for tree calibration and, as minage and maxage constraints, was transformed into absolute ages (Myr) using the upper (younger) bound of the interval, based on the stratigraphic time scale of Gradstein and Ogg (2004). The Langley Fitch ( Langley and Fitch, 1974 ) test of the molecular clock, as implemented in the program r8s version 1.71 ( Sanderson, 2003, 2004 ) was conducted. Penalized likelihood, implemented in r8s, requires a user-defined parameter to specify the level of molecular rate smoothing to be implemented in dating analysis. To identify the smoothing magnitude ( λ ) that best describes the available data, we used a cross validation procedure that calculates the predictive error associated to molecular rate and time estimates across the full tree, derived from sequentially removing minage and maxage constraints ( Sanderson, 2004 ). Cross validations were performed for relaxed and constrained maxage implementations, using one randomly selected phylogram among those with highest PP topology. Each cross validation tested 16 smoothing magnitudes ranging from log λ 10 = 2 to 5.5 at 0.5 intervals, which comprise a broad spectrum of substitution regimes. Penalized likelihood dating was conducted on all the phylograms with highest PP topology, using relaxed and constrained maxage constraints and implementing in each case the optimal smoothing magnitude

4 352 American Journal of Botany [Vol. 96 Fig. 1. Constrained dated phylogenetic tree for angiosperm orders. The tree is a graphical summary at the order level of a 265-terminal dated tree representing 52 angiosperm orders and outgroups. Ages correspond to the mean of 46 phylograms with highest posterior probability (PP) topology, dated with penalized likelihood, imposing a 130 Myr maximal age constraint to the angiosperm crown node. Clades subtended by orange branches are supported by <0.95 PP. Dashed lines correspond to orders inserted in the dated tree. Green ovals represent minimal age (minage) constraints, and numbers below them correspond to numbers in Appendix S2 (see Supplemental Data with the online version of this article) and the age (Myr) they provide. Two or more minage

5 January 2009]Magallón and Castillo Angiosperm diversification 353 found in the respective cross validation. Penalized likelihood analyses used a TN algorithm with bound constraints with five initial starts and three perturbed restarts with perturbations of magnitude 0.05 in random directions. The mean age, standard deviation, and 95% confidence interval of every node in the tree were derived from the point estimates of age in each of the phylograms with highest PP topology. Diversification rates Diversification rates were calculated using method-of-moments estimators ( Rohatgi, 1976 ) in the context of a birth-anddeath model ( Kendall, 1948 ) that consider the species diversity and age of a clade. These estimators provide absolute estimates of the rate of diversification of a clade, they are conditional on the survival of the clade to a given time t, in this case, the present, and they can differentially estimate the diversification rate of a stem clade or of a crown clade ( Doyle and Donoghue, 1993 ; Magall ó n and Sanderson, 2001 ). These conditional estimators of absolute diversification rates correspond to eqs. 6 and 7 in Magall ó n and Sanderson (2001 ) : for stem groups and r 1 ˆε = log[ n (1 ε ) +ε] t 1 1 = ε + ε t 2 1 (1 ) ( 2 + ε nnε 8 ε+ 2 nε+ n ) log rˆ ε log n(1 ) 2 for crown groups. The relative extinction rate ( ε ) is defined as ε = μ /λ. The variable t corresponds to a time after the origin of the clade, here the present, and n is the standing species diversity of the clade at time t. Because absolute speciation and extinction rates for angiosperms and angiosperm clades are unknown, diversification rates were estimated assuming that the relative extinction rate is bounded within ε = 0.0, which implies no extinction, and ε = 0.9, which implies a very high relative extinction rate. Whereas ε = 0.0 represents an absolute lower bound for the relative extinction rate, the selection of ε = 0.9 as an upper bound is arbitrary. It is nevertheless justified by the observation that as ε approaches 1, the magnitudes of the rates of speciation ( λ ) and extinction ( μ ) increase rapidly, exceeding values of one event per million year. These values approximately represent the empirical maximum estimated from real data for a variety of animal taxa ( Stanley, 1979 ; Hulbert, 1993 ). Also, for large clades, values of ε larger than 0.9 correspond to clades having less than a 10% chance of surviving to the present, which in the case of angiosperms, with a living diversity of about species, seems unlikely ( Magall ó n and Sanderson, 2001 ). Therefore, we estimated the diversification rate of each clade considering ε = 0.0 and 0.9. Standing species diversity in angiosperm orders was obtained from the APW ( Stevens, 2001 ). In addition to angiosperm orders, absolute diversification rates were estimated for nonnested crown clades appearing every 10 Myr since the onset of angiosperm crown group diversification. This approach aimed to evaluate diversification rate from a standpoint that relies on the temporal distribution of branching events on a tree. The identification of nonnested crown nodes was based on the timing of angiosperm lineage diversification according to constrained dating. To correctly quantify the number of living species derived from a node in the tree, we needed to consider the species diversity of orders (and unplaced families) that were not sampled in the phylogenetic analysis. Unsampled orders were intercalated in the dated tree according to their position in the order-level phylogeny in the APW ( Stevens, 2001 ; Fig. 1 ). The stem group age of every intercalated order was estimated as the midpoint between the ages of dated nodes immediately above (younger) and below (older) it. Orders (or families) of unresolved phylogenetic position in the APW ( Stevens, 2001 ) were placed in a polytomy ( Fig. 1 ). We estimated diversification rates of angiosperm orders using relaxed and constrained dates; their stem group age (for 72 orders: 52 sampled, 20 inserted) and their crown group age (for 41 orders), with a relative extinction rate ( ε ) of 0.0 and of 0.9. Diversification rates of nonnested crown clades were also estimated using ε = 0.0 and 0.9. Estimated diversification rates were plotted against the age of the respective clade ( Figs. 2, 3 ). We attempted to implement topology-based, whole-tree statistical tests for detecting significantly different diversification rates in the angiosperm tree and to identify the branches of the angiosperm tree in which diversification rate shifts have occurred (Symmetree program; Chan and Moore, 2005 ). Nevertheless, the tests could not be conducted due to the large number of species encompassed in several terminals of the angiosperm order-level tree (B. R. Moore, U.C. Berkeley, personal communication). RESULTS Phylogenetic relationships The concatenated sequences of the five molecular markers ( atpb, rbcl, matk, 18S nrdna, and 26S nrdna) are 9789 base pairs (bp) in length and are available for all the genera in the data set. The best-fit model for each of the four data partitions (i.e., 1 st and 2 nd codon positions of rbcl and atpb ; 3rd positions of rbcl and atpb ; matk ; and 18S- 26S nrdna) included six nucleotide substitution parameters, among-site rate variation, and a proportion of invariable sites (GTR+I+ Γ ). After generations, the two independent Markov chains were estimated to have converged (standard split frequencies < 0.01). Plots of generation number vs. likelihood value indicated that in both runs, likelihood values stabilized approximately after generations; however, the trees sampled in the initial generations (10% of the total) were excluded as burn-in. The 50% MR consensus of post burn-in trees (available in TreeBASE SN4019 ) contains five unresolved polytomies: within Alismatales, among major core eudicot lineages, within Saxifragales, and within Ericales (two polytomies). The topology with the highest PP was selected as a working phylogenetic hypothesis for molecular dating. An order-level summary is shown in Fig. 1, and the full tree is available in TreeBASE (SN4019 ). Most relationships are in agreement with independent assessments (e.g., Soltis et al., 1999 ; Stevens, 2001 ), and relatively unusual relationships, for example, the sister group relationship between Amborellales and Nymphaeales, are weakly supported. Unless otherwise indicated, the following relationships are supported by 0.95 PP. The deepest split within angiosperms separates a branch including Amborellales and Nymphaeales (0.74 PP) from all other members of the clade. Austrobaileyales is the sister to core angiosperms, which are divided into a branch that includes Chloranthales plus monocots (0.57 PP) and eumagnoliids, and a branch that includes Ceratophyllales and eudicots. Ranunculales is the sister to all other eudicots, followed by a branch that includes Sabiales and Proteales (0.73 PP). Buxales is the sister to core eudicots. The 50% majority consensus tree contains a trichotomy involving Gunnerales, Dilleniales, and a clade that includes all other core eudicot lineages. In the highest PP topology, Gunnerales and Dilleniales are sister taxa ( < 50% PP). The deepest split within the clade that includes all other core eudicot constraints on the same branch in the summarized order-level tree constrained different nodes in the 265-terminal phylogenetic tree (Appendix S2, see Supplemental Data with the online version of this article). The number of living species represented by each order is indicated after the order s name. Purple ovals with white numbers indicate nonnested crown clades appearing every 10 Myr intervals (vertical bands), corresponding to numbered clades in Table 2. Some order names were abbreviated because of lack of space.

6 354 American Journal of Botany [Vol. 96 Fig. 2. Diversification rate through time for angiosperm order-level clades. (A) Stem group diversification rate derived from relaxed dating. (B) Crown group diversification rate derived from relaxed dating. (C) Stem group diversification rate derived from constrained dating. (D) Crown group diversification rate derived from constrained dating. Diversification rates were obtained assuming a relative extinction rate ( ε ) of 0.0 (circles), and of 0.9 (solid triangles). Because estimated crown group diversification rates of Malvales are substantially higher than all others (see Results and Discussion), they were not included in the graphs. lineages separates a clade (0.86 PP) that includes Saxifragales, Vitales, and rosids, and another clade that includes Santalales, Berberidopsidales, Caryophyllales, and asterids ( Fig. 1 ). Age estimation The age and phylogenetic position of the 49 minage constraints are shown in Fig. 1 and described in Appendix S2 (see Supplemental Data with the online version of this article). Among the phylograms sampled by the two Markov chains, 46 are topologically identical to the topology with highest PP. Dating was conducted on these 46 phylograms. The Langley Fitch test of the molecular clock indicated that relaxed and constrained phylograms departed significantly from rate constancy ( P = 0 in all tests). Not surprisingly given the number of taxa and the branch length heterogeneity in the phylogram, the relaxed and constrained cross validation procedures returned several failed optimizations. Nevertheless, both showed relatively small differences in the raw error associated to the range of tested smoothing magnitudes ( λ ). A general positive relation between raw error and smoothing magnitude was found, except for the highest smoothing magnitudes in the relaxed cross validation (results available from the authors). In both cross validations, the lowest smoothing magnitude that was tested (which passed the optimization) has the smallest associated raw error, and hence, relaxed and constrained dating analyses were conducted implementing a smoothing parameter of log λ 10 = 2 (λ = 0.01). Ages of nodes derived from relaxed dating were substantially older than those obtained in the constrained analysis, except within eudicots, where ages are similar in both analyses. Both sets of ages were used to estimate diversification rates. The mean age and 95% confidence interval estimated for the stem group and crown group of angiosperm orders according to relaxed and constrained dating are shown in Table 1. The chronogram resulting from constrained dating is shown in Fig. 1. The relaxed chronogram is available from the authors. Nonnested crown group clades Twenty orders (and unplaced families) were inserted in the constrained dated tree based on their position in the APW order-level tree ( Stevens, 2001 ) and a stem group age intermediate between that of immediately deeper and shallower dated nodes ( Fig. 1 ). In many cases, the bounding nodes are separated by a short time gap;

7 January 2009]Magallón and Castillo Angiosperm diversification 355 Fig. 3. Diversification rate through time for nonnested crown clades appearing at every 10-Myr interval. The 13 nonnested clades are defined in Table 2. Diversification rates were obtained assuming a relative extinction rate ( ε ) of 0.0 (circles) or 0.9 (solid triangles). Vertical dashed lines represent boundaries between 10-Myr intervals. thus, the possibility of a large error in the age assigned to the intercalated nodes is small. Diversification rates were estimated for the inserted orders (Table 1); however, these should be regarded as especially tentative due to the unavailability of a direct estimate of age, and in some cases, uncertainty in phylogenetic position. Thirteen nonnested crown clades appearing every 10 Myr and in six intervals from 130 to Myr were identified (including angiosperms as a whole; Table 2). The three intervals between 120 and Myr gave rise to the largest number of nonnested crown clades (three, two, and five, respectively), while the first ( ) and two last intervals ( ) each gave rise to one ( Fig. 1, Table 2). The phylogenetic equivalence and species diversity (including inserted orders) of nonnested crown clades are shown in Table 2. Diversification rates Diversification rates calculated for angiosperm orders based on their stem group (72 orders: 52 sampled and 20 inserted) and crown group age (41 orders), derived from ages obtained with relaxed and constrained dating, and relative extinction rate ( ε ) of 0.0 and 0.9 are shown in Table 1. The absolute (crown group) diversification rates estimated for angiosperms as a whole are Myr 1 with ε = 0.9, and Myr 1 with ε = 0.0 using relaxed dating, and Myr 1 with ε = 0.9, and Myr 1 with ε = 0.0 using constrained dating. Angiosperm orders with the highest stem group diversification rates are Lamiales ( Myr 1 ), Gentianales ( Myr 1 ), Asterales ( Myr 1 ) and Solanales ( Myr 1 ). Orders with the lowest stem group rates are Amborellales (0 Myr 1 ), Acorales ( Myr 1 ), Ceratophyllales ( Myr 1 ), and Berberidopsidales ( Myr 1 ). Among the orders for which crown group age was available, Malvales was identified as having the highest diversification rate ( Myr 1 ), followed somewhat distantly by Lamiales ( Myr 1 ), Brassicales ( Myr 1 ), Asterales ( Myr 1 ), and Sapindales ( Myr 1 ). Crown group clades with the lowest rates are Berberidopsidales ( Myr 1 ), Austrobaileyales ( Myr 1 ), Canellales ( Myr 1 ), and Chloranthales ( Myr 1 ). All else being equal, diversification rates derived from constrained dating were typically higher than those derived from relaxed dating, and those obtained with ε = 0.0 were higher than those obtained with ε = 0.9. In clades for which stem group and crown group diversification rate were estimated, the two rates were of similar magnitude. A general positive relationship between the stem group and crown group diversification rate for any given clade was found, and a tendency for one rate to be larger than the other was not observed. Diversification rates of the 13 nonnested crown clades appearing every 10 Myr implementing ε = 0.0 and ε = 0.9, are shown in Table 2. The diversification rates estimated for nonnested crown clades appearing between 130 and Myr are similar to those of angiosperms as a whole. Among these, only the clade corresponding to the most recent common ancestor (MRCA) of Pandanales and Dioscoreales ( Myr interval) and the MRCA of Commelinales and Zingiberales ( Myr interval), have somewhat lower rates ( Myr 1 and Myr 1, respectively; Table 2, Fig. 3 ). However, the diversification rates of the two youngest nonnested crown clades are substantially higher than all the rest (Table 2, Fig. 3 ). Diversification rates for the MRCA of Vahliaceae and Solanales ( Myr interval) range from to Myr 1, and for the MRCA of Lamiales and Solanales ( Myr interval) from to Myr 1. As observed for angiosperm orders, diversification rates obtained with ε = 0.0 were higher than those obtained with ε = 0.9. Diversification through time The relationships of diversification rate and clade age show that the highest diversification rates are found among younger orders, whether considering relaxed or constrained dating, stem or crown group diversification rates, or high or low relative extinction rates ( Fig. 2 ). The diversification rate of nonnested crown clades is approximately constant, except for the two youngest nonnested crown clades, which exhibit appreciably higher diversification rates ( Fig. 3 ). DISCUSSION Penalized likelihood dating Penalized likelihood analyses included a comprehensive representation of angiosperms at the order level and incorporated a substantial amount of fossil-derived information. The 130 Myr maxage constraint to the angiosperm node in constrained dating exerted a determinant influence on age estimates across the tree. Constrained dates were much younger than relaxed dates; however, in some cases, they were substantially older than the earliest fossil record of particular angiosperm lineages ( Fig. 1 ). Other studies (e.g., Wikstr ö m et al., 2001 ; Bell et al., 2005 ; Magall ó n and Sanderson, 2005 ) have obtained ages for angiosperms or angiosperm clades that, as in the relaxed analysis, are substantially older than their oldest fossils. Here, we followed the approach of Schneider et al. (2004), who performed alternative dating analyses by fixing or unfixing the age of angiosperms at 132 Myr. These authors also found that ages obtained in the unfixed analysis were much older than those in the fixed analysis and the angiosperm fossil record. In both relaxed and constrained analyses, the timing of appearance of orders is more or less continuously distributed through a delimited time interval. In relaxed dating, this interval

8 356 American Journal of Botany [Vol. 96 Table 1. Species diversity, age, and diversification rate at two relative extinction rates (ε) for angiosperm orders. The two subheadings above each column describe the first and second row, respectively, for each order listing. Age Diversification rate Order Standing species diversity Constrained stem group group ε = 0.0 Constrained stem group ε = 0.0 ε = 0.9 Constrained stem group ε = 0.9 ε = 0.0 group ε = 0.0 ε = 0.9 group ε = 0.9 Angiosperms ( ) ( ) ( ) ( ) Amborellales na 0 0 na na ( ) na 0 0 na na ( ) Nymphaeales ( ) ( ) ( ) ( ) Austrobaileyales ( ) ( ) ( ) ( ) Chloranthales ( ) ( ) ( ) ( ) Acorales na na na ( ) na na na ( ) Alismatales ( ) ( ) ( ) ( ) Petrosaviales* (na) na na na (na) na na na Pandanales ( ) ( ) ( ) ( ) Dioscoreales ( ) ( ) ( ) ( ) Liliales ( ) ( ) ( ) ( ) Asparagales ( ) ( ) ( ) ( ) Dasypogonaceae* (na) na na na (na) na na na Arecales* (na) na na na (na) na na na Poales ( ) ( ) ( ) ( ) Commelinales na na na ( ) na na na ( ) Zingiberales ( ) ( ) ( ) ( )

9 January 2009]Magallón and Castillo Angiosperm diversification 357 Table 1. Continued Age Diversification rate Order Standing species diversity Constrained stem group group ε = 0.0 Constrained stem group ε = 0.0 ε = 0.9 Constrained stem group ε = 0.9 ε = 0.0 group ε = 0.0 ε = 0.9 group ε = 0.9 Canellales ( ) ( ) ( ) ( ) Piperales ( ) ( ) ( ) ( ) Magnoliales ( ) ( ) ( ) ( ) Laurales ( ) ( ) ( ) ( ) Ceratophyllales na na na ( ) na na na ( ) Ranunculales ( ) ( ) ( ) ( ) Sabiales na na na ( ) na na na ( ) Proteales ( ) ( ) ( ) ( ) Trochodendrales* (na) na na na (na) na na na Buxales ( ) ( ) ( ) ( ) Gunnerales na na na ( ) na na na ( ) Dilleniales ( ) ( ) ( ) ( ) Saxifragales ( ) ( ( ) ( ) Vitales ( ) ( ( ) ( ) Picramniaceae* (na) na na na (na) na na na Myrtales* (na) na na na (na) na na na Geraniales na na na ( ) na na na ( ) Crossosomatales ( ) ( )

10 358 American Journal of Botany [Vol. 96 Table 1. Continued Age Diversification rate Order Standing species diversity Constrained stem group group ε = 0.0 Constrained stem group ε = 0.0 ε = 0.9 Constrained stem group ε = 0.9 ε = 0.0 group ε = 0.0 ε = 0.9 group ε = ( ) ( ) Sapindales ( ) ( ) ( ) ( ) Huerteales* (na) na na na (na) na na na Brassicales ( ) ( ) ( ) ( ) Malvales ( ) ( ) ( ) ( ) Zygophyllales* (na) na na na (na) na na na Oxalidales ( ) ( ) ( ) ( ) Celastrales ( ) ( ) ( ) ( ) Malpighiales ( ) ( ) ( ) ( ) Cucurbitales ( ) ( ) ( ) ( ) Fagales ( ) ( ) ( ) ( ) Fabales na na na ( ) na na na ( ) Rosales ( ) ( ) ( ) ( ) Santalales ( ) ( ) ( ) ( ) Berberidopsidales ( ) ( ) ( ) ( ) Caryophyllales ( ) ( ) ( ) ( ) Cornales ( ) ( ) ( ) ( ) Ericales ( ) ( )