Meliaceae) Shed New Light on the Origins of Rainforest Hyperdiversity

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Recently evolved diversity and convergent radiations of rainforest mahoganies (Meliaceae) shed new light on the origins of rainforest hyperdiversity

Erik J. M. Koenen1,2, James J. Clarkson3, Terence D. Pennington4 and Lars W. Chatrou2 1Institute of Systematic Botany, University of Zurich, Zollikerstrasse 107, 8008 Zurich,€ Switzerland; 2Biosystematics Group, Wageningen University, Droevendaalsesteeg 1, 6708 PB

Wageningen, the Netherlands; 3Molecular Systematics Section, Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3DS, UK; 4Herbarium, Library, Art & Archives, Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3AB, UK

Summary Author for correspondence: Tropical rainforest hyperdiversity is often suggested to have evolved over a long time-span Erik J. M. Koenen (the ‘museum’ model), but there is also evidence for recent rainforest radiations. The mahoga- Tel: +41 (0) 44 634 84 16 nies (Meliaceae) are a prominent plant group in lowland tropical rainforests world-wide but Email: [email protected] also occur in all other tropical ecosystems. We investigated whether rainforest diversity in Received: 8 December 2014 Meliaceae has accumulated over a long time or has more recently evolved. Accepted: 15 April 2015 We inferred the largest time-calibrated phylogeny for the family to date, reconstructed ancestral states for habitat and deciduousness, estimated diversification rates and modeled New Phytologist (2015) 207: 327–339 potential shifts in macro-evolutionary processes using a recently developed Bayesian method. doi: 10.1111/nph.13490 The ancestral Meliaceae is reconstructed as a deciduous species that inhabited seasonal habitats. Rainforest clades have diversified from the Late Oligocene or Early Miocene Key words: diversification rate, evolutionary onwards. Two contemporaneous Amazonian clades have converged on similar ecologies and radiations, extinction, Meliaceae, molecular high speciation rates. dating, phylogenetics, species turnover, Most species-level diversity of Meliaceae in rainforest is recent. Other studies have found tropical rainforest hyperdiversity. steady accumulation of lineages, but the large majority of plant species diversity in rainforests is recent, suggesting (episodic) species turnover. Rainforest hyperdiversity may best be explained by recent radiations from a large stock of higher level taxa.

Introduction forests that are found scattered throughout the Neotropics, Africa and Madagascar (Kier et al., 2005). There are also striking differ- Biodiversity is unevenly distributed, geographically as well as ences in diversity between continents: tropical Africa has rela- across lineages of the tree of life. Across most groups of organ- tively low plant species richness compared with South-East isms, a latitudinal diversity gradient is observed, where high-lati- Asia and the Neotropics. This diversity anomaly suggests that tude (or high-altitude) frost-prone regions presumably are less historical factors, for example, higher extinction rates in Africa conducive to diversification and/or more prone to extinction than (Kissling et al., 2012; Bardon et al., 2013), could play an tropical regions (Davies et al., 2004; Pyron & Wiens, 2013). As important role in shaping the distribution of biodiversity. plants are fundamental constituents and key primary producers The evolution of rainforest hyperdiversity is not well under- of terrestrial ecosystems, their diversity distribution is likely to stood and different hypotheses have been put forward. Under the determine or at least reflect the distribution of total terrestrial so-called ‘museum model’ of diversification (Wallace, 1878; diversity to a large extent. Important climatic predictors of vascu- Stebbins, 1974), history is central to explaining rainforest hyper- lar plant diversity at the global scale are related to energy and diversity: rainforests are viewed as old ecosystems which have water availability (Kreft & Jetz, 2007), which explains the latitu- been prevalent throughout much of the Cenozoic, and the model dinal gradient and predicts that the highest diversity is found in implies constant diversification rates with a low extinction frac- the wet tropics, where the highest number of plant species can tion, thought to be related to the ecological stability of rainfor- co-exist and therefore evolve. In the tropics, both the amount ests, leading to gradual accumulation of diversity over a long and timing of precipitation strongly influence the distribution of time period. Fossil evidence shows that modern-type rainforests ecosystems, and have a strong effect on the distribution of plant are indeed at least as old as the early Cenozoic (Wing et al., species richness (Kreft & Jetz, 2007). The wettest and least sea- 2009). Phylogenetic methods have also provided insights, show- sonal lowland evergreen rainforests are generally more species- ing that certain lineages, such as Malpighiales (Davis et al., rich than the seasonal, drier savannas or seasonally dry deciduous 2005), Annonaceae (Couvreur et al., 2011a; Chatrou et al., 2012;

Ó 2015 The Authors New Phytologist (2015) 207: 327–339 327 New Phytologist Ó 2015 New Phytologist Trust www.newphytologist.com New 328 Research Phytologist

Pirie & Doyle, 2012), Arecaceae (Couvreur et al., 2011b) and Meliaceae and increase taxon sampling. Sequences of ycf1 were Menispermaceae (Wang et al., 2012), that are nowadays common newly generated for several taxa in subfamily Melioideae. The in rainforests are indeed old with ancient diversification or steady few studies that have used this marker suggest that it is one of the accumulation of lineages, lending support to the museum model. most variable and phylogenetically informative plastid regions An alternative hypothesis is the ‘recent cradle’ model, where (Neubig et al., 2009; Parks et al., 2009; Chaowasku et al., 2014). diversification rates have not remained constant, but most diver- Detailed laboratory methods are included in Methods S1 and sity is more recently evolved. Several young radiations of rainfor- GenBank accession numbers in Table S2. est lineages have been documented (Richardson et al., 2001; Kay We used BAYESPHYLOGENIES v2.0 with reversible-jump Markov et al., 2005; Erkens et al., 2007; S€arkinen et al., 2007), support- chain Monte Carlo (MCMC) to model rate heterogeneity across ing the recent cradle model. However, we are not aware of any sites (Pagel & Meade, 2004) and estimate an a posteriori parti- studies that compared diversification processes over longer time- tioning scheme. Three individual runs were performed until the scales across multiple lineages spanning both wet and seasonally MCMC reached stationarity (11–13 million generations), dry ecosystems. employing a GTR + Γ model of nucleotide substitution, with The mahogany family (Meliaceae) is a medium-sized angio- two chains for each analysis (Metropolis-coupling of ‘cold’ and sperm family of tropical trees and shrubs (c. 702 species in 53 ‘hot’ chains) and sampling parameter estimates, tree topology genera; see Supporting Information Table S1) in the Sapindales. and rate heterogeneity patterns every 5000 generations. Phyloge- The family is well known for its high-quality timbers, traded as netic analysis is routinely performed with partitioned data matri- ‘mahogany’. Most Meliaceae are characterized by compound ces to unlink parameter estimation and/or to use different models leaves, functionally unisexual flowers (usually dioecious) and cap- of nucleotide substitution for different parts of the alignment. sular fruits, with either winged seeds (in subfamily Cedreloideae) Often partitioning is performed on a per-gene and/or per-codon- or seeds enveloped by a fleshy layer facilitating zoochory (in position basis, but it is unrealistic that all sites within a gene or Melioideae) (Pennington & Styles, 1975). The family mirrors each codon position would evolve similarly because of different global tropical diversity patterns, with higher diversity in rainfor- functional constraints across genes. The phylogenetic mixture ests (540 species) than in seasonal tropical vegetation (162 species model implemented in BAYESPHYLOGENIES integrates over all pos- distributed in semi-deciduous forests, savannas and dry deciduous sible partitioning schemes using reversible-jump MCMC and forests, and a few species in deserts (Nymania capensis) and groups sites together in ‘patterns’ of partitions, often leading to mangroves (Xylocarpus spp.)). The family has lower diversity in significantly better model fit (Pagel & Meade, 2004). Model tropical Africa (102 species) compared with tropical Asia (303 parameters are estimated per partition and the MCMC chain will species) or the Neotropics (189 species) and Meliaceae are also converge on a posterior distribution of patterns. This method has present in diverse habitats on Madagascar with an estimated been shown to improve phylogeny estimation and was used to diversity of 107 native species (c. 94.4% endemic; Madagascar extract an a posteriori partitioning scheme (Xi et al., 2012). We Catalogue, 2013) that are still poorly known. Among-lineage used the Perl script of Xi et al. (2012) to extract site patterns from variation in diversification rate in Meliaceae is apparent from a a sample of 1521 post-burn-in MCMC generations. Subse- more than ten-fold difference in species numbers between the two quently, we used a custom Python script to estimate an optimal subfamilies Cedreloideae (59 species) and Melioideae (643 species) partitioning scheme by assigning each site to the partition in which are sister clades (Muellner et al., 2003, 2006), and from the which it was sampled most frequently during the MCMC analy- large disparities in species numbers per genus (Table S1). Studying sis. This partitioning scheme was then used for molecular dating the diversification history of the family using comparative phyloge- analyses (see next paragraph). A majority-rule consensus tree was netic methods might therefore provide insights into the evolution summarized using the sumtrees.py script in DENDROPY (Sukuma- of tropical biodiversity across ecosystems and continents. Using a ran & Holder, 2010), with 10% discarded as burn-in after analy- new time-calibrated phylogeny for Meliaceae, we address the sis of the parameter output file in TRACER v1.5 (Rambaut & following questions: (1) Was the ancestral Meliaceae an evergreen Drummond, 2007). or deciduous species and did it inhabit rainforests or not? If not, We estimated a time-calibrated phylogeny using BEAST v1.8 when did Meliaceae first adapt to rainforests? (2) Do evergreen (Drummond et al., 2012). The same concatenated alignment was rainforest lineages diversify faster than lineages in seasonal habitats? used and the matrix was partitioned as described in the previous (3) Can the diversity of Meliaceae in tropical rainforests best be paragraph. Analyses were run with a GTR + Γ model, a birth- explained by steady accumulation of lineages over a long time death model for the tree prior and an uncorrelated log-normal period or have rates of diversification been more dynamic? relaxed clock model. The MCMC chain was run for 30 million generations with two independent runs. Convergence and burn- Materials and Methods in were checked using TRACER. Four fossils which we believe can be placed reliably on particular nodes were selected for calibra- We assembled a supermatrix of five loci: the internal transcribed tion. First, the stem node of Cedreloideae was calibrated at 47.8 spacer (ITS) region of the nuclear genome and chloroplast genes million yr ago (Ma) based on a fossil fruit and seeds from the rbcL, matK, rps16 and ycf1. Available sequences of ITS, rbcL, London Clay fossil site, UK (Chandler, 1964). The septicidal matK and rps16 for Meliaceae were downloaded from GenBank. dehiscence of the capsular fruit and winged seeds provide diag- Additional ITS sequences were generated to include all genera of nostic synapomorhies for Cedreloideae and this is the oldest fossil

New Phytologist (2015) 207: 327–339 Ó 2015 The Authors www.newphytologist.com New Phytologist Ó 2015 New Phytologist Trust New Phytologist Research 329 that can be reliably assigned to the subfamily. This fossil was reported remarkably similar ages of c. 71 Ma. A standard devia- described as Toona sulcata and previously interpreted as interme- tion of 2.55 was set, reflecting the uncertainty of Wang et al.’s diate between Toona and Cedrela and placed on the crown node age estimate with 95% of the distribution falling between 76 and of tribe Cedreleae (Muellner et al., 2009). However, fruits of 66 Ma. (2) For a more conservative secondary calibration point, extant species of Toona and Cedrela are more elongate than the we used the stem age of Sapindales from Magallon & Castillo globose fossil fruits and these genera have extant geographic dis- (2009) as a hard maximum bound of 98.43 Ma. (3) To avoid tributions (Australasia and the Neotropics, respectively) very dis- using secondary calibrations altogether and rely solely on fossil tant from the fossil site, making the placement in Cedreleae evidence, we used the age of the first tricolpate pollen (127.2 Ma; doubtful. Hence, we use a more conservative interpretation and Clarke et al., 2011) as a hard maximum bound. This is perhaps use the age of the fossil as a minimum constraint on the Cedreloi- the only angiosperm fossil that can be used as a hard maximum deae stem. Second, the stem age of Cedreleae was constrained at age constraint as it is a plant part that is produced in abundance, a minimum age of 28.1 Ma, based on early Oligocene fossil fossilizes easily and appears all over the fossil record shortly after leaves and seeds from the Florissant fossil site in Colorado, USA its first appearance (Magallon & Castillo, 2009; Clarke et al., (MacGinitie, 1953). Although the fossils were described as 2011). Consequently, it seems unlikely that eudicots will be Cedrela lancifolia, these could represent a stem relative of the much older. Obviously, the age of eudicots is older, and probably Cedreleae, and by calibrating the stem node of the tribe we reflect significantly so, than that of Sapindales. The third calibration this more cautious interpretation. Third, we calibrated the stem therefore overestimates node ages but provides a maximum age node of Swietenia with a minimum age of 22.5 Ma based on a for Meliaceae that is exclusively based on fossil evidence. We used well-preserved flower of Swietenia miocenica from Late Oligo- TREEANNOTATOR (BEAST package) to find the maximum clade cene-Early Miocene amber from Chiapas, Mexico (Castaneda-~ credibility (MCC) tree and to summarize the posterior samples Posadas & Cevallos-Ferriz, 2007). This flower can be confidently of trees produced by BEAST. assigned to Swietenia as it shares the diagnostic staminal tube To reconstruct ancestral states for several well-supported clades appendages with the three extant species of the genus. The fourth in the phylogeny, we used BAYESTRAITS (Pagel & Meade, 2007). calibration point is provided by Dysoxylum fossils from a small Analyses were run using 100 trees from the posterior sample of maar lake deposit in New Zealand (Conran et al., 2014; D. E. the BEAST analysis, with outgroups pruned. All Meliaceae taxa Lee, University of Otago, pers. comm.), including leaves, flowers were scored for habitat preference (lowland rainforest/seasonal or with in situ pollen and fruits. The age of these deposits is well montane tropical vegetation) and deciduousness (deciduous/ever- constrained at 23 Ma. There is one extant species of Dysoxylum in green), based on the literature (de Wilde, 1968, 2007; Penning- New Zealand (Dysoxylum spectabilis), but here we use the fossil ton & Styles, 1975; Mabberley, 1979; Pennington et al., 1981; more conservatively as a minimum stem age constraint for the Pannell, 1992; Styles & Bennett, 1992; Mabberley et al., 1995; clade that also includes Pacific island Dysoxylum species and the Louppe et al., 2008; Pennington & Muellner, 2010; Koenen & monotypic genus Cabralea, as our phylogeny (Fig. 1) suggests de Wilde, 2012; Pennington & Clarkson, 2013), with ambigu- that New Zealand may have been the ancestral area of this clade. ous taxa scored as polymorphic. The maximum likelihood (ML) All four fossil calibration points were set with uniform priors option was chosen with 10 ML attempts per tree and the results with the fossil ages as a minimum age and the maximum age con- summarized from the best attempt for each tree, over all 100 strained to the same age as the root height prior (see further trees. We also tested for state-dependent diversification in rain- below in this paragraph). The use of nonuniform prior distribu- forest with the BiSSE method (Maddison et al., 2007), imple- tions has been advocated by some (e.g. Ho & Phillips, 2009), but mented in the R package DIVERSITREE (FitzJohn, 2012), with the shape of the chosen prior distribution is often subjective. ambiguous taxa scored as nonrainforest. Choosing an informed prior distribution based on palaeobiologi- We used Bayesian analysis of macro-evolutionary mixtures cal evidence (e.g. using the method of Nowak et al., 2013) would (BAMM) v2.0 (Rabosky, 2014) to infer speciation rates across the be better, but this is not possible given the sparse fossil record of phylogeny, using a birth–death model with rate shifts. BAMM uses Meliaceae. We ran the analyses without data to check the shape reversible-jump MCMC to average over multiple rate shift mod- of the effective prior distributions that BEAST creates by generat- els, where shifts are changes between different models. Within ing a joint prior (Warnock et al., 2012). The effective priors were these models, rates are estimated separately and can be nonlinear, not uniform but placed greater prior probability on ages closer to and therefore shifts can be seen as ‘regime’ shifts, rather than the minimum age, caused by interactions with the root height purely a shift in speciation and/or extinction rate(s). To account prior. The maximum age prior on the root can have a strong for nonrandom incomplete sampling, we estimated total diver- influence on age estimates. An overly restrictive maximum age sity, and hence sampling fractions, for each genus in the Melia- may a priori dismiss true ages of deeper nodes, while an overly lax ceae from the literature, augmented by our own taxonomic prior may result in unduly old ages of these nodes. To account knowledge (de Wilde, 1968, 2007; Pennington & Styles, 1975; for these difficulties, we ran three analyses with different maxi- Mabberley, 1979; Pennington et al., 1981; Pannell, 1992; Mab- mum age priors. (1) A normal prior was used to calibrate the root berley et al., 1995; Louppe et al., 2008; Pennington & Muellner, (Sapindales minus Biebersteiniaceae, Kirkiaceae and Nitrariaceae) 2010; Koenen & de Wilde, 2012; Madagascar Catalogue, 2013; with the crown age for Sapindales as found by Magallon & Ca- Pennington & Clarkson, 2013). These sampling fractions were stillo (2009), Wang et al. (2009) and Zanne et al. (2013), who all assigned to the sampled tips in the phylogeny as species-specific

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Fig. 1 Chronogram of Meliaceae with ancestral reconstructions and tip states indicated. Pie charts above nodes show results of BAYESTRAITS optimization of presence/absence in rainforest and pie charts below nodes refer to reconstructions for evergreen or deciduous species, with colors referring to the different states as indicated in the key. Coloured circles behind tips indicate the character states of sampled extant species and coloured squares indicate geographical area, as indicated in the key. Black and white symbols refer to calibration points employed in the dating analyses as indicated in the lowermost box of the key. Geological eras are indicated on the time-scale (Pl, Pliocene; Q, Quaternary), the time-scale being in millions of years ago (Ma). Numbers on nodes refer to clades listed in Table 1. The tree displayed here is the maximum clade credibility (MCC) tree of Scenario 2.

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et al., 2013). Our phylogeny includes 24 of the species recognized by Kenfack, but for the BAMM analyses we pruned species that are not supported by population genetic analyses (Scotti-Saintagne et al., 2013) or continuing taxonomic work on the Central Afri- can flora (J. J. F. E. de Wilde, pers. comm.), leaving 12 Carapa species (two Neotropical and 10 African). To see what effect this has, we also ran an analysis without pruning these species. We also ran analyses with extinction rates fixed to low and high levels (0.05 and 0.2 lineages/Ma, respectively, reflecting the lower and upper limits of the estimated rates), to test if the diversification rate heterogeneity that is inferred is robust with regard to the estimation of extinction rates. BAMM was run on the MCC trees of each dating scenario, with outgroups pruned. We ran BAMM for 10 million generations and, after checking for convergence, discarded the first 10% as burn-in. We used the R package BAMM- tools (Rabosky et al., 2014) to summarize rates over each branch of the phylogeny (visualized as so-called ‘phylorate’ plots), and to plot the credible shift set (CSS) with sampling frequencies of shift configurations and the shift set with the maximum a posteriori (MAP) probability. We used the plotRateThroughTime function to plot speciation rates from the posterior sample. For compari- son, we also created a lineage-through-time (LTT) plot using the R package APE (Paradis et al., 2004).

Results We sampled 254 species of Meliaceae (c. 34%) from all genera with an additional 30 outgroup accessions representing all major families in the Sapindales, giving a total of 284 taxa. After exclud- ing sites with > 99% missing data (data only present for one or two taxa), the total aligned length was 5207 bp; matrices are available from http://purl.org/phylo/treebase/phylows/study/ TB2:S17516. BAYESPHYLOGENIES analyses found an a posteriori pattern with five partitions to best fit the rate heterogeneity across the alignment. The 50%-majority rule consensus tree (Fig. S1; http:// purl.org/phylo/treebase/phylows/study/TB2:S17516) shows higher level relationships that are congruent with the findings of previous studies (Muellner et al., 2003, 2008a), that is, mono- phyly of the two subfamilies, nonmonophyly of tribes Trichilieae and Turraeae (but most members of these tribes in a combined clade, here referred to as the TT clade; node 11 in Fig. 1), and Aglaieae nested in Guareeae (the combined clade here referred to as the GA clade; node 14 in Fig. 1). Relationships between Melia- ceae, Rutaceae and Simaroubaceae have been difficult to resolve Fig. 1 (Continued). (Muellner et al., 2007; Soltis et al., 2011). Here we report a sister-group relationship between Meliaceae and Simaroubaceae sampling probablities as implemented in BAMM, which corrects with 0.93 pp. The topology indicates an Old World origin for for unsampled diversity in the estimation of speciation and the family, as the Palaeotropical taxa in the family are paraphylet- extinction rates. In the case of nonmonophyletic genera, multiple ic with respect to Neotropical lineages (Figs 1, S1). Neotropical genera were lumped (as in Table S1), with the exception of the Swietenia, Carapa, Trichilia and Guarea+Ruagea have a disjunct poorly resolved Dysoxylum where each tip was assigned the sam- African sister group, as do Cedreleae, but the Neotropical pling fraction of the genus as a whole. The taxonomy of Carapa Cedrela is sister to the Australasian Toona. Cabralea canjerana, was recently revised by Kenfack (2011), who recognized 27 spe- widespread in Neotropical humid forests, is sister to a clade of cies. However, population genetic analyses demonstrated that this Pacific island Dysoxylum species, and Schmardaea microphylla, a may be an overestimate (Duminil et al., 2012; Scotti-Saintagne morphological outlier from montane forests in the Andes (Styles

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& Bennett, 1992), is sister to the Indian endemic Chukrasia distribution. Apparently the fossil calibrations favor an older age tabularis. Several genera in the Melioideae are shown to be non- of Sapindales than ages found in angiosperm-wide dating studies monophyletic: (1) Lepidotrichilia volkensii, a species of East Afri- (c. 71 Ma; Magallon & Castillo, 2009; Wang et al., 2009; Zanne can montane forests, does not group with the only sampled et al., 2013). When we allow the root to become older by using Malagasy species of the genus, but is instead sister to another uniform priors with hard maximum bounds (Scenarios 2 and 3; Malagasy genus, Astrotrichilia; (2) Humbertioturraea and Table 1), the crown age of the Sapindales becomes unrealistically Calodecaryia are nested in Turraea; (3) Pterorhachis and old, especially when the tricolpate pollen age is used. The MCC Pseudobersama are nested in Trichilia; (4) the affinities of many tree of the second dating scenario was chosen for all further analy- Dysoxylum species are uncertain but the genus is nonmonophylet- ses, but BAMM analyses were conducted on MCC trees of all three ic, at least in terms of the well-supported sister relationship scenarios. between Cabralea and a clade of Pacific Dysoxylum species for- The combination of inhabiting lowland rainforests and the merly segregated as the genus Didymocheton; (5) Aglaia is non- presence of evergreen leaves is most prevalent in extant Melia- monophyletic, as found by Grudinski et al. (2014a); (6) Leplaea, ceae (Fig. 1). However, BAYESTRAITS reconstructions (Fig. 1) sug- segregated recently from Guarea (Koenen & de Wilde, 2012), is gest that this was not the ancestral ecology of the family, but monophyletic in BEAST analyses (Figs S2–S4) but not in the BA- rather that the most recent common ancestor (MRCA) of Melia- YESPHYLOGENIES analysis (Fig. S1). Neither Leplaea nor Neoguarea ceae was a deciduous species that inhabited seasonal (e.g. savan- (another recent Guarea segregate; Koenen & de Wilde, 2012) nas or dry forests) or montane tropical vegetation, both with a groups with Guarea, whose sister clade (1.00 pp) is Ruagea, con- probability of 0.84. Evergreen species appear early in the evolu- firming the re-circumscription of the previously nonmonophylet- tion of Melioideae. However, the reconstructions for the deeper ic Guarea. We also conclusively demonstrate that the Neotropical nodes of Melioideae are more ambiguous than in Cedreloideae, Guarea and South-East Asian Chisocheton are not sister genera and a scenario of multiple losses and gains in the former sub- (Figs 1, S1). These genera share the highly unusual character of family is possible. The fact that the genus Melia and Azadirachta paripinnate leaves with an apical bud (pseudogemmula) that indica are deciduous (which together with Owenia are sister to allows for indeterminate growth (Pennington & Styles, 1975; all other Melioideae), as well as Quivisianthe and Turraea, sug- Steingraeber & Fisher, 1986; Mabberley et al., 1995), which thus gests that deciduousness was potentially labile during the early evolved twice independently. evolution of Melioideae, suggesting that the subfamily was not Chronograms (MCC trees) of the BEAST analyses under the predominantly a rainforest clade during its early evolution. three different root calibrations (Figs S2–S4, hereafter referred to However, all species in the GA clade are evergreen and the as Scenarios 1–3) and age estimates for selected clades (Table 1) deciduous state has been lost completely in this clade. With few show that the three different root calibrations mostly affect ages exceptions, species in the GA clade are found in lowland ever- of the deeper nodes (Table 1) (MCC trees are available from green rainforests. Apart from the GA clade, clades that are char- http://purl.org/phylo/treebase/phylows/study/TB2:S17516). The acterized by a predominance of rainforest occurrence and age of Sapindales under Scenario 1 is estimated at 78.8 Ma evergreen species are Trichilia, the Heynea and Walsura clade, (Table 1), which is outside the 95% credible interval of the prior and Carapa. Trait reconstructions suggest that Melioideae have

Table 1 Median age estimates (with 95% HPD interval), in millions of years ago (Ma), for selected nodes under three different root calibration scenarios

Scenario 1 Scenario 2 Scenario 3 Clade (normal crown) (uniform stem) (tricolpate pollen)

1. Sapindales (w/o Biebersteiniaceae, 78.8 (74.3–83.1) 97.0 (92.6–98.4) 120.4 (108.1–127.2) Kirkiaceae and Nitrariaceae – root node) 2. Meliaceae + Rutaceae + Simaroubaceae 73.5 (67.8–79.3) 87.0 (79.6–94.6) 104.7 (91.0–118.1) 3. Meliaceae 59.3 (53.9–64.8) 68.4 (61.4–75.4) 80.4 (69.1–91.6) 4. Cedreloideae 38.6 (33.0–45.8) 42.5 (35.7–50.9) 48.5 (39.6–59.3) 5. Cedreleae + Entandrophragma 29.8 (28.1–33.6) 30.9 (28.1–36.1) 33.5 (28.1–40.4) 6. Cedreleae 9.9 (7.2–13.2) 11.1 (8.2–15.0) 12.9 (9.2–17.6) 7. Carapa 7.4 (5.7–9.2) 8.2 (6.3–10.4) 9.1 (6.9–11.7) 8. Melioideae 53.9 (48.9–59.4) 61.8 (54.4–68.6) 72.6 (62.2–82.9) 9. Heynea + Walsura 15.1 (9.8–21.4) 16.9 (10.7–24.5) 19.6 (12.1–28.6) 10. Clade with Turraeeae, Trichilieae p.p., 34.3 (31.2–37.8) 38.2 (34.5–42.3) 43.9 (37.9–50.0) Guareeae and Aglaieae 11. TT clade (Turraeeae + Trichilieae p.p.) 32.0 (29.1–35.5) 35.7 (32.1–39.6) 41.0 (35.4–47.1) 12. Trichilia s.l. (incl. Pseudobersama and Pterorhachis) 22.2 (18.1–26.9) 24.4 (20.2–29.5) 28.2 (22.6–34.5) 13. Shift clade Trichilia 8.0 (6.1–10.3) 8.8 (6.6–11.3) 10.0 (7.5–12.9) 14. GA clade (Guareeae + Aglaieae) 25.6 (24.0–27.6) 27.1 (24.7–30.1) 30.3 (26.2–34.8) 15. Aglaieae (incl. Dysoxylum arborescens) 21.2 (19.1–22.9) 21.9 (19.9–23.9) 23.9 (21.0–27.2) 16. Guarea + Ruagea 19.3 (16.6–22.1) 21.0 (18.3–24.1) 23.6 (19.9–27.6) 17. Shift clade Guarea 6.7 (5.3–8.2) 7.4 (5.9–9.1) 8.4 (6.5–10.5)

New Phytologist (2015) 207: 327–339 Ó 2015 The Authors www.newphytologist.com New Phytologist Ó 2015 New Phytologist Trust New Phytologist Research 333 been present in rainforests since at least the Eocene, but the crown ages of the aforementioned four clades all fall in the Oli- gocene or early Miocene, except for Carapa which appears to be younger (Table 1). The BiSSE analysis shows higher speciation rates for rainforest taxa compared with nonrainforest taxa (Fig. S5a), while extinction rates largely overlap (Fig. S5b). The BiSSE method has been shown to have low statistical power (Davis et al., 2013) and extremely high Type I error rates (Rabosky & Goldberg, 2015). Although these results suggest that rainforest lineages have experienced higher speciation rates, this should not be interpreted as implying a causal relationship. The BAMM phylorate plot of speciation rate (Fig. 2) shows striking rate heterogeneity across the family. The clade that combines the TT clade and GA clade (node 10, Fig. 1) shows elevated rates as a whole compared with the rest of the family (but the increase along its stem is not present in the MAP configuration; the marginal probability of a shift on that particular branch is 0.15). A subsequent increase to a higher rate is found for the GA clade as well as two more recent increases to even higher rates for subclades of Guarea and Trichilia. In the MAP configuration, these three clades are subtended by regime shifts (Figs 2, S6), and they are hereafter referred to as ‘shift clades’. Further rate heterogeneity is found in the TT clade, with overall decreasing speciation rates, but a modestly increased rate in the predominantly Afro-Malagasy genus Turraea (including Humbertioturraea and Calodecaryia), with a marginal probability of a shift of 0.10. These rate changes are represented by shifts in the CSS (Fig. S6), in which the MAP configuration is the most sampled in the MCMC analysis (f = 0.34). Running the analysis without pruning the doubtfully distinct Carapa species (see Materials and Methods) leads to a slightly increased speciation rate for the genus, but this increase is not among the shifts in the MAP configuration (results not shown). Running the analyses with extinction rates estimated or fixed to low or high levels does not change the overall pattern of among-lineage diversification rate heterogeneity (Fig. S7), but the background net diversification rate is estimated to be higher with high extinction (Fig. S7d). Running the analyses on the MCC trees of the different dating scenarios does not change the overall pattern, only the absolute rate estimates. The large differences in speciation rates across the phylogeny are especially evident from the rate-through-time plot (Fig. 3). The speciation rate of the GA clade is circa twice the background rate and the shift clades within Guarea and Trichilia show large spikes in their speciation rates, Fig. 2 Phylorate plot with branches colored by speciation rate (lineages/ which then rapidly slow down towards the present. The back- Myr) as indicated by the scale bar, representing a summary of the full ground rate is constant through time, showing that the higher post-burn-in Markov chain Monte Carlo (MCMC) sample of the Bayesian rates towards the present observed in the global rate (inset Fig. 3) analysis of macro-evolutionary mixtures (BAMM) analysis. Red circles are caused by the higher rates of the shift clades. The LTT plot, by indicate the positions of regime shifts in the maximum a posteriori (MAP) configuration (the MAP configuration is also shown in the upper left panel contrast, shows constant lineage accumulation over time (Fig. S8). of Supporting Information Fig. S6). Numbers beneath the shifts indicate the marginal probability of a shift occurring along that branch. The tree Discussion displayed here is the maximum clade credibility (MCC) tree of Scenario 2. How the interplay between environmental and historical factors make inferences about macro-evolutionary processes and habitat has shaped the distribution of biodiversity remains poorly under- and trait evolution. Integrating our findings on temporal dynam- stood. We inferred the most comprehensive time-calibrated phy- ics of diversification, ecological span and geographic distribution logeny for Meliaceae to date and used comparative methods to of Meliaceae and drawing comparisons to other angiosperm clades

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Fig. 3 Rate-through-time plots for speciation rate (lineages/Myr) with 95% conﬁdence interval indicated by shaded areas. Blue, the rate across the phylogeny without the shift clades (‘background rate’); green, the rate for the GA clade (the combined clade of Guareeae and the nested Aglaieae); yellow and red, the shift clades within Guarea and Trichilia, respectively. Note that the increase and subsequent decrease visible in the GA clade (green) in the last c. 7.5 million yr is caused by the Guarea shift clade, which is included in the GA clade. The inset shows the global rate across Meliaceae with all shift clades included. These plots are derived from the Bayesian analysis of macro-evolutionary mixtures (BAMM) analysis that was conducted on the maximum clade credibility (MCC) tree of Scenario 2.

and palaeobotanical evidence provides new insights into the evo- results cast doubt on their inference of Eocene Boreotropical dis- lution of lowland evergreen tropical rainforest hyperdiversity. persal across the Northern Atlantic. While Old World/New Our dating analyses provide a new view of temporal evolution World disjunctions are indeed substantially too young (Oligo- of Meliaceae, with implications for its biogeographic and diversi- cene or younger) for vicariant explanations, they are even too fication history. We placed carefully selected fossils on the stem recent to fit with an Eocene Boreotropical dispersal scenario of clades with which they share apomorphies, because these stem (Morley, 2003). Our results indicate more recent trans-Atlantic ages should not pre-date the fossils, but the fossils can pre-date dispersal from Africa to the New World for the disjunctions the crown group. This results in younger ages for several clades mentioned in the Results section, with the exception of Cabralea than previously reported (Table S1; Muellner et al., 2006, (trans-Pacific dispersal) and Schmardaea (the only disjunction 2008b, 2010). We found age estimates for Meliaceae between that could perhaps be explained by the Boreotropics hypothesis). 59.3 and 80.4 Ma. Previously, Muellner et al. (2006) found ages The MRCA of Meliaceae is at least as old as the Paleocene for the family ranging from 76.3 to 103.7 Ma, which is not com- (Table S1), and so are modern-type tropical rainforests (Wing patible with ages found for Sapindales by other studies (c. 71 Ma; et al., 2009). If the MRCA of Meliaceae occurred in rainforest Magallon & Castillo, 2009; Wang et al., 2009; Zanne et al., and diversity in the family gradually accumulated, the 2013). Especially striking is the much younger estimate for Cedr- diversification patterns in Meliaceae would be congruent with the eleae (9.9–12.0 Ma) compared with a study focused just on the museum model. However, the ancestral Meliaceae was probably tribe (48.4 or 54.8 Ma; Muellner et al., 2010). In that study, the deciduous and inhabited seasonal or montane tropical habitats Toona sulcata fossil was placed on the crown node of Cedreleae, (Fig. 1). Clades that occur almost exclusively in rainforest (Cara- while we place it on the stem node of Cedreloideae (see the pa (12 species), Heynea and Walsura (18), Trichilia (107) and the Materials and Methods section), which is more compatible with GA clade (363)) date back only to the Late Oligocene or Early the other fossil calibration points. The ages for Guarea + Ruagea Miocene and BAMM results show increased speciation rates for the (19.3–23.6 Ma) are similar to those (14.1–24.0 Ma) found by GA clade and progressive increases for subclades of Guarea and Muellner et al. (2006). For Aglaieae, we find only slightly youn- Trichilia (Figs 2, 3) to even higher rates. The Late Oligocene and ger ages (21.2–23.9 Ma) than Grudinski et al. (2014b), who Early Miocene are thought to be periods of overall lower tropical reported an age of 24.3 Ma. The Old World origin of the family diversity (Morley, 2000; Jaramillo et al., 2006), suggesting major was previously suggested by Muellner et al. (2006); however, our turnover. The increases we find may be related to Miocene

New Phytologist (2015) 207: 327–339 Ó 2015 The Authors www.newphytologist.com New Phytologist Ó 2015 New Phytologist Trust New Phytologist Research 335 climate change (Zachos et al., 2001) or geological processes such Malagasy Meliaceae are not densely sampled in our phylogeny, as orogeny (Antonelli et al., 2009). We show that the large major- but we do note that the three largest genera on Madagascar ity of species diversity of Meliaceae in rainforests is recent and the (Turraea, including the nested Humbertioturraea and Calodecaryia result of higher speciation rates compared with nonrainforest lin- (c.46species),Malleastrum (24 species) and Astrotrichilia (16 eages (Figs 2, 3, S5). Melioideae may have been present in rain- species)) are also the youngest lineages present on the island. All forests as far back as the Eocene (Fig. 1), but in that case those three originated in the late Miocene and include several rainforest lineages did not diversify at that time. Alternatively, and perhaps species (Leroy & Lescot, 1996; Callmander et al.,2012). more likely, they were also species-rich, but experienced Studies in typical rainforest families Annonaceae, Arecaceae and subsequent extinction and are now replaced by more recently Menispermaceae have endorsed the museum model, with gradual evolved diversity, following a model of (episodic) species turn- and more or less constant lineage accumulation (Couvreur et al., over. 2011a,b) or with an early burst shortly after the K–Pg boundary The rate-through-time plots (Fig. 3) illustrate nicely how followed by constant diversification (Wang et al., 2012). What changes in the global speciation rate over the phylogeny are sets our study apart from these studies is that we densely sample explained by the dynamics of a few specific clades, all of them several species-rich genera, use the recently developed BAMM strongly associated with rainforests (Fig. 1), rather than being method, and account for unsampled species diversity. Couvreur caused by a global effect. The two shift clades of Guarea and et al. (2011a,b) and Wang et al. (2012) defined an age cut-off Trichilia (Fig. 2) converged on almost identical, much acceler- after which their LTT plots are not realistic because of incom- ated rates over the last 10 Ma (Fig. 3). Both clades have their cen- plete sampling, but they note that rates need to have increased ters of diversity in Amazonia, where they occur as understorey towards the present to explain current species richness. Our LTT trees in primary evergreen rainforest. Species in these clades are plot suggests constant lineage accumulation (Fig. S8), compara- also very similar in reproductive biology, reflected by their similar ble to the studies on Annonaceae, Arecaceae and Menisperma- floral and fruit morphologies and bird-dispersed seeds. These ceae. But with BAMM, we do detect recent increases in speciation radiations are not only ecologically and phenotypically conver- rates, as would be necessary to reach present diversity, suggesting gent, but remarkably also convergent in location, timing and spe- that the apparent rate constancy observed in LTT plots is an arte- ciation rates. These similarities strongly imply a common driver. fact caused by incomplete taxon sampling. Another study in An- We hypothesize that their initial high speciation rates are related nonaceae shows that the third largest genus of Neotropical trees, to the opportunity to radiate presented by the expanding lowland Guatteria, radiated recently (Erkens et al., 2007). Also in other rainforests of Amazonia, following the demise of wetland ecosys- recent radiations in Neotropical rainforests (Inga (Richardson tems (known as the Pebas system) that are thought to have been et al., 2001), Costus (Kay et al., 2005), Renealmia (S€arkinen et al., prevalent across western Amazonia until c. 10 Ma (Antonelli 2007) and Protieae (Fine et al., 2014)) most diversification et al., 2009; Hoorn et al., 2010). occurred contemporaneously with the Guarea and Trichilia Three large genera in the GA clade have their centers of radiations identified here. diversity in Malesia, but unlike Neotropical lineages these do Recency of species-level diversity seems to be a common not show such recent increases in speciation rates. Aglaia, the pattern across Neotropical ecosystems (Simon et al., 2009; Hoorn largest genus in the family, is well sampled here and experienced et al., 2010; De-Nova et al., 2012; Hughes et al., 2013). In South- more or less constant or decreasing rates since its origin in the East Asian rainforests, Malesian Begonia represents another exam- Miocene (Figs 1, 2). Dysoxylum and Chisocheton are too poorly ple of a recent radiation (Thomas et al., 2012). On a global scale, sampled to detect separate diversification processes within these succulents (Arakaki et al., 2011) and grasses (Edwards et al., genera. The Asian wet tropics harbor a rich diversity of Melia- 2010; Spriggs et al., 2014) apparently radiated from the Miocene ceae, with just the aforementioned three large genera together onwards, which was interpreted as an effect of global cooling and constituting c. 35% of total diversity in the family (Table S1), the spread of seasonally dry ecosystems. However, with so many diversifying with an elevated but decreasing speciation rate since examples of recent species-level diversification across disparate the Oligocene. ecosystems, a universal model of species turnover could offer a Meliaceae are less species-rich in African rainforests than in the more general explanation of the origins of (tropical) diversity that Neotropics and South-East Asia, a pattern that has long been rec- places less emphasis on extrinsic factors such as the geological and ognized across the entire rainforest flora (Gentry, 1982; Antonelli climatic settings of individual radiations, or on intrinsic factors & Sanmartın, 2011). Africa might have experienced higher such as traits associated with adaptations to particular ecosystems. extinction rates (Kissling et al., 2012), but fossil evidence for this It is tempting to link individual radiations to certain intrinsic and is scarce (Pan et al., 2006). It was also suggested that the Neo- extrinsic factors, but this will only explain which lineages can seize tropics and SE-Asia rather have experienced increased diversifica- the opportunity to radiate in a given environment. We propose tion rates compared with Africa (Baker & Couvreur, 2013), but that diversification across ecosystems is highly dynamic and background rates and rates in Africa may have been underesti- linked to environmental change (Xing et al., 2014), rather than mated as a result of underestimating extinction. In fact, the lower climatic stability as suggested by the museum model. diversity in Africa can also be predicted based on environmental McKenna & Farrell (2006) have suggested that the museum data alone (Kreft & Jetz, 2007). See Couvreur (2014) for an elab- and cradle models are not mutually exclusive; Couvreur et al. orate review on the topic of lower diversity in African rainforests. (2011b) also suggested a mixed model of steady processes and

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recent diversification. However, a model of high species turnover Acknowledgements rather suggests that steady (‘museum-like’) processes caused by stable conditions did not prevail over long time periods, but that The authors thank Ria Vrielink-van Ginkel for assistance in labo- also extinction rates have been highly dynamic, instead of persis- ratory work and Andrew Meade for providing the unpublished tently low as the museum model postulates. This is in line with version 2 of BAYESPHYLOGENIES and the program SITEBREAK- fossil evidence which shows that Eocene rainforests were even DOWN. We thank Daphne Lee for sharing information on the more diverse than contemporary rainforests (Wilf et al., 2003; unpublished Dysoxylum fossils, and Hans de Wilde for unpub- Jaramillo et al., 2006, 2010), followed by a period of decreasing lished taxonomic information on African taxa. We thank Guy diversity until the Early Miocene. This implies both ancient rapid Atchison, Renske Onstein, Yanis Bouchenak-Khelladi and diversification and high past extinction rates. Yaowu Xing for useful discussion and are very grateful to Colin Extinction events are not observed in phylogenies, causing low Hughes, who provided invaluable comments on an earlier version power in estimation of extinction rates (Rabosky, 2010). We of the manuscript. We also thank Bill Baker, Thomas Couvreur have therefore not interpreted the extinction rate estimates from and two anonymous referees for useful comments on the manu- our analyses, but net diversification rates show an identical script. Fieldwork in Gabon was financially supported by the pattern to the speciation rate plot (Fig. 2), both when extinction Alberta Mennega Stichting and the Systematics Research Fund (Linnean Society/Systematics Association). rates are estimated by BAMM and when they are fixed at low or high levels (Fig. S7). Inaccurate extinction rate estimation will affect net diversification as well as speciation rates. Estimating References macro-evolutionary rates from phylogenies at deeper time-scales Antonelli A, Nylander JAA, Persson C, Sanmartın I. 2009. Tracing the impact will be more challenging, and underestimated extinction can lead of the Andean uplift on Neotropical plant evolution. Proceedings of the National to too low background rates. Indeed, fixing extinction rates at a Academy of Sciences, USA 106: 9749–9754. high level has a strong effect on the background rate, with much Antonelli A, Sanmartın I. 2011. Why are there so many plant species in the – higher rates during the early evolution of Meliaceae (Fig. S7d). Neotropics? Taxon 60: 403 414. Arakaki M, Christin PA, Nyffeler R, Lendel A, Eggli U, Ogburn RM, Spriggs E, Past high speciation and extinction rates are not inferred in Moore MJ, Edwards EJ. 2011. 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Recent assembly of the Cerrado, a neotropical plant diversity hotspot, by sis.

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Figs S2–S4 MCC trees for the three different root calibration Table S2 GenBank numbers and voucher information for previ- scenarios. ously published and newly generated DNA sequence data

Fig. S5 BiSSE profile plots. Methods S1 Laboratory methods for DNA extraction, PCR amplification and sequencing. Fig. S6 Credible shift set (CSS) of BAMM analysis. Please note: Wiley Blackwell are not responsible for the content Fig. S7 Phylorate plots of BAMM analysis or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be Fig. S8 Lineage-though-time plot. directed to the New Phytologist Central Office.

Table S1 Estimates of species numbers per genus and the sampling fractions assigned in the BAMM analysis

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