Molecular Phylogenetics and Evolution 95 (2016) 116–136

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Molecular Phylogenetics and Evolution

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Divergence times, historical biogeography, and shifts in speciation rates of Myrtales q ⇑ Brent A. Berger a,b, , Ricardo Kriebel b, Daniel Spalink b, Kenneth J. Sytsma b a Department of Biological Sciences, St. John’s University, 8000 Utopia Parkway, Queens, NY 11432, USA b Department of Botany, University of Wisconsin-Madison, 430 Lincoln Dr., Madison, WI 53706, USA article info abstract

Article history: We examine the eudicot order Myrtales, a clade with strong Gondwanan representation for most of its Received 9 June 2015 families. Although previous phylogenetic studies greatly improved our understanding of intergeneric Revised 3 September 2015 and interspecific relationships within the order, our understanding of inter-familial relationships still Accepted 4 October 2015 remains unresolved; hence, we also lack a robust time-calibrated chronogram to address hypotheses Available online 14 November 2015 (e.g., biogeography and diversification rates) that have implicit time assumptions. Six loci (rbcL, ndhF, matK, matR, 18S, and 26S) were amplified and sequenced for 102 taxa across Myrtales for phylogenetic Keywords: reconstruction and ten fossil priors were utilized to produce a chronogram in BEAST. Combretaceae is BEAST identified as the sister clade to all remaining families with moderate support, and within the latter clade, BioGeoBEARS BAMM two strongly supported groups are seen: (1) Onagraceae + Lythraceae, and (2) Melastomataceae + the Diversification Crypteroniaceae, Alzateaceae, Penaeaceae clade along with Myrtaceae + Vochysiaceae. Divergence time Gondwana estimates suggest Myrtales diverged from Geraniales 124 Mya during the Aptian of the Early Phylogenetics Cretaceous. The crown date for Myrtales is estimated at 116 Mya (Albian–Aptian). BioGeoBEARS showed significant improvement in the likelihood score when the ‘‘jump dispersal” parameter was added. South America and/or Africa are implicated as important ancestral areas in all deeper nodes. BAMM analyses indicate that the best configuration included three significant shifts in diversification rates within Myrtales: near the crown of Melastomataceae (67–64 Mya), along the stem of subfamily Myrtoideae (Myrtaceae; 75 Mya), and along the stem of tribe Combreteae (Combretaceae; 50– 45 Mya). Issues with conducting diversification analyses more generally are examined in the context of scale, taxon sampling, and larger sets of phylogenetic trees. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction hypothesis testing (Davis et al., 2014; Ruhfel et al., 2014; Soltis et al., 2011; Zeng et al., 2014; Magallón et al., 2015). In turn, Resolving deep-level phylogenetic relationships has often been model-based approaches have been developed to more rigorously difficult due to confounding issues of ancient, rapid lineage diver- estimate biogeographic history (Lagrange: Ree et al., 2005; BEAST: sification, a lack of clear morphological synapomorphies, a propen- Drummond et al., 2006; BioGeoBEARS: Matzke, 2013) and to assess sity for morphological and molecular homoplasy, and organismal shifts in diversification rates (MEDUSA: Alfaro et al., 2009; BAMM: extinction (e.g., Davis et al., 2005; Deng et al., 2015; Givnish Rabosky, 2014; Rabosky et al., 2014). The use of these advances has et al., 2009; Schönenberger et al., 2005). A lack of a well-resolved allowed more detailed insight into clades lacking resolution of phylogenetic framework has made it difficult to estimate biogeo- early diverging lineages (e.g., Asteraceae: Panero et al., 2014), with graphic ancestral ranges and assess shifts in species diversification histories shaped by ancient intercontinental disjunctions (e.g., rates within such groups. However, increased amounts of phyloge- campanulids; Beaulieu et al., 2013), and with lineage specific shifts netic/phylogenomic data and improved analytical tools to evaluate in speciation and/or extinction rates (e.g., Bromeliaceae: Givnish these data are providing more robust phylogenetic frameworks for et al., 2014; hummingbirds: McGuire et al., 2014).

1.1. Biogeographic hypotheses of Southern Hemisphere disjunct groups q This paper was edited by the Associate Editor Akiko Soejima. ⇑ Corresponding author at: Department of Biological Sciences, St. John’s Univer- sity, 8000 Utopia Parkway, Queens, NY 11432, USA. Fax: +1 608 262 4490. Of particular interest for re-examination using these improved E-mail address: [email protected] (B.A. Berger). phylogenetic approaches are groups exhibiting a Southern http://dx.doi.org/10.1016/j.ympev.2015.10.001 1055-7903/Ó 2015 Elsevier Inc. All rights reserved. B.A. Berger et al. / Molecular Phylogenetics and Evolution 95 (2016) 116–136 117

Hemisphere affinity and whose current distributions, shifts into focusing on the order or including placeholder taxa, support a different habitat or biome types, and thus rates of species monophyletic Myrtales regardless of the genic region used (see diversification may be influenced by the continental breakup of Soltis et al., 2011; Sytsma et al., 2004). Although previous phyloge- Gondwana during the Cretaceous (Crisp et al., 2009; Donoghue netic studies greatly improved our understanding of intergeneric and Edwards, 2014). The distribution patterns and implied biogeo- and interspecific relationships within the order, our understanding graphical events of Southern Hemisphere organisms have been of inter-familial relationships still remains unresolved, and thus, vigorously debated since the late 1960s, but a strong vicariance we also lack a strong time-calibrated chronogram to address voice (e.g., Axelrod, 1970; Cracraft, 1988; Raven and Axelrod, hypotheses (e.g., biogeography and diversification rates) that have 1974; Rosen, 1978) had emerged for explaining present-day dis- implicit time assumptions. An earlier r8s (Sanderson, 2002) analy- junct patterns in a wide diversity of plant and animal groups sis for the order used multiple fossil calibration points and a two- (e.g., Edwards and Boles, 2002; Haddrath and Baker, 2001; genome data set, but did not account for time estimate variation at Murphy et al., 2001; Sequeira and Farrell, 2001; Swenson et al., nodes and was based on a single fixed topology (Sytsma et al., 2001; Vinnersten and Bremer, 2001). Recent molecular phyloge- 2004). Advances in the last decade for estimating chronograms, netic studies that have included dating methods have provided availability of considerably more molecular data, and documenta- support for the vicariance model for some Southern Hemisphere tion of many more Myrtales fossils mandate a more thorough anal- groups (e.g., Bukontaite et al., 2014; Korall and Pryer, 2013; Mao ysis of the Myrtales as done here. et al., 2012; Murienne et al., 2013; Wilf and Escapa, 2014). How- Especially problematic in Myrtales has been the placement of ever, others have questioned the sole reliance on Gondwanan the pantropical Combretaceae (Conti et al., 1996, 1997; Sytsma vicariance as explanatory for many plant and animal clades show- et al., 2004). Two alternative hypotheses exist: (1) Combretaceae ing Southern Hemisphere disjunct patterns (Crisp and Cook, 2013; is sister to the clade comprising Onagraceae + Lythraceae, which Sanmartín and Ronquist, 2004). These studies have included sup- is then sister to the rest of Myrtales (Conti et al., 1996, 1997); or port for the Boreotropics Hypothesis (Lavin and Luckow, 1993)or (2) Combretaceae is sister to all other members of Myrtales Laurasian rather than Gondwanan origin of some of these plant (Sytsma et al., 2004). The placement of Combretaceae is of consid- clades (Baker and Couvreur, 2013; Davis et al., 2002; Renner erable importance because its position directly influences clade et al., 2001; Zerega et al., 2005). Most studies, however, have ages, hypotheses addressing dispersal/vicariance scenarios, species revealed clade dates that are indicative of long-distance dispersals diversification rates, and character reconstructions. Since Conti (sometimes associated with vicariance) rather than solely vicari- et al. (1996, 1997) proposed the first interfamilial relationships ance for Southern Hemisphere disjunctions of both plants and ani- of Myrtales based on rbcL, most ordinal level phylogenetic studies mals (Armstrong et al., 2014; Barker et al., 2007; Beaulieu et al., have continued to utilize a limited number of taxa and gene 2013; Chacón and Renner, 2014; Christenhusz and Chase, 2013; regions (often the same GenBank accessions) as placeholders for Cook and Crisp, 2005; Friedman et al., 2013; Gallaher et al., Combretaceae (e.g., Magallón, 2010; Rutschmann et al., 2007; 2014; Gamble et al., 2011; Givnish and Renner, 2004; Givnish Sytsma et al., 2004; Wang et al., 2009). The continued use of the et al., 2000, 2004; Knapp et al., 2005; Müller et al., 2015; de same 2–5 taxa and 1–2 plastid regions has limited our ability to Queiroz, 2005; Rowe et al., 2010; Sytsma et al., 2004; Thomas place Combretaceae and confidently address other hypotheses. et al., 2014). To circumvent this issue (as summarized by Sanderson et al., 2010), our analysis increases sampling, both in terms of number 1.2. Myrtales as a model group for assessing Southern Hemisphere of taxa and breadth across the family. biogeography and rates of species diversification Thus, we develop here a more rigorous time-calibrated phylo- genetic framework for Myrtales based on a three-genome Here we examine the eudicot order Myrtales, a clade that has approach with nearly 98% coverage of gene regions for each sam- strong Gondwanan representation for most of its families, although pled taxon. Taxa sampling is designed to cover all major clades one family (Onagraceae) is most diverse in Laurasia. APGIII (2009) within each family. We then use this new historical framework recognizes nine families in Myrtales, including: Melastomataceae of Myrtales to: (1) evaluate phylogenetic relationships within (188 genera/4618 species), Myrtaceae (131/5638), Onagraceae and among the nine families; (2) estimate when and where major (22/667), Lythraceae (31/522), Combretaceae (14/570), lineages of Myrtales originated with BEAST and BioGeoBEARS anal- Vochysiaceae (7/217), Penaeaceae (9/29), Crypteroniaceae (3/10), yses; (3) examine the biogeographic processes that may have con- and Alzateaceae (1/2). Four large families (Melastomataceae, tributed to extant distributions, especially several different Myrtaceae, Lythraceae, and Combretaceae) are pantropical in distri- disjunct patterns in the Southern Hemisphere; and (4) test for bution, while the Vochysiaceae possess an amphi-Atlantic disjunct shifts in speciation and extinction rates across Myrtales and within distribution pattern. The order is the third most species rich lineage each of the five major family clades using BAMM analyses. of the Superrosidae clade of angiosperms (12,264 spp.), varies tremendously in habit (including herbaceous herbs, lianas, trees, and mangroves), floral form, and fruit type (berry, capsule, drupe 2. Material and methods and samara), and exhibits high species diversifications in several fleshy-fruited and dry-capsular clades (e.g., within Melastomat- 2.1. Taxon and gene sampling, and phylogenetic analyses aceae and Myrtaceae) (Dahlgren and Thorne, 1984). Despite this ecological and morphological variation, as well as the presence of Sampling was performed in an effort to maximize diversity species rich subclades within Myrtales, no detailed analysis of across the nine currently recognized families (APGIII, 2009); thus, diversification rates, shifts in these rates, or correlation with evolu- we based our sampling of 102 taxa on available sequences and pre- tionary traits has been performed across the order or within family vious phylogenetic studies (see Supplementary Information clades. Table S1). Outgroups (15 species) were selected from Vitales (Vitis), A well-resolved and temporally calibrated phylogenetic frame- Crossosomatales (Crossosoma), Malvales (Thymelaea), Brassicales work of Myrtales is essential to address questions and hypotheses (Arabidopsis, Carica), and Geraniales (California, Erodium, Francoa, relating to ancestral range estimation, importance of vicariance Geranium, Hypseocharis, Melianthus, Monsonia, Pelargonium, Vivia- and/or dispersal models, and character evolution relative to diver- nia). Geraniales is the sister order to Myrtales, with the Crossoso- sification. All previous molecular phylogenetic studies, either matales, Malvales, and Brassicales representing three orders of 118 B.A. Berger et al. / Molecular Phylogenetics and Evolution 95 (2016) 116–136 the Malvid clade within the Rosidae sister to these two orders et al., 2010). BUCKy uses the Bayesian concordance approach (APGIII, 2009; Soltis et al., 2011). Vitis was used as the ultimate (Ane et al., 2007), which integrates over gene tree uncertainty outgroup in all phylogenetic reconstructions. When possible, the without speculating what causes discordance. For the BUCKy anal- same species, and often the same DNA samples from previous ysis, we used the posterior distribution of the chloroplast, mito- studies, were used to expand gene sampling. When unavailable, chondrial, and nuclear ribosomal gene trees produced with sequences of closely related species were generated or obtained MrBayes. The 18S and 26S data sets were analyzed separately as from GenBank to serve as generic placeholders. Species, voucher/- a conservative approach given the differing best models of collection information, and GenBank accession numbers are pro- sequence evolution. The primary concordance tree and concor- vided in SI Table S1. dance factors were estimated with the discordance parameter (a) Six loci (rbcL, ndhF, matK, matR, 18S, and 26S) were amplified of 1 and 100. Both analyses were run with two Markov chain and sequenced for taxa across Myrtales and Geraniales using pri- Monte Carlo searches (MCMC) of 100,000 generations with mers shown in SI Table S2. Each locus was selected based on utility 10,000 generations burn-in. Both runs converged on the same pri- in large-scale studies across angiosperms (see Soltis et al., 2011), mary concordance tree with identical concordance factors. The the Rosidae (Wang et al., 2009; Zhu et al., 2007), and family- final analysis was conducted with a = 1 and two MCMC chains of wide studies in Myrtales (Biffin et al., 2010; Bult and Zimmer, 1,000,000 generations following a burn-in of 100,000 generations. 1993; Clausing and Renner, 2001; Conti et al., 1993, 1996, 1997, 2002; Gadek et al., 1996; Huang and Shi, 2002; Graham et al., 2.2. Fossil priors and BEAST analyses 2005; Levin et al., 2003, 2004; Lucas et al., 2005; Maurin et al., 2010; Rutschmann et al., 2004, 2007; Schönenberger and Conti, Divergence time estimates were performed in BEAST v.1.8.0 2003; Sytsma et al., 2004; Tan et al., 2002; Thornhill et al., 2012; (Drummond et al., 2012), incorporating an uncorrelated lognor- Wilson et al., 2005). 307 sequences from GenBank were added to mal clock and Yule speciation process. In order to accommodate the dataset to improve sampling and to serve as placeholders for rate heterogeneity, chloroplast markers and matR were parti- key lineages essential for biogeographic reconstruction and assess- tioned into three codon positions. We ran two independent anal- ing rates of species diversification. yses of 50,000,000 generations each. To verify effective sampling Total genomic DNA was extracted from silica-dried leaf mate- of all parameters and to assess convergence of independent rial and herbarium specimens using a DNeasyTM Plant Mini Kit (Qia- chains, we examined output log files in Tracer v.1.6 (Rambaut gen, Valencia, California). All samples received an extended heat et al., 2014). After removing 20% of samples as burn-in, indepen- treatment (30 min at 65 °C instead of 10 min) to reduce secondary dent runs were combined and a maximum clade-credibility compound co-precipitation. PCR methods followed those described (MCC) tree was constructed using TreeAnnotator v.1.8.0 previously (Wilson et al., 2001; Huang and Shi, 2002; Sytsma et al., (Drummond et al., 2012). We offset the minimum ages of 14 2004). PCR products were sequenced using BigDye Terminator kits nodes across the phylogeny using a combination of fossil (see (Applied Biosystems, Foster City, California) on an Applied Biosys- below) and secondary priors (Table 1). All fossil priors were tems 3730xl automated DNA sequencer at the University of placed under a lognormal distribution with a mean of 1.5 and a Wisconsin-Madison Biotechnology Center. Contiguous alignments standard deviation of 1, allowing for the possibility that these were edited and assembled using Geneious Pro v.6.1.8 (http:// nodes are considerably older than the fossils themselves. Second- www.geneious.com, Kearse et al., 2012). Sequences were aligned ary priors were placed under a normal distribution and the root initially using MAFFT v.6.864 (Katoh and Toh, 2008) with subse- under a uniform distribution. Because of the aforementioned quent adjustments made by eye in Geneious. Gaps were treated topological variation with regard to Myrtales and Geraniales, we as missing data. constrained the topology using the most likely tree obtained from Best fitting models of sequence evolution for each locus were GARLI. No age offset was placed on this prior. determined using the Bayesian Information Criterion (BIC) in Eleven fossil priors were utilized in this study. In Melastomat- jModeltest v.2.1.4 (Darriba et al., 2012; Guindon and Gascuel, aceae, we used a leaf fossil (Hickey, 1977) to offset the most recent 2003). The BIC was used for model selection based on its ability common ancestor (MRCA) of Melastomataceae s.s. (minus tribe to outperform other model-selection criteria (Luo et al., 2010). Kibessieae) and a seed fossil as a Melastomeae crown prior Models selected included TVM+I+G for rbcL, TVM+G for matK, matR, (Clausing and Renner, 2001; Renner et al., 2001; Renner, personal and ndhF, GTR+I+G for 26S, and SYM+I+G for 18S. Maximum likeli- communication; Rutschmann et al., 2007; Sytsma et al., 2004; hood (ML) and Bayesian inference (BI) analyses were performed Wehr and Hopkins, 1994). The placement of the second fossil using the CIPRES Science Gateway v.3.3 (www.phylo.org; Miller was supported by morphological characters, as extant Melas- et al., 2010). ML analyses were conducted using default parameters tomeae exhibit cochleate seeds with a round operculum and tuber- in GARLI v.2.01 (Zwickl, 2006). One thousand bootstrap (BS) repli- culate testa, whereas all other Melastomataceae have straight or cates were conducted using the same parameters applied for ML cuneate seeds that lack operculi and differ in ornamentation searches. BI was performed using MrBayes v.3.2.3 (Alteker et al., (Renner et al., 2001). In Myrtaceae, we used two Myrtaceidites 2004; Huelsenbeck and Ronquist, 2001; Ronquist and (=Syncolporites) fossilized pollen grains to place priors on or near Huelsenbeck, 2003). Preliminary BI analyses revealed topological the crown of the family. Myrtaceidites lisimae is the oldest fossil variation in the placement and relationships of outgroup taxa (data in Myrtaceae (from Gabon, Africa; Boltenhagen, 1976; Muller, not shown), especially with regard to Geraniales; therefore, the sis- 1981). While the assignment of M. lisimae to the Myrtaceae is clear, ter relationship of Myrtales to Geraniales was constrained (APGIII, its affinity to either subfamily is ambiguous and likely represents a 2009; Ruhfel et al., 2014; Soltis et al., 2011). All BI analyses were stem lineage placement (Rutschmann et al., 2007; Sytsma et al., run for 15,000,000 generations with four chains in two parallel runs 2004); therefore, we placed it conservatively on the crown of the sampling every 1500 generations. Proper mixing was determined family. We placed Myrtaceidites mesonesus on the first node in from using Tracer v.1.6 (Rambaut et al., 2014) and 20% of trees were dis- the crown of subfamily Myrtoideae (i.e., excluding Lophostemon; M. carded as burn-in prior to constructing a majority rule consensus mesonesus) following the suggestion of Thornhill et al. (2012). The tree using TreeAnnotator v.1.8.0 (Drummond et al., 2012). third prior across Myrtaceae, placed on the stem node of Eucalyp- Congruence among plastid (rbcL, ndhF, matK), mitochondrial tus, represents the earliest record of meso-fossils attributed to (matR), 18S rDNA, and 26S rDNA was assessed by estimating con- the eucalypt clade from South America (Gandolfo et al., 2011; cordance factors (CF; Baum, 2007) using BUCKy v.1.4.3 (Larget Thornhill et al., 2012). In Lythraceae, we used fossilized pollen B.A. Berger et al. / Molecular Phylogenetics and Evolution 95 (2016) 116–136 119

Table 1 Details of fossil calibrations used in BEAST analysis. Age estimates are based on 2009 Geologic Time Scale (Walker and Geissman, 2009). Clades designated with (⁄) indicate secondary calibrations obtained from the literature.

Fossil (clade) Minimum age Coalescent node Reference(s) Prior (Ma) Root⁄ 100 Vitis + Osbeckia See Sytsma et al. (2014) Uniform 125–100 Malvidae crown⁄ 109.5 Crossosoma + Osbeckia Wang et al. (2009) Normal 109.5 (5.33) Caricaceae/Brassicaceae crown⁄ 98.65 Arabidopsis + Carica Cardinal-McTeague et al. Normal 98.65 (unpublished) (3.7) Geraniales crown⁄ 87 Viviania + Geranium Sytsma et al. (2014) Normal 87.0 (5.6) Pelargonium crown⁄ 28.4 Pelargonium a + Pelargonium Sytsma et al. (2014) Lognormal 1.5 h (1.0) Eucalyptus frenguelliana Gandolfo & Zamaloa, sp. n. 51.69 Stockwellia + Eucalyptus Gandolfo et al. (2011) Lognormal 1.5 (1.0) Myrtaceidites lisamae van der Hammen ex 83.5 Heteropyxis + Myrtus See Sytsma et al. (2004) Lognormal 1.5 Boltenhagen (1.0) Myrtaceidites mesonesus Cookson & Pike 61.7 Osbeckia + Myrtus Couper (1960) Lognormal 1.5 (1.0) Melastomeae seeds 23.0 Rhexia + Osbeckia See Renner et al. (2001) Lognormal 1.5 (1.0) Acrovena laevis Hickey 48.6 Bellucia + Osbeckia See Renner et al. (2001) Lognormal 1.5 (1.0) Koninidites aspis Pocknall & Mildenhall 21.4 Fuchsia pa + Fuchsia c Berry et al. (1990); Lee et al. (2013) Lognormal 1.5 (1.0) Lythrum elkensis sp. n./Peplis eaglensis sp. n. 81.0 Lythrum + Peplis Grímsson et al. (2011) Lognormal 1.5 (1.0) Lagerstroemia patelii Lakhanpal & Guleria 56.0 Lagerstroemia + Duabanga See Graham (2013) Lognormal 1.5 (1.0) Punicoxylon eocenicum Privé-Gill 40.4 Punica + Galpinia See Graham (2013) Lognormal 1.5 (1.0) Sonneratiaoxylon preapetalum Awasthi 63.8 Sonneratia + Trapa See Graham (2013) Lognormal 1.5 (1.0) Dilcherocarpon combretoides gen. et. sp. n. 93.5 Laguncularia + Terminalia k Manchester and O’Leary (2010) Lognormal 1.5 (1.0)

grains attributed to Lythrum and Peplis as an offset for the crown of 2.3. Inserting additional terminals for biogeographical and the two lineages (Grímsson et al., 2011). We also used the oldest diversification analyses described Lagerstroemia leaf impression fossil, the silicified wood of Punicoxylon, and the oldest confirmed Sonneratiaoxylon wood To improve lineage sampling for biogeographical and diversifi- as conservative offsets on the stem nodes of Lagerstroemia, Punica, cation analyses, we added five tips to the MCC tree from BEAST. and Sonneratia, respectively (Graham, 2013). In Onagraceae, fossil These five taxa were too incomplete for our gene sampling, but pollen of Koninidites aspis (the type palynomorph of Diporites aspis their phylogenetic placements within families are well known. was recently found to be a fungal sporophyte, thus, new typologi- The five new tips added included: (1) Xylonagra Donn. Sm. & Rose cal nomenclature was assigned; Berry et al., 1990; Lee et al., 2013) in Onagraceae (Levin et al., 2003, 2004); (2) Syncarpia Ten. and (3) was used to offset the split between Old World and New World Xanthostemon F. Muell. in Myrtaceae to represent tribes Syncar- Fuchsia (Berry et al., 2004; Rutschmann et al., 2007; Sytsma pieae and Xanthostemoneae, respectively (Thornhill et al., 2012); et al., 2004). and (4) Astronia Blume and (5) Merianthera Kuhlm. in Melastomat- Although previous large-scale studies (Bell et al., 2010; Smith aceae to represent the Astronieae and the clade comprised of et al., 2011; Wikström et al., 2001) have included putative Combre- ‘‘Merianthera and its allies” + ‘‘Bertolonieae 2” + Cyphostyleae taceous fossils assigned to Esgueiria (Friis et al., 1992; Takahashi (Goldenberg et al., 2012). The taxa were added to the BEAST tree et al., 1999) to offset the Combretaceae node, the fossil differs sig- using the bind.tip function of the phytools package (Revell, 2012) nificantly from extant taxa, especially with regard to stylar for R (R Development Core Team, 2014). This function rescales an branches, and its placement cannot be assigned confidently (E.M. ultrametric tree after new tips are added so the tree remains ultra- Friis, personal communication; Friis et al., 1992; Magallón et al., metric. Similarly, we added these five tips to a random subset of 1999). In lieu of Esgueiria, we use the fin-winged fruits of Dilchero- 100 trees obtained from the posterior distribution of post-burnin carpon combretoides (Manchester and O’Leary, 2010) as an offset on BEAST trees for further biogeographical analyses. the stem node of tribe Combreteae. Morphologically, the epigynous fruits resemble those of extant Combretum and some Terminalia 2.4. Ancestral range estimation in BioGeoBEARS (Gere, 2014; Maurin et al., 2010), possessing four lateral wings arising in two perpendicular planes. The presence of abundant par- Ancestral range estimation (ARE) for Myrtales was done using allel veins and the absence of a fimbrial vein on the fruit is an the nested DEC and DECj models in BioGeoBEARS (Matzke, 2013, extremely rare combination of characters outside of the family 2014) in R v3.1.1. Similar to the Dispersal-Extinction-Cladogenesis (Manchester and O’Leary, 2010). Although winged fruits are (DEC) program LaGrange (Ree and Smith, 2008; Ree et al., 2005), reported in all genera of Combretaceae (Johnson and Briggs, the DEC model in BioGeoBEARS evaluates ML parameters for ana- 1984), the occurrence of winged fruits in the genus Strephonema genetic events involving range expansion and extinction and for (subfamily Strephonematoideae) has not been confirmed and the cladogenetic events involving sympatry and vicariance. Unlike fruits of tribe Laguncularieae appear to be secondarily derived LaGrange, however, BioGeoBEARS with the DECj model can also from bracteoles (Stace, 2007). parameterize cladogenetic ‘‘founder-events” (Templeton, 1980) 120 B.A. Berger et al. / Molecular Phylogenetics and Evolution 95 (2016) 116–136 by incorporating the J parameter for ‘‘jump-dispersals”. This J sensitive and diversity-dependent, allowing rate shifts to occur parameter allows for a daughter lineage to immediately occupy anywhere on a branch based on the posterior tree density. BAMM via long-distance dispersal a new area that is different from the accounts for non-random and incomplete taxon sampling in the parental lineage. DECj models have been shown to be significantly phylogenetic trees by allowing all non-sampled species to be asso- better than DEC models for island groups (Matzke, 2014) and for ciated with a particular tip or more inclusive clade. Terminals and inter-continental distributions, but not for some more localized proportion of extant species sampled for each are provided in SI continental groups (e.g., western North American Salvia; Walker Table S4. For example, only one of eight species of Circaea (Ona- et al., 2015). graceae) was sampled, and thus BAMM recognized sampling at this We identified six broad geographic areas, modified after Buerki tip as only 0.125. Tips were assigned to the smallest possible tax- et al. (2011), important in the context of the distributions of the onomic unit, which often corresponded to genera or tribes within families in Myrtales (see Fig. 3): (1) Eurasia (from western Europe, families. For example, all 873 species representing a number of Mediterranean Africa, to temperate Asia); (2) Sub-Saharan Africa genera of the tribe Eucalypteae (Myrtaceae) were identified as including Madagascar; (3) Southeast Asia, including India, Indo- belonging to a clade defined by the most recent common ancestor China, the Malaysian Peninsula, the Philippines, Sumatra, Borneo of our three sampled genera (Eucalyptus, Angophora, and Stock- and the Inner Banda Arc; (4) Australia, including New Guinea, wellia). Species numbers were obtained from published sources New Caledonia and New Zealand, as well as the Pacific Islands (Forster, 1994; Jordaan et al., 2011; Maurin et al., 2010; Renner, (e.g. Hawaii); (5) North America; and (6) South America, including 1993; Stace, 2007; Stevens, 2001; The Plant List, 2014). Terminals most of Mexico, Central America and the West Indies. Because our and proportion of extant species sampled for each are provided in study focused on family-level relationships, all tip taxa within SI Table S4. A total of 12,264 species in Myrtales are recognized, Myrtales were coded as present or absent for each of the six areas updating previous numbers from Stevens (2001) and Soltis et al. based on extensive literature available for each family. In instances (2011). where a tip taxon represented a more diverse and widespread lin- Priors for BAMM were generated using the R package BAMM- eage, we coded these tips to cover the maximal distribution of the tools v.2.0.2 (Rabosky, 2014) by providing the MCC tree from lineage. Due to the difficulty of scoring under-sampled outgroup BEAST and total species numbers across the order (12,264). Two orders for geographic areas, we restricted outgroup taxa only to independent MCMC chains of 100,000,000 generations were run the Geraniales as the sister clade to Myrtales. Geraniales has an in BAMM and convergence was assessed by computing the effec- ancestral area of Africa, South America, or combined Africa + South tive sample sizes of log likelihoods, as well as the number of shift America (Fiz et al., 2008; Palazzesi et al., 2012; Sytsma et al., 2014). events present in each sample using the R package coda v. 0.16-1 Our sampling of three families in the order included all possibili- (Plummer et al., 2006). After removing 10% of trees as burn-in, ties: African Melianthaceae, South American Vivianiaceae, and we analyzed the BAMM output using BAMMtools and computed likely combined African + South American Geraniaceae. the 95% credible rate shift configurations using the Bayes factors In BioGeoBEARS we allowed the inferred ancestors to occupy up criterion for including nodes as core shifts set to 5. This procedure to all six areas. Dispersal probabilities between pairs of areas and computes Bayes factors evidence associated with a rate shift for areas allowed were specified for four separate time slices (SI every branch in the phylogeny and excludes all unimportant nodes Table S3) based on known geological events that have been simi- using the Bayes factor criterion (see BAMM documentation). Large larly analyzed elsewhere (e.g., Buerki et al., 2011; Drew and Bayes factors values are taken as strong evidence for a shift on a Sytsma, 2012; Sessa et al., 2012; Sulman et al., 2013). We esti- branch in the tree. We also estimated the rate shift configuration mated ancestral ranges on the MCC tree from BEAST, pruned of with the highest maximum a posteriori (MAP) probability (‘‘the all outgroups except Geraniales, with the addition of five terminals. best shift configuration”) after having excluded all none core shifts. The resulting ML score for the more parameter-rich DECj model Additionally, we ran independent BAMM analyses on each of was tested for significance against the resulting ML score of the the five largest families within Myrtales (Combretaceae, Lythra- DEC model. We explored the degree to which differences in tree ceae, Melastomataceae, Myrtaceae, and Onagraceae) to test if topology and branch lengths yield different ARE for nodes by BAMM identified different shifts when each family was analyzed developing a script to run BioGeoBEARS on the 100 randomly separately. The same parameters and analytical procedures were selected PP trees from the BEAST analysis (with five species added used for family-specific analyses except the number of generations for each) under both DEC and DECj models. Additionally, we con- was reduced to 50,000,000. Family level phylogenies were ducted biogeographical stochastic mapping analyses in BioGeo- obtained by extracting the corresponding families from the Myr- BEARS on the single best BEAST tree (with five species added) tales phylogeny. under both DEC and DECj models. This permitted measuring the probability of each class of cladogenetic event (vicariance, sympa- try, subset-sympatry, and jump dispersals) given the DEC and DECj 3. Results models, distribution data, and phylogeny. 3.1. Phylogenetic relationships and timing of clade formation in 2.5. Estimating speciation and extinction rates and identifying rate Myrtales shifts in species diversification in BAMM A total of 362 sequences were generated for this study: 31 rbcL, We used the program BAMM (Rabosky, 2014; Rabosky et al., 28 ndhF,80matK, 63 18S, 62 26S and 98 matR (see SI Table S1). The 2014) to: (1) estimate rates of speciation, extinction, and net diver- aligned, partitioned data set consisted of 7337 characters, with sification for clades, (2) conduct rate-through-time analysis of 1626 being parsimony-informative. ML and BI trees highlight sev- these rates, and (3) identify and visualize shifts in species rates eral important phylogenetic results at the broad scale within Myr- across the Myrtales phylogeny. BAMM is a Bayesian approach that tales (Fig. 1). First, Combretaceae is identified as the sister clade to uses reversible jump Markov chain Monte Carlo (RJMCMC) sam- all remaining families with moderate support (ML-BS/BI- pling to explore shifts in macro evolutionary regimes assuming PP = 69/0.84). Second, within the latter clade, two strongly sup- they occur across the branches of a phylogeny under a compound ported groups are observed: (1) Onagraceae + Lythraceae form a Poisson process, and explicitly accommodates diversification rate subclade with strong support (ML-BS/BI-PP = 100/1.0), and (2) variation through time and among lineages. BAMM is both time- Melastomataceae + the CAP clade and Myrtaceae + Vochysiaceae B.A. Berger et al. / Molecular Phylogenetics and Evolution 95 (2016) 116–136 121

Osbeckia */0.90 Melastoma */0.99 Pterolepis 89/1.0 */0.99 Tibouchina Heterocentron Arthrostemma Rhexia 95/1.0 Rhynchanthera */0.94 Lavoisiera 91/0.99 Medinilla Blakea Bertolonia 85/0.97 92/0.99 Tococa 89/1.0 Miconia Clidemia Leandra 77/0.97 Macrocentrum Graffenrieda

Meriania Melastomataceae Henriettea Bellucia Mouriri Memecylon Pternandra e Pternandra c 89/1.0 Penaea Olinia 72/0.93 Rhynchocalyx Alzatea Crypteronia Metrosideros c Metrosideros m Syzygium Backhousia Tristania Tristaniopsis 0.2 substitutions/site */0.90 Myrtus Eugenia Uromyrtus Lindsayomyrtus 77/0.99 Eucalyptus Angophora Stockwellia Leptospermum

Kunzea Myrtaceae CAP Chamelaucium Melaleuca */1.0 Callistemon Osbornia Lophostemon Psiloxylon Heteropyxis Ruizterania */0.93 */0.92 Qualea 69/0.84 Vochysia Erisma Erismadelphus Trapa Vochys. 90/1.0 Sonneratia */1.0 */1.0 Lagerstroemia Duabanga Lawsonia Ammania Peplis 81/1.0 Lythrum Decodon Heimia Punica 86/1.0 Galpinia Lythraceae Cuphea Adenaria Oenothera 93/1.0 Clarkia Epilobium Gongylocarpus */0.99 Lopezia Fuchsia pr raceae

Fuchsia c g Fuchsia pa Circaea

Hauya Ona Ludwigia Terminalia k Terminalia c 83/1.0 Terminalia i Terminalia p Terminalia u Terminalia b 89/1.0 Terminalia a Buchenavia 85/1.0 Conocarpus Bucida Combretum iv Combretum c Combretum in Combretum r Combretum f 81/1.0 Macropteranthes Combretaceae 71/0.98 Lumnitzera Laguncularia Strephonema p Strephonema m Viviania Melianthus Francoa */1.0 77/0.97 Geranium California Erodium Monsonia Pelargonium a

Pelargonium h Geraniales Hypseocharis Carica */1.0 Arabidopsis Thymelaea Crossosoma Vitis

Fig. 1. Maximum likelihood tree of Myrtales and Geraniales based on six loci shown as a cladogram (phylogram inset). Tree is rooted with four other Rosidae orders (Vitis is the ultimate outgroup). Support values are provided for each branch: (ML-BS/BI-PP). Bold black branches are ML-BS P 95% and BI-PP = 1.0. Aside from the node designating the divergence of Combretaceae from all other Myrtales, support values 670% ML-BS are designated by a (⁄). Support values 670% ML-BS and 60.9 BI-PP are collapsed. Families are color-coded (as in other figures) to assist comparisons. 122 B.A. Berger et al. / Molecular Phylogenetics and Evolution 95 (2016) 116–136 are both highly supported (ML-BS/BI-PP = 100/1.0), as is the entire Most of the major family clades also have either South America subclade (ML-BS/BI-PP = 100/1.0). Third, all families within Myr- (Melastomataceae, Vochysiaceae, Onagraceae, Lythraceae) or tales are monophyletic with high support (ML-BS/BI- Africa (Myrtaceae, CAP clade) as ancestral. Australia, including PP = 100/1.0). And fourth, our sub-sampling scheme corroborates islands east of the Wallace Line and Polynesia, is an important bio- most large-scale studies addressing intergeneric relationships geographic area early in the Late Cretaceous or Paleocene radiation (see Section 4). Bayesian concordance analysis using BUCKy of subfamily Myrtoideae (Myrtaceae), Melastomataceae, Cryptero- resulted in a primary concordance tree with the same overall niaceae, and Combretaceae. Eurasia and Southeast Asia appear to topology as the BI and ML phylogenies (SI Fig. S1). Concordance play more minor and recent roles in most families, with the excep- factors were moderate to high for all families (>0.5) and these val- tion of an early diversification (Late Cretaceous) in Lythraceae in ues decreased toward the backbone of the tree. Eurasia. More recent migrations in the Paleogene over adjacent Divergence time estimates suggest Myrtales diverged from or still connected areas are seen between South America and Aus- Geraniales 123.6 Mya (Table 2; Fig. 2) during the Aptian of the tralia (e.g., Melastomataceae, Onagraceae, and Lythraceae) and Early Cretaceous. The crown date for Myrtales is estimated at between Australia, Southeast Asia, and Eurasia (Melastomataceae, 116.4 Mya (Albian–Aptian). Stem lineages of the four major Myrtaceae, and Combretaceae). Long distance dispersal and estab- clades of Myrtales are all present by the end of the Albian lishment (LDDE) events between South America and Africa and (109.3 Mya): (1) Combretaceae, (2) Lythraceae + Onagraceae, (3) over oceanic water barriers during the Paleogene occur in the Melastomataceae + the CAP clade, and (4) Myrtaceae + Vochysi- CAP clade, Vochysiaceae, and Combretaceae. aceae. Stem lineages of all extant families are present by 90.7 Mya (mid-Turonian). Crown dates of modern families range 3.3. Speciation and extinction rates and identification of three rate from 102.6 Mya for the Combretaceae (Albian) to 38.8 Mya for shifts in species diversification in Myrtales the Vochysiaceae (late Eocene). After discarding the burn-in, we confirmed convergence of the 3.2. Biogeographical patterns of diversification in Myrtales MCMC chains in the BAMM analyses, as well as effective samples sizes >900 for both the number of shifts and log likelihoods. BAMM In BioGeoBEARS, significant improvement in the likelihood analyses strongly supported a diversity-dependent speciation pro- score of the model was seen when the ‘‘jump dispersal” parameter cess across Myrtales with a net diversification rate (speciation was added (DECj) vs. without (DEC), as indicated by a likelihood minus extinction) of 0.065 species/Myr (Table 3). The highest spe- ratio test (DEC LnL À341.97, DECj LnL = À287.70, df = 1, ciation rates are seen at nodes within Combretaceae (0.69 species/ P = 2.05eÀ25). Because of the significant improvement in the like- Myr), Melastomataceae (0.45 species/Myr), and Myrtaceae lihood of the model when the jump dispersal parameter is added, (0.32 species/Myr) (Table 3). These three families also show the only ARE under the DECj model is presented. The resulting param- highest extinction rates (0.54, 0.25, 0.15 species/Myr, respectively). eters of the DECj models included: anagenetic dispersal rate Onagraceae, Lythraceae, and the CAP clade exhibit the lowest spe- d = 0.0049; extinction rate e = 1.0eÀ08; cladogenetic dispersal rate ciation and extinction rates within the order. Rate-through-time j = 0.096. Biogeographical stochastic mapping, given the parame- plots for the 120 Myr history of Myrtales revealed a decreasing ters of the DECj model, indicated that 60% of all cladogenetic speciation rate until 60–70 Mya, at which point speciation began events are sympatric, 32% are subset-sympatry, 7% are vicariance, to increase, with the highest average rate (0.28 species/Myr) seen and 1% involve founder-event dispersals. at present (Fig. 4). Extinction rates were low (0.06 species/Myr) ARE for Myrtales and Geraniales using BioGeoBEARS is shown in until 60–70 Mya and steadily climbed until the present (0.19 spe- Fig. 3 (area or combined area with highest frequency shown for cies/Myr). each node and corner). The chronogram illustrates ARE in the con- The 95% credible set of rate shift configurations sampled with text of geological age and continental drift during the Cretaceous BAMM included 271 distinct configurations of which the configu- and Paleogene. The MCC tree indicates a combined South Amer- rations with the highest probability included three to four shifts. ica + Africa ARE for the ancestor of Myrtales + Geraniales and for Fig. 5 represents the single best configuration and includes three the crowns of both Myrtales and Geraniales, with these diversifica- shifts detected within Myrtales: (1) near the crown of Melastomat- tions occurring in the early Cretaceous when the two continents aceae (63 Mya) after the divergence of Pternandra, (2) along the were attached (Fig. 3). The only difference in ARE between the stem of subfamily Myrtoideae (Myrtaceae; 76 Ma), and (3) along BEAST tree and the 100 PP BEAST trees is in the crown node of Myr- the stem of tribe Combreteae (Combretaceae; 43 Mya). Although tales (South America only 85% of the trees, as shown in Fig. 3). the specific placements are at different times, all three rate shifts Thus, ARE of Myrtales is robust at least in the context of likely occur on branches that span the Cretaceous–Paleogene boundary alternative topologies or branch lengths. The frequencies of other (K–Pg = 66 Mya; formerly K–T boundary). Bayes factor evidence possible areas (or area combinations) at each node and corner was strongest (>800) for the shifts in Combretaceae (BF = 22,921) are provided in SI Fig. S2. and Melastomataceae (BF = 3240). The shift at the base of the Myr-

Table 2 Estimated ages (Mya) of nodes within Myrtales using BEAST analysis and comparable node dates based on r8s (Sytsma et al., 2004). Stem of Myrtales constrained to 120 Mya in r8s analysis. Combretaceae and Lythraceae are only family crowns not comparable between the two analyses as BEAST analysis included earlier diverging lineages (thus older crown ages) than sampled in the r8s analysis (younger crown ages).

Node Clade BEAST age (95% HPD) r8s age (Sytsma et al., 2004) Stem Crown Stem Crown 1 Myrtales 123.6 (124.8–122) 116.4 (118.8–113.7) 120 111 2 Combretaceae 116.4 (118.8–113.7) 102.6 (106.5–98.9) 111 46 3 Onagraceae 104.6 (109.1–100.2) 85.4 (96.2–73.3) 93 82 4 Lythraceae 104.6 (109.1–100.2) 95.5 (99.7–91.7) 93 60 5 Vochysiaceae 101.1 (107.0–95.1) 38.9 (51.8–28.0) 93 32 6 Myrtaceae 101.1 (107.0–95.1) 85.0 (87.6–83.6) 93 76 7 CAP clade 90.7 (98.8–82.4) 52.7 (66.4–40.4) 82 52 8 Melastomataceae 90.7 (98.8–82.4) 64.5 (74.81–56.1) 82 56 B.A. Berger et al. / Molecular Phylogenetics and Evolution 95 (2016) 116–136 123

Fig. 2. Maximum clade credibility (MCC) tree of Myrtales obtained from BEAST analysis. Mean divergence time estimates are shown with 95% highest posterior density (HPD; blue bars). Clades I–VII represent crown radiations of extant families. Mean age plus 95% HPD are as follows: I – 64.5 Ma (74.8–56.1 Ma); II – 52.7 Ma (66.4–40.4 Ma); III – 85.0 Ma (87.6–83.6 Ma); IV – 38.8 Ma (51.8–28.0 Ma); V – 95.5 Ma (97.7–91.7 Ma); VI – 85.4 Ma (96.2–73.3 Ma); and VII – 102.6 Ma (106.5–98.9 Ma). Black circles indicate calibration points 1–7 (see Table 2 for additional information). The shaded box corresponds to the maximum/minimum age range for the divergence of all extant Myrtalean stem lineages (based on 95% confidence intervals). rprinoto 0 Ptes(fls hn10 htteae o obndae)hdtehgetM rbblt.Istgoe eitcnietlposit continental depict globes Inset probability. all ML four highest to the up had area, area) combined after combined (or (modified (or area boundary area the the K–Pg for that the coded 1.0) and color than are Cretaceous less corner Late (if and the trees node PP each 100 at of Boxes out inset. proportion map the on probability. shown ML areas highest biogeographical six the for coded 3. Fig. 124 neta ag siain(R)o h ytlscrnga ihBoeBAS(E+ oe) ra ftpseis(rgnr)aesonlf ftx na taxa of left shown are genera) (or species tip of Areas model). (DEC+J BioGeoBEARS with chronogram Myrtales the on (ARE) estimation range Ancestral IFg S2 Fig. SI rvdsalAEprnd n onrwt isdsgaigpoaiiyo ahae o obndae) ubr ett oe niaethe indicate nodes to next Numbers area). combined (or area each of probability designating pies with corner and node per ARE all provides ..Bre ta./MlclrPyoeeisadEouin9 21)116–136 (2016) 95 Evolution and Phylogenetics Molecular / al. et Berger B.A. cts,1998 Scotese, ). osa h einn of beginning the at ions wd ihthe with owed) e,color- mes,

Geraniales Combretaceae Onagraceae Lythraceae Vochys. Myrtaceae CAP Melastomataceae B.A. Berger et al. / Molecular Phylogenetics and Evolution 95 (2016) 116–136 125

Table 3 Estimated speciation, extinction, and net diversification rates (with 95% CI) for selected crown nodes in Myrtales based on BAMM analysis. Ages are Mya. Rates are species/Myr. Clades are ordered by magnitude of speciation rate. Three clades with significant shifts in speciation rates are indicated in bold (see Figs. 4–6). Symbols ‘‘ + ” or ‘‘À” indicate one younger or older node, respectively.

Clade Age Speciation Extinction Net diversification Median 95% CI Median Median Median 95% CI Combretaceae shift + 17.0 0.69 0.38–1.08 0.54 0.20–0.94 0.15 0.14–0.19 Combretaceae shift 33.2 0.57 0.33–0.87 0.44 0.16–0.76 0.12 0.11–0.16 Miconieae (Melastomataceae) 27.1 0.45 0.23–0.72 0.25 0.05–0.51 0.20 0.18–0.21 Melastomataceae shift + 42.4 0.40 0.22–0.65 0.25 0.04–0.51 0.15 0.11–0.15 Melastomataceae shift 62.2 0.39 0.21–0.65 0.24 0.04–0.51 0.15 0.14–0.17 Melastomataceae 64.5 0.36 0.20–0.59 0.23 0.04–0.48 0.13 0.11–0.15 Combretaceae shift À 94.5 0.32 0.19–0.48 0.24 0.10–0.41 0.07 0.07–0.09 Myrteae (Myrtaceae) 18.4 0.30 0.12–0.60 0.15 0.01–0.39 0.15 0.11–0.21 Combretaceae 102.6 0.28 0.17–0.41 0.21 0.09–0.36 0.07 0.05–0.08 Myrtaceae shift 67.3 0.22 0.12–0.39 0.13 0.02–0.31 0.09 0.08–0.10 Myrtaceae shift + 64.1 0.22 0.12–0.40 0.13 0.01–0.32 0.09 0.09–0.09 Syzigieae (Myrtaceae) 22.5 0.22 0.11–0.40 0.13 0.01–0.33 0.09 0.08–0.11 Myrtaceae 85.0 0.21 0.11–0.37 0.12 0.02–0.30 0.08 0.07–0.09 Myrtales 116.4 0.19 0.14–0.26 0.13 0.07–0.20 0.06 0.06–0.07 Onagreae (Onagraceae) 45.7 0.14 0.04–0.35 0.08 0.01–0.21 0.06 0.03–0.14 Onagraceae 85.4 0.10 0.05–0.17 0.07 0.01–0.14 0.03 0.03–0.03 Lythraceae 95.5 0.09 0.04–0.16 0.07 0.01–0.14 0.02 0.02–0.03 CAP clade 52.7 0.08 0.03–0.14 0.07 0.01–0.14 0.02 0.01–0.03 Fuchsia (Onagraceae) 23.0 0.08 0.030.16 0.06 0.01–0.14 0.02 0.02–0.02

0.4 0.4 0.4 ) 0.3 0.3 r 0.3 ) λ

0.2 0.2 0.2 Combretaceae Melastomataceae extinction rate (µ) speciation rate ( Myrtaceae net diversification rate (

0.1 0.1 0.1

0.0 0.0 0.0

120 100 80 60 40 20 0 120 100 80 60 40 20 0 120 100 80 60 40 20 0 time before present (Mya) time before present (Mya) time before present (Mya)

Fig. 4. Rates-through-time plots of speciation, extinction, and net diversification in Myrtales utilizing BAMM. Curved black lines represent the median values with the 95% confidence intervals shown in gray. Arrows point to the three significant shifts in rates of speciation identified in Fig. 5. taceae (excluding Heteropyxis and Psiloxylon) received a Bayes fac- clade comprised of Clidemia, Miconia and Tococa (Fig. 6). Bayes factor tor of 300, which does not closely approach the 800 value of over- evidence for this shift was low (BF = 116). Lastly in Myrtaceae, the whelming support for a rate shift. 95% credible set of rate shift configurations sampled included 23 The family level analyses (Figs. 5 and 6) recovered shifts in spe- distinct configurations of which the configurations with the highest ciation rates that are similar but not identical to the ordinal level probability included one, two, or no core shifts. The best configura- analyses. Like in the large analyses, shifts were detected in the Com- tion included a single core shift on the crown of the Myrteae clade bretaceae, Myrtaceae, and Melastomataceae. In Combretaceae, the (Fig. 6). Bayes factor evidence for this shift was 84. 95% credible set of rate shift configurations sampled with BAMM included 2 distinct configurations of which the configurations with the highest probability included 1–2 core shifts. The best shift con- 4. Discussion figuration included a single core shift on the same branch as the ordinal level analyses (Fig. 6). Bayes factor evidence for this shift 4.1. Myrtales: a model Cenozoic clade for understanding the role of was high (BF = 3158). In the Melastomataceae, the 95% credible dispersal, vicariance, cladogenesis, and extinction in formation of set of rate shift configurations sampled included 3 distinct configu- Southern Hemisphere biodiversity rations of which the configurations with the highest probability included one or no core shifts. The best configuration detected a sin- Our results, which are based on the largest assemblage of both gle shift within the tribe Miconieae, on the branch leading to the taxa and loci for the order Myrtales, provide strong evidence for 126 B.A. Berger et al. / Molecular Phylogenetics and Evolution 95 (2016) 116–136

Fig. 5. Speciation rates in Myrtales utilizing BAMM. Chronogram of Myrtales with branch lengths colored according to speciation rate. Red circles indicate significant increases in speciation rates present in the best shift configuration. Right panel shows speciation rates in seven family clades when each are evaluated separately. The blue curve is the speciation rate for the entire Myrtales, the respective colored curve is for the specific family. B.A. Berger et al. / Molecular Phylogenetics and Evolution 95 (2016) 116–136 127

A Terminalia k B Osbeckia C Metrosideros c Terminalia c Melastoma Metrosideros m Pterolepis Terminalia i Syzygium Tibouchina Backhousia Terminalia p Heterocentron Arthrostemma Tristania Terminalia u Rhexia Tristaniopsis Terminalia b Rhynchanthera Myrtus Terminalia a Lavoisiera Eugenia Merianthera Uromyrtus Buchenavia Medinilla Lindsayomyrtus Conocarpus Blakea Eucalyptus Bucida Bertolonia Angophora Tococa Combretum i Miconia Stockwellia Combretum c Clidemia Syncarpia Leptospermum Quisqualis Leandra Macrocentrum Kunzea Combretum r Graffenrieda Chamelaucium Combretum f Meriania Melaleuca Henriettea Callistemon Macropteranthes Bellucia Lumnitzera Osbornia Astronia Xanthostemon Laguncularia Mouriri Memecylon Lophostemon Strephonema p Pternandra e Psiloxylon Strephonema m Pternandra c Heteropyxis 100 80 60 40 20 0 60 50 40 30 20 10 0 80 60 40 20 0

0 0.8

Fig. 6. Speciation rates for three families in Myrtales utilizing BAMM. Chronograms of each family with branch lengths colored according to speciation rate. Red circles indicate significant increases in speciation rates present in the best shift configuration. (A) Combretaceae. (B) Melastomataceae. (C) Myrtaceae. phylogenetic relationships among and within the nine families, for 2007), and multiple representatives from each of the largest sub- the ancestral areas, timing, and biogeographical mode of this tribes (subfam. Combretoideae tribe Combreteae subtribe Com- remarkable radiation, and for the specific subclades that have bretinae and subtribe Terminaliinae; Stace, 2007). Based on the undergone significant shifts in speciation. We show for the first ML and BI analyses, Combretaceae is sister to the rest of the order time, stronger support for the placement of Combretaceae as sister with moderate support (Fig. 1). In addition to molecular phyloge- to all remaining Myrtales. This relationship and other early clado- netic results, the position of Combretaceae is supported by recent genetic events within Myrtales signal the importance of both South fossil evidence considered the earliest occurrence of Myrtales America and Africa (western Gondwana) as ancestral areas in the (Manchester and O’Leary, 2010). early biogeographic diversification of the order during the early Importantly, this new phylogenetic framework for Myrtales (or Cretaceous. This biogeographic diversification involves both vicari- that based on sets of plausible PP trees) will be critically important ant and dispersal (including jump or founder) events among conti- in re-evaluating the prodigious wealth of morphological and nents. Lastly, we find three significant shifts in speciation rates anatomical data that has accumulated for the order, such as that within three families (Myrtaceae, Melastomataceae, Combre- found in the hallmark work of Johnson and Briggs (1984) who taceae) and all on branches spanning the K–Pg boundary. With examined ‘‘cladistically” 77 characters across Myrtales. For exam- the wealth of previously published phylogenetic, fossil, biogeo- ple, they highlight the unusual and distinctive presence of fibrous graphical, ecological, and evolutionary studies, the Myrtales is seed exotegmen as an example of convergence between the Com- now one of the better-known Southern Hemisphere dominant lin- bretaceae and the Lythraceae + Onagraceae clades based on a mor- eages of organisms. phology derived phylogram. However, under the new DNA-based framework with Combretaceae sister to the remaining families, 4.2. Phylogenetic perspective of evolution within Myrtales this feature now can be interpreted as the plesiomorphic state retained in these three families but subsequently lost in the larger Our results provide new insight into phylogenetic relationships clade sister to Lythraceae + Onagraceae. We are now using updated within Myrtales and support an ancient radiation of the order clo- Myrtales relationships to better understand morphospace evolu- ser to the diversification of the eudicot angiosperms than has pre- tion of quantitative features such as pollen size and shape viously been suggested (Bell et al., 2010; Magallón and Castillo, (Kriebel et al., 2015). 2009; Moore et al., 2010; Smith et al., 2010; Sytsma et al., 2004; Wang et al., 2009; Wikström et al., 2001). Seven major clades rep- 4.3. Origin and biogeography of Myrtales resenting the major families (the CAP clade is considered a single lineage) are resolved with high support. Aside from the position The biogeographical scenario presented here is the first for the of Combretaceae, all other interfamilial relationships are congruent entire order Myrtales. Several families or closely related clades with previous studies. have been examined in more detail, notably Myrtaceae, Vochysi- Prior analyses of relationships within Myrtales have provided aceae, Combretaceae, Melastomataceae, and the CAP clade an improved understanding of both the phylogenetic relationships (Berger, 2012; Conti et al., 2002; Gere, 2014; Maurin et al., 2010; among the families and the biogeography of the order; however, Renner et al., 2001; Rutschmann et al., 2004; Sytsma et al., the placement of Combretaceae has remained unresolved (Conti 2004). ARE at greater taxonomic depth presents both opportunities et al., 1996, 1997, 2002; Soltis et al., 2011; Sytsma et al., 2004). to gain biogeographical insight concerning early cladogenetic The lack of adequate taxon sampling of the Combretaceae (Figs. 1 events but also issues due to the lack of sampling within families and 2) contributed to the uncertainty in its phylogenetic placement and thus loss of biogeographical signal. This trade-off is clearly in these previous studies. In our study, we explicitly improved observed with Myrtales where on one hand, ARE provides a consis- sampling in Combretaceae by including representatives of Stre- tent geographical area (South America and/or Africa) for the stem, phonema (subfam. Strephonematoideae; Stace, 2007), the ‘man- crown, and other deeper nodes of the order (and sister order Gera- grove’ clade (subfam. Combretoideae tribe Laguncularieae; Stace, niales), while on the other hand, a lack of dense taxonomic sam- 128 B.A. Berger et al. / Molecular Phylogenetics and Evolution 95 (2016) 116–136 pling within families, despite adequate coverage of major within- between 106 and 84 Mya. Thus, the early diversification of Myr- family subclades, generates ARE of some family crown nodes that tales, including all the major stem clades, are west Gondwana in are at odds with previous family-level studies (see below for fur- origin, date to no younger than 85–90 Mya, and are largely sup- ther discussion). One possible solution to this biogeographic chal- portive of vicariant explanations. lenge, which we are now exploring, is to use a supermatrix-derived Recent fossil evidence assigned to Combretaceae (Manchester chronogram that is framed on the well-supported Myrtales topol- and O’Leary, 2010) represents the earliest occurrence of the order ogy presented here that uses a smaller data set but with nearly and establishes a minimum date for tribe Combreteae that is older complete taxon-gene coverage. Additionally, it is increasingly rec- than the crown date used for the family in several previous studies ognized that extant taxa indicate persistence in time only, not nec- (Bell et al., 2010; Magallón et al., 1999; Smith et al., 2010; essarily in area, and that many clades have been subject to Wikström et al., 2001). Our analyses estimate the stem age of Com- considerable extinction without an attendant fossil record (Crisp bretaceae (=crown of Myrtales) at 116 Mya, which is closely et al., 2011; Kooyman et al., 2014). Thus, the following discussion aligned with the recent estimate for the crown of Myrtales of biogeographic history in Myrtales concedes that limitations in obtained when examining flowering plant diversification full taxon sampling and in our knowledge of extinction events do (117 Mya; Magallón et al., 2015). Of the remaining Myrtales lin- not permit unambiguous interpretations. eages, two major clades were established shortly thereafter in the early Albian–late Aptian (115 Mya): (1) Onagraceae + Lythraceae 4.3.1. Early historical biogeography of Myrtales and (2) Melastomataceae s.l. + CAP + Myrtaceae + Vochysiaceae. The biogeography of Myrtales is often attributed to a Gond- Separation of Melastomataceae s.l. + CAP from Myrtaceae wanan origin (Conti et al., 1997; Johnson and Briggs, 1984; + Vochysiaceae occurred mid-Albian (109 Mya) with all major Muller, 1981; Raven and Axelrod, 1974; Rutschmann et al., 2007; extant lineages represented by the Turonian (91 Mya). As in pre- Sytsma et al., 2004). However, phylogenetic uncertainty (Conti vious studies (APGIII, 2009; Conti et al., 1996, 1997; Rutschmann et al., 1996, 1997; Sytsma et al., 2004), particularly with respect et al., 2007; Soltis et al., 2011; Sytsma et al., 2004), Onagraceae to the position of Combretaceae, has been a hindrance. Improved + Lythraceae form a well-supported sister clade, which diverged support for the placement of Combretaceae and other clades 105 Mya in the Albian. This date represents the oldest estimate shown by this study provides a good phylogenetic framework for for the clade by 12 Myr (93 Mya in Sytsma et al., 2004) and may hypothesis testing and ancestral range estimation. Importantly, be attributed to new fossil findings, particularly of Lythrum and despite the use of a larger number of fossils, different genes, and Peplis pollen from the Lower Campanian (82–81 Mya; Grímsson different analytical approaches to estimate a chronogram, the et al., 2011). These estimates agree with ages obtained in other dates we infer here are largely consistent with previous dates for fossil-calibrated studies of major clades of eudicots, including the Myrtales evolution (Table 2) based on fixed fossil constraints in Campanulids (Beaulieu et al., 2013), Saxifragales (Jian et al., r8s (Sytsma et al., 2004). Most of the stem and crown dates 2008), Malpighiales (Davis et al., 2005), Geraniales (Sytsma et al., obtained with r8s, other than clades not comparably sampled at 2014), Sapindales (Buerki et al., 2010), and Dipsacales (Bell and their bases, fall within the 95% credibility ages obtained with Donoghue, 2005). BEAST (Table 2). Thus, we argue that downstream analyses utiliz- ing this Myrtales chronogram(s) (e.g., biogeography, species diver- 4.3.2. Historical biogeography of Combretaceae sification, character evolution) have a stronger phylogenetic and Combretaceae began differentiating 116 Mya with the split temporal framework from which to interpret those results. into two extant subfamilies occurring 104 Mya. While the stem The BioGeoBEARS analyses support a west Gondwana (South date of the Combretaceae is older than previously proposed ages America, Africa) origin of the Myrtales (stem age 124 Mya, crown for the crown of Myrtales (88.2 Mya, Magallón and Sanderson, age 116 Mya) and not east Gondwana (Antarctica, Australia, 2001; 107 Mya, Wang et al., 2009; 107 Mya, Wikström et al., India, Madagascar). ARE does suggest other possible areas (or com- 2001; 108 Mya, Magallón and Castillo, 2009; 111 Mya, Sytsma bined areas) at the oldest nodes within Myrtales, but the dominant et al., 2004), the estimates presented here better approximate ARE shown in Fig. 3 have fairly high likelihood scores. The west the ages of the order and the family for three reasons: (1) the inclu- Gondwana origin for Myrtales has previously been suggested by sion of the West African Strephonema (subf. Strephonematoideae) Raven and Axelrod (1974), as well as Johnson and Briggs (1984), allows more accurate estimation of the stem lineage of Combre- and is also seen in the Geraniales, which is the sister group to Myr- taceae (Conti et al., 1996, 1997; Sytsma et al., 2004); (2) improved tales (Sytsma et al., 2014). Diversification of all the major stem sampling provides support for Combretaceae as sister to the rest of clades within Myrtales are also west Gondwana in origin and date the order; and (3) recent fossil descriptions attributed to Combre- to no younger than 85–90 Mya. taceae from the Albian–Cenomanian (112–93.5 Mya; Manchester Although dates obtained in our analysis are all younger than age and O’Leary, 2010) pushes back the crown date of the family to estimates for the initial breakup of Gondwana (180–150 Mya; Ali at least 103 Mya. and Aitchison, 2008; Chatterjee et al., 2013; Jokat et al., 2003; Based on BioGeoBEARS analysis, the crown of Combretaceae McLoughlin, 2001; Scotese et al., 1988; White et al., 2013) into was in an ancestral area comprising Africa + South America west Gondwana and east Gondwana, subsequent continental con- (Fig. 2), which is consistent with a Gondwanan vicariant origin. nections, separations, and tectonic shifts allowed for persistent but Subsequent diversification within the family involved repeated intermittent contact and floral exchange into the Paleogene area shifts, often involving more recent dispersals based on conti- (Hallam, 1994). Raven and Axelrod (1974) argued that it appears nental separations (Fig. 3). We note that the long branch leading to likely that upwards of 80 large angiosperm families were widely the core Combretaceae (minus the African trees of Strephonema distributed in tropical regions before the Gondwanan continents and the mangrove clade) has an ARE of South America for both separated. According to Pitman et al. (1993), the beginning of the the stem and crown nodes. Recent studies with denser sampling separation of Africa from South America occurred just before within the core Combretaceae (Berger, 2012; Gere, 2014; Maurin 135 Mya in the Early Cretaceous (Valanginian). The early phase et al., 2010) have ARE of Africa for the same nodes, suggesting that of rifting lasted from 135 to about 106 Mya (middle Albian) but, biogeographic signal for these nodes is impacted by taxon sam- due to the rotation of Africa away from South America, tropical pling. Family level ARE such as within Combretaceae, however, West Africa was still in contact with northeastern South America. often cannot, or do not, take into account the third branch leading Complete separation of the two continents was achieved sometime into the crown node – that coming from the sister clade. This might B.A. Berger et al. / Molecular Phylogenetics and Evolution 95 (2016) 116–136 129 be especially problematic in biogeographic studies centering on and seeds from the lower Eocene beds in England and Eocene Rus- Combretaceae as our results provide support that the sister clade sia. While one of the major clades (Trapa, Sonneratia, Duabanga, is the remainder of Myrtales. Lagerstroemia, Ammania, Lawsonia, Lythrum, Peplis, and Decodon) provides clear signal of movement from North America into Africa, 4.3.3. Historical biogeography of Onagraceae + Lythraceae Eurasia, and eventually into the South Pacific for some lineages, the While multiple phylogenetic studies have corroborated most other (Galpinia, Punica, Cuphea, Adenaria, and Hemia) indicates tribal and generic relationships across Onagraceae (Bult and LDDE from South America into Sub-Saharan African with move- Zimmer, 1993; Conti et al., 1993; Levin et al., 2003, 2004), the bio- ment northward in Eurasia during the Eocene by a single lineage geographical history of the family has remained speculative. A (Punica). Gondwanan origin was suggested by Raven and Axelrod (1974) and Raven (1988) based on the pattern of extant plant distribu- 4.3.4. Historical biogeography of Melastomataceae + CAP clade tions and plate tectonics. Conti et al. (1997) provided the first phy- Renner et al. (2001) suggested a Paleogene (Paleocene/Eocene), logenetic context invoking a West Gondwanan origin, while non-Gondwana (north of Tethys Sea) diversification for Melastom- Sytsma et al. (2004) first estimated divergence times using Penal- ataceae s.s. based exclusively on ndhF. They estimated the crown ized Likelihood (PL) and suggested a Late Cretaceous origin for diversification 53 Mya with substantial diversification within Onagraceae + Lythraceae during the Albian (99 Mya) with subse- the family beginning 30 Ma. While an alternative biogeographic quent divergence of the two families by 93 Mya (Table 2). In con- hypothesis based on plate tectonics was put forward suggesting trast to Sytsma et al. (2004), our data support an earlier Early a much older age of Melastomataceae (Morley and Dick, 2003), Cretaceous origin (115 Mya) for the stem lineage of Ona- Renner (2004) provided additional evidence refuting most claims. graceae + Lythraceae with subsequent splitting of the families by Sytsma et al. (2004) further supported the findings of Renner the end of the Albian (105 Mya). Both the timing of these events et al. (2001) using rbcL + ndhF and suggested a crown date for and the pattern of cladogenesis provide credence for a west Gond- Melastomataceae of 56 Mya. In both Renner et al. (2001) and wanan origin of Onagraceae with LDDE to the humid Boreotropics Sytsma et al. (2004), Early Eocene fossil leaves (Hickey, 1977) were of North America from South America during the Late Cretaceous used to constrain the crown of Melastomataceae s.s. including tribe to Paleocene likely playing a role in diversification of the clade Kibessieae. However, based on the leaf description (Hickey, 1977) (Wolfe, 1975). Within the family, two dispersal events back to and personal correspondence (S. Renner, personal communica- South America from North America coincide with the cooling and tion), we used the fossils to offset the crown of Melastomataceae degradation of the Boreotropical flora during the Early Eocene Cli- s.s. without tribe Kibessieae; hence the older dates for the crown matic Optimum/Paleocene–Eocene Thermal Maximum (EECO/ Melastomataceae s.l. should be viewed as minimum ages as PETM), 55–50 Mya (Zachos et al., 2001). Additionally, as cooling opposed to maximum ages. of the planet and subsequent drying intensified with the onset of Although some phylogenetic uncertainty remains with regard glaciation during the Oligocene 33 Mya (McLoughlin, 2001), to the earliest diverging lineages of the family (Pternandra or Mour- new lineages, including Circaea and Fuchsia, evolved from a South iri + Memecylon), biogeographic reconstruction with just extant American lineage (29 Mya) and moved northward and south- taxa included supports a South American origin for the family with ward, respectively. Unlike previous studies (Berry et al., 2004; its crown diversification at 64 Mya. Across Melastomataceae, a Xie et al., 2009), we did not constrain the age of Circaea + Fuchsia minimum of five dispersal events occurred. The shift from South (41.5 Mya; based on Sytsma et al., 2004) and obtained an age esti- America to Australasia of the Pternandra clade occurred between mate more than 10 Myr younger, but much more in agreement 64 and 8 Mya. Whether this migration was facilitated by Southern with bioclimatic events. Consistent with previous studies, Circaea Hemisphere connections of South America, Antarctica, and Aus- exhibits a North American–Eastern Asian disjunct pattern (Xie tralia or by more direct LDDE is unclear. The spread of Pternandra et al., 2009), while Fuchsia demonstrates a pattern of LDDE from across tropical Australasia during the Paleogene was likely via dis- South America to the South Pacific (Berry et al., 2004). persal of fleshy capsules by frugivorous bats (Bolmgren and Previous phylogenetic studies of Lythraceae resolved most rela- Eriksson, 2010; Hodgkison et al., 2003; Tan et al., 2000), a mam- tionships among extant genera and supported an Old World origin malian lineage undergoing diversification at this time (Gunnell of the family (Conti et al., 1997; Graham et al., 2005; Huang and and Simmons, 2005; O’Leary et al., 2013). During the Eocene Shi, 2002; Morris, 2007). However, deep-level relationships of the (38 Mya), widespread dispersal of the Medinilla lineage into family have remained unresolved, likely reflecting an early, rapid sub-Saharan Africa, Madagascar, Eurasia, and Southeast Asia radiation (Morris, 2007). While our sampling represents a subset occurred, but limited sampling prevents any type of dispersal pat- of major lineages from the family, we do find evidence of rapid tern from being recognized. Near the Oligocene–Miocene border diversification near the base of Lythraceae. Lythraceae diverged (27–24 Mya), movement of three lineages out of South America from Onagraceae by the end of the Albian (105 Mya) with the occurred, including Rhexia moving into North America crown date for the family estimated at 96 Mya with two major (27 Mya), a clade within tribe Melastomeae dispersing to the clades established within 5 Myr. The fossil record of Lythraceae is Old World (Sub-Saharan Africa, Eurasia vs. Southeast Asia are diverse (wood, leaves, flowers, pollen and seeds) and widespread recovered equally as the most likely area), and LDDE of Memecylon in the northern hemisphere (Graham, 2013; Graham and from South America to the South Pacific (21 Mya). Graham, 1971, 2014) indicative of a Boreotropical distribution. Bio- Evidence previously presented by Conti et al. (2002, 2004) and geographical estimation provides strong support for a South Amer- Rutschmann et al. (2004, 2007) described the Gondwanan breakup ican ancestor of the Onagraceae + Lythraceae clade, suggesting as critical to the diversification of the CAP clade, with emphasis on movement from South America into North America early in the the isolation and rafting of the Indian subcontinent and subsequent history of the family (90 Mya). Fossils attributed to the family dispersal of Crypteroniaceae out of India. Our analyses strongly supporting this distribution include (reviewed in Graham, 2013): support (ML-BS/BI-PP = 100/1.0) the relationship between the Late Cretaceous (82–81 Mya) fossil pollen grains from Park County, Southeast Asian Crypteroniaceae with the west Gondwanan clade, Wyoming, USA, and western Siberia, Russia; leaf impressions and as well as the split between the South American Alzateaceae and wood from the Deccan intertrappean beds of western India (Late South African Penaeaceae (including Rhynchocalycaceae and Olini- Cretaceous or Early Paleocene 66–63.7 Mya); pollen grains from aceae; ML-BS/BI-PP = 100/1.0). While the ideal for calibration is to the Paleocene in France; fruits from the Eocene London Clay Flora; assign a fossil as close to the node to be estimated as possible, no 130 B.A. Berger et al. / Molecular Phylogenetics and Evolution 95 (2016) 116–136 current fossil evidence can confidently be assigned to the CAP clade nomenon in the Mascarenes (Heads, 2011; Le Pechon et al., 2015; (Mid-Miocene heterocolpate pollen assigned to Crypteroniaceae is Nowak et al., 2014; Renner et al., 2010). difficult to distinguish from Melastomataceae s.l. and Penaeaceae; The difficulties surrounding the biogeographic history of subf. Muller, 1975). In lieu of this limitation, we view that the use of Psiloxyloideae and the likely extinction of early lineages of Gond- multiple fossils across Myrtales provides a robust context to better wanan Myrtaceae elsewhere (Crisp et al., 2011; Kooyman et al., approximate the stem (91 Mya) and crown (53 Mya) dates of 2014; Thornhill et al., 2015) may impact our ability to precisely this lineage. Although our study used a subset of taxa from the determine the area of origin for the crown of the Myrtaceae and CAP clade, the estimated stem age (91 Ma) is compatible with the subsequent spread of the family whether via vicariance or dis- previously published estimates that utilized more intensive taxon persal. BioGeoBEARS interprets, with our sampling, an origin of sampling (141–106 Mya, Conti et al., 2002; 109–62 Mya, Myrtaceae in Africa (consistent perhaps with location of the oldest Rutschmann et al., 2004; 87.25–72.15 Mya, Rutschmann et al., known fossil) and subsequent LDDE to Australia. However, we can- 2007) and thus supports the ‘‘out-of-India” hypothesis previously not explicitly exclude the Antarctic, Australian, or South American proposed. parts of Gondwana from the ancestral area of Myrtaceae, with the evidence for this subsequently removed by extinction. As with other Gondwanan plant families, the biogeographic history of the 4.3.5. Historical biogeography of Myrtaceae Myrtaceae family may be more complex than an all-LDDE or all- Strong support (ML-BS/BI-PP = 100/1.0) for the inclusion of vicariance explanation (Crisp et al., 2011; Thornhill et al., 2015). Heteropyxis and Psiloxlon within Myrtaceae (now considered sub- Our results support a K–Pg boundary origin 67 Mya for subf. Myr- family Psiloxyloideae) agrees with previous results (Biffin et al., toideae in the part of Gondwana that broke off to form Australia. 2010; Conti et al., 1996, 1997; Gadek et al., 1996; Sytsma et al., This date is very similar to that found (64 Mya) by Thornhill 2004; Thornhill et al., 2012, 2015; Wilson et al., 2005). The split et al. (2012) based on another set of fossils, although later than of subfamily Psiloxyloideae from the rest of Myrtaceae (subfamily the 75 Mya date from Biffin et al. (2010) and Thornhill et al. Myrtoideae) occurred 85 Mya, which is in accordance with the (2015). Dispersal events to Africa, the Mediterranean, and the estimate of 83 Mya obtained by Sytsma et al. (2004). Similarity Americas occurred much more recently during the Miocene. between the estimates for the crown date is likely due to the place- Although our phylogenetic analyses did not fully resolve relation- ment of the fossil Myrtaceidites lisimae (=Syncolporites) as a mini- ships along the backbone of subf. Myrtoideae (Paleocene–Eocene), mum date for the split between the two subfamilies; however, all nodal inferences clearly support an Australian/South Pacific ori- where Sytsma et al. (2004) used a fixed date with a tight confi- gin. The rapid and extensive radiation of Myrtaceae within Aus- dence interval, we used a lognormal distribution to allow the pos- tralia (often unresolved in phylogenetic analyses) occurs from sibility for the clade to be much older. We note that the use of M. the Eocene into the Miocene as cooling and aridification intensified lisimae at the crown of Myrtaceae or M. mesonesus at the crown of (Byrne et al., 2008; Crisp and Cook, 2013; McLoughlin, 2001). Our subf. Myrtoideae as suggested by Thornhill et al. (2012) (or both sampling of South American taxa of fleshy-fruited myrtoids is lim- together) provides very similar dates for the Myrtaceae radiation. ited or includes genera of wider geographical distribution (e.g., Biogeographical estimations including subf. Psiloxyloideae have multiple areas attributed to a single placeholder); hence, there is been problematic for two reasons in Myrtaceae: (1) the restricted a lack of strong dispersal signal to the Neotropics relative to that ranges of Heteropyxis (SE Africa) and Psiloxylon (Mauritius and seen in Sytsma et al. (2004), which sampled broadly across the Réunion; part of the volcanic Mascarene Island chain) are consid- family, and in Thornhill et al. (2015), which recognized up to nine ered west Gondwanan, while the earliest diverging lineages of LDDE events in lineages with succulent fruits. The two main fleshy- subf. Myrtoideae are more indicative of an east Gondwanan origin fruited lineages (Syzygieae and Myrteae) appear to achieve their (Australian); and (2) the timing of the split between the two sub- pantropical distributions via dispersal during the Miocene. Phylo- families of Myrtaceae is younger than would be expected for a genetic relationships found strong support for tribal associations Gondwanan vicariance event (complete separation by 100– for Eucalypteae + (Leptospermeae + Chamelaucieae), Osbornieae 95 Mya; Pitman et al., 1993). The current distribution of subf. + Melaleuceae, and Metrosidereae + Backhousieae, all of which Psiloxyloideae reflects the growing challenge of biogeographical are also supported by Thornhill et al. (2012). studies that merge molecular systematics with stochastic models of area change. On one hand, sampling of extant taxa from across their distribution ranges is good; on the other hand, the overall his- 4.3.6. Historical biogeography of Vochysiaceae tory of both the lineages and the areas (especially with regard to Vochysiaceae is one of the more intriguing groups within Myr- volcanic island chains) is potentially more complex than current tales because it is so morphologically distinct that it was not placed data can reconstruct. We estimate the split between the two gen- in the order until Quirk (1980) and van Vliet and Baas (1984) era at 18 Mya, 20 Myr younger than estimates (40–38 Mya) described the presence of bicollateral vascular bundles and ves- obtained by Sytsma et al. (2004). Even with the reduced divergence tured pits—two anatomical synapomorphies of Myrtales. This age, the issue remains that Mauritius, oldest of the Mascarene anatomical association was subsequently corroborated by earlier Islands, is probably no older than 8 Myr (Bonneville et al., 1988; molecular phylogenetic studies (Conti et al., 1996, 1997; Sytsma Hantke and Scheidegger, 1998) suggesting that the island endemic et al., 2004). The amphi-atlantic distribution of the family has been Psiloxylon must have occupied other areas during the Paleogene of interest for decades and has prompted many discussions of (Sytsma et al., 2004). We recognize two plausible scenarios: (1) vicariance (e.g., Axelrod, 1970) versus dispersal (e.g., Smith, Psiloxylon dispersed directly to Mauritius sometime after its emer- 1973; Thorne, 1972, 1973) as explanatory for the disjunct pattern. gence from the Indian Ocean; and (2) Psiloxylon was perhaps situ- Sytsma et al. (2004) provided the first phylogenetic context using a ated on older islands of the chain now submersed and is restricted matK + ndhF chronogram including six American taxa and one spe- to the oldest emergent island. Because our evidence is no more cies of the African genus Erismadelphus. Those results indicated a conclusive than Sytsma et al. (2004), we also support the scenario separation from the Myrtaceae 93 Mya with a crown group radi- of an early Gondwanan origin in Africa, followed by a dispersal of ation for extant taxa 36–33 Mya. Our data demonstrate a similar unknown age to the Mascarenes (no earlier than 18 Mya), with pattern of an ancient divergence from the Myrtaceae (101 Mya) subsequent island hopping and extinction events. The presence followed by a long period of time until the crown radiation of old lineages on young islands is a repeated and perplexing phe- 39 Mya. B.A. Berger et al. / Molecular Phylogenetics and Evolution 95 (2016) 116–136 131

With regard to phylogenetic relationships, Sytsma et al. (2004) Eocene Thermal Maximum at 56.3 Mya. Whether either of these indicated a moderately supported sister relationship between two major climatic events with profound vegetation changes American Erisma and African Erismadelphus (ML-BS = 85), which (Jaramillo et al., 2010; McElwain and Punyasena, 2007) influenced was then sister to a clade comprised of the remaining five Neotrop- the shifts in diversification within Myrtales remains unknown. ical species (ML-BS < 50). The origin of African taxa out of a South The first and oldest shift in speciation rates within Myrtales, American clade was consistent with previous morphological and which occurred 76 Mya on the branch leading to the subf. Myr- molecular studies (Litt, 1999, 2003; Litt and Stevenson, 2003). In toideae (after divergence of the African lineage comprising this study with more limited taxon sampling but expanded loci Heteropyxis and Psiloxylon), coincides with the likely LDDE of its sampling, we found the African genus Erismadelphus sister to all ancestors to Australia from Africa (Fig. 3). A paraphyletic assem- Neotropical Vochysiaceae (BI-PP = 0.92). However, the African/ blage of largely Australian, capsular-fruited, and arid-adapted American split at the base of the family (rather than more internal) tribes (Xanthostemoneae, Lophostemoneae, Osbornieae, Melaleu- is still in agreement with previous studies as the stem lineage of ceae, Lindsayomyrteae, Syncarpieae, Eucalypteae, Leptospermeae, the family supports a South American origin. The timing of the and Chamelaucieae) are the first diverging clades within subf. Myr- South American – African split in the late Eocene indicates a LDDE toideae. Notably, a large, Southern Hemisphere clade containing all event in Vochysiaceae across an Atlantic Ocean that was then other tribes, including the fleshy-fruited and mesic-adapted taxa, almost 2/3rd formed, one of a number of similar South Ameri- diverged in the Paleocene and rapidly diversified starting at the can–African disjunct patterns best explained by LDDE rather than Eocene/Oligocene boundary. Thus, the shift in species diversifica- vicariance (Givnish and Renner, 2004; Givnish et al., 2000, 2004). tion in Myrtaceae is correlated with a shift to a new biogeographic area and seemingly to one of the strongest biome/niche shifts in 4.4. Myrtales exhibits three significant shifts in species diversification the Myrtales – tropical forest to arid communities. This niche shift, rates a pattern seen in other major radiations (Crisp et al., 2009; Donoghue and Edwards, 2014; Kissling et al., 2012; Linder et al., An important question now being addressed more critically in 2014), would be possible however only in the mid-Miocene evolutionary biology is the nature of the processes that lead to sig- (15 Mya) when initial aridification of Australia occurred after nificant shifts in speciation and/or extinction rates within clades South America broke free of Antarctica and initiated the Antarctic (e.g., Alfaro et al., 2009; Antonelli et al., 2015; Biffin et al., 2010; circumpolar current (Byrne et al., 2008). Thus, our dates (late Cre- Givnish et al., 2014; Linder et al., 2014; McGuire et al., 2014; taceous/early Paleocene) and even earlier dates of Ladiges et al. Rabosky et al., 2014; Smith et al., 2011). Relevant issues in detect- (2003), Thornhill et al. (2015) for the crown radiation of subf. Myr- ing significant rate shifts include incorporating extinction, phylo- toideae surprisingly suggest a rapid radiation of taxa all now lar- genetic uncertainty, phylogenetic scale, sampling density, gely adapted and confined to an arid biome that would not correlation and/or causality of biotic or niche attributes driving appear in Australia for at least another 40 Myr. the rate shifts. The program BAMM, as now implemented, can What biotic features of the Myrtaceae may be correlated with address a number of these issues. The largely Southern Hemi- this shift in species diversification? Ancestral state reconstruction sphere Myrtales is an ideal candidate for such studies considering of fruit type onto the phylogeny of Myrtales (unpublished data) its relatively old origins in the Cretaceous, a widespread biogeo- allowed us to test the hypothesis that birds or bats may have graphical distribution, occupation of diverse habitats, considerable played a role in this dispersal event, which was then followed by morphological diversity, the presence of both species-rich and the shift in speciation rate. We found no support for this hypothe- depauperate clades, and a now well-supported and dated phy- sis since the common ancestor of subf. Myrtoideae unambiguously logeny. Our analyses were conducted at two scales: (1) across Myr- had dry fruit, with fleshy fruits evolving much later some 30 mil- tales with 102 species, but carefully chosen to represent all the lion years ago. Other studies have also suggested the common major distinct clades within families, and (2) within individual ancestor and early divergent lineages of Myrtaceae to be dry families or family groups. fruited (Biffin et al., 2010). In that study focused on the subclade The baseline or average rate of net diversification for Myrtales is of the family with two, probably unrelated, fleshy fruit tribes, not striking (0.06–0.07 species/Myr; Table 3). This rate is very sim- increased rates of speciation were correlated with the acquisition ilar to those estimated by Magallón and Sanderson (2001) using of fleshy, bird-dispersed fruits. A second set of factors that may different approaches (0.079–0.097 species/Myr), and comparable be involved in the diversification of Myrtaceae include the evolu- to average rates they report for Eudicots and Angiosperms. tion and radiation of the most diverse group of bees, the corbicu- Antonelli et al. (2015) provided estimates for net diversification lates (Martins et al., 2014), and subsequently 50 Mya the in Myrtales (and 16 other lineages) from a supermatrix Angios- allodapines in Australia (Chenoweth and Schwarz, 2011). Bees perm tree, and while not strictly comparable to our study as they are the most common pollinators of Australian Myrtaceae today separated out rates based on biogeographical area, their results (Beardsell et al., 1993) and further studies should explicitly test are in line with those presented here (in species/Myr): African the impact of bee pollinators on subf. Myrtoideae. Interestingly, tropics (0.111), Asian tropics (0.051), American tropics (0.009). In the shift in speciation rate detected in Myrtaceae when examining general, and demonstrated in rates-through-time analysis the entire order was not detected in the Myrtaceae-only analysis (Fig. 4), Mytales exhibited long-term speciation rate decline for (Fig. 6). This is likely due to its occurrence toward the base of the the first 60 Myr and extinction rate increase throughout its entire Myrtaceae-only phylogeny. A supermatrix chronogram of Myr- 116 Myr diversification. However, at 65 Mya speciation (and taceae may be necessary to obtain sufficient taxon sampling to net diversification) increases rapidly (Fig. 4). Around this time more rigorously test the number and placement(s) of significant three significant rate shifts are seen within the Myrtales (Fig. 5) rate shifts and examine the potential role of these and other biotic and, though they are placed at different ages (76, 64, and abiotic factors in promoting diversification within the family. 43 Mya), all occur on branches that span the K–Pg boundary The second shift in species rates within Myrtales occurred (66 Mya). Antonelli et al. (2015) presented intriguing evidence, 64 Mya (branch age of 64.5–62.2 Mya) at the base of Melastom- based on their angiosperm-wide meta-analysis of net diversifica- ataceae, after the split between the genus Pternandra (tribe Kibes- tion rates in the context of biogeography, that a peak in angios- sieae) and the rest of the family (Fig. 4), and clearly occurred in a perm speciation and in biogeographical shifts between tropical South American lineage based on our phylogenetic estimate and regions is correlated to another climatic event – the Paleocene– ARE (Fig. 3). This shift corresponds with the suggested synapomor- 132 B.A. Berger et al. / Molecular Phylogenetics and Evolution 95 (2016) 116–136 phy for the subfamily Melastomatoideae of libriform fibers fruited (as in the Melastomataceae and Myrtaceae clades showing (Renner, 1993). This shift was not detected in the Melastomataceae significant speciation rate shifts). However, one feature this clade only BAMM analysis that shows support for a shift within tribe shares is winged fruits, although lacking in some members and Miconieae, after the divergence from Leandra. Both of these result- convergent with Macropteranthes (Manchester and O’Leary, 2010) ing shifts in speciation rates are somewhat unexpected because in the mangrove clade. Additionally, this clade shows pronounced fleshy-fruited clades such as the Miconieae are so diverse that levels of dispersal events between the three main tropical areas we would have predicted an association between diversification (Berger, 2012) and these events may have spurred repeated diver- rates and fleshy fruits within the Melastomataceae. This result sification in newly occupied areas. could also be due to the effects of scaling, sampling and phyloge- The analyses presented here, both at the ordinal and family netic resolution discussed below, which can affect the placement levels in Myrtales, reinforce the importance of scale, sampling, of shifts such as the one we expected in the common ancestor of and phylogenetic resolution when examining and interpreting pat- the tribe Miconieae. Future studies with higher taxon sampling terns of diversification. First, we demonstrate that scale influences in the Melastomataceae are needed to test this hypothesis. the best shift configuration despite the same taxa being analyzed. A second possible explanation for the shift in speciation rate For example, the rate shifts seen in Melastomataceae are different toward the base of the Melastomataceae is the evolution of porici- than that seen when the Melastomataceae are examined in the dal anthers that provide a pollen reward via buzz-pollination and context of the entire Myrtales (Fig. 6). Second, increased taxon are visited by female bees (Buchmann, 1983; De Luca and sampling (and thus larger numbers of terminals that can be scored Vallejo-Marín, 2013). Most of the 6000 species of Melastomataceae for extant species richness) should provide more precise identifica- are thought to be buzz-pollinated and poricidal anthers were tion of rate shift placements. And third, most diversification anal- inferred to have evolved early in the evolution of the family yses assume a single tree as representative of the group being (Clausing and Renner, 2001; Renner, 1989, 1993). Since only studied. Specifically, Biffin et al. (2010) acknowledge this issue as female bees can access the pollen reward offered by flowers in they evaluated rate shifts in speciation among the fleshy-fruited the family, buzz-pollination is considered a highly specialized Myrtaceae, two main lineages whose relationships to each other mechanism of pollination and likely contributed to the radiation are not yet known with certainty. Future studies should focus on of the family. Such is the strength of the relationship between two approaches. One is taking a supermatrix approach (e.g., these melastomes and their bee pollinators, that the Melastomat- Antonelli et al., 2015), although this approach entails assumptions aceae have been suggested to be trapped in an adaptive peak with (and thus trade-offs) using a single tree and estimating branch their bee pollinators, comparable to what is suggested for the lengths with a high percentage of missing data. A second approach Malpighiaceae and Solanaceae (Biffin et al., 2010; Renner, 1989). should examine PP distributions of trees (as we did in biogeo- Whether this close relationship between bees and the Melastomat- graphic analyses with BioGeoBEARS) and summarizing signal in aceae has resulted in the observed species rate shift is unclear. rate shifts across many trees. We note that a BAMM analysis con- Buzz pollination has rarely evolved within Myrtales aside from ducted on one of the most different 100 PP trees examined in this the Melastomataceae (but see Proença, 1992). For this reason, we study (Combretaceae sister to Onagraceae + Lythraceae), the same presently lack statistical power to test for the relationship between three significant rate shifts in speciation were uncovered within poricidal anthers/buzz-pollination and an increase in speciation Myrtales although at different points on their respective branches rate. (data not shown). Thus, these results should be viewed as coarse- A recent study looking at shifts in diversification rates associ- grained, and future studies using supermatrix approaches (e.g., ated with the transition from the forest to the fynbos habitat in Kriebel et al., 2015) and suites of alternative but likely trees may South Africa found a shift within the CAP clade that was not provide refinement on the placement of and processes leading to detected in our study (Onstein et al., 2014). The difference in shifts significant shifts in speciation rates within Myrtales. is likely due to the different strategies in taxon and gene sampling employed by both studies and highlights the sensitivity in detect- Acknowledgments ing shifts in diversification rates associated with scale. The final and third shift in speciation rate within Myrtales, and The authors thank Reinaldo Aguilar, Eladio Cruz, Roger Blanco the best supported with Bayes factor evidence, occurred within Segura, Álvaro Herrera, Aarón Rodríguez Contreras, Francisco Javier Combretaceae at 43 Mya. The shift occured after the earlier diver- Rendon Sandoval, Shirley Graham, Tim Curran, Craig Costion and gence of the west African tree genus Strephonema and the largely the Organization for Tropical Studies in aiding with field collec- pantropical mangrove clade. Although placed at 43 Mya, the tions; Olivier Maurin, Michelle van der Bank, Elena Conti, Frank branch exhibiting the rate shift spans from 94.5 to 33.2 Mya Rutschmann, Fabian Michelangeli, and the Royal Botanical Gar- (Fig. 5). Our biogeographical reconstruction indicated that the shift dens, Kew, for DNA samples; Jim Solomon, Jeremy Bruhl, and Ian occurred in South America, although Africa was important earlier Telford for curatorial assistance with herbarium samples; Bill in family differentiation. Greater taxonomic sampling of the Haber for the photograph of Alzatea verticillata; and Sarah Friedrich pantropical Combretum and Terminalia clade (subtribes Combreti- for graphical art assistance. The first author also thanks David nae and Terminaliinae) derived from this shift in speciation rate Baum, Cécile Ané, Tom Givnish, and Ken Cameron for helpful com- suggests Africa as a more likely ARE for this branch (Berger, ments and guidance toward completion of this dissertation 2012; Gere, 2014; Maurin et al., 2010). If so, the three significant research, and the Botany Department at the University of shifts within Myrtales at this scale involved three different South- Wisconsin-Madison for financial support. ern Hemisphere continents: Africa, South America, and Australia. The Combretaceae occupy a diversity of ecological niches and exhibit striking habit diversity (liana, mangrove, shrub, tree). The Appendix A. Supplementary material shift in speciation rate occured after the tree genus Strephonema and a mainly mangrove clade had already diverged. The clade Supplementary data associated with this article can be found, in demonstrating the increased rate of speciation, the Combretum the online version, at http://dx.doi.org/10.1016/j.ympev.2015.10. (272 spp.) and Terminalia (232 spp.) lineages, are rarely fleshy 001. B.A. Berger et al. / Molecular Phylogenetics and Evolution 95 (2016) 116–136 133

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