origin and evolution of chrysobalanaceae: insights into the evolution of plants in the neotropics

Origin and evolution of Chrysobalanaceae: insights intothe evolution of plants in the Neotropics

LÉA BARDON1, JULIETTE CHAMAGNE1,2, KYLE G. DEXTER1,3,CYNTHIA A. SOTHERS4, GHILLEAN T. PRANCE4 and JÉRÔME CHAVE1*

1Laboratoire Evolution et Diversité Biologique, UMR 5174, CNRS, Université Paul Sabatier, 31062Toulouse, France2Institute of Evolutionary Biology and Environmental Studies, University of Zurich,Winterthurerstrasse 190, CH-8057 Zürich, Switzerland3Royal Botanic Garden Edinburgh, 20a Inverleith Row, Edinburgh EH3 5LR, UK4Herbarium, Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3AB, UK

Received 15 December 2011; revised 1 June 2012; accepted for publication 15 June 2012

Some plant families show a striking imbalance in species diversity between the Neotropics and the Palaeotropics.The woody plant family Chrysobalanaceae is a typical example of this pattern, with 80% of the 531 species in theNeotropics. In order to test alternative interpretations for this pattern, we generated a dated phylogenetichypothesis for Chrysobalanaceae, using DNA sequence data from one nuclear and six plastid markers. Using amaximum likelihood approach, we jointly inferred ancestral areas and diversification rates in the Neotropics andPalaeotropics. We found that Chrysobalanaceae most probably originated in the Palaeotropics about 80 Mya. Thefamily dispersed into the Neotropics at least four times beginning 40–60 Mya, with at least one back-dispersal tothe Palaeotropics. Members of Chrysobalanaceae have experienced higher extinction, speciation and net diversi-fication rates in the Neotropics. Hence, the high species diversity of Chrysobalanaceae in the Neotropics appearsto be primarily caused by a higher speciation rate in this region. Several recent studies have shown highdiversification rates in Neotropical plant families, but have focused on Andean-centred taxa. Ours is the first studyto find a similar pattern in a family for which the centre of diversity is in eastern and central Amazonia. © 2012The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 19–37.

ADDITIONAL KEYWORDS: ancestral state reconstruction – diversification rates – fossil calibration –Neotropical plant biodiversity – phylogeny.

INTRODUCTION

Tropical forests house the majority of the 60 000–80 000 tree species in the world and, among tropicalforests, those of the Neotropics stand out as the mostdiverse, with > 20 000 tree species (Fine & Ree, 2006).Although the diversification and dispersal of plantlineages among tropical regions have probably shapedthis extant pattern, evolutionary hypotheses toexplain the exceptional Neotropical plant diversityremain poorly developed and tested (Raven &Axelrod, 1974; Stebbins, 1974; Gentry, 1982). If morelineages have been present longer in the Neotropics,

then higher Neotropical plant diversity could simplyreflect greater time for diversification. An alternative,but not mutually exclusive, hypothesis is that diver-sification rates are higher in the Neotropics, whichcould be a result of lower extinction rates and/orhigher speciation rates, as originally proposed byGentry (1982), who speculated that the uplift of theAndes was the foremost cause of higher speciationrates in the Neotropics, although late Cretaceousplant migration between North and South Americacould also have played a role in the assembly of theNeotropical flora. In order to test these hypotheses,studies are needed that track the origins and inter-continental dispersals of plant lineages and comparediversification, speciation and extinction rates among*Corresponding author. E-mail: [email protected]

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Botanical Journal of the Linnean Society, 2013, 171, 19–37. With 3 figures

© 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 19–37 19

tropical regions. Here, we use a molecular phyloge-netic approach to address the origin, dispersal anddiversification history of the pantropical plant familyChrysobalanaceae. This family is emblematic of thetropical diversity imbalance, with 80% of the 531species being found in the Neotropics.

The number of phylogenetic hypotheses availablefor plant groups has grown considerably in recentyears and, with fossil evidence, they have begun toshed light on the history of tropical plant lineages.For instance, numerous studies have shown thatintercontinental dispersal was rampant following thebreakup of Gondwana and must be accounted forwhen comparing diversity among tropical regions(Tiffney, 1985; Manchester, 1999; Morley, 2000, 2003;Sanmartin, Enghoff & Ronquist, 2001; Pennington &Dick, 2004). It is now known that Melastomataceaediversified in Eurasia before spreading to North andSouth America during the early Eocene (Renner,Clausing & Meyer, 2001). Likewise, it has been sug-gested that Rubiaceae (Antonelli et al., 2009), Guat-teria Ruiz & Pav. (Annonaceae) (Erkens, Maas &Couvreur, 2009) and Begonia L. (Begoniaceae)(Goodall-Copestake et al., 2010) probably originatedin the Palaeotropics and used the boreotropical routeto reach South America.

Many of these pantropical plant lineages show aconsistent pattern of being more diverse in the Neo-tropics, particularly in South America. If a lineage ismore diverse in the Neotropics, but originated else-where, then diversification rates must have beenhigher in the Neotropics to explain higher Neotropicaldiversity. This higher net diversification could be aresult of higher speciation rates (a cradle hypothesis)or lower extinction rates (a museum hypothesis). Insupport of the Neotropical ‘cradle’ hypothesis, thecomplex geological history of the Neotropics may haveyielded more frequent opportunities for allopatric spe-ciation. The uplift of the northern Andes since theearly Neogene (c. 23 million years ago (Mya)) yieldeda variety of new habitats, changed river flows andaltered climatic conditions (Burnham & Graham,1999; Linder, 2008; Antonelli et al., 2009; Hoorn et al.,2010). Since c. 3 Mya, the closure of the Isthmus ofPanama and the emergence of glacial/interglacial cli-matic oscillations have also played a role in drivinghigher speciation rates and thus shaping Neotropicalbiodiversity patterns (Gentry, 1982; Richardson et al.,2001; Bennett, 2004). In favour of the Neotropical‘museum’ hypothesis, we note that the Guianan andBrazilian cratons, geological formations dating backto around 2 giga years ago (Gya), have been above sealevel and were in tropical latitudes over much of theTertiary. Thus, South America has almost alwaysoffered ample space for tropical plant species tosurvive and diversify, at least over the past 65 million

years (Myr). If a larger block of rainforest was morecontinuously present through time, this may explainthe high diversity of the Neotropics in comparisonwith other tropical regions (Fine & Ree, 2006).

In order to contribute to a broader understandingof why the Neotropics have exceptional plant diver-sity, we studied the biogeography and modes ofdiversification in the pantropical plant familyChrysobalanaceae. The pantropical distribution ofChrysobalanaceae, its abundance and diversity inNeotropical forests, and its well-understood taxonomy(Prance, 1972; Prance & White, 1988; Prance &Sothers, 2003) make it an excellent family to studythe origins of high Neotropical plant diversity. It iscurrently unknown whether the high Neotropicaldiversity of the family is a result of a longer residencetime in the Neotropics, greater speciation rates and/orlower extinction rates. Here, we construct a datedphylogenetic hypothesis for the family, including 17 ofthe 18 genera, and 74 species (14% of the total diver-sity), based on DNA sequencing of six plastid markersand the nuclear marker ITS, and several fossil cali-bration points. We determine the biogeographicalorigin of the family and subsequent intercontinentaldispersal events. Further, whilst reconstructingancestral geographical states (Neotropical or Palaeo-tropical), we jointly estimated the rates of speciation,extinction and migration based on a maximum like-lihood technique. The results are discussed in thelight of recent evidence for patterns of Neotropicalplant diversification.

MATERIAL AND METHODSFAMILY DESCRIPTION AND SPECIES SAMPLING

Chrysobalanaceae (sensu Matthews & Endress, 2008;Yakandawala, Morton & Prance, 2010) is a tropicalwoody plant family in Malpighiales (Davis et al.,2005; APG III, 2009). The sister family is the mono-typic Euphroniaceae. Members of Chrysobalanaceaeare trees and shrub species often ecologically domi-nant in the New World tropics where they occupyboth moist and dry forest biomes (Prance, 1972). Themost recent taxonomic treatment of the familyincludes 531 species and 18 genera (Prance & White,1988; Prance & Sothers, 2003). Among these, 423species (80%) and eight genera are found in SouthAmerica. In floristic studies across the Amazon,members of Chrysobalanaceae comprise up to 10% ofthe trees in Amazonian forest plots and reach theirhighest diversity in the central and eastern Amazon(Prance & White, 1988; Hopkins, 2007). However,there is a greater generic diversification in the Pal-aeotropics, with 11 genera in Africa and seven inAsian tropics. A genus-level phylogenetic analysis has

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© 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 19–37

been conducted for the family based on the sequenc-ing of two DNA regions (rbcL and ITS), but it lackedsufficient resolution and sampling to assess the bio-geographical origins of the family or to calculatediversification rates (Yakandawala et al., 2010).

Tissue samples were collected throughout thetropics: South America (Guyana, French Guiana, Ven-ezuela, Brazil), Africa (Gabon, Cameroon, Benin,Central African Republic, Guinea, Senegal, Demo-cratic Republic of Congo), South-East Asia (Indonesia,Malaysia, New Caledonia), Madagascar and LaRéunion. The great majority of the collections fromSouth America were collected for the purpose of thisproject in the Guianas. Collections from Africa werecontributed by tissue exchange agreements with Uni-versité Libre de Bruxelles, the Royal BotanicGardens, Kew and Université de la Réunion, and ourown collections in the Central African Republic. Col-lections in South-East Asia were mostly obtainedby exchange agreements with the Royal BotanicGardens, Kew, and with the Botanic Garden ofSandakan, Malaysia. Appendix 1 reports the full listof samples and their collection locations. In addition,several sequences were retrieved from GenBank thatwere used in an earlier phylogenetic study of thefamily by Yakandawala et al. (2010).

DNA EXTRACTION, AMPLIFICATION AND SEQUENCING

New DNA for this study was extracted with aBiosprint 15 (Qiagen) auto-extractor, following theprotocol provided by the supplier (see Appendix 2).Some of the DNA extracts used in the analysis wereobtained through the DNA bank at the Royal BotanicGardens, Kew. The extraction protocol differed inincluding a standard cetyltrimethylammoniumbromide (CTAB)–chloroform extraction followed byethanol precipitation and washing, and then bydensity gradient cleaning and dialysis.

We initially attempted to amplify 30 markers forwhich primers have already been published in theliterature: 15 plastid intergenic spacers or introns(Hamilton, 1998; Shaw et al., 2007), two plastid genes(Savolainen et al., 2000; Dunning & Savolainen,2010), 13 low-copy nuclear genes (Strand, Leebens-Mack & Milligan, 1997; Li et al., 2008) and one mito-chondrial gene (Davis & Wurdack, 2004). We failed toamplify any of the low-copy nuclear genes or themitochondrial gene. Six plastid markers were finallyselected for this analysis: psbD-trnT, psbA-trnH, atpI-atpH, the ndhA intron, matK and rbcLa. In additionto these markers, the nuclear ITS region was ampli-fied (see Appendix 3 for details on primers).

DNA amplification was performed using thepolymerase chain reaction (PCR) protocols reported inAppendix 2. PCR products were cleaned and sequenced

on an ABI3730XL Automated DNA sequencer (Geno-screen, Lille, France). The two complementary DNAstrands were manually corrected and assembled usingSequencher version 4.8 (Gene Codes Corporation,2003). The total DNA matrix was aligned for eachmarker with the MUSCLE alignment program (Edgar,2004). The resulting sequence matrix was furthermanually edited in MEGA 4 (Tamura et al., 2007). Thenewly generated sequences are available on GenBankand EMBL (accessions JQ898692 to JQ899029, seeAppendix 1). (See also Table 1 for a summary of thesestatistics.)

PHYLOGENETIC RECONSTRUCTION

Sequences were assembled into three datasets. Thefirst dataset contained sequences obtained for the sixplastid regions: atpI-atpH, psbD-trnT, psbA-trnH,ndhA intron, matK and rbcLa. The second datasetincluded the ribosomal intergenic spacer ITS. Thealigned plastid and nuclear datasets included 6177 bpand 720 bp, respectively (Table 1). The third datasetincluded all sequences concatenated for the sevenmarkers (i.e. datasets 1 plus 2). For each marker, thenumber of available sequences is reported in Table 1.

Maximum likelihood analyses were conductedusing RaxML software version 7.0.4 (Stamatakis,Hoover & Rougemont, 2008) run on the completedataset including the seven markers through theCIPRES supercomputer cluster (http://www.phylo.org). Our alignment was separated into four parti-tions: ITS, matK, rbcLa and noncoding plastid DNAregions. Branch support was assessed using a rapidbootstrap procedure (Stamatakis et al., 2008). Branchlength in the phylogram denoted overall branch diver-gence. We also performed a tree reconstruction using

Table 1. Summary data on the sequence matrix used inphylogenetic tree reconstruction of Chrysobalanaceae.‘Length’ is the amplicon length retained for the analysis inthe consensus matrix, Ng is the number of sequencesnewly generated for the analysis, Nt is the total number ofsequences, including those retrieved from public domainsequence databases, and P is the percentage of availablesequences in the complete dataset

Amplicon Length Ng Nt P (%)

ITS 720 57 71 90matK 854 55 56 73rbcLa 650 60 72 90psbA-trnH 415 45 45 58ndhA 1304 47 47 61atpI-atpH 1258 45 45 56psbD-trnT 1696 29 29 38Total 6897 338 362

PHYLOGENETICS OF CHRYSOBALANACEAE 21


RaxML based on the ITS dataset only (nine of 77 ITSsequences were missing). We then compared the ITS-based tree with that based on the complete dataset totest whether our missing plastid sequences mayresult in tree topology issues, such as the presence of‘wildcard’ taxa (Platnick, Griswold & Coddington,1991). We finally performed a tree reconstructionbased on the plastid DNA dataset only and comparedthe resulting tree with that based on the ITS datasetto explore potential inconsistencies among topologies.The last two trees gave similar results, and so thetree based on the plastid DNA dataset only is notshown here.

In a second step, we constructed a dated phyloge-netic tree using BEAST software (Drummond &Rambaut, 2007). First, we constructed an initial phy-logenetic tree obtained using the PhyML version3.0.1 maximum likelihood phylogeny reconstructionprogram (Guindon et al., 2010), as implemented inseaview4 software (Gouy, Guindon & Gascuel, 2010).The consensus phylogram was then roughly datedusing PATHd8 version 1.0 (Britton et al., 2007) basedon four calibration points (three fossils and one tem-poral constraint on the root; see below). This tree thenserved as input for a combined analysis of divergencetimes and phylogenetic topology using the softwareBEAST version 1.6.1 (Drummond & Rambaut, 2007).The advantage of this preliminary procedure is toprovide BEAST with a phylogenetic hypothesis nottoo far from the most likely region for the parameters,hence avoiding floating-point computing errors. Sub-stitution and clock models were unlinked, and eachmarker evolved independently. For all markers, wechose the most parameterized molecular evolutionmodel, the general time-reversible (GTR) model. Thisassumes site heterogeneity modelled by a Gammadistribution, and takes into account proportions ofinvariant sites. Divergence times were estimatedunder a log-normal uncorrelated relaxed clock methodfor each partition and using the Yule model of spe-ciation. Several preliminary BEAST runs were per-formed using one Markov Chain Monte-Carlo(MCMC) for 1 000 000 generations to adjust theoperators for optimal mixing of the MCMCs. Then,ten independent runs of 20 000 000 generations wereconducted with sampling every 2000th generation forthe combined dataset.

The burn-in part of the MCMC was discarded (10%of the total number of generations). Post burn-in treeswere merged using LogCombiner version 1.6.1(Drummond & Rambaut, 2007) and performance wasevaluated using Tracer version 1.5 (Drummond &Rambaut, 2007). Mean evolutionary rates and diver-gence times were calculated using TreeAnnotatorversion 1.6.1 after the removal of 25% burn-in,keeping target heights concerning the node heights

option. The effective sampling sizes (ESSs) of eachparameter were checked at the end of each simula-tion, and were considered to be of good quality when> 200.

AGE CONSTRAINTS

Phylogenetic analyses and clock-independent datingestimates of Davis et al. (2005) provided a minimumage for the stem node of Chrysobalanaceae s.s. of~60 Myr (corresponding to the age found for thecrown age of Chrysobalanaceae s.s.) and a maximumage of ~90 Myr (corresponding to the age found forthe split between Chrysobalanaceae s.s./EuphroniaMart. and Dichapetalum Thouars/Trigonia Aubl.).The mean age found for the split between Chryso-balanaceae s.s. and Euphronia guianensis (R.H.Schomb.) Hallier f. was ~78 Myr. Here, we used thisdate for the Chrysobalanaceae–Euphroniaceae splitwhich was constrained by a normal probabilitydistribution.

In addition, two South American macrofossils andone North American palynofossil were used asminimum age constraints for three internal clades.Several endocarp fossils have been reported for thegenus Parinari Aubl., notably from the publication ofTiffney, Fleagle & Brown (1994), who studied an Ethio-pian plant assemblage dated at 16.1 Mya, and Wijn-inga (1996) from Colombia. Here, we used the slightlyolder date of the fossil endocarp also attributed toParinari from the Cucaracha Formation, Panama (F.Herrera and S. Manchester, Department of Biology,Florida Museum of Natural History, University ofFlorida, Gainesville, FL, USA, pers. comm.), whichwas dated at 19.0–17.5 Mya. Second, a fossil of HirtellaL. from the Gandarela Basin, Minas Gerais, Brazil,published by Duarte & Mello Filha (1980), is based onleaf material only. The dating of this rock formation isalso less clear, but is thought to be of the Eocene period(56–34 Mya). Third, we used the pollen type attributedto Chrysobalanus L. by Wodehouse (1932) found in theshales of the Green River Formation, Colorado, USA,and used by Davis et al. (2005). This pollen attributedto Chrysobalanus was dated to the early to middleEocene (49–34 Mya), and we used the most recent ageestimate (34 Myr) to constrain the tree dating.

For fossil calibrations based on a minimum age fora split, Ho (2007) suggested a log-normal prior dis-tribution with the probability of the nodal agedecreasing with time. Log-normal distributions withan offset were chosen to calibrate splits between thegenera Chrysobalanus (BEAST parameters: mean,2.6; standard deviation, 0.5; offset, 34), Hirtella(mean, 2.6; standard deviation, 0.5; offset, 34) andParinari (mean, 2.9; standard deviation, 0.5; offset,17.5) and their sister group, respectively, in BEASTanalyses. The lower bounds of the age estimates

22 L. BARDON ET AL.


(in Myr) were chosen as offsets for the log-normaldistributions.

ANCESTRAL STATE RECONSTRUCTION AND INFERENCE

OF DIVERSIFICATION RATES

Here, we sought to assign extant species into Neotropi-cal and Palaeotropical species (Appendix 1). We codedthe Neotropical/Palaeotropical distinction as a binarycharacter, and inferred the state of the internal nodesof the consensus phylogenetic tree obtained by theBayesian reconstruction. For two species, Parinariexcelsa Sabine and Chrysobalanus icaco L., sequencesfrom both South America and Africa were available.

To carry out this analysis, we used the binary-statespeciation and extinction (BiSSE) method (Maddison,Midford & Otto, 2007) in the package ‘diversitree’(FitzJohn, Maddison & Otto, 2009) of the R statisticalsoftware 2.13.0 (R Development Core Team, 2009).BiSSE computes the probability of a phylogenetic treeand of the observed distribution of a binary characterstate (here Neotropical or Palaeotropical) among thetree tips, given a model of character evolution, spe-ciation and extinction (Maddison et al., 2007). Pre-cisely, this method jointly estimates six parameters:speciation and extinction rates l1and m1 in the Neo-tropical zone and l0 and m0 in the Palaeotropical zone,and two shift rates from the Neotropical to the Pal-aeotropical zone (q10), and from the Palaeotropical tothe Neotropical zone (q01). BiSSE maximizes the like-lihood of obtaining the observed tree, given particularvalues of speciation, extinction and transition rates.It is possible to fix parameters to be equal to eachother by setting constraints, so that alternativemodels of evolution can be tested and then comparedwith each other by means of the assessment of like-lihood or Akaike information criterion (AIC) values.We tested four alternative models. First, we fitted the‘free’ model in which all six parameters were esti-mated. Then, we fitted a model with equal speciationrates (l1 = l0), one with equal extinction rates(m1 = m0), one with equal speciation and extinctionrates (l1 = l0 and m1 = m0) and, finally, one with equalmigration rates (q10 = q01). Recent extensions of theBiSSE model allow parameter estimation from anincompletely sampled phylogeny and measures ofparameter uncertainty (FitzJohn et al., 2009). Theposterior probability distribution of the model param-eters was approximated by MCMC, using an expo-nential prior for parameters whose mean was equal totwice the character-independent diversification rate(FitzJohn et al., 2009). To infer the model parametersin BiSSE, we ran two independent MCMCs for 10 000steps. Ancestral states were also inferred in the diver-sitree package taking into account the different ratescalculated from the best model.

RESULTSPHYLOGENETIC RELATIONSHIPS IN

CHRYSOBALANACEAE

The phylogenetic trees obtained with the completedataset using a maximum likelihood reconstructionand a Bayesian method are displayed in Figures 1and 2, respectively. Some genera appear to be mono-phyletic: Maranthes Blume (two species), Dactylad-enia Welw. (two species), Couepia Aubl. (sevenspecies), Hirtella L. (13 species) and Parinari Aubl.(seven species). Several clades are well supportedwhatever the method: clades L1, L2 and L3 includethree species of Licania Aubl., clade L4 includes fourLicania spp. and clade L5 includes 12 Licania spp.However, it is impossible to draw conclusions concern-ing the monophyly of Licania because of the weaksupport for basal nodes. We also define clade N whichincludes only Neotropical species (the genera Hirtella,Couepia and Licania), apart from Afrolicaniaelaeosperma Mildbr. Two of the three species of Mag-nistipula Engl. included in this analysis form a clade,but this clade excludes the accession of Magnistipulatamenaka (Capuron) F.White. Clade M includes onlyPalaeotropical species. We also define clade P, whichincludes Parinari, plus Neocarya macrophylla(Sabine) Prance ex F.White.

The topologies obtained with plastid markers only(result not shown) and the nuclear marker (Appendix4) were similar, although slight inconsistencies couldbe noticed, in particular with respect to the position ofsome Licania spp. The phylogenetic tree obtainedfrom the ITS dataset was consistent with the topologyobtained with the full dataset, further suggesting thatthe poor placement of some of the species in theplastid DNA tree (Magnistipula tamenaka, Acioaedulis Prance) is the result of a wildcard taxon effect.Hereafter, we discuss analyses using the phylogenetictrees generated from the combined dataset.

BIOGEOGRAPHY OF CHRYSOBALANACEAE

Figure 2 represents the dated phylogenetic tree withthe representation of ancestral states (Neotropical orPalaeotropical) as obtained by the BiSSE analysis(the 95% highest posterior distributions, or HPDs, forages are indicated on the phylogenetic tree presentedin Appendix 5). The most recent common ancestor(MRCA) of the family appears to have been native tothe Palaeotropics (probability P = 1.00) and, as theage of the family was constrained at 78 Myr, based onthe broader analysis of Malpighiales by Davis et al.(2005), migration into South America after thebreakup of Gondwana is highly likely.

Clade N is an almost strictly Neotropical clade,except for Afrolicania elaeosperma, the MRCA of



Figure 1. Phylogenetic tree of Chrysobalanaceae obtained with the full dataset (plastid plus nuclear markers) inferredfrom a maximum likelihood phylogeny reconstruction method (RaxML). Branches supported by bootstrap values > 80%are thick and those with bootstrap values of 60–80% are marked by an asterisk. The dating points used for Figure 2 aremarked with a white star.

24 L. BARDON ET AL.


which appeared to be Neotropical (P = 0.92). Thisclade may have diversified around 47 Mya (HPD,40–54 Myr), suggesting that the arrival of Chrysobal-anaceae in the Neotropics occurred before this date.Further, Hirtella appears to have diversified about10 Mya (HPD, 6–14 Myr) and Couepia about 11 Mya(HPD, 7–14 Myr).

An inspection of clade P, including Parinari, showsthat the MRCA of clade P, and the ancestor of allParinari, was Palaeotropical (P = 1.00) and the splitbetween Parinari and Neocarya macrophylla is

inferred at 33 Mya (HPD, 26–45 Myr). Evidence fromthe genus in both Africa at 16.1 Mya and the Neo-tropics prior to 17 Mya suggests that the dispersalinto South America is older than these dates.

JOINT ESTIMATES OF DISPERSAL, SPECIATION AND

EXTINCTION RATES

We used the consensus phylogenetic tree produced byBEAST to test the different ‘BiSSE’ models. Usinglikelihood ratio tests and AIC scores, we determined

Figure 2. Dated phylogenetic tree from a Bayesian phylogeny reconstruction (BEAST version 1.6). Branches supportedby posterior probabilities > 0.95 are thick and those with posterior probabilities of 0.85–0.95 are tagged by a star. On thesame tree, an ancestral state reconstruction was inferred using a likelihood reconstruction method (binary-statespeciation and extinction, BiSSE). Grey (vs. black) colour represents Neotropical (vs. Palaeotropical) species or internalnodes. States for unambiguous nodes are not represented for the sake of clarity.



that the best model was the fully parameterizedmodel in which speciation, extinction and transitionrates were different between the Neotropics and thePalaeotropics (Table 2).

Figure 3 shows the posterior distributions of the sixparameters estimated, together with diversification(speciation minus extinction) rates for each geo-graphical area. Both speciation and extinction rateswere found to be significantly higher in the Neotrop-ics than in the Palaeotropics. The net diversificationrate was higher in the Neotropics, although the dif-ference was not significant. Likewise, migration fromthe Palaeotropics into the Neotropics was higherthan vice versa, although this difference was notsignificant.

DISCUSSIONPHYLOGENETIC RELATIONSHIPS IN

CHRYSOBALANACEAE

Overall, our analysis provides strong support for thevalidity of the morphological characters used todelimit genera in this family (as discussed previouslyin Prance & White, 1988). Chrysobalanaceae s.s. wasdivided by Prance & Sothers (2003) into 18 genera onthe basis of morphological data. Among the sevengenera for which at least two species have beensampled, five were confirmed as monophyletic on thebasis of our sampling. Good support for generaHirtella and Couepia was found, supporting a previ-ously held view that these genera are well defined ona morphological basis (Martius & Zuccarini, 1832 inPrance, 1972). The monophyly of Parinari is also wellsupported in our study, as is the clade grouping ofParinari with Neocarya macrophylla. Neocarya mac-

rophylla was called Parinari senegalensis Perr. ex DC.in the past and grouped with Parinari excelsa in oneof the two Parinari sections (Prance & White, 1988).

For two genera, Licania and Magnistipula, ouranalysis indicates that they may not be monophyletic.Yakandawala et al. (2010) also suggested that theyare nonmonophyletic on the basis of morphologicaldata, although they stated that further work usingmolecular data and more taxa was needed to confirmthese findings. Our new analysis remains inconclu-sive because of the weak support for deep nodes andthe limited taxonomic coverage of the family in ourphylogenetic analyses. Prance (1972) proposed thatLicania should be separated into four subgenera:Moquilea Aubl., Parinariopsis Huber, Angelesia(Korth.) Prance & F.White and Licania. Clade L1includes three species of subgenus Moquilea, whereasall species present in the Licania clades L2, L3 andL5 belong to subgenus Licania. Clade L4 lumps threespecies belonging to subgenus Licania and one ofsubgenus Moquilea. Thus, the subgeneric classifica-tion of Licania may be in need of revision. In thegenus Magnistipula, two of the three species includedin our analysis belong to subgenus Magnistipula andgroup together. The third species, M. tamenaka, fallsout in a separate clade; it is one of the two species ofMagnistipula from Madagascar and belongs to sub-genus Tolmiella F.White. It cannot be excluded thatthe odd position of M. tamenaka is a result of incom-plete sequencing of the markers. An alternative pos-sibility is that M. tamenaka belongs to a segregate ofgenus Magnistipula. Finally, we note that the twoaccessions of Acioa Aubl. did not fall together as asingle clade. However, Acioa edulis was representedby a single sequence and, in another phylogeneticstudy (C. Sothers, unpubl. data), A. guianensis Aubl.and Acioa edulis formed a well-supported clade;therefore, we believe that the results regarding thephylogenetic position of M. tamenaka and A. edulisare most probably a result of a wildcard taxon effect.

BIOGEOGRAPHY OF CHRYSOBALANACEAE

On the basis of age estimates of Davis et al. (2005),Chrysobalanaceae s.s. originated well after the lastknown connection between Africa and South America(~105 Mya). The ancestral state reconstruction showsthat the MRCA of Chrysobalanaceae was Palaeo-tropical. The stem age of clade N, which comprisesthe majority of South American species, estimated at49 Mya (HPD, 44–59 Myr), and the crown age, esti-mated at 47 ± 6 Mya, place the temporal boundarieson the first dispersal of Chrysobalanaceae in the NewWorld. This corresponds well with the late Palae-ocene climate maximum (LPCM, 58–52 Mya), duringwhich a boreotropical forest putatively existed in

Table 2. Maximum likelihood value and Akaike informa-tion criterion (AIC) scores for alternative diversificationmodels. The ‘free’ model represents an unconstrainedmodel (all parameters are independently estimated). The‘Equal.l’ model assumes equal speciation rates betweenthe Neotropics and the Palaeotropics. The ‘Equal.m’ modelassumes equal extinction rates. The ‘Equal.q’ modelassumes equal migration rates across the Neotropics andthe Palaeotropics. Finally, the ‘Equal.lm’ model assumesthat both the speciation and extinction rates are con-strained to be equal between the two regions

Model Log-likelihood AIC

Free -321.46 654.91Equal.l -331.16 672.31Equal.m -332.01 674.02Equal.q -331.20 672.41Equal.lm -333.58 675.15

26 L. BARDON ET AL.


North America and Europe and the two continentswere connected by the North Atlantic land bridge(NALB; Tiffney, 1985). Wodehouse (1932) identified amiddle Eocene palynofossil in the Green River For-mation, Colorado, USA as Chrysobalanus pollen, sug-gesting that North America contained suitablehabitat for Chrysobalanaceae 45 Mya. We thereforesuggest that Chrysobalanaceae first dispersed fromAfrica to the Neotropics via Europe and NorthAmerica during the LPCM. The family may havethen subsequently dispersed from North Americainto South America via an island corridor of theproto-Greater Antilles, thought to have been present45–35 Mya (Iturralde-Vinent & MacPhee, 1999). It isimportant to emphasize, however, that future fossildiscoveries could modify or even overturn this pro-posed scenario.

The genus Parinari contains evidence of furtherdispersals between the Palaeo- and Neotropics. TheMRCA of all Parinari spp. sampled was found inthe Palaeotropics around 8 Mya (HPD, 8–17 Myr),whereas the stem age of the genus was estimated atc. 33 Mya. As Parinari fossils older than 16 Myr have

been found in both Africa and the Neotropics, thissuggests that we have underestimated the age of theParinari MRCA. Further, dispersal of this genusprobably occurred from Africa to the Neotropics, andthe first event must have been older than 16 Myr. Thepresence of P. excelsa in both the Neotropics andAfrica could suggest that there may have been onemore recent dispersal event. However, our accessionsof P. excelsa from both continents fall into two sepa-rate clades, raising the possibility that P. excelsa inAfrica and the Neotropics are distantly related and donot represent a recent dispersal event.

The other well-known example of recent trans-oceanic dispersal is that of Chrysobalanus icaco. Inour analysis, we have included one accession fromAfrica (sometimes called Chrysobalanus ellipticus Sol.ex Sabine, but synonymized under Chrysobalanusicaco by Prance & White, 1988) and one accessionfrom South America. Our dating of their split rangesbetween 4 and 13 Mya. When discussing possiblerecent trans-oceanic dispersal events, the cladeincluding genus Chrysobalanus deserves additionalscrutiny. The position of Afrolicania elaeosperma,

Figure 3. Posterior probability distributions of the rates of speciation, extinction, diversification and transition (species-Myr-1) generated in the binary-state speciation and extinction (BiSSE) analysis. Horizontal bars indicate the 95%

credibility interval. Estimates are shown for the Neotropics (in grey) and the Palaeotropics (in black).



which falls clearly into the Neotropical clade N, isalso an interesting result, because it must be asecondary dispersal from the Neotropics into thePalaeotropics.

Another group is poorly supported (bootstrap value,36; posterior probability, 0.74), but includes AcioaAubl., Licania subgenus Angelesia, Hunga Pranceand Exellodendron Prance. If confirmed, this cladewould form an interesting boreotropical group from abiogeographical standpoint: Exellodendron (fivespecies) and Acioa (four species) are found only inSouth America, Hunga (11 species) is found only inPapua New Guinea and New Caledonia, and Licaniasubgenus Angelesia (three species) is widespread inSouth-East Asia (New Guinea, Thailand, Philippines).This supports the hypothesis that Licania splendens(Korth.) Prance may be a different genus, and that itmay bear some affinities with Hunga. This also sug-gests that a Pacific dispersal route may have beenpossible. A better sampling of this group should yieldmore results.

The genus Maranthes Blume was, for a long time,placed in the genus Parinari (Prance & White, 1988),but is here placed in clade M, also containing generaDactyladenia and Grangeria Comm. ex Juss. Clade Mand Maranthes are found to be Palaeotropical in ourancestral state reconstruction analysis. However, onespecies, Maranthes panamensis (Standl.) Prance &F.White, is found in Central America. It would beimportant to include an accession of this species infuture analyses to confirm that M. panamensis repre-sents a recent trans-oceanic dispersal event in theMaranthes clade.

Prance & White (1988) suggested that long-distance oceanic dispersal may be common in Chryso-balanaceae, particularly in the light of the fact thatsix genera have species that can disperse via flota-tion. Our results suggest that this is indeed the case,as many of the more recent dispersal events do notseem to be explicable by dispersal via continents orland bridges.

DIVERSIFICATION IN THE NEOTROPICS

Differences in species richness between the Neotrop-ics and Palaeotropics could be explained by differ-ences in the time of species colonizations and/ordifferences in diversification rates between theseregions (Moore & Donoghue, 2007). Our study showsthat Chrysobalanaceae originated in the Palaeotrop-ics, ruling out longer residence time in the Neotropicsas an explanation for greater species richness there.Our diversification analyses showed that extinctionrates have been higher in the Neotropics than thePalaeotropics, which rules out a ‘museum’ hypothesisof low extinction. This result is somewhat surprising

because wide-scale aridification leading to dispropor-tionate extinctions is often invoked to explain why theAfrican flora is less diverse than that of SouthAmerica.

The only hypothesis to explain the higher Neo-tropical diversity of Chrysobalanaceae that was sup-ported by our analyses was the ‘cradle’ hypothesis ofhigher speciation rates in the Neotropics. The recentappearance and radiation of species-rich genera, suchas Hirtella and Couepia (~12 Mya), lend support tothe idea of a high speciation rate for Chrysobal-anaceae in the Neotropics. This result suggests thatthe current composition of Neotropical rainforestswas essentially set up during the Neogene (~23–5 Mya; Potter & Szatmari, 2009). It is tempting toexplain this high speciation rate by invoking theuplift of the Andes, as described by Gentry (1982).This uplift began for the northern Andes around23 Mya and reached peaks of intensity during thelate to middle Miocene (~12 Mya) and early Pliocene(~4.5 Mya) (Richardson et al., 2001; Erkens et al.,2009; Hoorn et al., 2010). However, the large majorityof species of Chrysobalanaceae are in the central andeastern Amazon, and this family is predominantly alowland rainforest group, with a few outliers in otherhabitats and few at high altitudes. Thus, it is likelythat the rise of the Andes did not play a direct rolein speciation events for Chrysobalanaceae. A similarscenario may be invoked for other Amazonian-centred Gondwanan families of woody trees, such asCaryocaraceae, Humiriaceae, Lecythidaceae, Sapota-ceae and Vochysiaceae.

An alternative explanation is that the instability ofthe South American climate during the Plioceneand/or Pleistocene may have led to recurrent phasesof retraction of species of Chrysobalanaceae intorefugia, thus causing allopatry and acting as a spe-ciation pump (Haffer, 1969; Prance, 1974). However, itis not clear that the palaeoclimate was less stable inSouth America than in Africa or other tropical regionsover the last 10 Myr. More detailed phylogenetichypotheses, including speciation events spanning thisepoch, would be needed to adequately test thisscenario.

Our best model allowed for different transitionrates between regions, with the transition rate fromthe Palaeotropics to the Neotropics being higherthan the reverse. This asymmetric dispersal mayreflect the prevailing direction of past ocean currents(Renner, 2004). Some of the recent long-distance dis-persal events appear to have occurred from Africa toSouth America (e.g. Dick, Abdul-Salim & Berming-ham, 2003, for Symphonia globulifera L.f.). If plantlineages experience higher speciation rates in areas towhich they have recently dispersed (sensu Moore &Donoghue, 2007), the observed asymmetric dispersal

28 L. BARDON ET AL.


may provide an additional explanation for thehigher species diversity of Chrysobalanaceae in theNeotropics.

CONCLUDING REMARKS

The current patterns in the diversity of Chrysobal-anaceae can be explained by multiple dispersal eventsbetween the Palaeotropics and the Neotropics andhigher speciation rates in the Neotropics. Our biogeo-graphical scenario for this family is comparable withthat developed by Tiffney (1985) and Morley (2000,2003), although more recent dispersal events prob-ably represent long-distance dispersal across oceans,and we are currently unable to provide a finer grainedscenario within the Palaeotropics. The causes of thehigh speciation rate of Chrysobalanaceae in the Neo-tropics remain unknown. Given the abundance ofspecies of Chrysobalanaceae in eastern and centralAmazonia and in drier habitat types, it is possiblethat the observed diversification is related to thecomplex geological and environmental history ofSouth America and not necessarily directly to theuplift of the Andes.

ACKNOWLEDGEMENTS

We thank Alexandre Antonelli and Toby Penningtonfor their invitation to contribute to this issue, FabianyHerrera and Steven Manchester for sharing unpub-lished dated fossils, and Bruce Tiffney for useful com-ments. We thank Joan Pereira, Colin Maycock(Herbarium of the Forest Reserve Centre at Sepilok,Malaysia), Olivier Hardy and Ingrid Parmentier forsharing their African collections and Maxime Réjou-Méchain and the ARF project of the Central AfricanRepublic for providing two samples from the Mbaïkiexperimental plot. This work has benefited from‘Investissement d’Avenir’ grants managed by AgenceNationale de la Recherche (CEBA: ANR-10-LABX-0025; TULIP: ANR-10-LABX-0041). It was partlyfunded by Agence Nationale pour la Recherche(BRIDGE project), the Programme Interdisciplinairede Recherche AMAZONIE (CNRS) and the Fondationpour la Recherche sur la Biodiversité (ATROPHYproject).

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32

32 L. BARDON ET AL.


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34 L. BARDON ET AL.


APPENDIX 2LABORATORY PROTOCOLS: DNA EXTRACTION AND

AMPLIFICATION

DNA extractionTo extract DNA, leaf material was placed into 2-mLEppendorf tubes with three autoclaved balls and wasground to dust with a Bio/Tissue Lyser. Cell lysis wasperformed as follows: 800 mL of cetyl trimethylammo-nium bromide (CTAB) and 10 mL of proteinase K wereadded to all tubes, which were then incubated for 2 hat 55 °C. Tubes were then centrifuged for 10 min at20 817 g (14 000 rpm). DNA was extracted from thesupernatant using the auto-extractor Biosprint 15(Qiagen), following the protocol provided by the sup-plier. This protocol involves a precipitation in isopro-panol, rinsing in pure ethanol and conservation insterile water.

DNA amplificationDNA amplification was performed using classicpolymerase chain reaction (PCR) protocols. A total of1 mL of DNA of each extract was added to 10 mL ofPCR buffer, 1 mL of deoxynucleoside triphosphate(dNTP), 1 mL of each primer at a concentration of20 mM, 0.2 mL of Taq polymerase and 35.8 mL ofsterile water. Then, DNA extracts were placed into athermal cycler to realize a PCR. For markers newlyamplified on Chrysobalanaceae, the first PCR protocoltested was that provided in the original publication. If

needed, the second protocol tested involved templatedenaturation at 93 °C for 1 min, followed by 35 cyclesof denaturation at 93 °C for 10 s, primer annealing at50 °C for 1 min and primer extension at 68 °C for1 min, followed by a final extension step of 2 min at68 °C (henceforth called LongRange). The protocolwas occasionally optimized by adding MgCl2 and per-forming temperature gradients. A touch down ampli-fication was programmed for the ITS region. Cyclingconditions were 20 decreasing cycles (30 s at 94 °C,30 s at the hybridization temperature and 45 s at72 °C) for which the hybridization temperature was62 °C at the first cycle and then decreased by 0.6 °Cevery cycle until it reached 50 °C. This process elimi-nates poor-quality strands during the amplification.This protocol was achieved by 20 normal cycles at ahybridization temperature of 50 °C to amplify all theremaining strands. For trnH-psbA, cycling conditionsinvolved template denaturation at 80 °C for 5 min,followed by 35 cycles of denaturation at 94 °C for 30 s,primer annealing at 56 °C for 30 s and primer exten-sion at 72 °C for 1 min, followed by a final extensionstep of 10 min at 72 °C. Finally, for rbcLa, a firstdenaturation stage at 95 °C for 1 min was followed by35 amplification cycles (1 min of denaturation at95 °C, 30 s of hybridization at 50 °C and 1 min ofstrand polymerization at 72 °C) and 7 min at 72 °C toachieve the polymerization of all the strands. In a fewcases, we used more than one tissue sample to gen-erate sequences for a species.

APPENDIX 3

Amplified region names, associated primer names and sequences, amplification protocols used and referencesfor primer sequences.

Region Characteristics Primer name and sequence (5’–3’)Amplificationprotocol Reference

rpl14-rps8-infA-rpl36

Plastid intergenic spacer rpl14: GGRTTGGAACAAATTACTATAATTCG LongRange Shaw et al. (2007)rpl36: AAGGAAATCCAAAAGGAACTCG

psbD-trnT Plastid intergenic spacer psbD: CTCCGTARCCAGTCATCCATA LongRange Shaw et al. (2007)trnT (GGU)-R: CCCTTTTAACTCAGTGGTAG

psaI-accD Plastid intergenic spacer accD: AATYGTACCACGTAATCYTTTAAA LongRange Shaw et al. (2007)psaI-75R: AGAAGCCATTGCAATTGCCGGAAA

atpI-atpH Plastid intergenic spacer atpI: TATTTACAAGYGGTATTCAAGCT LongRange Shaw et al. (2007)atpH: CCAAYCCAGCAGCAATAAC

rps16-trnK Plastid intergenic spacer rpS16x2F2: AAAGTGGGTTTTTATGATCC LongRange Shaw et al. (2007)trnK (UUU) x1: TTAAAAGCCGAGTACTCTACC

ndhA intron Plastid intron ndhAx1: GCYCAATCWATTAGTTATGAAATACC LongRange Shaw et al. (2007)ndhAx2: GGTTGACGCCAMARATTCCA

psbJ-petA Plastid intergenic spacer psbJ: ATAGGTACTGTARCYGGTATT LongRange Shaw et al. (2007)petA: AACARTTYGARAAGGTTCAATT

ndhJ-trnF Plastid intergenic spacer ndhJ: ATGCCYGAAAGTTGGATAGG LongRange Shaw et al. (2007)TabE: GGTTCAAGTCCCTCTATCCC

trnH-psbA Plastid intergenic spacer trnH RKr: ACTGCCTTGATCCACTTGGC Hamilton (1998)psbA FKr: CGAAGCTCCATCTACAAATGG

matK Plastid gene 1R_KIM: ACCCAGTCCATCTGGAAATCTTGGTTC Dunning & Savolainen (2010)3F_KIM: CGTACAGTACTTTTGTGTTTACGAG

rbcLa Plastid gene rbcL 1F: ATGTCACCACAAACAGAAAC Savolainen et al. (2000)rbcL 724R: TCGCATATGTACCTGCAGTAGC

ITS Ribosomal intergenic spacer ITS juliette1f: AGTGTTCGGATCGCGC UnpublishedITS juliette 1r: GCCGTTACTAGGGGAATCCT Unpublished



APPENDIX 4

Phylogenetic tree for Chrysobalanaceae obtained with the ITS dataset inferred from a maximum likelihoodmethod (RaxML). Branches supported by bootstrap values > 80% are thick and those with bootstrap valuesbetween 60 and 80% are indicated with a star.

36 L. BARDON ET AL.


APPENDIX 5

Dated phylogenetic tree obtained with the full dataset and inferred using Bayesian phylogeny reconstructionsoftware (BEAST version 1.6), with the 95% highest posterior distributions (HPDs) for ages. Branches supportedby posterior probabilities > 0.95 are thick and those with posterior probabilities between 0.85 and 0.95 areindicated with a star.



origin and evolution of chrysobalanaceae: insights into the evolution of plants in the neotropics

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