sign in sign up
<<
Home , Boswellia frereana, Boswellia sacra, Bursera, Bursera fagaroides, Bursera hindsiana, Bursera lancifolia

TAXON 61 (2) • April 2012: 333–343 Becerra & al. • Monophyly of

The monophyly of Bursera and its impact for divergence times of

Judith X. Becerra,1 Kogi Noge,2 Sarai Olivier1 & D. Lawrence Venable3

1 Department of Biosphere 2, University of , Tucson, Arizona 85721, U.S.A. 2 Department of Biological Production, Akita Prefectural University, Akita 010-0195, Japan 3 Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, Arizona 85721, U.S.A. Author for correspondence: Judith X. Becerra, [email protected]

Abstract Bursera is one of the most diverse and abundant groups of and of the Mexican tropical dry forests. Its interaction with its specialist herbivores in the chrysomelid , is one of the best-studied coevolutionary systems. Prior studies based on molecular phylogenies concluded that Bursera is a monophyletic genus. Recently, however, other molecular analyses have suggested that the genus might be paraphyletic, with the closely related , nested within Bursera. If this is correct, then interpretations of coevolution results would have to be revised. Whether Bursera is or is not monophyletic also has implications for the age of Burseraceae, since previous dates were based on calibrations using Bursera fossils assuming that Bursera was paraphyletic. We performed a phylogenetic analysis of 76 and varieties of Bursera, 51 species of Commiphora, and 13 outgroups using nuclear DNA data. We also reconstructed a phylogeny of the Burseraceae using 59 members of the family, 9 outgroups and nuclear and chloroplast sequence data. These analyses strongly confirm previous conclusions that this genus is monophyletic. New calculations of the age of Burseraceae date the beginning of its diversification to at least 93 million years ago.

Keywords Bursera ; Burseraceae; divergence times; monophyly; phylogeny

Supplementary Material The alignment files are available in the Supplementary Data section of the online version of this article (http://www.ingentaconnect.com/content/iapt/tax).

INTRODUCTION & Venable, 1999a; Becerra & al., 2009). Phylogenetic relation- ships of Bursera and Blepharida have been central for testing Bursera Jacq. ex L. comprises about 105 species distrib- aspects of coevolution (Becerra, 2007; Becerra & al., 2009). uted from southern U.S. to Peru and the Caribbean. Most of Because of the tightness of their relationships and high degree the species (~ 85%) are endemic to , where they are of coadaptation, Bursera and Blepharida are now considered some of the most conspicuous woody members of the floras one of the model systems to study –herbivore coevolution of arid and semiarid regions (Rzedowski, 1978; Rzedowski (Hillis, 1997; Mitchell-Olds & Bergelson, 2000; Speight & al., & al., 2006). They are typically low- to medium-size trees. 2008; Futuyma, 2009; Futuyma & Agrawal, 2009). Further- The genus is relatively well known taxonomically, and it has more, because Bursera is highly adapted to the ecological and been divided into two subgenera of roughly equal size, B. subg. climatic conditions of the dry forests, and of great physiog- Bursera and subg. Elaphrium Jack. (McVaugh & Rzedowski, nomic importance in these forests, its evolution and diversifica- 1965; Rzedowski & Kruse, 1979). tion has been used as an indicator of the historical expansion of Bursera is an old tropical genus, whose distribution in the the tropical dry forests of Mexico (Rzedowski, 1978; Becerra, past extended across central North America. Fossil of 2005; Pennington & al., 2006). B. serrulata (Lesquereux) MacGinitie, a putative relative of the Several hypotheses have been advanced to identify the phy- modern B. tecomaca (DC.) Standl., are abundant in the early logenetic relationships within Bursera using molecular markers. Oligocene beds of Florissant, in Colorado (MacGinitie, 1953; The most comprehensive study included 66 Bursera species Rzedowski & Kruse, 1979). Fossil pollen of Bursera has also and 5 outgroups, and was reconstructed using sequences from been reported from these beds (Meyer, 2003). Another fossil the internal transcribed spacer (ITS) region, the external tran- species belonging in the subg. Elaphrium, Bursera inaequi- scribed spacer (ETS) region, the 5.8S coding region, and the 5S lateralis MacGinitie, is known from the Eocene Green River, non-transcribed (5S-NTS) region of the nuclear ribosomal DNA Colorado-Wyoming, suggesting that the genus had began diver- (Becerra & Venable, 1999b; Becerra, 2003a). This molecular sifying by at least 45 million years (Ma) ago (MacGinitie, 1969). phylogeny shed light not only on the relationships of the main Bursera has been the subject of several ecological, chemi- lineages but also on the relations between many of the individual cal, and evolutionary studies. These have a long history species. Analyses strongly supported the monophyly of Bursera, of interaction with the phytophagous beetle genus Blepharida as well as monophyly of its two subgenera. It also suggested that Chevrolat (Chrysomellidae) (Becerra, 1997, 2003b; Becerra Bursera is sister to Commiphora Jacq.

333 Becerra & al. • Monophyly of Bursera TAXON 61 (2) • April 2012: 333–343

Other studies using molecular markers as well as morpho- MATERIALS AND METHODS logical characteristics have also explored relationships of Bursera and its allied genera within Burseraceae (Gillett, 1980; Harley Phylogenetic analysis of Bursera and Commiphora & Daly, 1995; Clarkson & al., 2002; Weeks & al., 2005). Burs- era + Commiphora is well supported as a monophyletic group, Taxonomic sampling. — Our taxonomic sample consisted Bursereae, subtribe Burserinae. Molecular dating analyses using of 76 species and varieties of Bursera (see Appendix), including fossils of Bursera and L. (a more distantly related 42 species of subg. Bursera and 34 of subg. Elaphrium. This genus) to calibrate a phylogeny of Burseraceae indicated that the sampling represents about 72% of the genus and its entire range family began to diversify after 58 Ma ago (Weeks & al., 2005). of geographic distribution (i.e., all the main regions of distri- Recently, however, the monophyly of Bursera was chal- bution of the genus in Mexico, including Baja , and lenged. A phylogenetic analysis of 50 Burseraceae species also Central and South America, and the Caribbean). Fifty-one including 10 species of Commiphora, and 12 Bursera spe- species of Commiphora were sampled also representing all the cies based on sequences of the ETS region, the chloroplast major regions of geographic distribution of the genus (includ- psbA-trnH spacer region, and the rps16 intron, suggested that, ing , , South America, and ). Because while Commiphora is monophyletic, Bursera is not (Weeks C. leptophloeos (Mart.) J.B. Gillett has a disjunct distribution in & al., 2005). In this phylogeny Bursera is either paraphyletic, South America, we sampled both regions with possibly distinct with Commiphora nested within Bursera and with B. subg. populations. Our outgroups were chosen so as to sample taxa Elaphrium in a basal position and subtending the sister pair of that are closely related to Commiphora and Bursera as well as subg. Bursera and Commiphora; or these three clades form a some that are more distantly related, but still with sequences polytomy. Another phylogenetic reconstruction that included that are straightforward to align. They included seven species 37 species of Commiphora and 13 Bursera species could not of three Burseraceae genera that are closely related to Burseri- support Bursera’s monophyly using the same markers as Weeks nae ( Bu r m . f., Crepidospermum Hook. f., & al. (2005) with Burseraceae (Weeks & Simpson, 2007). Ac- Gaertn.), three species of two more distantly related genera cording to Weeks & al. (2005) the discrepancies of their results (Canarium, Roxb. ex Colebr.) as well as three spe- with previously published phylogenies supporting the mono- cies of . phyly of Bursera were due to their more comprehensive sam- DNA sequencing. — To reconstruct the phylogeny we used pling of Commiphora species compared to the earlier studies the ITS1 and ITS2 and the 5.8S coding region (ITS region), as (Becerra, 2003a) which included only five species. However, well as part of the ETS region of nuclear ribosomal DNA. We the paraphyly of Bursera was inferred using only 13 species have used these markers for Bursera and Commiphora previ- (Weeks, & al., 2005), whereas previous studies indicating ously, and they provided nearly full resolution at the deeper and monophyly included 66 species. Therefore, it seems that none medium branching levels in both genera (Becerra & Venable, of the phylogenetic studies carried out to date have used a suf- 1999b; Becerra, 2003a). ficiently comprehensive taxonomic sampling of both Bursera Many of the sequences of Bursera and some of Commi­ and Commiphora to test for the monophyly of Bursera. phora used in this study were already available in GenBank Whether Bursera is or is not a monophyletic genus has im- from our previous studies and also from studies by others (Ap- portant consequences. First, conclusions about the evolutionary pendix). For species not considered in previous studies and interactions between Bursera and its herbivores could change for which sequences were thus not available (a total of 49), we depending on the phylogenetic hypotheses considered. Sec- collected fresh material from the field, botanical gardens, ond, because the time of origin and diversification of Bursera- and private collections. Voucher specimens of all Bursera and ceae was calibrated with Bursera fossils (Weeks & al., 2005), Commiphora taxa used in this study have been deposited at changes within its phylogeny (i.e., Bursera paraphyly) might ARIZ. affect inferences regarding the age of the family. Third, the DNA extraction from leaf samples was performed using the current would have to be updated to accommodate DNeasy Plant-Mini-Kit (Qiagen, Valencia, California, U.S.A.) the new molecular systematic relationships of Bursera. following the manufacturer’s instructions. For DNA ampli- The main goal of this study was to perform a molecular fication and sequencing we followed the protocols described phylogenetic analysis using improved and more comprehensive in Becerra & Venable (1999b) and Becerra (2003a). Sequenc- taxonomic sampling to address the monophyly of Bursera. We ing was carried out by an ABI 3730xl DNA analyzer (Applied present a phylogeny of 76 species and varieties of Bursera, Biosystems, Foster City, California, U.S.A.) at the Genomic 51 species of Commiphora, and 13 other Burseraceae species Analysis & Technology Core of the University of Arizona. DNA as outgroups based on nuclear sequence data. To further ex- sequences were assembled using Sequencher v.4.0 (Gene Codes plore the relationship between these two genera we also recon- Corp., Ann Arbor, Michigan, U.S.A.) Voucher information and structed a phylogeny of 59 Burseraceae species that includes GenBank accession numbers are found in the Appendix. 15 species of Commiphora and 15 species of Bursera and 9 DNA sequences obtained in this study were added to the outgroups using nuclear and chloroplast sequence data. Lastly, sequences obtained from GenBank and aligned, first using we time-calibrate this Burseraceae reconstruction with Bursera Clustal W v.2.0 (Larkin & al., 2007), and then correcting mis- fossils as well as recently discovered Canarium fossils to as- alignments by eye with MacClade v.4.0 (Maddison & Mad- semble the diversification history of Burseraceae. dison, 2000).

334 TAXON 61 (2) • April 2012: 333–343 Becerra & al. • Monophyly of Bursera

Parsimony analysis. — Phylogenies were inferred from DNA sequences. — To reconstruct the phylogeny we used the sequence matrix using maximum parsimony (MP) analysis the ITS region, part of the ETS region, and the rps16 intron. with PAUP* v.4.0 b et a 10 (Swofford, 2002). The ITS region Sequences were available in GenBank. They were aligned first and the ETS datasets were analyzed first separately and then using Clustal W v.2.0 (Larkin & al., 2007) and then correcting in combination. Indels of more than one base pair (bp) were misalignments by eye with MacClade v.4.0 (Maddison & Mad- treated as single characters resulting from one mutational dison 2000). The following species did not include ITS sequence event. Heuristic searches were conducted with 1000 random data: Bursera spinescens Urb. & Ekman, Canarium pilosum replicates, based on branch swapping and bisection recon- A.W. Benn., C. vulgare Leenh., C. littorale Blume, C. indi- nection (TBR), and unordered and equally weighted characters. cum L., C. zeylanicum (Retz.) Blume, Protium unifoliolatum Individual branch support was determined by performing a MP Merr., griffithi (Hook. f.) Engl., S. apiculata Benn, Dac- bootstrap analysis with 1000 replicates. ryodes klaineana (Pierre) H.J. Lam, D. buettneri (Engl.) Lam, Likelihood analysis. — The phylogenetic relationships of Boswellia neglecta S. Moore, B. frereana Birw., Trattinickia species also were inferred under the assumption of maximum glaziovii Swart, and T. cf. burserifolia Mart. These species did likelihood (ML) using PAUP* v.4.0 with the combined ITS and not include rps16 data: Bursera biflora (Rose) Standl., B. mo- ETS dataset. The best-fit model of nucleotide substitution for the relensis Ramirez, Commiphora kua Vollesen, C. lamii H. Per- dataset including outgroups was identified by ModelTest v.3.7 rier, C. simplicifolia H. Perrier, Tratinickia glaziovii, Boswellia (Posada & Crandall, 1998) under the Akaike information crite- hildebrandtii Engl., and Dodonaea viscosa. Crepidospermum rion (AIC) as the GTR + I + G model. The specified substitution prancei Daly did not include ETS sequence data. rate matrix was 0.7125, 1.7919, 0.9789, 0.4565, 2.4803, 1.000. Parsimony analysis. — The analysis was conducted as Nucleotide frequencies were A = 0.2272, C = 0.3259, G = 0.3025, above for the Bursera-Commiphora reconstruction but using and T = 0.1444. The assumed proportion of invariable sites was the combined ITS, ETS, and rps16 datasets. 0.0921 and a gamma distribution was assumed for rates at vari- Likelihood analysis. — The phylogenetic relationships of able sites with a shape parameter (alpha) of 0.7331. Heuristic species were inferred under the assumption of maximum like- searches were conducted with 500 random replicates based on lihood (ML) also using PAUP* v.4.0 with the three datasets branch swapping and tree bisection reconnection (TBR), and combined. The best-fit model of nucleotide substitution for unordered and equally weighted characters. Node support was the dataset including outgroups was identified by ModelTest estimated with an ML bootstrap analysis with 500 replicates. v.3.7 (Posada & Crandall, 1998) under the Akaike information Bayesian inference. — A Bayesian analysis was also criterion (AIC) as the GTR + I + G model. The specified sub- performed using MrBayes v.3.1.2n (Huelsenbeck & Ronquist, stitution rate matrix was 1.227, 2.2561, 0.4138, 0.8538, 3.1846, 2001). The ITS + ETS dataset was partitioned to incorporate the 1.000. Nucleotide frequencies were A = 0.2692, C = 0.2181, G best-fitting model of evolution for each region. The best fitting = 0.1975, and T = 0.3152. The assumed proportion of invari- model of sequence evolution was chosen for each marker using able sites was 0.3180, a gamma distribution was assumed for ModelTest v.3.7 (Posada & Crandall, 1998). The AIC criterion rates at variable sites with a shape parameter (alpha) of 0.6765. was implemented. Evolutionary models that best fitted the ITS Heuristic searches were conducted with 500 random replicates and ETS entire datasets were GTR + I + G and TVM + I + G, re- based on branch swapping and TBR, and unordered and equally spectively. The Markov chain Monte Carlo (MCMC) analysis weighted characters. Node support was estimated with an ML consisted of two independent runs each with 106 generations, bootstrap analysis with 500 replicates. four chains (three heated, one cold), and 10 trees saved every Bayesian inference. — A Bayesian analysis was performed 500 generations. The analysis was discontinued after the two using MrBayes v.3.1.2n (Huelsenbeck & Ronquist, 2001). The simultaneous runs reached the same distribution (the average ITS + ETS + rps16 dataset was partitioned to incorporate the best- standard deviation of split frequencies dropped to below 0.01). fitting model of evolution for each region. Evolutionary models The chains achieved stability within the first quarter of the sam- that best fitted the ITS and ETS entire datasets according to ples, so the 5000 trees generated in the burn-in were discarded. ModelTest v.3.7 (Posada & Crandall 1998) were GTR + I + G, The remaining trees were used to generate a 50% majority-rule TVM + I + G, and GTR + G, respectively. The mcmc analysis consensus tree and to calculate posterior probabilities. consisted of two independent runs each with 106 generations, four chains (three heated, one cold), and 15 trees saved every Phylogenetic reconstruction of Burseraceae 500 generations. The analysis was discontinued after the two simultaneous runs reached the same distribution. Six thousand We performed a phylogenetic analysis of Burseraceae two hundred trees generated in the burn-in were discarded. using 59 species of this family using nuclear and chloroplast The remaining trees were used to generate a 50% majority-rule data. The taxonomic sampling consisted of all of the species consensus tree and to calculate the posterior probabilities. analyzed in Weeks & al. (2005) except for members of Triomma Hook. f., which were not sampled. We extended the number of Divergence time estimation Commiphora and Bursera taxa to 15 species for each genus. As outgroups we used 8 species of Anacardiaceae, the sister Divergence times were estimated using fossil calibrations. group of Burseraceae (Wang & al., 2009), and one, Dodonaea Several fossils attributed to Burseraceae are available includ- viscosa (L.) Jacq., belonging to Sapindaceae. ing some recently discovered ones that are well-preserved. We

335 Becerra & al. • Monophyly of Bursera TAXON 61 (2) • April 2012: 333–343

used the two fossils that are most satisfactorily attributed to support. This was expected since the molecular markers were specific clades. Forty-seven million years old fossil endocarps chosen for their high level of resolution at the higher and me- identified as Canarium sp. have been recently reported from dium branching levels of these genera (Becerra, 2003a). the exceptionally well-preserved biota of the Middle Eocene In all three bootstrap analyses (MP, ML, Bayesian) Com- Messel oil shale in the Messel Pit near Darmstadt, Germany miphora and Bursera were each supported as monophyletic, (Collinson & al., 2010) and previously also from sediments with 100 MP, 100 ML, and 100 Bayesian and 98 MP, 96 ML, from a maar lake at Eckfeld/Eifel, Germany (Wilde & Fran- and 100 Bayesian percentage bootstrap support, respectively. kenhäuser, 1988). The other fossil consists of leaves identified Within Bursera two well-supported clades coincide with the as B. serrulata, a common member of the early Oligocene (34 two recognized subgenera (bootstrap support of 95, 100, and Ma) beds of Floissant, Colorado, that resembles closely the 100% for subg. Bursera and 99, 100, and 100% for subg. Elaph- extant B. tecomaca (subg. Elaphrium; MacGinitie, 1953). The rium). The analyses also recovered four well-resolved clades Canarium fossil was used to impose a minimum age constraint that have been traditionally recognized within subg. Bursera on the node of the Canariae (defined by the most recent com- (Becerra, 2003a). mon ancestor [MRCA] of C. vulgare and buettneri). Phylogenetic analysis of Burseraceae. — Parsimony anal- The B. serrulata fossil was used to impose the minimum age ysis of the combined ETS, ITS, and rps16 datasets yielded 1555 constraint of subg. Elaphrium, on the node determined by the trees of 2957 steps. Of the 2269 characters, 677 were parsimony- mrca of B. tecomaca and B. sarukhanii Guevara & Rzed. informative characters. Almost half of these phylogenetically Calibrations were done with the penalized likelihood informative characters were in the ITS region (a total of 313), method (PL, Sanderson, 2002) implemented in the program while the ETS region contributed 232 (34%) and the rps16 region r8s (Sanderson, 2003). Because several of the Burseraceae taxa had only 132 (19%). Maximum likelihood analysis resulted in a lacked ITS sequence information, only the ETS and rps16 da- single tree with a score of −ln(L) = 18,880. Here again, MP, ML, tasets were included in this analysis. Taxa for which ETS or and Bayesian analyses inferred a distribution of phylogenies rps16 information was missing were omitted. Beiselia mexi- with very similar topologies and support (Fig. 2). cana Forman had to be excluded from phylogeny calibrations. All analyses (MP, ML, Bayesian) confirmed that Burs- The sequences of this species are quite different from other eraceae is monophyletic. The analyses also recovered several Burseraceae and these discrepancies may cause misalignments well-resolved clades that have been recognized within the fam- with the other sequences that could artificially increase its age ily. There is good support for a monophyletic Protieae (98 MP, of divergence. 88 ML, and 100 Bayesian percentage bootstrap support) and When using the r8s program, the value of the smoothing a monophyletic Canarieae (85 MP, 100 ML, and 100 Bayesian factr (sf) was determined using the cross-validation procedure percentage bootstrap support). However, as reported by Weeks implemented in the program. This value was 100. The scale & al. (2005), Bursereae is not monophyletic and it is divided for rate penalty was logarithmic and terminal taxa with zero into a monophyletic Burserinae (90 MP, 86 ML, and 100 Bayes- branch lengths were deleted. Standard deviations of divergence ian percentage bootstrap support) that is sister to Protieae, and times of the deepest nodes were estimated following Baldwin the genus Boswellia that is sister to Canarieae. As in previous & Sanderson (1998) using 100 resampled data matrices. molecular phylogenetic analyses (Clarkson & al., 2002; Weeks We repeated our calibrations using a topology in which & al., 2005), Beiselia Forman is basal to all other taxa. In this Commiphora is nested within Bursera, as concluded by Weeks phylogeny the monophyly of both Commiphora and Bursera is & al. (2005), in order to compare the resulting age estimates again strongly supported, with 96 MP, 88 ML, and 100 Bayes- with those inferred using a phylogeny in which Bursera is ian bootstrap support for Commiphora; and 95 MP, 84 ML, monophyletic. and 100 Bayesian percentage bootstrap support for Bursera. Timing the diversification of Burseraceae. — Fig- ure 3 shows the time-calibrated phylogeny of Burseraceae. RESULTS According to this calibration, diversification of extant Burs- eraceae started at least at the beginning of the Late Cretaceous Phylogenetic analysis of Bursera and Commiphora. — period, about 93 Ma ago. With a topology in which Bursera The aligned sequences of the ETS region resulted in a matrix is paraphyletic, with Commiphora nested within Bursera, of 384 characters while the one of the ITS region included the minimum age of diversification of Burseraceae is 98 Ma 890 characters. Parsimony analyses of the combined datasets (± 18.2 Ma). Thus, while the monophyly of Bursera has a mod- yielded 780 trees of 3543 steps. Of the 384 ETS characters, 200 est effect on the estimated age of this family, the inclusion of the (52%) were phylogenetically informative, while 392 characters newly discovered Canarium fossil has a stronger impact on the (44%) of the ITS region were phylogenetically informative. age of diversification of Burseraceae, substantially increasing Maximum likelihood analysis resulted in one single tree with previous estimates of ~58 Ma. a score of −ln(L) = 20,280. The three analyses (MP, ML, and The increase in age of diversification of Burseraceae as Bayesian analyses) inferred a distribution of phylogenies with compared to the ages presented previously also increases es- almost identical topologies, thus only one phylogeny is shown timates of minimum ages of the tribes, subtribes, and genera (Fig. 1). All of the deeper nodes were highly supported by within the family. For example, tribe Protieae, that includes all three analyses while more recent divergences had lower Crepidospermum, Protium, and Tetragastris now appears

336 TAXON 61 (2) • April 2012: 333–343 Becerra & al. • Monophyly of Bursera

filicifolia Fig. 1. Fifty percent majority- macvaughiana rupicola rule consensus tree of Com- ceracifolia palmeri miphora and Bursera from stenophylla hindsiana asplenifolia Bayesian inference. Bayesian hintonii velutina (B), maximum parsimony copallifera excelsa (MP), and maximum likelihood bipinnata bicolor Bursera (ML) analyses yielded almost sarukhanii diversifolia submoniliformis subg. Elaphrium identical topologies. Numbers vejar-vazquezii sarcopoda on branches indicate MP/ML/B cuneata laxiflora bootstrap support. For the pur- bonetii 94/96/100 biflora pose of clarity support is only mirandae xochipalensis linanoe shown for deeper nodes. glabrifolia 99/100/100 coyucensis heteresthes citronella infernidialis penicillata epinnata tecomaca bolivarii chemapodicta schlechtendalii medranoana aptera fagaroides var. fagaroides ariensis fagaroides var. purpusii trifoliolata Bursera discolor odorata 98/96/100 80/60/95 confusa paradoxa palaciosii staphyleoides silviae occulta crenata kerberi denticulata lancifolia trimera Bursera 84/90/100 fragilis multijuga fagaroides var. elongata subg. Bursera suntui galeottiana morelensis arida rzedowski microphylla 95/100/100 laurihuertae grandifolia instabilis cinerea longipes acuminata attenuata 95/68/100 arborea simaruba frenningae spinescens africana glandulosa 95/92/100 ellembeckii chaetocarpa ovalifolia kua gowlello merkeri viminea pyracanthoides habessinica madagascariensis wightii schimperi berryi caudata campestris rostrata */60/100 samharensis foliacea tenuipetiolata wildii corrugata marlothii guidotii eminii kataf 89/74/99 edulis unilobata mollis mildbraedii karibensis mossambicensis neglecta 100/100/100 grandifolia coleopsis lamii capuronii Commiphora aprevalii mafaidoha ankaranensis krauseliana 100/100/100 gracilifrondosa saxicola monstruosa simplicifolia humbertii guillaumini orbicularis leptophloeos leptophloeos 94/100/100 Protium trifoliolatum Protium madagariense Crepidospermun prancei 99/98/100 Crepidospermun goudotianum Crepidospermun rhoifolium Outgroups */62/96 Tetragastris altissima Canarium album Boswellia sacra 100/100/100 Boswellia hildebrandtii Schinus molle Rhus trilobata mexicana

337 Becerra & al. • Monophyly of Bursera TAXON 61 (2) • April 2012: 333–343

Fig. 2. Fifty percent majority- 65/85/100 rule consensus tree of Bursera- Bursera spinescens ceae from Bayesian inference. 88/90/100 78/86/100 Bursera discolor Bayesian (B), maximum parsimony (MP), and maxi- 91/79/100 Bursera schlechtendalii mum likelihood (ML) analyses 81/77/100 Bursera morelensis 95/84/100 yielded identical topologies. 58/56/94 Numbers on nodes indicate MP/ ML/B bootstrap support. Bursera biflora Bursera sarukhanii 67/52/90 Bursera cuneata */67/100 96/81/100 Bursera copallifera BURSEREAE Bursera submoniliformis 88/66/98 Bursera tecomaca subtribe 90/86/100 Burserinae 62/56/93 Commiphora kua 88/90/100 58/*/97 78/52/97 Commiphora rostrata Commiphora eminii */*/96 Commiphora ugogensis Commiphora leptophloeos Commiphora edulis 68/75/100 Commiphora lamii 96/88/100 Commiphora angolensis Commiphora monstruosa 83/78/100 Commiphora simplicifolia Commiphora franciscana Protium guianense Protium pilosum 65/56/100 Protium unifoliolatum Protium trifoliolatum 98/88/100 Protium copal Protium madagascariense 81/63/100 PROTIEAE Crepidospermun rhoifolium Crepidospermun goudotianum 98/82/100 70/90/100 Crepidospermun prancei Tetragastris mucronata 95/100/100 Tetragastris altissima 100/100/100 Canarium album Canarium pilosum 90/71/100 Canarium vulgare Canarium indicum Canarium zeylanicum 85/100/100 Canarium littorale CANARIAEAE 95/85/100 100/73/100 53/59/76 Santiria apiculata Trattinnickia glaziovii */90/100 Trattinnickia cf. burserifolia */82/100 Dacryodes klaineana 70/90/100 Dacryodes buettneri 60/90/99 Boswellia sacra 100/100/100 Boswellia frereana BURSEREAE Boswellia hildebrandtii subtribe 94/78/100 Boswellia neglecta Beiselia mexicana Boswellinae 100/97/100 Rhus copallinum 88/79/100 Rhus trilobata */89100 Pistacia mexicana Heeria argentea 100/88/100 longifolia OUTGROUPS 85/67/71 Sorindeia madagascariensis Schinus molle Spondias mombin Dodonaea viscosa

338 TAXON 61 (2) • April 2012: 333–343 Becerra & al. • Monophyly of Bursera

Bursera sarukhanii Bursera copallifera Bursera cuneata Bursera hindsiana * Bursera tecomaca Bursera lancifolia Bursera microphylla Bursera fagaroides Bursera discolor Bursera simaruba Commiphora kua Commiphora wightii Commiphora africana Commiphora schimperi Commiphora rostrata Commiphora eminii Commiphora ugogensis Commiphora edulis Commiphora angolensis Commiphora leptophloeos Commiphora monstruosa Commiphora franciscana Crepidospermun rhoifolium Crepidospermun goudotianum Protium madaga Tetragastris mucronata Tetragastris altissima Protium pilosum Protium unifoliolatum Protium guianense 92.7 Protium copal Canarium vulgare Canarium indicum Canarium zeylanicum Canarium littorale Canarium album Canarium pilosum Santiria griffithii * Santiria apiculata Trattinnickia cf. burserifolia Dacryodes klaineana Dacryodes buettneri Boswellia sacra Boswellia frereana Boswellia neglecta Heeria argentea Sorindeia madagascariensis Rhus copallinum Pistacia mexicana Schinus molle

90 60 30 0 Ma

Fig. 3. Time-calibrated phylogeny of Burseraceae using ETS and rps16 markers. The asterisks (*) indicate the nodes where the dates of fossil calibration were applied. Red lines on branches show standard deviations.

339 Becerra & al. • Monophyly of Bursera TAXON 61 (2) • April 2012: 333–343

to have separated from Bursereae, subtribe Burserinae branches having a value greater than or equal to 90%. Results (that includes Bursera and Commiphora) at least 86 Ma ago with the ETS sequences were also troublesome. With only 137 (± 10.7 Ma). The new topology used for the calibration had a informative sites the most parsimonious tree had less than 12% particularly dramatic effect on the age of divergence of Com- of the individual branching events supported with parsimony miphora, for which previous literature indicated an age of bootstrap values of 90% or higher. The ETS-rps16 matrix da- 28 Ma (Weeks & al., 2005). According to our results, since it taset used in Weeks & al. (2005) had 360 phylogenetically is no longer nested within Bursera, it separated at least 61 Ma informative sites and also low branch support. Their evidence ago (± 5.6 Ma). This current calibration, however, agrees with for the non-monophyly in Bursera lacked parsimony bootstrap previous estimates that Commiphora started diversification support (62%) and was unsupported by Bayesian analysis. In after 28 Ma ago. our phylogenetic reconstruction of the two genera, the combi- nation of the ETS-ITS dataset provided 592 informative sites, nearly double those of the Weeks & al. (2005) dataset and more DISCUSSION than double those of the dataset of Weeks & Simpson (2007). The ITS region, not analyzed by Weeks & al. (2005), was par- Whether Bursera is or is not monophyletic has been a con- ticularly useful for resolving relationships at the generic level troversial theme for many years. Commiphora includes about here. A second reason might be that with 76 Bursera and 51 200 species distributed in the drier parts of tropical Africa, Commiphora species, our study comprised a much greater Arabia, India, and South America. They are trees with some number of sequences. However, our phylogeny of Bursera- traits shared with B. subg. Elaphrium like a non-exfoliating ceae includes only 15 species of each genus, and still supports , four-merous , and bilocular ovaries (Rzedowski Bursera’s monophyly. & Kruse, 1979). Their main two differences to Bursera are their Our findings are also congruent with previous results of calyx and spines. Commiphora species have a calyx closed in phylogenetic analyses of Bursera that suggested that ITS and the young bud so that the corolla is hidden, and they often have ETS provide good resolution at the generic level. However, spines. In Bursera the calyx opens in the bud leaving the corolla a faster evolving marker like the 5S-NTS can provide a bet- visible, and they almost never have spines (Gillett, 1980). Based ter resolution for more recent branching events in this genus on morphological characteristics, Rzedowski & Kruse (1979) (Becerra, 2003a). Indeed, relationships within subg. Elaphrium hypothesized that Bursera is diphyletic, with subg. Elaphrium reconstructed with ITS, ETS, and 5S-NTS provided better reso- giving rise to Commiphora. This contrasted with molecular lution and higher support for many of its nodes. These markers phylogenies of Becerra & Venable (1999b) and Becerra (2003a) also proved instrumental to solve relationships among lineages that strongly supported the monophyly of Bursera as well as of the Fagaroides group (Becerra & Venable, 1999) within subg. the monophyly of its two subgenera. Later, Weeks & al. (2005) Bursera (Becerra & al., unpub. data). proposed, based on nuclear and chloroplast sequence data, that Results from this study suggest that Burseraceae started Commiphora is sister to subg. Bursera. Other authors have sug- diversification at least 93 Ma ago (± 17.9). This new estimate gested that Bursera is not monophyletic because it includes spe- is considerably older than was previously calculated (58 Ma), cies that belong to Commiphora. Rzedowski & Palacios (1985) but is somewhat consistent with estimates of the age of Malvi- and Palacios (1984) suggested, based on pollen morphology, dae, the group to which and Burseraceae belong, that B. tecomaca and B. sarcopoda P.G. Wilson were closer at between 108 and 121 Ma (Bell & al., 2010) as well as other to Commiphora than to Bursera. Also, recent phylogenetic estimates of the age of Sapindales at about 100 Ma (Magallon analyses by Rosell & al. (2010) indicated that Cuban Commiph- & Castillo, 2009). The use of fossils in these efforts to date the ora glauca (Griseb.) M. Moncada Ferrera and C. angustata angiosperm phylogeny was very conservative in some cases. (C. Wright ex Griseb.) M. Moncada Ferrera belong in Bursera. For example, using 125 Ma for the first appearance of tricol- Our results strongly support previous arguments that Com- pate pollen as the age of the eudicot crown clade (Magallon miphora and Bursera are very closely related and that they are & Castillo, 2009) has been considered an underestimation of both monophyletic. They also indicate that Bursera tecomaca the origin of (Smith & al., 2010). Tricolpate pollen and B. sarcopoda fall into the clade of subg. Elaphrium rather first appeared in separated geographical areas and the grains than into Commiphora. themselves are not uniform in morphology, suggesting that the The phylogenetic analysis of Weeks & al. (2005) suggested tricolpate clade originated some time before its appearance in that Bursera was either paraphyletic with subg. Elaphrium in a the fossil record. Megafossils of Archaefructus Sun & al. sp. basal position and subtending the sister groups, subg. Bursera that resemble the Ranunculaceae genus Delphinium L. (123– and Commiphora, or that these three clades form a polytomy. 126 Ma) also suggest that the basal eudicots were already pres- Why do their results differ from ours? Several aspects of meth- ent and diverse by the latest Barremian and earliest Aptian odology may explain the discrepancies. First, these two studies (125–126 Ma ago; Sun & al., 2011). Constraints for the age of differ in the degree of resolution of the markers used. The com- Burseraceae were also conservative. A fossil with an age of 50 bined chloroplast datasets used in Weeks & Simpson (2007) Ma was used to mark the minimum age of separation between resulted in 1550 bp of which only 93 (6%) were informative Burseraceae and Anacardiaceae (Bell & al., 2010). This age is sites. Because of this, parsimony bootstrap support for diver- probably too young because fossils of close to that age have gence events was very low, with less than 18% of individual been discovered for specific derived clades within Burseraceae.

340 TAXON 61 (2) • April 2012: 333–343 Becerra & al. • Monophyly of Bursera

Table 1. Comparison of estimated ages of Bursera clades resulting from the present analysis and Becerra (2003b, 2005). Crown clade mean age from present study Age estimated by Becerra (2003b, 2005) Clade (± standard error) (± standard error) Bursera 57.1 (± 5.6) 74.2 (± 14.3) B. subg. Bursera 45.3 (± 5.3) 49.1 (± 11.7) B. subg. Elaphrium 34.0 (fixed) 38.4 (±8.5) MRCA B. lancifolia–B. discolor 31.0 (± 2.4) 37.7 (± 12.4) MRCA B. hindsiana–B. sarukhanii 15.4 (± 1.9) 19.0 (± 4.8) MRCA, most recent common ancestor.

Recognizing that both Bursera and Commiphora are AcKNOWLEDGEMENTS monophyletic has only a modest effect on the estimated age of diversification of Burseraceae. Under this assumption, the We thank Jason Eslamieh, Boris Vrskovy, and Jerzy Rzedowski for node of divergence of Bursera tecomaca and the diversifica- providing samples of some of the Commiphora and Bursera species used tion of subg. Elaphrium (used to calibrate the phylogeny) is in this study; Michael Sanderson and Brigitte Marazzi for help and advice now farther from the root of the tree than when Commiphora on analyses performed; and the three anonymous reviewers that provided is nested within Bursera. With this alternative topology, the di- very valuable comments and suggestions to an earlier manuscript. versification age of Burseraceae increases by 5 Ma. Divergence estimates are more affected by calibration with the recently dis- covered Canarium fossils. These fossils are more than double LITERATURE CITED the age of the Canarium fossil used by Weeks & al. (2005; 47 vs. 23 Ma), and thus substantially increase the estimated age Baldwin, B.G. & Sanderson, M.J. 1998. Age and rate of diversifica- of the family. tion of the Hawaiian silversword alliance (Compositae). Proc. Natl. Acad. Sci. U.S.A. 95: 9402–9406. Diversification time of the genus Bursera has also been Becerra, J.X. 1997. Insects on plants: Macroevolutionary chemical controversial. Becerra (2003b, 2005) found that the peak diver- trends in host use. Science 276: 253–256. sification of northwestern Mexican Bursera lineages occurred Becerra, J.X. 2003a. Evolution of Mexican Bursera (Burseraceae) in- 34–17 Ma ago, following the uprising of the Mexican Sierra ferred from ITS, ETS, and 5S nuclear ribosomal DNA sequences. Madre Occidental (34–15 Ma ago), while diversification of Molec. Phylogenet. Evol. 26: 300–309. southern Mexican lineages peaked at 13.5 Ma ago, tracking Becerra, J.X. 2003b. Synchronous coadaptation in an ancient case of herbivory. Proc. Natl. Acad. Sci. U.S.A. 100: 12804–12807. the uplift of the Neovolcanic axis. Becerra (2003b, 2005), how- Becerra, J.X. 2005. Timing the origin and expansion of the Mexican ever, used the separation of Africa and South America (95 Ma tropical dry forest. Proc. Natl. Acad. Sci. U.S.A. 102: 10919–10923. ago) to date the node of divergence of African and American Becerra, J.X. 2007. The impact of plant–herbivore coevolution on plant Commiphora and also the age of the B. serrulata fossil to date community structure. Proc. Natl. Acad. Sci. U. S.A. 104: 7843–7488. the node of divergence of B. tecomaca (and subg. Elaphrium). Becerra, J.X. & Venable, D.L. 1999a. Macroevolution of insect-plant Although divergence of South American Commiphora at the associations: The relevance of host biogeography to host affiliation. Proc. Natl. Acad. Sci. U.S.A. 96: 12626–12631. time of break-up of Gondwana is not tenable according to this Becerra, J.X. & Venable, D.L. 1999b. Nuclear ribosomal, DNA phy- study, because of the use of the B. serrulata fossil in previous logeny and its implications for evolutionary trends in Mexican and the present study, many of the divergence time estimates Bursera (Burseraceae). Amer. J. Bot. 86: 1047–1057. resulting from our analysis coincide closely with those ob- Becerra, J.X., Noge, K. & Venable, D.L. 2009. Macroevolutionary tained by Becerra (2003b, 2005) (Table 1). However, with the chemical escalation in an ancient plant–insect arms race. Proc. Natl. Acad. Sci. U.S.A. 43: 18062–18066. purported age of no more than 58 Ma for Burseraceae (Weeks Bell, C.D., Soltis, D.E. & Soltis, P.S. 2010. The age and diversifica- & al., 2005), diversification of Bursera would have started 30 tion of the angiosperms re-revisited. Amer. J. Bot. 97: 1296–1303. to 37 Ma ago and peaked well after the uplift of the Mexican Clarkson, J.J., Chase, M.W. & Harley, M.M. 2002. Phylogenetic re- mountainous systems. Results reported here using only fossils lationships in Burseraceae based on plastid rps16 intron sequences. confirm an early origin for both Bursera and Commiphora as Kew Bull. 57: 183–193. well as diversification times of Bursera concurrent with the Collinson, M.E., Manchester, S.R., Wilde, V. & Hayes, P. 2010. and floras from exceptionally preserved biotas in the Euro- formation of the Mexican mountain ranges. pean Paleogene. Bull. Geosci. 85: 155–161. Commiphora and Bursera are the most species-rich gen- Futuyma, D.J. 2009. Evolution, 2nd ed. Sunderland, Massachusetts: era in Burseraceae. They are also very important physiog- Sinauer. nomic components of the tropical dry forests of Africa and Futuyma, D.J. & Agrawal, A.A. 2009. Macroevolution and the bio- North America, where they often are among the dominant logical diversity of plants and herbivores. Proc. Natl. Acad. Sci. woody taxa. Having a robust understanding of their systemat- U.S.A. 106: 18054–18061. Gillett, J.B. 1980. Commiphora (Burseraceae) in South America and ics will support future studies that will help us comprehend its relationship to Bursera. Kew Bull. 34: 569–589. the reasons behind the evolutionary and ecological success Harley, M.M. & Daly, D.C. 1995. Burseraceae Kunth, Protieae March. of Burseraceae. em. Engl. World Pollen Spore Fl. 20: 1–44.

341 Becerra & al. • Monophyly of Bursera TAXON 61 (2) • April 2012: 333–343

Hillis, D.M. 1997. Biology recapitulates phylogeny. Science 276: 218–219. Rzedowski, J. & Kruse, H. 1979. Some evolutionary trends in Bursera Huelsenbeck, J.P. & Ronquist, F.R. 2001. MrBayes: Bayesian infer- (Burseraceae). Taxon 28: 103–116. ence of phylogeny. Bioinformatics 17: 754–755. Rzedowski, J. & Palacios, R. 1985. La presencia de Commiphora Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGetti- (Burseraceae) en México. Taxon 34: 207–210. gan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Rzedowski, J., Medina Lemos, R. & De Rzedowski, G.C. 2006. In- Lopez, R., Thompson, J.D., Gibson, T.J. & Higgins, D.G. 2007. ventario del conocimiento taxonómico, así como de la diversidad Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947–2948. y del endemismo regionales de las especies mexicanas de Bursera MacGinitie, H.D. 1953. Fossil plants of the Florissant beds, Colorado. (Burseraceae). Acta Bot. Mex. 70: 85–111. Publ. Carnegie Inst. Washington 599: 1–188. Sanderson, M.J. 2002. Estimating absolute rates of molecular evolution MacGinitie, H.D. 1969. The Eocene Green River flora of Northwestern and divergence times: A penalized likelihood approach. Molec. Colorado and Northeastern . Univ. Calif. Publ. Geol. Sci. Phylogenet. Evol. 19: 101–109. 83: 1–140. Sanderson, M.J. 2003. r8s: Inferring absolute rates of evolution and Maddison, D.R. & Maddison, W.P. 2000. MacClade: Analysis of phy- divergence times in the absence of a molecular clock. Bioinformat- logeny and character evolution, version 4. Sunderland: Sinauer. ics 19: 301–302. Magallon, S. & Castillo, A. 2009. Angiosperm diversification through Smith, S.B., Beaulieu, J.M. & Donoghue, M.J. 2010. An uncorre- time. Amer. J. Bot. 96: 349–365. lated relaxed-clock analysis suggests an earlier origin for flowering McVaugh, R. & Rzedowski, J. 1965. Synopsis of the genus Bursera L. plants. Proc. Natl. Acad. Sci. U.S.A. 107: 5897–5902. in western Mexico, with notes on the material of Bursera collected Speight, M.R., Hunter, M.D. & Watt, A.D. 2008. Ecology of insects: by Sesse & Mocino. Kew Bull. 18: 317–382. Concepts and applications. West Sussex, U.K.: Wiley-Blackwell. Meyer, H.W. 2003. The fossils of the Florissant. Washington, D.C.: Sun, G., Dilcher, D.L., Wang, H. & Chen, Z. 2011. A eudicot from the Smithsonian Books. Early Cretaceous of China. Nature 471: 625–628. Mitchell-Olds, T. & Bergelson, J. 2000. Biotic interactions – Genom- Swofford, D.L. 2002. PAUP*: Phylogenetic analysis using parsimony ics and coevolution – Editorial overview. Curr. Opin. Pl. Biol. 3: (*and other methods), version 4.0b10 ed. Sunderland: Sinauer. 273–277. Wang, H.C., Moore, M.J., Soltis, P.S., Bell, C.D., Brockington, S.F., Palacios, R. 1984. La morfología de los granos de pollen de las especies Alexandre, R., Davis, C.C., Latvis, M., Manchester, S.R. & mexicanas del género Bursera. Biotica 9: 153–182. Soltis, D.E. 2009. Rosid radiation and the rapid rise of angiosperm- Pennington, R.T., Richardson, J.E. & Lavin, M. 2006. Insights into dominated forests. Proc. Natl. Acad. Sci. U.S.A. 106: 3853–3858. the historical construction of species-rich biomes from dated plant Weeks, A. & Simpson, B.B. 2007. Molecular phylogenetic analysis phylogenies, neutral ecological theory and phylogenetic commu- of Commiphora (Burseraceae) yields insight on the evolution and nity structure New Phytol. 4: 605–616. historical biogeography of an “impossible genus”. Molec. Phylo- Posada, D. & Crandall, K.A. 1998. MODELTEST: Testing the model genet. Evol. 42: 62–79. DNA substitution. Bioinformatics 14: 817–818. Weeks, A., Daly, D.C. & Simpson, B.B. 2005. The phylogenetic history Rosell, J.A., Olson, M.E., Weeks, A., De-Nova, J.A., Lemos, R.M., and biogeography of the and family (Burs- Camacho, J.P., Feria, T.P., Gomez-Bermejo, R., Montero, J.C. eraceae) based on nuclear and chloroplast sequence data. Molec. & Eguiarte, L.E. 2010. Diversification in species complexes: Tests Phylogenet. Evol. 35: 85–101. of species orgin and delimitation in the Bursera simaruba clade of Wilde, V. & Frankenhäuser, H. 1988. The Middle Eocene plant tapho- tropical trees (Burseraceae). Molec. Phylogenet. Evol. 57: 798–811. coenosis from Eckfeld (Eifel, Germany). Rev. Palaeobot. Palynol. Rzedowski, J. 1978. Vegetación de México. México, D.F.: Limusa. 10: 7–28.

Appendix. Species included in the phylogenetic analyses, voucher specimen number, and GenBank accession numbers of the DNA sequences. Taxon; provenance; voucher; ITS, ETS, rps16 Beiselia mexicana Forman; Mexico; Michoacan; –; –, 224459583, 36939076. Boswellia frereana Birdw.; –, 37677988, 60116131; B. hildebrandtii Engl.; Kenya; –; 28190288, 29421969, –. B. neglecta S. Moore; –; –; –, 37677993, 37677992. B. sacra Flueck., Kenya; –; 28190287, 37677994, 36939070. Bursera acuminata Engl.; Mexico: Estado de Mexico; J. Becerra & D.L. Venable 228 (ARIZ); 28190276, 29421959, –. B. aptera Ramirez; Mexico: Puebla; J. Becerra & D.L. Venable 282 (ARIZ); 28190248, 29421930, –. B. arborea (Rose) L. Riley; Mexico: Colima; J. Becerra & D.L. Venable 247 (ARIZ); 28190275, 29421958, –. B. arida (Rose) Standl.; Mexico: Puebla; J. Becerra & D.L. Venable 127 (ARIZ); 28190261, 29421944, –. B. ariensis (Kunth) McVaugh & Rzed.; Mexico: Guerrero; J. Becerra & D.L. Venable 124 (ARIZ); 28190256, 29421939, –. B. asplenifolia Brandegee; Mexico: Oaxaca; J. Becerra & D.L. Venable 277 (ARIZ); 28190218, 29421901, –. B. attenuata (Rose) L. Riley; Mexico: Nayarit; J. Becerra & D.L. Venable 231 (ARIZ); 28190278, 29421961, –. B. bicolor (Willd. ex Schltdl.) Engl.; Mexico: Puebla; J. Becerra & D.L. Venable 275 (ARIZ); 28190220, 29421903, –. B. biflora (Rose) Standl.; Mexico: Puebla; J. Becerra & D.L. Venable 296 (ARIZ); 28190214, 29421897. B. bipinnata (DC.) Engl.; Mexico: Jalisco, J. Becerra & D.L. Venable 256 (ARIZ); 28190234, 29421917, –. B. bolivarii Rzed.; Mexico: Guerrero, J. Becerra & D.L. Venable 649 (ARIZ); 28190246, 29421929, –. B. bonetii Rzed.; Mexico: Guerrero; J. Becerra & D.L. Venable 298 (ARIZ); 28190213, 29421896, –. B. cera- sifolia Brandegee; Mexico: Sur J. Becerra & D.L. Venable 734 (ARIZ); 28190221, 29421904, –. B. chemapodicta Rzed. & E. Ortiz; Mexico: Guerrero; J. Becerra & D.L. Venable 304 (ARIZ); 28190252, 29421934, –. B. cinerea Engl.; Mexico: Morelos; J. Becerra & D.L. Venable 648 (ARIZ); 28190271, 29421954, –. B. citronella McVaugh & Rzed.; Mexico: Colima J. Becerra & D.L. Venable 642 (ARIZ); 28190242, 29421925, –. B. confusa Engl.; Mexico: Guerrero; J. Rzedowski 37788 (IE-BAJIO), JN882659, JN971038, –. B. copallifera (DC.) Bullock; Mexico: Guerrero; J. Becerra & D.L. Venable 113 (ARIZ); 28190240, 29421923, 36939081. B. coyucensis Bullock; Mexico: Guerrero; J. Becerra & D.L. Venable 312 (ARIZ); 28190243, 29421926, –. B. crenata Paul G. Wilson; Mexico: Michoacan; J. Becerra & D.L. Venable 215 (ARIZ); 28190263, 29421946. B. cuneata (Schltdl.) Engl.; Mexico: Guerrero; J. Becerra & D.L. Venable 309 (ARIZ); 28190232, 29421915, 36677949. B. denticulata McVaugh & Rzed.; Mexico: Michoacan; J. Becerra & D.L. Venable 318 (ARIZ); 28190269, 29421952, –. B. discolor Rzed.; Mexico: Guer- rero; J. Becerra & D.L. Venable 319 (ARIZ); 28190253, 29421935, 35299314. B. diversifolia Rose, Mexico: Guerrero J. Becerra & D.L. Venable 120 (ARIZ); 28190230, 29421913, –. B. epinnata (Rose) Engl.; Mexico: Baja California Sur; J. Becerra & D.L. Venable 745 (ARIZ); 28190223, 29421899. B. excelsa (Kunth) Engl.; Mexico: Sinaloa; J. Becerra & D.L. Venable 122 (ARIZ); 28190219, 29421902, –. B. fagaroides var. elongata McVaugh & Rzed.; Mexico: ; J. Becerra & D.L. Venable 502 (ARIZ); 28190255, 29421938, –. B. fagaroides var. fagaroides (H.B.K.) McVaugh & Rzed.; Mexico: Jalisco; J. Becerra & D.L. Venable 330 (ARIZ); 28190250, 29421932, 60116113. B. fagaroides var. purpusii (Brandegee) McVaugh & Rzed.; Mexico: Estado de Mexico; J. Becerra & D.L. Venable 333 (ARIZ); 28190251, 29421933. B. filicifolia Brandegee; Mexico; –; 28190211, 29421894, –. B. fragilis S. Watson; Mexico: Sinaloa; J. Becerra & D.L. Venable 504 (ARIZ); 28190267, 29421950. B. frenningae Correll; –; –; JN882665, 35288036, –. B. galeottiana Engl.; Mexico: Oaxaca; J. Becerra & D.L. Venable 239 (ARIZ); 28190260, 29421943. B. glabrifolia (Kunth) Engl.; Mexico: Guerrero; J. Becerra & D.L. Venable 342 (ARIZ); 28190244, 29421927, –. B. grandifolia (Schltdl.) Engl.; Mexico: Sonora; J. Becerra & D.L. Venable 339 (ARIZ); 28190273, 29421956 –. B. heteresthes Bullock; Mexico: Michoacan; J. Becerra & D.L. Venable 203 (ARIZ); 28190237, 29421920 –. B. hindsiana Engl. in DC.; Mexico; –; 28190238, 29421921, 36677952. B. hintonii Bullock; Mexico: Michoacan; J. Becerra & D.L. Venable

342 TAXON 61 (2) • April 2012: 333–343 Becerra & al. • Monophyly of Bursera

Appendix. Continued. 219 (ARIZ); 28190226, 29421909, –. B. inferdinalis Guevara & Rzed.; Mexico: Michoacan; J. Becerra & D.L. Venable 208 (ARIZ); 28190228, 29421911, –. B. in- stabilis McVaugh & Rzed.; Mexico: Jalisco J. Becerra & D.L. Venable 504 (ARIZ); 28190272, 29421955, –. B. kerberi Engl.; Mexico: Nayarit; J. Becerra & D.L. Ven- able 239 (ARIZ); 28190265, 29421948, –. B. lancifolia (Schltdl.) Engl.; Mexico: Puebla; J. Becerra & D.L. Venable 348 (ARIZ);28190264, 29421947, 35299318. B. laurihuertae Rzed. & Calderon; Mexico: Oaxaca; A. Saynes V 2662 (IE-BAJIO), JN882664, JN971065, –. B. laxiflora S. Watson; Mexico; –; 28190233, 29421916, –. B. linanoe (La Llave) Rzed., Calderon & Medina; Mexico: Puebla; J. Becerra & D.L. Venable 261 (ARIZ); 28190231, 29421914, –. B. longipes (Rose) Standl.; Mexico: Puebla; J. Becerra & D.L. Venable 384 (ARIZ); 28190277, 29421960, –. B. macvaughiana Cuevas & Rzed.; Mexico; –; 28190212, 29421895, –. B. me- dranoana Rzed. & E. Ortiz; Mexico: Hidalgo; J. Becerra & D.L. Venable 654 (ARIZ); JN882657, 29421937, –. B. microphylla A. Gray; Mexico: Sonora; J. Becerra & D.L. Venable 507 (ARIZ); 28190262, 29421945, 35299321. B. mirandae C.A. Toledo; Mexico: Guerrero J. Becerra & D.L. Venable 349 (ARIZ); 28190215, 29421898, –. B. morelensis Ramirez; Mexico: Puebla; J. Becerra & D.L. Venable 350 (ARIZ); 28190259, 29421942, 35299320. B. multijuga Engl.; Mexico: Nayarit; J. Becerra & D.L. Venable 237 (ARIZ); 28190268, 29421951, –. B. occulta McVaugh & Rzed., Mexico; Rzedowski 37974 (IE-BAJIO), JN882663, JN971042, –. B. odorata Brandegee; Mexico: Baja California Sur; J. Becerra & D.L. Venable 701 (ARIZ); 28190257, 29421940, –. B. palaciosii Rzed. & Calderon; Mexico: Colima; J. Becerra & L. Venable 1020 (ARIZ); JN882660, JN971039, –. B. palmeri S. Watson; Mexico: Queretaro; J. Becerra & D.L. Venable 163 (ARIZ); 28190225, 29421908, –. B. paradoxa Guevara & Rzed.; Mexico: Michoacan; J. Becerra and D.L. Venable 205 (ARIZ); 28190249, 29421931, –. B. penicillata (DC.) Engl.; Mexico: Nayarit; J. Becerra & D.L. Venable 253 (ARIZ); 28190222, 29421905, –. B. rupicola Cuevas; Mexico: Baja California Sur; J. Becerra & D.L. Venable 707 (ARIZ); 28190217, 29421900, –. B. rzedowskii C.A. Toledo; Mexico: Guerrero; J. Becerra & D.L. Venable 671 (ARIZ); 28190270, 29421953, –. B. sarcopoda P.G. Wilson: Mexico: Colima; J. Becerra & D.L. Venable 635 (ARIZ); 28190241, 29421924, –. B. sarukhanii Guevara & Rzed.; Mexico: Michoacan; J. Becerra & D.L. Venable 212 (ARIZ); 28190227, 29421910, 36939084. B. schlechtendalii Engl.; Mexico: Puebla; J. Becerra & D.L. Venable 509 (ARIZ); 28190254, 29421936, 35299324. B. silviae Rzed. & Calderon; Mexico; Oaxaca; G. Juarez G. 2365 (IE-BAJIO), JN882662, JN971042. B. simaruba (L.) Sarg.; Mexico; Sonora; J. Becerra & D.L. Venable 511 (ARIZ); 28190274, 29421957, –. B. spinescens Urb. & Ekman; –; –; JN882666, 35287999, 35299326. B. staphyleoides McVaugh & Rzed; Mexico; Rzedowski 37801 (IE- BAJIO), JN882661, JN971041, –. B. stenophylla Sprague & L. Riley; Mexico: Sonora; J. Becerra & D.L. Venable 513 (ARIZ); 28190235, 29421918, –. B. submo- niliformis Engl.; Mexico; Puebla; J. Becerra & D.L. Venable 264 (ARIZ); 28190229, 29421912, –. B. suntui C.A. Toledo; Mexico: Guerrero; J. Becerra & D.L. Ven- able 359 (ARIZ);28190258, 29421941, –. B. tecomaca (DC.) Standl.; Mexico: Guerrero; J. Becerra & D.L. Venable 366 (ARIZ); 28190245, 29421928, 35299312. B. trifoliolata Bullock; Mexico: Estado de Mexico; J. Becerra & D.L. Venable 369 (ARIZ);28190247, JN9710. B. trimera Bullock; Mexico: Guerrero; J. Becerra & D.L. Venable 167 (ARIZ);2 8190266, 29421949, –. B. vejar-vazquezii Miranda; Mexico: Puebla; J. Becerra & D.L. Venable 269 (ARIZ); 28190236, 29421919, –. B. velutina Bullock; Mexico; J. Becerra & D.L. Venable 377 (ARIZ); 28190239, 29421922, –. B. xochipalensis Rzed.; Mexico: Guerrero; J. Becerra & D.L. Venable 380 (ARIZ); 28190224, 29421907, –. Canarium album Leenh. –; –; 99030343, 55613449, 5513410. C. indicum L. Malasysia; Lai s.n. (BH); –, 37678018, 36939085. C. littorale Blume; Malaysia; Lai s.n. (BH); –, 37678021, 36939086. C. pilosum A.W. Benn; –; –, –, 37678023, 3693087. C. vulgare Malasysia; Lai s.n. (BH); –, 37678025, 36939088. C. zeylanicum Blume; Malasysia; Lai s.n. (BH); –, 37678028, 36939089. Commiphora africana (A. Rich.) Engl.; , Messina; J. Becerra & D.L. Venable 1003 (ARIZ); 28190280, 29421963 61677225. C. angolensis Engl. South Africa: Transvaal; Raal & Raal 801; –, 37677959, 36939090. C. ankaranensis (J.-F. Leroy) Cheek & Rakot; Madagascar; Becerra & Venable 1021 (ARIZ); JN882692, JN971062, –. C. aprevalii (Baill.) Guillaumin; Madagascar; Becerra & Venable 1022 (ARIZ); JN882701, 61677154, –. C. berryi Engl.; India; Becerra & Venable 1023 (ARIZ); JN882667, –. C. campestris Engl.; Ethiopia; Becerra & Venable 1025 (ARIZ); JN882698, 61677157, –. C. capuronii Bardot-Vaucoulon; Madagascar; Becerra & Venable 1026 (ARIZ); JN882691, JN971043, –. C. caudata Engl.; India; Becerra & Venable 1027 (ARIZ); JN882668, JN971043, –. C. chaetocarpa Engl.; Kenya; Becerra & Venable 1028 (ARIZ); JN882669, JN971044, –. C. coleopsis H. Perrier; Madagascar; Becerra & Venable 1029 (ARIZ); JN882690 JN8826, JN971061, –. C. corrugata Gillett & Vollesen; Ethiopia, Becerra & Venable 1030 (ARIZ); JN882670, JN971045, –. C. edulis Engl.; South Africa; Becerra & Venable 1031 (ARIZ); JN882706, 37677962 36939091. C. el- lenbeckii Engl.; Ethiopia; Becerra & Venable 1032 (ARIZ); JN882674, JN971048, –. C. eminii Engl.; Kenya; Becerra & Venable 1033 (ARIZ); JN882700, 37677965 36939092. C. franciscana Capuron; Madagascar: Tulear; Labat 2082 (MO); –, 37677967, 36939093, –. C. foliacea Sprague; Kenya; Becerra & Venable 1034 (ARIZ); JN882681, JN971054, –. C. glandulosa Schinz, South Africa, Becerra & Venable 1035 (ARIZ); JN882710, JN971064. C. gowlello Sprague, East Africa, Becerra & Venable 1082 (ARIZ); JN882674, JN971047, –. C. gracilifrondosa Dinter Ex Van Der Walt; South Africa; Becerra & Venable 1085 (ARIZ); JN882683, JN971056, –. C. grandifolia Engl.; Madagascar; Becerra & Venable 1037 (ARIZ); JN882671, 61677160, –. C. guidottii Chiov. ex Guid.; Kenya; Becerra & Venable 1036 (ARIZ); JN882679, JN971052, –. C. guillaumini H. Perrier, Madagascar; Becerra & Venable 1038 (ARIZ); JN882693, 61677161, –. C. habessinica Engl.; East Africa, Becerra & Venable 1039 (ARIZ); JN882673, JN971046, –. C. humbertii Perrier; Madagascar; Becerra and Venable 1087 (ARIZ); JN882694, –. C. karibensis Wild, South Africa; Becerra & Venable 1040 (ARIZ); JN882684, JN971057, –. C. kataf Engl.; Kenya; Becerra & Venable 1041 (ARIZ); JN882709, 61677162. C. krauseliana Heine; South Africa; Becerra & Venable 1083 (ARIZ) JN882676, JN971049, –. C. kua Vollesen; Kenya; Becerra & Venable 1042 (ARIZ); JN882696, 37677972, –. C. lamii H. Perrier; Madagascar; Becerra & Venable 1043 (ARIZ); JN882689, JN971060, –. C. leptophloeos (Mart.) J.B. Gillett; Brazil; –; 28190282, 37677974, –. C. leptophloeos (Mart.) J.B. Gillett; Bolivia; Becerra & Venable 1044 (ARIZ); 28190282, 37677975 36939095. C. madagascariensis Jacq; India; Becerra & Venable 1080 (ARIZ); JN882672. C. mafaidoha H. Perrier; Madagascar; Becerra & Venable 1045 (ARIZ); JN882703, 61677165, –. C. marlothii Engl.; South Africa; Becerra & Venable 1046 (ARIZ); JN882677, 61677167, –. C. merkeri Engl.; South Africa; Becerra & Venable 1047 (ARIZ); JN882678, JN971051, –. C. mildbraedii Engl.; Kenya; Becerra & Venable 1048 (ARIZ); JN882695, JN971063, –. C. mollis (Oliv.) Engl.; South Africa; Becerra & Venable 1049 (ARIZ); 28190283, 34396119, –. C. monstruosa (Perrier) Capuron; Madagascar; Becerra & Venable 1050 (ARIZ); 28190285, 61677168, 61677206. C. mossambicensis Engl.; South Africa; Becerra & Venable 1051 (ARIZ); JN882685, –. C. myrrha (T. Nees) Engl.; Kenya; Becerra & Venable 1052 (ARIZ); JN882706, 61677169, –. C. neglecta Verd.; South Africa; Becerra & Venable 1053 (ARIZ); JN882686, 61677170, –. C. orbicularis Engl.; Madagascar; Becerra & Venable 1088 (ARIZ); JN882697, 61677172, –. C. ovalifo- lia J.. B. Gillett; Kenya; Becerra & Venable 1054 (ARIZ); JN882688, JN971059. C. pyracanthoides Engl.; South Africa; –; 28190279, 61677174, –. C. rostrata Engl.; Kenya; Becerra & Venable 1055 (ARIZ); JN882704, 37677976 36939096. C. samharensis Schweinf.; Kenya; Becerra & Venable 1056 (ARIZ); JN882680, JN971053, –. C. saxicola Engl.; South Africa; Becerra & Venable 1086 (ARIZ); JN882687, JN971058, –. C. schimperi (O. Berg) Engl.; South Africa; Becerra & Venable 1057 (ARIZ); JN882702, 37677981, 36939097, –. C. simplicifolia H. Perrier; Madagascar; Becerra & Venable 1286 (ARIZ); 28190286, 37677979, –. C. tenuipetiolata Engl.; South Africa; J. Becerra & D.L. Venable 1552 (ARIZ); –, –, –. C. ugogensis Engl.; Tanzania; Lvett 1626 (MO); –, 37677983, 36939098. C. unilobata Gillett & Vollesen; Kenya; Becerra & Venable 1058 (ARIZ); JN882708, 61677178, –. C. viminea Burtt Davy; South Africa; Becerra & Venable 1059 (ARIZ); JN882682, JN971055, –. C. wightii (Arn.) Bhandari; India; –; 167077465, 37677986 36939099. C. wildii Merxm.; South Africa; Becerra & Venable 1060 (ARIZ); JN882705, 61677179, –. Crepidospermum goudotianum Triama and Planch. –; –; C. prancei D.C. Daly; Vasquez & Jaramillo 6232 (MO); Peru; 42475907, 62287789 60116137. C. rhoifolium (Benth.) Triana and Planch; Venezuela; Delgado 759 (NY); 42475908, 37678006, 36939102. Dacryodes klaineana (Pierre) H.J. Lam; –; –; –, 37678047, 36939105. D. buettneri (Engl.) Lam; –; –; –, 37678044, 36939103. D. edulis (G. Don) H.J. Lam; –; –; –, –, 369339104. Dodonaea viscosa (L.) Jacq.; –; –; 326381203, –,60116140. Heeria argentea Meisn.; –; –; 60116113, 60116112, 60116145. Pistacia mexicana Kunth; –; –; 88909741, 37678049, 36939116. Protium copal (Schltdl. & Cham.) Engl.; Mexico; Killen & al. 3136 (MO) –; 42475904, 62287795, 369339106. P. guianense (Aubl.) Marchand; Suriname: Sipaliwini; Miller & Hauk 9391 (MO); 422475893, 37678011, 36939107. P. madagascariense Engl.; Madagascar; Schatz & al. 3616 (MO); 42475910, 37678013, 36939108. P. pilosum (Cuatrec,) Daly; Brazil; Gentry 49069 (MO); –, 37678000, 36939113. P. trifoliolatum Engl.; –; –; 42475906, 62287818. P. unifoliolatum Engl.; Peru: Maynas; Rimachi 2948 (MO); –, 37678003, 36939114. Protorhus longifolia (Bernh.) Engl; –; –; 60116116, 126594357, 60116163. Rhus copallinum L.; –; –; 60116215, 60116215, 60116166. Rhus trilobata Nutt.; –; –; 55977597, 37678052, 36939117. Santiria apiculata A.W. Benn. –; –; 37678032, 369339109. S. griffithii Engl.; –; –; –, 37678035, 36939110. Schinus molle L.; –; –; 55977612, 55494979, 17385601. Sorindeia madagascariensis Thouars ex DC,; –; –; 60116122, 60116220, 60116180. Spondias mombin L.; Mexico; –; 28190289, –, –. Tetragastris altissima (Aubl.) Swart; Guyana: Waraputu; Polak 616 (MO); 224382234, 37678054, 3693911. T. mucronata (Rusby) Swart; Guyana: Waraputu; Polak 616 (MO); –, 37677998, 36939112. Trattinnickia cf. burserifolia Wild.; Brazil: Ducke Reserve; Hoffman &. al. 694 (MO); –, 36939115, 37678037. T. glaziovii Swart; Gentry & Revilla 69141 (MO); –, 37678041, 36939115.

343