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Molecular Phylogenetics and Evolution 60 (2011) 21–28
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Molecular Phylogenetics and Evolution
journal homepage: www.elsevier .com/ locate /ympev
Multi-gene analysis provides a well-supported phylogeny of Rosales
Shu-dong Zhang a, Douglas E. Soltis b, Yang Yang a, De-zhu Li a,⇑, Ting-shuang Yi a,⇑a Key Laboratory of Biodiversity and Biogeography; Plant Germplasm and Genomics Center, Germplasm Bank of Wild Species, Kunming Institute of Botany,Chinese Academy of Sciences, Kunming, Yunnan 650204, Chinab Department of Biology, University of Florida, Gainesville, FL 32611, USA
a r t i c l e i n f o a b s t r a c t
Article history:Received 9 June 2010Revised 7 April 2011Accepted 7 April 2011Available online 22 April 2011
Keywords:BarbeyaceaeDirachmaceaePhylogeneticsRosales
1055-7903/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.ympev.2011.04.008
⇑ Corresponding authors.E-mail addresses: [email protected] (D.-Z. Li),
(T.-S. Yi).
Despite many attempts to resolve evolutionary relationships among the major clades of Rosales, somenodes have been extremely problematic and have remained unresolved. In this study, we use two nuclearand 10 plastid loci to infer phylogenetic relationships among all nine families of Rosales. Rosales werestrongly supported as monophyletic; within Rosales all family relationships are well-supported with Ros-aceae sister to all other members of the order. Remaining Rosales can be divided into two subclades: (1)Ulmaceae are sister to Cannabaceae plus (Urticaceae + Moraceae); (2) Rhamnaceae are sister to Elaeagn-aceae plus (Barbeyaceae + Dirachmaceae). One noteworthy result is that we recover the first strong sup-port for a sister relationship between the enigmatic Dirachmaceae and Barbeyaceae. These two smallfamilies have distinct morphologies and potential synapomorphies remain unclear. Future studies shouldtry to identify nonDNA synapomorphies uniting Barbeyaceae with Dirachmaceae.
� 2011 Elsevier Inc. All rights reserved.
1. Introduction
Analyses of individual chloroplast (cpDNA) or nuclear loci, andmore recently concatenated data sets and supermatrices, haveproduced well-resolved phylogenetic trees of rosids (Rosidae, sen-su Cantino et al., 2007). As a result, most deep level phylogeneticrelationships within the Rosidae are now well understood (seeHilu et al., 2003; Ravi et al., 2007; Savolainen et al., 2000a,b;Soltis et al., 2000, 2005, 2007; Wang et al., 2009). However,relationships within some orders (sensu APG, 2009) remainuncertain; Rosales represent one such example. Rosales comprisenine families: Barbeyaceae, Cannabaceae, Dirachmaceae,Elaeagnaceae, Moraceae, Rhamnaceae, Rosaceae, Ulmaceae, andUrticaceae (APG, 1998, 2003, 2009). As reviewed elsewhere, thesefamilies are morphologically heterogeneous (Baas et al., 2000),and had been considered to be distantly related and placed in dif-ferent orders in traditional classifications (e.g., Cronquist, 1988;Dahlgren, 1989; Takhtajan, 1987; Thorne and Reveal, 2007; Wuet al., 2003). Based on sequence data from the plastid invertedrepeat (IR), Zhang et al. (2009) recently proposed that the familyCynomoriaceae may also be part of Rosales. However, other datasets have variously placed this enigmatic family of parasites with-in Santalales, Saxifragales, Myrtales, or Sapindales (reviewed inJian et al. (2008) and Nickrent et al. (2005)). Hence, the actualphylogenetic position of Cynomoriaceae is still not clear. Recent
ll rights reserved.
molecular studies in the Soltis’ lab using more nuclear and mito-chondrial loci further reveal the phylogenetic position of this fam-ily; but only IR data suggest an affinity with Rosales (Jian, Soltis,Matthews, unpublished data). We thus excluded Cynomoriaceaefrom the current study of Rosales.
Although the circumscription of Rosales has become clear,interfamily relationships within the order remained ambiguous(Thulin et al., 1998). Some phylogenetic studies suggest that Rosa-ceae may be sister to the rest of the order (e.g. Hilu et al., 2003;Savolainen et al., 2000b; Soltis et al., 2000, 2007), with the remain-ing families forming two distinct clades: one clade centered onUlmaceae and relatives and the second composed of Rhamnaceaeand relatives (see Richardson et al., 2000; Savolainen et al.,2000a; Sytsma et al., 2002). However, phylogenetic relationshipsamong several families, Barbeyaceae, Dirachmaceae, Elaeagnaceaeand Rhamnaceae, remained uncertain. Recently, Wang et al. (2009)published a well resolved phylogeny of Rosidae that revealed astrongly supported Rosales as sister to Cucurbitales plus Fagales.Most family level relationships within Rosales were resolved byWang et al. (2009) and were consistent with previous studies,but with higher bootstrap support values. However, two families,Barbeyaceae and Dirachmaceae, were not included in the analysesby Wang et al. and most families were represented by just one ortwo exemplars.
The main purpose of this study is to better resolve phylogeneticrelationships among members of Rosales, with particular emphasison the poorly understood families Barbeyaceae, Dirachmaceae, Ela-eagnaceae and Rhamnaceae. To accomplish these goals we con-structed a data set employing 12 genes.
22 S.-d. Zhang et al. / Molecular Phylogenetics and Evolution 60 (2011) 21–28
2. Materials and methods
2.1. Taxon sampling
We constructed a data set of 25 species (representing 25 gen-era) of Rosales. Sequences for five of these species are fromWang et al. (2009) and were downloaded from GenBank (Appen-dix A). The other 20 species are based on data provided in thisstudy. These species represent all nine recognized families of Ro-sales, and span much of the lineage diversity suggested in previ-ous phylogenetic studies (Hadiah et al., 2008; Potter et al., 2007;Richardson et al., 2000; Song et al., 2001; Sytsma et al., 2002;Wiegrefe et al., 1998; Zerega et al., 2005). For most of the taxaincluded here, the same DNA was used for all PCR and sequenc-ing. However, sequences for matK of Filipendula and 26S rDNA ofBroussonetia were sequenced in earlier studies (Mishima et al.,2002; Zerega et al., 2005) from different congeneric species oraccessions (Appendix A). The 18S rDNA and atpB sequences ofCannabis sativa were obtained from a different accession fromthat used by Wang et al. (2009); but other sequences of this spe-cies are from Wang et al. (2009). We failed to amplify atpB ofDirachma and rps4 of Filipendula and Gouania despite multipleattempts. Sequences of 13 genera of Fagales, Fabales andCucurbitales obtained by Wang et al. (2009) were used as out-groups. The choice of outgroups (i.e., other members of theNitrogen-Fixing clade) was based on recent molecular phyloge-netic studies of Rosidae (e.g., Ravi et al., 2007; Wang et al.,2009). Among the outgroups, eight sequences were unavailable(psbBTNH and rps4 of Anisophyllea; 26S rDNA of Cucumis; ndhFof Polygala; 18S rDNA, 26S rDNA and atpB of Quillaja; 26S rDNAof Stylobasium); sequences of five genera (Alnus, Begonia, Myrica,Polygala and Quercus) were amplified from different congenericspecies (see Appendix A).
2.2. DNA isolation, amplification and sequencing
In this study, we targeted ten plastid (rbcL, atpB, matK, thepsbBTNH region (= 4 genes), rpoC2, ndhF, and rps4) and two nucleargenes (18S and 26S rDNA) for all taxa. Primers for amplificationand sequencing are provided in Table 1.
Total genomic DNAs were extracted from silica-gel-dried leavesusing the DNeasy Plant Mini Kit (Qiagen Inc., Valencia, California,USA). Polymerase chain reaction (PCR) amplifications were per-formed in 25 ul reactions with approximately 10–50 ng of genomicDNA, 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mMof each dNTP, 0.1 lM of each primer, and 1U Taq polymerase (TaKa-Ra). The PCR parameters for all the DNA regions were as follows: a94 �C initial hot start for 10 min with 35 cycles of 94 �C for 30 s,50 �C (55 �C for the primers 544F and 1587R of 26S rDNA) for30 s and 72 �C for 120 s, and a final extension of 72 �C for 10 min.PCR products were isolated and purified using the DNA purificationkit (Sangon Inc., Shanghai, China) following the manufacturer’sinstructions. Cycle sequencing was carried out using the ABI PrismBigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Fos-ter City, California, USA) according to the recommendations of thehandbook, with slight modifications in some cases. Cycle sequenc-ing conditions were as follows: 30 cycles of 10 s denaturation(96 �C), 5 s annealing (50 �C), and 4 min elongation (60 �C). Sampleswere sequenced in an ABI 3730xl automated sequencer. We se-quenced both strands of DNA with overlapping regions to help en-sure that each base call is unambiguous. Electropherograms wereassembled; ambiguous bases were corrected; and consensus se-quences were generated with Sequencer (Gene Codes Crop., AnnArbor, Michigan, USA). Sequences generated in this study aredeposited in GenBank (Appendix A).
2.3. Phylogenetic analysis
Sequences were initially aligned using MAFFT (Katoh et al.,2002) and manually adjusted using Se-Al ver. 2.0 (Rambaut,2002) where necessary. Gaps were treated as missing data and re-gions (553–1101 bp) of questionable homology in rps4 were omit-ted from subsequent phylogenetic estimations. All data matriceshave been deposited in TreeBASE (study number SN 11237).
Given that the plastid genome behaves as a single linked regionand that the regions examined exhibited low levels of variation,the chloroplast markers were concatenated a priori. Topologicalincongruence between partitions (combined plastid vs. nuclearmarkers) was examined using the Incongruence Length Difference(ILD) test (Farris et al., 1995) as implemented in PAUP�4.0b10(Swofford, 2003) with 1000 heuristic searches after the removalof all invariable characters from the data set (Cunningham, 1997).
Phylogenetic analyses of the combined plastid, combined nucle-ar, as well as the total evidence data set were performed usingmaximum parsimony (MP) in PAUP�4.0b10 (Swofford, 2003),Maximum-likelihood (ML) using Garli v0.96 (Zwickl, 2006), andBayesian inference (BI) using MrBayes 3.1.2 (Ronquist andHuelsenbeck, 2003). For the parsimony analyses, heuristic treesearches were conducted with tree bisection–reconnection (TBR)branch swapping, with zero-length branches collapsed. The initialtrees for branch swapping were obtained by using 1000 randomstepwise taxon addition replicates, with 10 trees saved at eachstep. All character states were treated as unordered and equallyweighted, with gaps treated as missing data. Support was assessedusing the bootstrap (MPBS) (Felsenstein, 1985) by using 1000 rep-licates with the same settings as for heuristic searches.
For Bayesian and maximum-likelihood analyses, individual andcombined datasets were tested for the appropriate model of nucle-otide evolution with ModelTest 3.7 (Posada and Crandall, 1998).The model chosen by the Akaike information criterion (AIC) wasthen implemented for the analyses (Posada and Buckley, 2004).
For the Bayesian inference, one cold and three incrementallyheated Markov chain Monte Carlo (MCMC) chains were run for3,000,000 generations until the average deviation of split frequen-cies fell well below 0.01. Trees were sampled every 100 genera-tions. Since model partitioning is important for large datasets inthe Bayesian analysis (Brown and Lemmon, 2007), a partitionedBayesian analysis of the combined nuclear and plastid data setwas implemented by applying the previously determined modelsto each data partition. For each dataset, MCMC runs were repeatedtwice to avoid spurious results. Stationarity of the Markov chainwas ascertained by plotting likelihood values against number ofgenerations for apparent stationarity. The first 25% trees beforestationarity were discarded as burn in, and the remaining treeswere used to construct majority-rule consensus trees usingPAUP�4.0b10 (Swofford, 2003). The posterior probability (PP) valueof each topological bipartition was estimated by the frequency ofthese bipartitions across all sampled trees.
Maximum-likelihood analyses (ML) were conducted using Garliv0.96 (Zwickl, 2006). Default parameters were used for the Garlisearches except that significant topochange was set to 0.01. A totalof 100 ML bootstrap replicates (MLBS) were also performed byusing Garli. The trees obtained from Garli were used to constructmajority-rule consensus trees using PAUP�4.0b10 (Swofford, 2003).
3. Results
3.1. Characteristics of sequence data and inferred phylogenetic tree
A total of 17,706 aligned base pairs representing the 12 targetedgenes for 38 taxa (25 members of Rosales and 13 outgroup taxa)
Table 1Primers used for PCR and sequencing.
Gene/partition Primer name Primer sequence References
18S rDNA 15F TGC CAG TAS TCA TAT GCT TG Designed for this study911F TTC TTG GAT TTA TGA AAG ACG Designed for this study1000R GAC GGT ATC TGA TCG TCT TC Designed for this study1770R TAC GGA AAC CTT GTT ACG AC Designed for this study
26S rDNA 20F CCG CTG AAT TTA AGC ATA TC Designed for this study544F TGG TGA ACT ATG CCT GAG CG Designed for this study1810F GGA GAC CTG ATG CGG TTA TG AG Designed for this study2700F CTC TGC CAC TTA CAA TAC CC Designed for this study900R AAC GAT TTG CAC GTC AGT AT Designed for this study1587R CTT GAA AAT CCG GAG GAC Designed for this study2760R GCT TTC ACG GTT CGT ATT C Designed for this study3300R ACA GAA ATC TCG TGT GAA AC Designed for this study
atpB 110F TGC CTA ATA TTT ACA AYG CTC T Designed for this study1440R TCA TCR ATG TTA CCT ACC AA Designed for this study
matK FAmatK490F SAA ATC TTG GTT CAA AYC CTT CG Hilu et al. (2003)trnK2R AAC TAG TCG GAT GGA GTA G Johnson and Soltis (1995)
ndhF 100F RTG GGY TTT TCC WAG TRT TTT Designed for this study972R CTC GAT AAG ACC CCA TAC CT Designed for this study972F GTC TCA ATT GGG TTA TAT GAT G Olmstead and Sweere (1994)2153R GGA ATT CCA TCA ATT AYT CGT YTA TC Designed for this study
psbBTNH B60F CAT ACW GCT YTR GTT GCT GGT TGG Designed for this study63F GGA TTR CGY ATG GGA AAT ATT GAA AC Designed for this study67F GRG AYG TYT TTG CYG GTA TTG A Designed for this studyB64R CTT GCT GRA ART AKC CYT GAT CCC Designed for this studyB68R GTA GTY GGR TCY CCM AGT TTT TTG G Designed for this studyB71R CCW GGA GCT ACT TTA CCA TAT TC Designed for this study
rbcL 30F AGC AAG TGT TGG ATT CAA AG Designed for this study870R ACG RTG RAT GTG AAG AAG TA Designed for this study1400R CRG CMG CTA GTT CRG GAC TC Designed for this study
rpoC2 80F TAG ATC ACT TCG GAA TGG C Wang et al. (2009)910F ATY TGY CGA TTA TGT TAT GG Designed for this study1880F TTY TTG CTT ATT TYG ATG ATC Designed for this study2960F GGM ACT WTT CRT ACG TTS TTG A Designed for this study1000R GAT TGA CCC GCA ATA ATA CC Wang et al. (2009)2050R GAT TCR GGK AAA ATR TGC AC Designed for this study3030R AAT GGA YYC ATT TGA AAA C Designed for this study4070R GCT TCS GAT ATR AAA CTT TG Designed for this study4200R TTT CAG GCC TTT YAR CCA RTC Wang et al. (2009)
rps4 48F GGC TTT ACC RGG ACT AAC Wang et al. (2009)trnS TAC CGA GGG TTC GAA TC Souza-Chies et al. (1997)
Table 2Characteristics of individual and combined data sets of Rosales.
Dataset No. oftaxa
Alignedlength
Variablesites
Parsimony-informativesites
No. MPtrees
MP treelength
CI RI Model selected byAIC
GARLI MLscore
18S rDNA 37 1733 250 139 23 599 0.528 0.510 GTR + I + G �5834.322526S rDNA 35 3263 573 317 239 1457 0.510 0.450 GTR + I + G �12169.6252atpB 36 1242 345 237 18 874 0.542 0.617 GTR + I + G �6463.3948matK 38 1045 644 425 5 1922 0.521 0.592 GTR + G �10761.0902ndhF 37 2150 1064 730 5 3509 0.492 0.563 GTR + I + G �19698.5016psbBTNH 37 2340 760 466 4 1936 0.553 0.591 TVM + I + G �13364.9566rbcL 38 1289 372 236 6 1107 0.454 0.526 GTR + I + G �7603.5706rpoC2 38 4092 1898 1213 2 4619 0.574 0.627 GTR + I + G �30019.9586rps4 34 552 200 108 2 410 0.649 0.715 TVM + G �3017.3150Total nuclear
genes37 4996 823 456 6 2075 0.510 0.458 GTR + I + G �18150.4507
Total plastidgenes
38 12,710 5283 3415 1 14,565 0.528 0.585 GTR + I + G �92526.4124
Total evidence 38 17,706 6106 3878 1 16,684 0.525 0.570 GTR + I + G �111984.9963
S.-d. Zhang et al. / Molecular Phylogenetics and Evolution 60 (2011) 21–28 23
were analyzed in this study. Sequence characteristics of the gene/partition are presented in Table 2. The ILD test comparing the 18Sand 26S rDNA datasets suggested a P value of 0.51, indicating con-gruence between the two data sets. For this reason, the two nuclearregions were combined in analyses. The ILD test comparing thecombined nuclear vs. the combined plastid markers yielded a P va-
lue of 0.01, indicating significant incongruence between the twodata sets. The sensitivity of the ILD test is well-known (e.g., Darluand Lecointre, 2002; Hipp et al., 2004). Importantly, the treesresulting from the combined nuclear vs. the combined plastid datasets did not contain well-supported clades that were in conflict,and the nuclear data set didn’t resolve the relationships among
24 S.-d. Zhang et al. / Molecular Phylogenetics and Evolution 60 (2011) 21–28
families. Hence, the plastid and nuclear partitions were combinedin our analyses. MP, ML and BI analyses of the total evidence data-set yielded similar topologies within Rosales, with the later de-picted in Fig. 1.
3.2. Phylogenetic relationships of Rosales
Based on the samples included in our analyses, Rosales arestrongly supported as monophyletic (MPBS = 100%; MLBS = 100%;
Ba
H
Ceanot
Neillia
Dryas
Datisca
Myr
Juglans
Alnus
Stylobasium
Albizia
Quillaja
100/100/1.0
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-/64/0.89
Fig. 1. ML tree resulting from GARLI analysis of total evidence dataset (two nuclear genesBI analyses are shown above clades (MPBS/MLBS/PP, dash indicate the branches that ar
PP = 1.0). Within Rosales, most families were represented by twoto four samples (excepting the small families Barbeyaceae andDirachmaceae); the monophyly of each family was also stronglysupported. Rosaceae are sister to the rest of the order. The remainingRosales can be divided into two subclades: (1) Ulmaceae, Cannaba-ceae, Moraceae, and Urticaceae (MPBS = 100%; MLBS = 100%;PP = 1.0); (2) Rhamnaceae, Elaeagnaceae, Barbeyaceae, andDirachmaceae (MPBS = 82%; MLBS = 100%; PP = 1.0). In the first ofthese clades, Moraceae and Urticaceae were well-supported as
Ulmus
Zelkova
rbeya
Dirachma
Broussonetia
Ficus
Cudrania
Morus
Debregeasia
Pilea
Trema
Cannabis
Celtis
Elaeagnus
Hippophae
Gouania
ovenia
hus
Rhamnus
Spiraea
Sanguisorba
Filipendula
Begonia
Cucumis
Coriaria
Anisophyllea
ica
Quercus
Polygala
Moraceae
Urticaceae
Cannabaceae
Ulmaceae
Elaeagnaceae
Barbeyaceae
Dirachmaceae
Rhamnaceae
Rosaceae
RO
SAL
ES
CU
CU
RB
ITAL
ES
FAG
AL
ES
FAB
AL
ES
100/100/1.0
0/1.0
0/100/1.0
Boehmeria
Trema
Cannabis
Celtis
Cannabaceae
rbeya Barbeyaceae
ElaeagnusE
HippophaeHElaeagnaceae0/100/1.0
Spiraea
Sanguisorba
Filipendula
Rosaceae
, 10 plastid genes). The bootstrap values (%) of MP and ML analysis, and PP values ofe not supported by MPBS, MLBS or PP values).
S.-d. Zhang et al. / Molecular Phylogenetics and Evolution 60 (2011) 21–28 25
monophyletic (MPBS = 99%; MLBS = 100%; PP = 1.0); Mora-ceae + Urticaceae, in turn, formed a clade with Cannabaceae(MPBS = 100%; MLBS = 100%; PP = 1.0); finally, Ulmaceae were sisterto Cannabaceae, Moraceae, and Urticaceae (MPBS = 100%;MLBS = 100%; PP = 1.0). In the second of these clades, Rhamnaceaewere sister to the rest of this clade, which formed a well-supportedmonophyletic group (MPBS = 100%; MLBS = 100%; PP = 1.0). We re-cover the first strong support for a sister relationship between theenigmatic Dirachmaceae and the monotypic Barbeyaceae(MPBS = 54%; MLBS = 88%; PP = 1.0). Dirachmaceae plus Barbeya-ceae formed a clade with Elaeagnaceae (MPBS = 69%; MLBS = 67%;PP = 1.0).
4. Discussion
Matrices of concatenated sequences with many characters butlimited taxon sampling may greatly exacerbate phylogenetic arti-facts caused by long-branch attraction (Felsenstein, 1978; Soltiset al., 2004; Stefanovic et al., 2004). Rosales are very diverse,including 9 families 261 genera and 7725 species (Stevens,2001). Recent molecular studies have largely resolved phylogeneticrelationships within most families of Rosales (Hadiah et al., 2008;Potter et al., 2007; Richardson et al., 2000; Song et al., 2001;Sytsma et al., 2002; Wiegrefe et al., 1998; Zerega et al., 2005).Our sampling spanned the phylogenetic diversity of most familiesand was designed to mitigate as much as possible against long-branch attraction and related systematic errors. We performedphylogenetic analyses using MP, BI, as well as ML analyses, thelatter is relatively insensitive to the problem of long-branch attrac-tion (Huelsenbeck, 1997). Furthermore, different methods yieldedsimilar topologies. Our analysis thus likely does not suffer fromerrors related to taxon sampling.
This molecular study is the first to include all currently recog-nized families of Rosales. We found strong support for the mono-phyly of Rosales; our molecular data fully resolved phylogeneticrelationships within Rosales with strong internal support. Rosa-ceae were sister to all other Rosales, which was congruent withprevious studies (Hilu et al., 2003; Savolainen et al., 2000b; Soltiset al., 2000; Sytsma et al., 2002; Wang et al., 2009). The remainderof the order comprises two subclades; (1) Ulmaceae are sister toCannabaceae plus (Urticaceae + Moraceae); (2) Rhamnaceae aresister to Elaeagnaceae plus (Barbeyaceae + Dirachmaceae). Theclose relationship among members of the first of these subclades(Ulmaceae, Cannabaceae, Urticaceae, Moraceae) agrees with previ-ous classifications; these families were included in Urticales. In aprevious molecular analysis, Sytsma et al. (2002) referred to thisclade as the Urticalean rosids. The phylogenetic relationshipsamong Ulmaceae, Cannabaceae, Urticaceae, Moraceae recoveredhere are consistent with previous molecular studies (Sytsmaet al., 2002; Wang et al., 2009), further supporting the relationshipsof (Ulmaceae, (Cannabaceae, (Moraceae, Urticaceae))). Circum-scription of Ulmaceae and Cannabaceae and the relationshipsamong these four families are discussed in detail by Sytsma et al.(2002) who recovered the same topology.
Considering the second subclade recovered here (Rhamnaceae,Elaeagnaceae, Barbeyaceae and Dirachmaceae), phylogenetic rela-tionships among these families were not resolved or supportedin previous molecular studies (Richardson et al., 2000; Savolainenet al., 2000b; Soltis et al., 2007; Thulin et al., 1998). Our moleculardata strongly support a clade formed by these four families, withinwhich Rhamnaceae are sister the remaining families; Elaeagnaceaeare then sister to a clade of Barbeyaceae + Dirachmaceae.
One of the most important findings of this study is the sistergroup relationship of Barbeyaceae + Dirachmaceae. The mono-typic Barbeyaceae (Barbeya oleoides Schweinf.) are distributed
in the northeastern corner of Africa and adjacent areas of Arabia(Thulin et al., 1998). This species was first described by Schwein-furth (1891) as a member of Urticaceae s.l. and has been in-cluded in the family Ulmaceae (Engler, 1897; Engler and Diels,1936; Melchior, 1964); more often it has been placed in itsown family Barbeyaceae (see Thulin et al., 1998). Morphologicaland anatomical evidence suggested uncertain affinities forBarbeyaceae (Bouman and Boesewinkel, 1997; Dickison andSweitzer, 1970), however the sieve tube elements of the second-ary phloem, vestiture and pollen morphology suggested thatBarbeyaceae may be ‘‘an independent offshoot’’ (Dickison andSweitzer, 1970; see also Tobe and Takahashi, 1990). Phylogeneticanalyses using 22 morphological characters plus DNA sequencessuggested a close relationship between Barbeyaceae andElaeagnaceae with relatively high support (Thulin et al., 1998).The phylogenetic position of Barbeyaceae has been addressedin only a few previous molecular studies. Swensen (1996)suggested Barbeyaceae belonged to a clade composed ofElaeagnaceae, Rhamnaceae and Urticales. Using 18S rDNA, rbcLand atpB data, Soltis et al. (2000, 2007) found Barbeyaceaeformed a clade with Elaeagnaceae, but Dirachmaceae were notincluded in those studies.
Dirachma (Dirachmaceae) comprises two species endemic tothe Isle of Socotra and central Somalia (Link, 1991). Dirachmaceaehave unique flower architecture with distinctive nectaries, fruitmorphology, and anemochorous seed dispersal, which have tradi-tionally given this family an isolated status; its affinities with othertaxa were uncertain when condidering morphology (Link, 1991).Dirachma has traditionally been included in Geraniaceae (e.g.Cronquist, 1981, 1988; Dahlgren, 1989; Takhtajan, 1987). Thisgenus is more often treated as a member of its own familyDirachmaceae, and associated with Geraniaceae in Geraniales orwith Tiliaceae in Malvales (see Boesewinkel and Bouman, 1997;Thulin et al., 1998). Based on ovules, seed and vegetative anatom-ical characters, Boesewinkel and Bouman (1997) and Baas et al.(2001) suggested a close relationship between Dirachmaceae andRhamnaceae. A possible sister relationship between these twofamilies was weakly supported with molecular data (Richardsonet al., 2000; Savolainen et al., 2000b; Thulin et al., 1998), andadditional analyses were required.
A close relationship of Barbeyaceae with Dirachmaceae as foundhere had been proposed previously, albeit without support, basedon rbcL and trnL-F sequences (Thulin et al., 1998). These two fam-ilies have distinct morphologies and potential synapomorphies re-main unclear. The two families do share an indumentum withunicellular and non-glandular trichomes, absence of a nectary disk,superior ovary, and straight embryo (Ronse De Craene and Miller,2004; Thulin et al., 1998), but these characters also are shared byother members of Rosales. Interestingly, Barbeyaceae andDirachmaceae do share a similar geographic range in northeasternAfrica. Future studies should try to identify nonDNA synapomor-phies uniting Barbeyaceae with Dirachmaceae.
Our molecular data also reveal that Elaeagnaceae are sister toBarbeyaceae plus Dirachmaceae. Elaeagnaceae include three gen-era, Elaeagnus, Hippophae and Shepherdia. Most authors tradition-ally placed Elaeagnaceae near to Proteaceae, Rhamnaceae,Thymelaeaceae, or Penaeaceae; Elaeagnaceae have been includedin different orders in different classifications (see Jansen et al.,2000). The placement of Elaeagnaceae in a clade composed of Bar-beyaceae, Dirachmaceae and Rhamnaceae is supported by recentmolecular studies (Richardson et al., 2000; Savolainen et al.,2000b; Soltis et al., 2007; Sytsma et al., 2002; Thulin et al., 1998;Wang et al., 2009), however the relationships between Elaeagna-ceae and other three families were not well resolved or supported.Wood anatomical features also suggest a rather isolated position ofElaeagnaceae in Rosales (Jansen et al., 2000). Our molecular data
26 S.-d. Zhang et al. / Molecular Phylogenetics and Evolution 60 (2011) 21–28
strongly support a sister relationships between Elaeagnaceae andBarbeyaceae plus Dirachmaceae.
Rhamnaceae contain about 50 genera and approximately 900species (Richardson et al., 2000). Rhamnaceae have been placednear to diverse groups including Vitaceae and Leeaceae on the ba-sis of shared floral features (Cronquist, 1988; Takhtajan, 1980) andElaeagnaceae on the basis of shared vegetative characteristics(Hutchinson, 1959; Takhtajan, 1997; Thorne, 1992). A close rela-tionship of Rhamnaceae with Barbeyaceae, Dirachmaceae and Ela-eagnaceae was first suggested based on rbcL sequences (Soltiset al., 1995), and further supported in subsequent morphologicaland molecular phylogenetic studies (Richardson et al., 2000;Savolainen et al., 2000b; Soltis et al., 2007; Sytsma et al., 2002;Thulin et al., 1998; Wang et al., 2009). Richardson et al. (2000)suggested a sister relationships between Rhamnaceae and theDirachmaceae and Barbeyaceae clade. Soltis et al. (2007) revealedthat Rhamnaceae are sister to a clade of Barbeyaceae andElaeagnaceae with very weak support. Our results are in agreementwith these earlier studies, but provide the first strong support ofRhamnaceae as sister to a clade of Elaeagnaceae and (Barbeyaceaeplus Dirachmaceae).
Acknowledgments
We thank Anthony Miller, Xun Gong and Meiqing Yang for helpwith obtaining samples, the DNA Bank of the Royal Botanic Gar-dens, Kew for offering DNAs, and anonymous reviewers and Asso-ciate Editor Barbara Whitlock for constructive comments. Thelaboratory experiments were done in the Molecular Biology Exper-iment Center, Kunming Institute of Botany. This study was sup-ported by the National Natural Science Foundation of China(Grant No. 40830209) and CAS Knowledge Innovation Program(Grant No. KSCX2-YW-R-136) and the Angiosperm Tree of Life Pro-ject (NSF EF-0431266).
Appendix A
Taxa, specimen numbers, herbaria where the specimens aredeposited, and GenBank accession numbers for sequence used inthis study. A dash indicates the sequence is not available for thespecies. Data in square bracket indicated different species or acces-sion used. Accession numbers are given in the following order: 18S,26S, atpB, matK, rbcL, ndhF, psbBTNH, rpoC2, rps4. Abbreviations ofherbaria follow Index Herbariorum (Holmgren et al., 1990).
Rosales: – Barbeyaceae: Barbeya oleoides Schweinf., Collenette1/93 (K); JF317358, JF317379, JF317398, JF317418, JF317437,JF317457, JF317477, JF317497, JF317517. Cannabaceae: Cannabissativa L., W. Hahn s.n. (WIS); JF317360 [M.Q. Yang 003 (KUN)],EU002151, JF317400 [M.Q. Yang 003 (KUN)], AF345317,AJ402933, AY289250, EU002390, EU002481, EU002301. Celtis tetr-andra Roxb., S.D. Zhang 090047 (KUN); JF317361, JF317380,JF317401, JF317420, JF317439, JF317459, JF317479, JF317499,JF317519. Trema angustifolia (Planch.) Blume, X. Gongzw2009082601 (KUN); JF317376, JF317395, JF317415, JF317434,JF317454, JF317474, JF317494, JF317514, JF317532. Dirachma-ceae: Dirachma socotrana Schweinf. ex Balf.f., A. Miller LB20 (E);JF317364, JF317383, –, JF317423, JF317442, JF317462, JF317482,JF317502, JF317522. Elaeagnaceae: Elaeagnus bockii Diels, S.D.Zhang 090030 (KUN); JF317366, JF317385, JF317405, JF317425,JF317444, JF317464, JF317484, JF317504, JF317524. Hippophaerhamnoides L. subsp. sinensis Rousi, S.D. Zhang and H.J. He 081532(KUN); JF317370, JF317389, JF317409, JF317428, JF317448,JF317468, JF317488, JF317508, JF317526. Moraceae: Broussonetiapapyrifera Vent., T.S. Yi and S.D. Zhang 080645 (KUN); JF317359,AY686805 [G. Weiblen 1176 (MIN)], JF317399, JF317419,
JF317438, JF317458, JF317478, JF317498, JF317518. Cudrania tricu-spidata Bureau ex Lavallée, S.D. Zhang 090045 (KUN); JF317362,JF317381, JF317402, JF317421, JF317440, JF317460, JF317480,JF317500, JF317520. Ficus tikoua Bureau, T.S. Yi and S.D. Zhang080142 (KUN); JF317367, JF317386, JF317406, JF317426,JF317445, JF317465, JF317485, JF317505, JF317525. Morus indicaL., unknown; L24398 [M. alba L., Nickrent 2924 (SIU)], AF479232[M. nigra L., Nickrent 2924 (SIU)], NC008359, NC008359,NC008359, NC008359, NC008359, NC008359, NC008359. Rhamn-aceae: Ceanothus prostratus Benth., D. Soltis 2712 (FLAS); U42799,AF479102, AF209558, AF049815, U06795, EU002210, EU002393,EU002484, EU002304. Gouania mauritiana Lam., Chase 904 (K);JF317369, JF317388, JF317408, JF317427, JF317447, JF317467,JF317487, JF317507, –. Hovenia trichocarpa Chun and Tsiang var.robusta (Nakai and Y. Kimura) Y.L. Chen and P.K. Chou, S.D. Zhang090046 (KUN); JF317371, JF317390, JF317410, JF317429,JF317449, JF317469, JF317489, JF317509, JF317527. Rhamnus utilisDecne., S.D. Zhang 090890 (KUN); JF317374, JF317393, JF317413,JF317432, JF317452, JF317472, JF317492, JF317512, JF317530.Rosaceae: Dryas octopetala L., S.D. Zhang 070897 (KUN);JF317365, JF317384, JF317404, JF317424, JF317443, JF317463,JF317483, JF317503, JF317523. Filipendula vulgaris Moench, S.D.Zhang 070898 (KUN); JF317368, JF317387, JF317407, AB073684[F. multijuga Maxim., unknown 010531 (TI)], JF317446, JF317466,JF317486, JF317506, –. Neillia thibetica Bureau and Franch., S.D.Zhang and H.J. He 081314 (KUN); JF317372, JF317391, JF317411,JF317430, JF317450, JF317470, JF317490, JF317510, JF317528. San-guisorba sitchensis C.A. Mey., T.S. Yi and S.D. Zhang 080431 (KUN);JF317375, JF317394, JF317414, JF317433, JF317453, JF317473,JF317493, JF317513, JF317531. Spiraea tomentosa L., D. Soltis 2691(FLAS); U42801, AF479103, AJ235608, AF288127, L11206,EU002259, EU002451, EU002541, EU002362. Ulmaceae: Ulmusmacrocarpa Hance, T.S. Yi and S.D. Zhang 080312 (KUN); JF317377,JF317396, JF317416, JF317435, JF317455, JF317475, JF317495,JF317515, JF317533. Zelkova serrata Makino, R.G. Olmstead 2001-2(WTU); U42819, AF479099, AF209699, AF345328, AF206835,EU002273, EU002465, EU002556, –. Urticaceae: Debregeasia sae-neb (Forssk.) Hepper and J.R.I. Wood, T.S. Yi Yi09052 (KUN);JF317363, JF317382, JF317403, JF317422, JF317441, JF317461,JF317481, JF317501, JF317521. Pilea cadierei Gagnep. and Guillau-min, T.S. Yi Yi09051 (KUN); JF317373, JF317392, JF317412,JF317431, JF317451, JF317471, JF317491, JF317511, JF317529.Boehmeria macrophylla Hornem. var. scabrella (Roxb.) D.G. Long,T.S. Yi Yi09053 (KUN); JF317378, JF317397, JF317417, JF317436,JF317456, JF317476, JF317496, JF317516, JF317534.
OutgroupsCucurbitales: – Anisophylleaceae: Anisophyllea fallax Scott-
Elliot, L. Gautier 3256 (MO); AY968390, AY968401, AY968424,AY968444, AF027109, AY968487, –, EU002470, –. Begoniaceae:Begonia cucullata Willd., D. Soltis 2696 (FLAS); AF008950 [B. oxylobaWelw. ex Hook. f., Brouillet 1670-86 (unknown)], AY968403 [B. oxy-loba Welw. ex Hook. f., Hughes s.n. (L. Forrest 279) (E)], AY968426[B. oxyloba Welw. ex Hook. f., Hughes s.n. (L. Forrest 279) (E)],AY968445 [B. oxyloba Welw. ex Hook. f., Hughes s.n. (L. Forrest279) (E)], L01888, EU002200, EU002384, EU002475, EU002294.Coriariaceae: Coriaria nepalensis Wall., M.W. Chase 6416 (K);AY968394, AY968407, AY968429, AB016460, AY968522,EU002214, EU002399, EU002490, EU002310. Cucurbitaceae: Cuc-umis sativa L., unknown; AF206894 [unknown], –, NC007144,NC007144, NC007144, NC007144, NC007144, NC007144,NC007144. Datiscaceae: Datisca cannabina L., M.W. Chase 2745(K); AF008952, AY968410, AJ235450, AY491654, L21939,EU002219, EU002403, EU002495, EU002315.
Fabales: – Fabaceae: Albizia julibrissin Durazz., D. Soltis 2633(FLAS); U42536, X66754, AF209524, AY386855, Z70147,EU002195, EU002379, EU002468, EU002288. Polygalaceae:
S.-d. Zhang et al. / Molecular Phylogenetics and Evolution 60 (2011) 21–28 27
Polygala cruciata L., M.W. Chase 155 (K); U42797 [P. pauciflora Muhl.ex Willd., Doyle 1567 (BH)], AF479233, AJ235568, AY386842[P. californica Nutt. ex Torr. and A. Gray, Wojciechowski and Steele887 (ASU)], L01945, –, EU002443, EU002533, EU002355. Quillaja-ceae: Quillaja saponaria Molina, M.W. Chase 10931 (K); –, –, –,AY386843, U06822, EU002255, EU002446, EU002536, EU002358.Surianaceae: Stylobasium spathulatum Desf., Brummitt, Georgeand Oliver 21242 (K); AF207030, –, AF209681, EU604032,U06828, EU002263, EU002455, EU002545, EU002366.
Fagales: – Betulaceae: Alnus sinuata (Regel) Rydb., R.G. Olm-stead 2005-5 (WTU); AY263900, AF479106 [A. glutinosa (L.) Gaertn.,unknown], AY147101, AB060053 [A. firma Siebold and Zucc., un-known], AY263926, EU002196, EU002380, EU002469, EU002289.Fagaceae: Quercus nigra L., M.J. Moore 311 (FLAS); AY147108 [Q.oxyodon Miq., Chen and Ma 961142 (XS)], AY094177 [Q. suber L., un-known], EU002167, EU002188, EU002284, EU002254, EU002445,EU002535, EU002357. Juglandaceae: Juglans nigra L., D. Soltis2520 (WS); AF206943, AF479105, AF209609, AF118036,AF206785, EU002237, EU002425, EU002515, EU002337. Myrica-ceae: Myrica cerifera L., D. Soltis 2699 (FLAS); AF206967,AF479247, AJ235537, AY191715 [M. gale L., KLW 9788 (NSW)],AF119179, EU002243, EU002433, EU002523, EU002345.
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