Phylogenetic analysis of the genus Hexachlamys (Myrtaceae) based on...

Phylogenetic analysis of the genus Hexachlamys(Myrtaceae) based on plastid and nuclear DNAsequences and their taxonomic implications

FERNANDA DA CRUZ1,2, ANDREIA C. TURCHETTO-ZOLET1,2*, NICOLE VETO2,CLÁUDIO AUGUSTO MONDIN3, MARCOS SOBRAL4, MAURÍCIO ALMERÃO2 andROGÉRIO MARGIS1,2

1Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Federal do RioGrande do Sul (UFRGS), Porto Alegre, Brazil2Centro de Biotecnologia and Departamento de Biofísica, UFRGS, Porto Alegre, Brazil3Pontifícia Universidade Católica do Rio Grande do Sul, PUCRS, Porto Alegre, Brazil4Departamento de Ciências Naturais, Universidade Federal de São João del-Rei, São João del-Rei,Brazil

Received 17 April 2012; revised 21 August 2012; accepted for publication 22 January 2013

Myrtaceae are one of the most species-rich families of flowering plants in the Neotropics. They include severalcomplex genera and species; Hexachlamys is one of the complex genera. It has not been recognized as a distinctgenus and has been included in Eugenia, based on morphological grounds. Therefore, molecular systematic studiesmay be useful to understand and to help to solve these relationships. Here, we performed a molecular phylogeneticanalysis using plastid and nuclear data in order to check the inclusion of Hexachlamys in Eugenia. Plastid (accD,rpoB, rpoC1, trnH-psbA) and nuclear (ITS2) sequence data were analysed using Bayesian and maximumparsimony methods. The trees constructed using ITS2 and trnH-psbA were the best able to resolve the relation-ships between species and genera, revealing the non-monophyly of Hexachlamys. The molecular phylogeneticanalyses were in agreement with previous morphological revisions that have included Hexachlamys in Eugenia.These results reinforce the importance of uniting knowledge and strategies to understand better issues ofdelimitation of genera and species in groups of plants with taxonomic problems. © 2013 The Linnean Society ofLondon, Botanical Journal of the Linnean Society, 2013, ••, ••–••.

ADDITIONAL KEYWORDS: chloroplast DNA (cpDNA) – internal transcribed spacer (ITS) – molecularphylogeny – Myrtaceae.

INTRODUCTION

Myrtaceae are a pantropical family of floweringplants with 142 genera and > 5500 species (Wilson,2011) and are one of the most species-rich families inthe Neotropics (Lucas et al., 2007). Representatives ofthis family have great ecological significance in forestecosystems and are also economically important tothe pharmaceutical, food, cosmetic and perfumeryindustries (Barroso & Peron, 1994). Despite theimportance of the family, the identities of several

species and delimitation of some genera are stilldebatable (Lucas et al., 2005).

The pantropical tribe Myrteae (sensu Wilson et al.,2005) contain approximately 54 genera and 1887species of trees and shrubs, of which approximately35 genera and 1700 species are Neotropical (Wilson,2011). All Brazilian Myrtaceae (224 genera andapproximately 970 species; Sobral et al., 2012) belongto tribe Myrteae.

Hexachlamys O.Berg was erected by Berg(1855–1856) for a single species, H. humilis O.Berg,collected in southern Brazil. It is distributed fromBolivia and Paraguay to south-eastern and southern*Corresponding author. E-mail: [email protected]

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Botanical Journal of the Linnean Society, 2013, ••, ••–••. With 4 figures

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Brazil, Argentina and Uruguay (McVaugh, 1968;Legrand & Klein, 1977). Berg distinguished Hexach-lamys from Eugenia L. based on the number andpersistence of calyx lobes (six and deciduous in Hex-achlamys vs. four and persisting in Eugenia) and theembryo morphology (hypocotyl visible and exsertedvs. cryptic). Such characters were not considered assignificant by some other authors; for example,Bentham (1868) and Niedenzu (1893), who consideredH. humilis to be a species of Eugenia. Nevertheless,the genus has been accepted by most students of theMyrtaceae throughout the 20th century (e.g. Legrand,1968; McVaugh, 1968; Legrand & Klein, 1977;Rotman, 1982; Mattos, 1983). In a synopsis of theBrazilian genera of Myrtaceae, Landrum & Kawasaki(1997) reported that Hexachlamys was closely relatedto Eugenia and should eventually be united with it, aconcept that has been accepted by some authors (e.g.Mattos, 1995; Sobral, 2003; Sobral et al., 2012).

Regarding the generic distinctions identified byBerg (1855–1856), it is true that embryo morphologyis suggestive of generic distinctness in Myrtaceae;since the work of Berg, three basic types of embryomorphology in American Myrtaceae have been recog-nized: one group with well-developed cotyledons andhypocotyl (informally, the Myrcia DC. group), anotherwith a well-developed hypocotyl and inconspicuouscotyledons (informally, the Psidium L. group) and athird with well-developed cotyledons and a small orcryptic hypocotyl (informally, the Eugenia group), butsee Lucas et al. 2007 for a reappraisal of embryotypology and phylogenetic relations in AmericanMyrteae). The embryo morphology of Hexachlamys,with an embryo with a small but visible hypocotyl(see illustration in Rotman, 1982), although fitting inthe Eugenia group, is not characteristic of mostEugenia. Nevertheless, embryo morphology in thisgenus is variable; most species have embryos withcotyledons firmly fused in an homogeneous mass, butsome species (e.g. E. beaurepaireana (Kiaersk.) D.Le-grand and E. pyriformis Cambess.) have embryoswith plano-convex, completely separate cotyledons. Ifthe embryo variation is accepted in a narrow sense todraw generic boundaries, it would also be wortherecting a new genus for these anomalous Eugeniaspp., and Kausel (1956) proposed a new genus, Pseu-domyrcianthes Kausel, to accomodate these and otherspecies. For the moment, genera erected on the samegrounds of the embryo distinction between Hexach-lamys and Eugenia, such as Pseudomyrcianthes orParamyrciaria Kausel, comprising species formerlyincluded in Myrciaria O.Berg but with separate coty-ledons (Kausel, 1966; Rotman, 1982; Sobral, 1991),have been relegated to the synonymy of other genera(McVaugh, 1968; Landrum & Kawasaki, 1997); con-sidering this, the features of Eugenia and Hexach-

lamys embryos do not seem distinctive enough tokeep the two genera separate.

Several Hexachlamys spp. have been reported tohave a variable number of calyx lobes, from fourto seven (Legrand, 1936, under E. myrcianthesNied.; Rotman, 1982: Sobral, 2003, under Eugenia).Although it is true that no Eugenia spp. except thoseoccasionally assigned to Hexachlamys are reported tohave more than four calyx lobes, this only character isnot sufficient to draw a generic limit (for instance,Psidium includes species with four to five calyx lobesand species with calyx lobes fused into one unit; seeMcVaugh, 1963); additionally, were it a Eugenia withfive calyx lobes, it would be probably included underHexachlamys by American myrtologists.

The deciduous calyx stressed by Berg (1855–1856)has not received attention from other Myrtaceae stu-dents who discussed Hexachlamys (e.g. McVaugh,1968; Legrand & Klein, 1977; Rotman, 1982;Landrum & Kawasaki, 1997). The plate in Berg’streatment of Brazilian species (Berg, 1857–1859)clearly depicts fruits with a circular scar attributableto the fall of calyx lobes. Unfortunately, fruits fromthe two remaining isotypes of H. humilis (at herbariaBR and US) are lost, and so it is no longer possible toverify if this feature was really present in the speci-mens examined by Berg. Recent gatherings of fruitingspecimens of H. humilis (filed under E. anomalaD.Legrand) do not show this feature; nevertheless,fruits with a circular scar were depicted by Rotman(1982), although this feature was not referred to inher description. Thus, the presence of a deciduouscalyx in Hexachlamys remains an open question.

A feature of the species assigned to Hexachlamysand absent from the protologue, as Berg did notexamine flowering specimens, is the presence of tri-chomes in the locule walls (see illustrations inRotman, 1982). This has been recorded in severalunrelated angiosperm families, including Araceae,Connaraceae, Sapotaceae, Styracaceae and Urticaceae(Dickison, 1993), and is poorly understood, althoughthere is evidence that they play a role in directingpollen tubes (Steyn, Robbertse & Coetzer, 1991).Intra-ovarial trichomes appeared independently inseveral unrelated lineages of American Myrtaceae; forexample, in most species of Neomitranthes Kausel exD.Legrand, a Brazilian genus of 16 species (Sobralet al., 2012), some Eugenia spp. included by Berg inhis section Dichotomae O.Berg (e.g. E. ischnoscelesO.Berg and E. pyriformis Cambess.; Berg, 1857–1859)and in most Hexachlamys spp. As is frequent in thetaxonomic history of American Myrtaceae, this char-acter was once considered important enough for erect-ing a genus, Pilothecium (Kiaersk.) Kausel (Kausel,1960), which is presently considered a synonym ofEugenia (Legrand, 1975).

2 F. DA CRUZ ET AL.

© 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, ••, ••–••

Most Hexachlamys spp. have been sporadicallytreated in floristic studies (Legrand, 1968; Legrand &Klein, 1977; Rotman, 1982; Mattos, 1983; Romagnolo& Souza, 2004). However, there is no thorough system-atic treatment of all species included in the genus.There are presently 20 names in the genus (Govaertset al., 2012; subspecific names not considered); mostspecies are from Brazil, except H. boliviana D.Legrand(Eugenia boliviana (D.Legrand) Mattos), H. bolivien-sis Kausel (a synonym of H. boliviana), H. rojasiana(D.Legrand) D.Legrand [a synonym of E. anomala; anisotype at SI and the protologue (Legrand, 1962) wereexamined] and H. tapiraguayensis (Barb.Rodr. exChod. & Hassl.) Mattos (also a synonym of E. anomala;Sobral, 2003). From the 16 names recorded for Brazil,seven species are recognized under Eugenia by Sobralet al. (2012): E. anomala (synonyms: H. humilisO.Berg, H. anomala (D.Legrand) D.Legrand and H. to-ledoi Mattos], E. dysenterica DC. (= H. macedoi D.Le-grand), E. geraensis (D.Legrand & Mattos) Mattos(= H. geraensis D.Legrand & Mattos), E. hamiltonii(Mattos) Mattos [= H. emrichii Mattos, H. hamiltoniiMattos, H. itararensis Mattos and H. sehnemiana(Mattos) Mattos], E. handroi (Mattos) Mattos[= H. handroi Mattos, H. itatiaiensis Mattos andH. kleinii (D.Legrand) Mattos], E. myrcianthes Nied.[= H. edulis (O.Berg) Kausel & D.Legrand, H. excelsa(Cambess.) Mattos, H. minarum Mattos & D.Legrand)and E. subterminalis DC. (= H. legrandii Mattos),although two of these species, E. dysenterica andE. subterminalis, clearly do not belong to the speciesgroup formerly delimited as Hexachlamys.

So far, the taxonomic discussions about Hexach-lamys have been based only on morphological char-acters. Therefore, studies of molecular systematicsmay be useful to help in its taxonomic delimitation. Abetter understanding of taxonomically complexgroups is needed and many recent studies haveshown the importance of molecular systematics in theelucidation of taxonomic relationships (Karehed &Bremer, 2007; Karehed et al., 2008; Peterson,Levichev & Peterson, 2008; Van Ee, Riina & Berry,2011; Wilson, 2011). Plastid markers, such as psbA-trnH, matk, rpl16, rbcL among others (van derMerwe, Van Wyk & Botha, 2005; Wilson et al., 2005;Lucas et al., 2007; Ji et al., 2008; Englund et al., 2009)and nuclear internal transcribed spacers (ITS)(Karehed et al., 2008; Englund et al., 2009; Zarreiet al., 2009; Kim et al., 2010) have been widely used inphylogenetic studies in plants.

In this study we performed a molecular phyloge-netic analysis including species of Hexachlamys andEugenia, using plastid and nuclear (nrDNA) markersin order to check the inclusion of Hexachlamys inEugenia, as has been proposed based on morphologi-cal characters.

MATERIAL AND METHODSSAMPLE COLLECTION

The study samples were collected as leaf materialfrom either herbaria specimens or field samples(Fig. 1). Six species formerly included in Hexachlamyswere included in this study, two collected in the fieldand four from herbaria. Eighteen Eugenia spp. wereused; for outgroup taxa, Myrciaria cuspidata O.Berg,Myrcia glabra (O.Berg) D.Legrand, Myrcia brasilien-sis Kiaersk., Myrcia palustris DC., Myrcianthes cis-platensis (Cambess.) O.Berg, Myrcianthes gigantea(D.Legrand) D.Legrand and Psidium sp. wereselected (see also Supporting Information, AppendixS1). The genera used as outgroups were, untilrecently, considered members of the three differentsubtribes of Myrteae in which the American generahave traditionally been included: subtribes MyrciinaeO.Berg (Myrcia), Eugeniinae O.Berg (Myrcianthesand Myrciaria) and Myrtinae O.Berg (Psidium).However, this subtribal circumscription is not phylo-genetically sound, according to the results of Lucaset al. (2007), and the authors of that study haveproposed an arrangement of the American Myrteaeinto six informal groups. Following their arrange-ment, Myrcia would be placed in the Myrcia group,Myrcianthes in the Eugenia group, Myrciaria in thePlinia group and Psidium in the Pimenta group. Inmost cases, more than one individual was sampled foreach species. Samples were collected as silica geldried leaves from natural populations and as leavesfrom herbaria. Voucher specimens were collected anddeposited in the ICN Herbarium of Department ofBotany at the Federal University of Rio Grande doSul (UFRGS) (see also Supporting Information,Appendix S1). The Supporting Information (AppendixS1)shows a list of Hexachlamys spp. with their alter-natives names in Eugenia.

DNA EXTRACTION, AMPLIFICATION AND SEQUENCING

Total genomic DNA was isolated using the 2 ¥ cetyltrimethylammonium bromide (CTAB) method (Doyle& Doyle, 1987). For the samples taken from herbariawe used the GenomiPhi V2 DNA amplification Kit(GE Healthcare). In this study, we tested 15 potentialDNA regions (Table 1); however, because of the diffi-culty of amplification for herbaria samples, only fourplastid regions (accD, psbA-trnH, rpoC1 and rpoB)and one nrDNA (ITS2) region were considered appro-priate for our analysis.

All primer sequences used for polymerase chainreaction (PCR) amplification and sequencing and theexpected sizes of the resulting fragments are pre-sented in Table 1. The PCR protocol was conductedusing 10 ng of genomic DNA, 2.5 mM magnesium

MOLECULAR PHYLOGENETIC OF HEXACHLAMYS 3


chloride (MgCl2), 0.25 mM deoxyribonucleotide tri-phosphates (dNTP) mix, 1 ¥ PCR buffer, 0.05 U ofPlatinum Taq DNA polymerase (Invitrogen) and0.25 mM of each primer, in a total volume of 20 mL. Allprimers were initially tested, and the following PCRconditions were able to amplify all fragments: aninitial hot-start step at 94 °C for 5 min, followed by 35cycles with denaturation at 94 °C for 20 s, an anneal-ing temperature of 50 °C for 30 s, and 45 s of elonga-tion at 72 °C. All PCR products were visualized byelectrophoresis on 1.5% agarose gels stained withSYBR Gold (Invitrogen) and precipitated using 3 M

sodium acetate (1:10) and 95% ethanol beforesequencing. Amplified PCR products were sequencedwith the dideoxy chain-termination method usingBig-Dye according to the manufacturer’s instructionsand run on an ABI-3100 automatic sequencer(Applied Biosystems). Both DNA strands were fullysequenced.

EDITING AND SEQUENCE ALIGNMENT

Sequences were individually checked by eye, and alldetected polymorphisms were checked with the origi-nal electropherograms. If possible polymorphismswere still unclear, independent PCR reactions were

carried out to confirm them. Sequence identities werecertified using the BLASTn algorithm against plantDNA sequences deposited at NCBI (http://www.ncbi.nlm.nih.gov). Nucleotide sequences were alignedusing MUSCLE (Edgar, 2004) implemented inMEGA5 (Molecular Evolutionary Genetics Analysis)version 5.0 (Tamura et al., 2011). Sequences gener-ated in this study were deposited in GenBank, andaccession numbers are found in the Supporting Infor-mation (Appendix S1).

PHYLOGENETIC ANALYSES

Four separate analyses were carried out. The first(analysis A) analysis used three concatenated plastidmarkers (accD, rpoB and rpoC1) and included 18Eugenia spp. and six Hexachlamys spp. The second(analysis B) analysis used four concatenated plastidmarkers (accD, rpoB, rpoC1 and trnH-psbA) and onlyfour Hexachlamys spp. and 17 Eugenia spp. The third(analysis C) analysis used concatenate nrDNA andchloroplast DNA (cpDNA) for 17 Eugenia spp. andfour Hexachlamys spp. The fourth (analysis D) wasperformed using only the nrDNA marker andincluded six Hexachlamys spp. and 18 Eugenia spp.For all four analyses, species from four genera

Figure 1. Map showing the collection sites of Eugenia spp. and Hexachlamys spp. used in this study. The circles andtriangles represent samples collected from the field and from herbaria, respectively. RS, Rio Grande do Sul State; SP, SãoPaulo State; MT, Mato Grosso State.

4 F. DA CRUZ ET AL.


http://www.ncbi.nlm.nih.gov

http://www.ncbi.nlm.nih.gov

Tab

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(Myrcia, Myrciaria, Myrcianthes and Psidium) wereused as outgroups. Maximum parsimony (MP) andBayesian analyses (BA) were used for phylogeneticinference. The number of Hexachlamys spp. variedbetween analyses because of difficulties in the ampli-fication of DNA for samples obtained from herbaria.

MAXIMUM PARSIMONY ANALYSIS

PAUP* (version 4.10b; Swofford, 2007) was used forparsimony analysis following widely used methods,including successive approximations, weighting andbootstrapping (Fay et al., 2000, Clarkson et al., 2004)(the bootstrap did not use the relative weights). Inanalyses A, B, C and D, tree searches were performedunder the heuristic search criterion with 1000random sequence additions and tree–bisection–reconnection (TBR) branch swapping, permitting tentrees to be held at each step (Multrees on), whichreduced the time spent searching suboptimal ‘islands’of trees (Chase et al., 2006). All of the shortest treescollected in the 1000 replicates were swapped on tocompletion without a tree limit. To evaluate internalsupport, 1000 bootstrap (BS) replicates were carriedout with equal weights, TBR branch swapping withfive trees held at each step and simple taxon addition(Felsenstein, 1985). The following descriptions for cat-egories of bootstrap support were used: weak, 50–74;moderate; 75–84; well supported, 85–100% (Chaseet al., 2000).

BAYESIAN ANALYSIS

Further phylogenetic analyses were performed usingBayesian inference as implemented in MrBayes(version 3.12; Ronquist & van der Mark, 2005).MrModeltest (version 2.2; Nylander, 2005) was usedto determine the best model of DNA substitution foreach partition, evaluating all models against the

defaults of the program. The GTR + I + G model (ageneral time-reversible model with a proportion ofinvariable sites and a gamma-shaped distribution ofrates across sites) was chosen for the four data sets asthe best fitting among the 24 models compared. Thus,all four data sets were assigned a model of six sub-stitution types (n ¼ 6) with a proportion of invariablesites. Two independent runs of 3 000 000 generations,each with two Monte Carlo Markov chains (MCMC),were run in parallel (starting each from a randomtree). Markov chains were sampled every 100 genera-tions, and the first 25% of the trees were discarded asburn-in. The remaining trees were used to compute amajority rule consensus tree (MrBayes command all-compat), the posterior probability (PP) of clades andbranch lengths. Convergence of the two runs wasassessed by checking the average standard deviationof split frequencies (below 0.01) and the potentialscale reduction factor (PSRF, very close to 1.00 for allparameters).

RESULTSSEQUENCE CHARACTERISTICS OF ITS AND THE FOUR

PLASTID DNA REGIONS

Sequence characteristics for each of the five markersand the three data sets (analysis A, B and C) areshown in Table 2. The total aligned length for nrDNA(ITS2) sequences was 418 bp, which included 160variable and 81 (19.38%) potentially parsimony-informative characters. The trnH-psbA intergenicspacer sequences showed variation at 243 of 563 sitesand 85 (15.10%) potentially parsimony-informativecharacters. For accD, the total aligned lengthsequences was 221, with 85 variable and 52 (23.53%)potentially parsimony-informative characters. Thetotal aligned length for rpoB sequences was 407 bp,which included 49 variable and 45 (11.06%) poten-

Table 2. DNA site variation for each marker and for the data sets used in the phylogenetic analyses

Total sitesaligned (N)

Variablesites (N)

Potentiallyparsimonyinformativesites (N)

Potentiallyparsimonyinformativesites (%)

accD 221 85 52 23.53rpoB 407 49 45 11.06rpoC1 434 59 49 11.29psbA-trnH 563 243 85 15.10(Analysis A) accD + rpoB + rpoC1 1062 193 151 14.22(Analysis B) accD + rpoB + rpoC1 + trnH-psbA* 1505 405 138 9.17(Analysis C) accD + rpoB + rpoC1 + trnH-psbA + ITS2* 1933 566 221 11.43(Analysis D) ITS2 418 160 81 19.38

*For the combined data set in analysis B and C, only four Hexachlamys spp. were used.

6 F. DA CRUZ ET AL.


tially parsimony-informative characters. The rpoC1sequences contained variation at 59 out 434 sites and49 (11.29%) parsimony-informative characters.

PHYLOGENETIC ANALYSES

All plastid and nrDNA regions showed polymor-phisms among the studied species. In general, resultsfrom the BA and MP tree analyses produced similartopologies and revealed that Hexachlamys spp. didnot form a monophyletic group (Figs 2–4); Hexach-lamys spp. were grouped with Eugenia spp. with highsupport.

PLASTID DATA SET (ANALYSIS A)

The total aligned length for the combined sequences inanalysis A (accD, rpoB and rpoC1) was 1062 charac-ters, which included 193 variable sites and 151(14.22%) potentially parsimony-informative charac-

ters. For this data set, MP and BA produced similartopologies; both recovered unresolved topologies withmoderately to highly supported branches (Fig. 2A andsee also Supporting Information, Appendix S2A). TheMP analysis produced 51 equally most-parsimonioustrees with a consistency index (CI) of 0.87 and aretention index (RI) of 0.94. The topologies of the MPand BA analyses revealed that the six Hexachlamysspp. did not form a monophyletic group. However,these markers were not very effective at resolvingrelationships among species.

PLASTID DATA SET (ANALYSIS B)

The combined sequences in analysis B (accD, rpoB,rpoC1 and trnH-psbA) included 1505 characters,which included 405 variable sites and 138 (9.17%)potentially parsimony-informative characters. BA andMP produced similar and well-resolved topologieswith highly supported branches (Fig. 2B and see also

Figure 2. Maximum parsimony analysis. A, phylogenetic analysis of combined accD, rpoB and rpoC1 sequences (analysisA). B, phylogenetic analysis of combined accD, rpoB and rpoC1 and trnH-psbA sequences (analysis B). Bootstrap values> 50% are shown above the branches.



Supporting Information, Appendix S2B). The MPanalysis produced 81 equally most-parsimonioustrees (CI = 0.798, RI = 0.818). The presence of thetrnH-psbA intergenic spacer in this data set caused itto produce trees with better resolution than analy-sis A, which only contained three markers (accD,rpoB and rpoC1). In both analyses (BA and MP), thefour Hexachlamys spp. did not form a monophyleticgroup. The Eugenia/Hexachlamys clade was well sup-ported (BS = 90%).

PLASTID AND NRDNA COMBINED (ANALYSIS C)

The combined plastid and nrDNA sequences in analy-sis C (accD, rpoB, rpoC1 and trnH-psbA + ITS2) con-sisted of 1933 characters, which included 566 variablesites and 221 (11.43%) potentially parsimony-informative characters. Both MP and BA analysesresulted in similar topologies (Fig. 3 and see alsoSupporting Information, Appendix S3). The MPanalysis produced 35 equally most-parsimonious

trees (CI = 0.76, RI = 0.80). In this analysis, asobserved in analysis B, Hexachlamys spp. did notform a monophyletic group. Hexachlamys emrichiigrouped with E. pyriformis and E. beaurepaireanawith moderate support (PP = 0.81) and the Eugenia/Hexachlamys clade was well supported (PP = 0.97).

NRDNA (ITS2) DATA SET (ANALYSIS D)

The nrDNA (ITS2) sequences alone were used toperform this phylogenetic analysis. The MP analysisproduced 81 equally most parsimonious trees(CI = 0.72 and RI = 0.83). In both analyses, the sixHexachlamys spp. did not form a monophyletic group.This analysis shows strong support for the groupingof H. boliviana with E. uniflora and E. dysenterica(PP = 1, BS = 100%) and the Eugenia/Hexachlamysclade was again well supported (PP = 0.89). The sepa-rate analysis of nrDNA was effective for resolving therelationships between species and genera (Fig. 4 andsee also Supporting Information, Appendix S4).

Figure 3. Bayesian inference analysis based on combined data of four plastid DNA (accD. rpoB.rpoC1 and trnH-psbA)and nrDNA (ITS2) regions (analysis C). Posterior probabilities values > 50% are shown above the branches.

8 F. DA CRUZ ET AL.


DISCUSSIONThe present study showed that the sequence datafrom both biparentally (nrDNA) and maternally(plastid) inherited genomes were all informative tosome degree, supporting the non-monophyly of Hex-achlamys. However, regarding the effectiveness inresolving phylogenetic relationships at the level of thegenus and species, ITS2 was better than plastid DNA(trnH-psbA, accD, rpoB and rpoC1). It has been shownthat at least some nuclear sequences evolve morequickly than plastid DNA, which could provide betterresolution of relationships in younger taxa or taxawith slow evolutionary rates (Karehed et al., 2008).Here, the phylogenetic analysis using nuclear ITS2region showed a well-supported clade (PP = 0.89)grouping Eugenia spp. and Hexachlamys spp. (namedas Eugenia/Hexachlamys clade in Fig. 4 and the Sup-porting Information, Appendix S4). The efficacy of theITS2 region has been also described in other studies(Schultz et al., 2005; Schultz, 2009; Muellner, Schaefer

& Lahaye, 2011). Moreover, Chen et al. (2010) showedthat the ITS2 region is an accurate and reliable toolfor species authentication, as its rate of successfulidentification was 92.7% at the species level. Thecombination of nuclear ITS2 sequences with plastidDNA (trnH- psbA, accD, rpoB and rpoC1) regions alsoproduced a well-resolved tree, showing a well-supported clade (PP = 0.97) grouping Eugenia spp.and Hexachlamys spp. (Fig. 3). It demonstrates thatthe combination of different markers may be impor-tant for elucidating the phylogenetic and taxonomicrelationships.

The phylogenetic analyses using the plastid (accD,rpoB and rpoC1) sequences produced unresolved treeswith many polytomies (Fig. 2A and see also SupportingInformation, Appendix S2A), showing that thesemarkers were ineffective in resolving the relationshipsin the taxa studied. It is probable that the high geneticsimilarity that resulted in these unresolved trees wascaused by the low rate of sequence divergence in these

Figure 4. Bayesian inference analysis obtained from ITS2 (analysis D). Posterior probabilities values > 50% are shownabove the branches.



genes, allied to the recent and rapid speciation of thesetaxa. The same result was also demonstrated in astudy of Meliaceae, in which these markers were alsonot effective in resolving species, and even the combi-nation of multiple loci did not improved the perform-ance (Muellner et al., 2011). When the intergenicspacer trnH-psbA was combined with accD, rpoB andrpoC1 genes (Fig. 2B and see also Supporting Infor-mation, Appendix S2B) we observed a better resolutionbetween species in relation to those performed onlywith accD, rpoB and rpoC1. However, this data set wasnot so efficient to resolve the relationships amonggenera, as a well-supported clade (BS = 90%) includingMyrcianthes spp. in the Eugenia/Hexachlamys cladewas observed. These results may suggest that theseplastid regions have not had enough time for diver-gence to separate these genera, which belong to thesame subtribe, Eugeniniae, of Myrteae (Sobral et al.,2012). Furthermore, we found some difficulties in PCRamplification and sequencing of trnH-psbA in samplesobtained from herbaria.

In the present study, we found some discordancebetween plastid and nrDNA gene trees. For example,in the plastid analysis (Fig. 2A), H. boliviana groupedwith H. hamiltonii and E. dysenterica (BS = 100),whereas in the nuclear analysis (Fig. 4) it was closelyrelated with E. dysenterica and E. uniflora (PP = 1).Such incongruence can be explained by reticulateevolution, which has been demonstrated in manystudies (Rieseberg et al., 1991; Soltis et al., 1991;Ferris et al., 1993; Jackson et al., 1999; Peng & Wang,2008; Wang et al., 2012).

CONCLUSIONS

We conducted a molecular phylogenetic analysis ofspecies previously classified as Hexachlamys andseveral Eugenia spp. Our results, together with thelittle morphological differentiation between thesegenera, corroborate the proposed synonymization ofHexachlamys under Eugenia. This findings reinforcesthe importance of the use of molecular approaches inhelping to understand issues of taxonomic delimita-tion. To further understand the evolutionary relation-ships in Eugenia, one of the largest genera inMyrtaceae, more studies including molecular andmorphological approaches are needed.

ACKNOWLEDGEMENTS

This work was supported by CNPq (Conselho Nacionalde Desenvolvimento Científico e Tecnológico) andFAPERGS (Fundação de Amparo a Pesquisa do Estadodo Rio Grande do Sul). F.D.C. received a master’sfellowship from CNPq, A.C.T.-Z. received PNPD/CAPES fellowships, M.A. received PDJ/ CNPq fellow-

ships, N.V. received IC fellowships and R.M. is therecipient of CNPq research fellowships. We would liketo thank the herbarium ICN at the Department ofBotany of the Federal Universidad do Rio Grande doSul (UFRGS), the herbarium Alarich Rudolf HolgerSchultz of the Museu de Ciências Naturais from theFundação Zoobotânica of Rio Grande do Sul (FZB) andthe herbarium Maria Eneyda P. K. Fidalgo from theInstitute of Botany of São Paulo (SP) for providing theHexachlamys samples for this study. We also thankthe administrators of Park Saint Hilaire and Pró-Mataof Rio Grande do Sul for their permission to collectEugenia and Hexachlamys in these areas.

REFERENCES

Barroso GM, Peron MV. 1994. Myrtaceae. In: de LimaMPM, Guedes-Bruni RR, eds. Reserva Ecológica de Macaéde Cima, Nova Friburgo: RJ. aspectos florísticos das espéciesvasculares, Vol. 1. Rio de Janeiro: Jardim Botânico, 261–302.

Bentham G. 1868. Notes on Myrtaceae. Journal of theLinnean Society (Botany) 10: 101–166.

Berg OC. 1855–1856. Revisio Myrtacearum Americae.Linnaea 27: 1–799.

Berg OC. 1857–1859. Myrtaceae. Flora Brasiliensis 14:1–656.

Chase MW, De Bruijn AY, Cox AV, Reeves G, Rudall PJ,Johnson MAT, Eguiarte LE. 2000. Phylogenetics ofAsphodelaceae (Asparagales): an analysis of plastid rbcLand trnL-F DNA sequences. Annals of Botany 86: 935–951.

Chase MW, Fay MF, Devey DS, Maurin O, Rønsted N,Davies TJ, Pillon Y, Petersen G, Seberg O, TamuraMN, Asmussen CB, Hilu K, Borsch T, Davis JI, Ste-venson DW, Pires JC, Givnish TJ, Sytsma KJ,McPherson MA, Graham SW, Rai HS. 2006. Multigeneanalyses of monocot relationships: a summary. In: Colum-bus JT, Friar EA, Hamilton CW, Porter JM, Prince LM,Simpson MG, eds. Monocots: comparative biology and evo-lution. Claremont: Rancho Santa Ana Botanic Garden,63–75.

Chen SL, Yao H, Han JP, Liu C, Song JY, Shi LC, ZhuYJ, Ma XY, Gao T, Pang XH, Luo K, Li Y, Li XW, JiaXC, Lin YL and Leon C. 2010. Validation of the ITS2region as a novel DNA barcode for identifying medicinalplant species. PLoS ONE 5: 8.

Clarkson JJ, Knapp S, Garcia VF, Olmstead RG, LeitchAR, Chase MW. 2004. Phylogenetic relationships in Nico-tiana (Solanaceae) inferred from multiple plastid DNAregions. Molecular Phylogenetics and Evolution 33: 75–90.

Dickison WC. 1993. Floral anatomy of the Styracaceae,including observations on intra-ovarian trichomes. Botani-cal Journal of the Linnean Society 112: 223–255.

Doyle JJ, Doyle JL. 1987. Isolation of plant DNA from freshtissue. Focus 12: 13–15.

Edgar RC. 2004. MUSCLE: multiple sequence alignmentwith high accuracy and high throughput. Nucleic AcidsResearch 32: 1792–1797.

10 F. DA CRUZ ET AL.


Englund M, Pornpongrungrueng P, Gustafsson MHG,Anderberg AA. 2009. Phylogenetic relationships andgeneric delimitation in Inuleae subtribe Inulinae (Aster-aceae) based on ITS and cpDNA sequence data. Cladistics25: 319–352.

Fay MF, Rudall PJ, Sullivan S, Stobart KL, de BruijnAY, Reeves G, Qamaruz-Zaman F, Hong WP, Joseph J,Hahn WJ, Conran JG, Chase MW. 2000. Phylogeneticstudies of Asparagales based on four plastid DNA regions.In: Wilson KL, Morrison DA, eds. Monocots: systematics andevolution. Melbourne: CSIRO, 360–371.

Felsenstein J. 1985. Confidence limits on phylogenies: anapproach using the bootstrap. Evolution 39: 783–791.

Ferris C, Oliver RP, Davy AJ, Hewitt GM. 1993. Nativeoak chloroplasts reveal an ancient divide across Europe.Molecular Ecology 2: 337–344.

Govaerts R, Sobral M, Ashton P, Barrie F, Holst BK,Landrum LR, Matsumoto K, Mazine FF, NicLughadhaE, Proença C, Soares-Silva LH, Wilson PG, Lucas EJ.2012. World checklist of Myrtaceae. The Board of Trustees ofthe Royal Botanic Gardens, Kew. Published on the Internet.Available at: http://www.kew.org/wcsp/ Accessed 2012–08–20.

Hamilton MB. 1999. Four primer pairs for the amplificationof chloroplast intergenic regions with intraspecific variation.Molecular Ecology 8: 521–523.

Jackson HD, Steane DA, Potts BM, Vaillancourt RE.1999. Chloroplast DNA evidence for reticulate evolutionin Eucalyptus (Myrtaceae). Molecular Ecology 8: 739–751.

Ji SG, Huo KK, Wang J, Pan SL. 2008. A molecular phy-logenetic study of Huperziaceae based on chloroplast rbcLand psbA-trnH sequences. Journal of Systematics and Evo-lution 46: 213–219.

Karehed J, Bremer B. 2007. The systematics of Knoxieae(Rubiaceae) – molecular data and their taxonomic conse-quences. Taxon 57: 668–668.

Karehed J, Groeninckx I, Dessein S, Motley TJ, BremerB. 2008. The phylogenetic utility of chloroplast and nuclearDNA markers and the phylogeny of the Rubiaceae tribeSpermacoceae. Molecular Phylogenetics and Evolution 49:843–866.

Kausel E. 1956. Beitrage zur Systematik der Myrtaceen.Arkiv für Botanik ser. 2 3: 491–516.

Kausel E. 1960. Zur Systematik von Pilothecium Kiärskou.Arkiv für Botanik ser. 2 4: 401–405.

Kausel E. 1966. Lista de las mirtaceas y leptospermaceasargentinas. Lilloa 32: 323–388.

Kim JH, Kim DK, Forest F, Fay MF, Chase MW. 2010.Molecular phylogenetics of Ruscaceae sensu lato and relatedfamilies (Asparagales) based on plastid and nuclear DNAsequences. Annals of Botany 106: 775–790.

Kress WJ, Erickson DL. 2007. A two-locus Global DNAbarcode for land plants: the coding rbcL gene complementsthe non-coding trnH-psbA spacer region. Plos One 2: 508.

Kress WJ, Wurdack KJ, Zimmer EA, Weigt LA, JanzenDH. 2005. Use of DNA barcodes to identify flowering plants.Proceedings of the National Academy of Sciences of theUnited States of America 102: 8369–8374.

Landrum LR, Kawasaki ML. 1997. The genera of Myrta-ceae in Brazil: an illustrated synoptic treatment and iden-tification keys. Brittonia 49: 508–536.

Legrand CD. 1936. Las mirtáceas del Uruguay. Anales delMuseo de Historia Natural de Montevideo, sér. 2 4: 1–71.

Legrand CD. 1962. Algunas especies y variedades nuevas demirtaceas de Paraguay y Argentina. Boletin de la SociedadArgentina de Botánica 10: 1–10.

Legrand CD. 1968. Las mirtaceas del Uruguay. III. Boletinde la Facultad de Agronomía de Montevideo 101: 1–80.

Legrand CD. 1975. Sobre Pilothecium (Kiaersk.) Kausel.Bradea 2: 33–40.

Legrand CD, Klein RM. 1977. Mirtáceas – Campomanesia,Feijoa, Britoa, Myrrhinium, Hexachlamys, Siphoneugena,Myrcianthes, Neomitranthes, Psidium. In: Reitz R, ed. Florailustrada Catarinense. Itajaí: Herbário Barbosa Rodriques,571–730.

Lucas EJ, Belsham SR, Lughadha EMN, Orlovich DA,Sakuragui CM, Chase MW, Wilson PG. 2005. Phyloge-netic patterns in the fleshy-fruited Myrtaceae – preliminarymolecular evidence. Plant Systematics and Evolution 251:35–51.

Lucas EJ, Harris SA, Mazine FF, Bellsham SR,Lughadha EMN, Telford A, Gasson PE, Chase MW.2007. Suprageneric phylogenetics of Myrteae, the generi-cally richest tribe in Myrtaceae (Myrtales). Taxon 56: 1105–1128.

Mattos JR. 1983. Myrtaceae do Rio Grande do Sul. Roessléria5: 169–359.

Mattos JR. 1995. Novidades taxonômicas em Myrtaceae – IX.Loefgrenia 105: 1–3.

McVaugh R. 1963. Tropical American Myrtaceae II. Notes ongeneric concepts and descriptions of previously unrecog-nized species. Fieldiana: Botany 29: 392–532.

McVaugh R. 1968. The genera of American Myrtaceae – aninterim report. Taxon 17: 354–418.

Muellner AN, Schaefer H, Lahaye R. 2011. Evaluation ofcandidate DNA barcoding loci for economically importanttimber species of the mahogany family (Meliaceae). Molecu-lar Ecology Resources 11: 450–460.

Niedenzu F. 1893. Myrtaceae. In: Engler, HGA and PrantilKAE, eds. Die natürlichen. Pflanzenfamilien ed. III, Vol. 7.Leipzig: W. Engelmann, 57–105.

Nylander JA. 2005. MrModeltest ver. 2.2. Program distrib-uted by the author. Uppsala: Evolutionary Biology Centre,Uppsala University.

Oxelman B, Magnus L, Berglund D. 1996. Chloroplastrpsl6 intron phylogeny of the tribe Sileneae (Caryophyl-laceae). Plant Systematics and Evolution 206: 393–410.

Peng D, Wang XQ. 2008. Reticulate evolution in Thujainferred from multiple gene sequences: implications for thestudy of biogeographical disjunction between eastern Asiaand North America. Molecular Phylogenetics and Evolution47: 1190–1202.

Peterson A, Levichev IG, Peterson J. 2008. Systematics ofGagea and Lloydia (Liliaceae) and infrageneric classifica-tion of Gagea based on molecular and morphological data.Molecular Phylogenetics and Evolution 46: 446–465.



http://www.kew.org/wcsp/

Rieseberg LH, Beckstrom-Sternberg SM, Liston A, AriasDM. 1991. Phylogenetic and systematic inferences fromchloroplast DNA and isozyme variation in Helianthus sect.Helianthus (Asteraceae). Systematic Botany 16: 50–76.

Romagnolo MB, Souza MC. 2004. Os gêneros CalycorectesO.Berg, Hexachlamys O.Berg, Myrcianthes O.Berg, Myrci-aria O.Berg e Plinia L. (Myrtaceae) na planícia alagável doalto rio Paraná, Brasil. Acta Botanica Brasilica 18: 613–627.

Ronquist FHJ, van der Mark P. 2005. MrBayes 3.1 manual.Available at: http://mrbayes.csit.fsu.edu/manual.php

Rotman AD. 1982. Los géneros Calycorectes, Hexachlamys,Myrciaria, Paramyrciaria, Plinia y Siphoneugena en la floraargentina (Myrtaceae). Darwiniana 24: 157–185.

Schultz J, Gerlach D, Muller T, Wolf M. 2005. A commoncore of secondary structure of the internal transcribed spacer2 (ITS2) throughout the Eukaryota. RNA 11: 361–364.

Schultz J, Maisel S, Gerlach D, Muller T, Wolf M. 2005.A common core of secondary structure of the internal tran-scribed spacer 2 (ITS2) throughout the Eukaryota. RNA 11:361–364.

Schultz J, Wolf M. 2009. ITS2 sequence-structure analysisin phylogenetics: a how-to manual for molecular systemat-ics. Molecular Phylogenetics and Evoution 52: 520–523.

Sobral M. 1991. Sinopsis de las especies reconocidas delgénero Paramyrciaria Kausel (Myrtaceae). In: Spichiger, REand Bocquet GF, eds. Notulae ad floram paraquaiensem.Candollea 46: 512–521.

Sobral M. 2003. A família das Myrtaceae no Rio Grande doSul. São Leopoldo: Ed. Unisinos., 216.

Sobral M, Proença C, Souza M et al. 2012. Myrtaceae inLista de Espécies da Flora do Brasil. Jardim Botânico do Riode Janeiro. Available at: http://floradobrasil.jbrj.gov.br/2012/index?mode=sv&group=Root_.Angiospermas_&family=&genus=neomitranthes&species=&author=&common=&occurs=1&region=&state=&phyto=&endemic=&origin=&vegetation=&last_level=subspecies&listopt=1

Soltis DE, Soltis PS, Collier TG, Edgerton ML. 1991.Chloroplast DNA variation within and among genera of theHeuchera group (Saxifragaceae) – evidence for chloroplasttransfer and paraphyly. American Journal of Botany 78:1091–1112.

Steyn EMA, Robbertse PJ, Coetzer LA. 1991. Intra-ovarian trichomes in Bequaertiodendron magalimontanum:location, origin, structure and possible function in reproduc-tive processes. South African Journal of Botany 57: 191–197.

Swofford D. 2007. PAUP*: phylogenetic analysis using par-simony, ver. 4.10b. Sunderland: Sinauer Associates.

Taberlet P, Coissac E, Pompanon F, Gielly L, Miquel C,Valentini A, Vermat T, Corthier G, Brochmann C,Willerslev E. 2007. Power and limitations of the chloro-plast trnL (UAA) intron for plant DNA barcoding. NucleicAcids Research 35: e14.

Tamura K, Peterson D, Peterson N, Stecher G, Nei M,Kumar S. 2011. MEGA5: molecular evolutionary geneticsanalysis using maximum likelihood, evolutionary distance,and maximum parsimony methods. Molecular Biology andEvolution 28: 2731–2739.

Van der Merwe MM, Van Wyk AE, Botha AM. 2005.Molecular phylogenetic analysis of Eugenia L. (Myrtaceae),with emphasis on southern African taxa. Plant Systematicsand Evolution 251: 21–34.

Van Ee BW, Riina R, Berry PE. 2011. A revised infragenericclassification and molecular phylogeny of New World Croton(Euphorbiaceae). Taxon 60: 791–823.

Wang N, Milne RI, Jacques FMB, Sun BL, Zhang CQ,Vang JB. 2012. Phylogeny and a revised classification ofthe Chinese species of Nyssa (Nyssaceae) based on morpho-logical and molecular data. Taxon 61: 344–354.

White TJ, Bruns T, Lee S, Taylor J. 1990. Amplificationand direct sequencing of fungal ribosomal RNA genes forphylogenetics. In: Innis MA, Gelfand DH, Shinsky JJ, WhiteTJ, eds. PCR Protocols: A Guide to Methods and Applica-tions. San Diego: Academic Press, 315–322.

Wilson CA. 2011. Subgeneric classification in Irisre-examined using chloroplast sequence data. Taxon 60:27–35.

Wilson PG, O’Brien MM, Heslewood MM, Quinn CJ.2005. Relationships within Myrtaceae sensu lato based on amatK phylogeny. Plant Systematics and Evolution 251:3–19.

Wojciechowski MF, Lavin M, Sanderson MJ. 2004. Aphylogeny of legumes (Legumenosae) based on analyses ofthe plastid matK gene resolves many well-supported subc-lades within the family. American Journal of Botany 91:1846–1862.

Zarrei M, Wilkin P, Fay MF, Ingrouille MJ, Zarre S,Chase MW. 2009. Molecular systematics of Gagea andLloydia (Liliaceae; Liliales): implications of analyses ofnuclear ribosomal and plastid DNA sequences for infrage-neric classification. Annals of Botany 104: 125–142.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:

Appendix S1. The species names, localities, GenBank accession numbers and voucher numbers for the taxaused in this study.Appendix S2. Bayesian inference analysis (analyses A and B).Appendix S3. Maximum parsimony analysis based on combined data of four plastid DNA (accD. rpoB.rpoC1and trnH-psbA) and nrDNA (ITS2) regions (analysis C).Appendix S4. Maximum parsimony analysis obtained from ITS2 (analysis D).

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