Summary. A first phylogenetic hypothesis of Neotropical Eugenia, including associated genera and species fromAfrica and the Pacific was produced using DNA sequence data from the nuclear (ITS, ETS) and plastid (psbA-trnH)genomes. This analysis aimed to investigate the validity of the currently recognised infrageneric groups withinEugenia as well as suites of supporting morphological characters, to determine relationships between groups andproduce a framework for future taxonomic research. A total of 70 samples were analysed and the resulting topo-logy confirms the inclusion of Neotropical genera Calycorectes, Hexachlamys, Phyllocalyx and Stenocalyx in Eugenia topreserve the monophyly of Eugenia. Within Eugenia s.l., nine clades are identified as morphologically diagnosablegroups and are assigned to two genera (Eugenia and Myrcianthes). Their morphological synapomorphies are disc-ussed. Some lineages identified by previous classifications are also supported. Preliminary phylogenetic resultspresented here combined with morphology point towards the need for a new subgeneric classification for Eugenia.
Key Words. Brazil, Calycorectes, Hexachlamys, Mata Atlântica, Phyllocalyx, South America, Stenocalyx.
IntroductionEugenia L. is a taxonomically difficult and ecologicallyimportant genus of trees or shrubs with a pantropicaldistribution. It is the largest genus of NeotropicalMyrtaceae, comprising c. 1000 species (World Checklistof Selected Plant Families (WCSP 2013), distributed fromSouthern Mexico, Cuba and the Antilles to Uruguayand Argentina, with c. 60 species in Africa and an evensmaller representation in Southeast Asia and thePacific (c. 14 and 35 species, respectively). Eugenia isthe most species-rich genus in terms of tree species inthe ombrophilous dense forest, which surrounds theAtlantic coast of Brazil, also widely known as “MataAtlântica” forest (Oliveira-Filho & Fontes 2000). Somespecies with edible fruits are cultivated in tropical andsubtropical regions (e.g. E. brasiliensis Lam. —
“grumixama”, E. involucrata DC. — “cereja-do-rio-grande”, E. pyriformis Cambess. — “uvaia”, E. stipitataMcVaugh — “araçá-boi”, E. uniflora L. — “pitanga”).Eugenia species are remarkable in their number and intheir morphological homogeneity, factors that haveresulted in taxonomic neglect and a reputation as ataxonomically complex group. Evolutionary relation-
ships within Eugenia are unclear and there arecurrently no reliable and unequivocal criteria availableto divide the group into manageable entities forsystematic study. Species are regularly unidentified ormis-named in ecological inventories or surveys of theforests in which they are most diverse, causing seriousproblems for biodiversity management.
Eugenia can be distinguished from the other generaof tribe Myrteae DC. by the generally 4-merousflowers, with free calyx-lobes, separate in the flowerbud, with a hypanthium usually not prolonged nortubular beyond the summit of the bilocular,multiovulate ovary and an embryo with cotyledonsfused in a solid and homogeneous mass (Holst et al.2003; Kawasaki 1984, 1989). The prevailing inflores-cence type within the group has been often referredto as a “bracteate shoot” with flowers in the axes of thebracts, sometimes with an abbreviated rachis orsolitary flowers (McVaugh 1958).
Traditionally, Eugenia was included in subtribeEugeniinae O. Berg (sensu Berg 1856), that alsoincluded genera Calycorectes, Hexachlamys, Myrcianthes,Siphoneugena, Neomitranthes, Plinia and Myrciaria. Berg’s
Accepted for publication 4 March 2014.1 Universidade Federal de São Carlos, campus Sorocaba, Rod. João Leme dos Santos km110, Sorocaba, SP 18052-780, Brazil. e-mail: [email protected] Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Av. Pádua Dias 11, Piracicaba, SP 13418-900, Brazil.3 DCNAT - UFSJ, São João del-Rei, MG, Brazil.4 Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3DS, UK.5 Herbarium, Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3AB, UK.
subtribes were distinguished by the embryo type,Eugeniinae being characterised by the eugenioidembryo with thick, fleshy cotyledons, these more-or-less plano-convex and separate or slightly connate, orfused into a homogenous mass and the hypocotylindistinct or distinct and much shorter than thecotyledons (Landrum & Kawasaki 1997). Lucas et al.(2007) were the first to demonstrate using a molecularphylogenetic approach that Eugeniinae is not mono-phyletic. They found that the genera Neomitranthes,Plinia, Myrciaria and Siphoneugena form one clade, notsister to another clade comprising Eugenia andMyrcianthes. Calycorectes and Hexachlamys were notincluded in that study. The main characteristicdistinguishing Eugenia and Hexachlamys is the pres-ence of predominantly 5-merous flowers inHexachlamys while Eugenia is mostly 4-merous.Merosity, although useful for distinguishing species,is not always a useful character for defining taxa atthe generic level (Sobral 2003). Several authorshave promoted synonymisation of Hexachlamys un-der Eugenia (Niedenzu 1893; Proença 1991;Landrum & Kawasaki 1997; Sobral 2003) as genericlimits between Eugenia, Hexachlamys and Calycorectesare unclear, but more taxonomic studies wereneeded to clarify them. Cruz et al. (2013) showedthat sequence data from nuclear (nrDNA) andplastid genomes did not support a monophyleticHexachlamys and the inclusion of this genus withinEugenia. In the present study we refer to Eugenias.s. in the sense of Landrum & Kawasaki (1997)including Hexachlamys and Calycorectes. Key morpho-logical synapomorphies that distinguish and groupthe clades are discussed below.
Old World EugeniaVan der Merwe et al. (2005) presented a molecularphylogenetic analysis of Eugenia with an emphasis onSouthern African taxa and reported the existence oftwo natural supraspecific groups, referred to as groupsX and Y, supported by morphological characters suchas stomata structure (Van Wyk et al. 1982), seed (VanWyk 1982; Van Wyk & Botha 1984), bark (Van Wyk1985), inflorescence position and fruit morphology(Van Wyk & Lowrey 1988). Based on seed struc-ture, Van Wyk & Botha (1984) suggested thatspecies later included in group X are most closelyrelated to New World species of Eugenia, a hypoth-esis supported by the molecular evidence of vander Merwe et al. (2005). Van der Merwe et al.(2005) found that group X is the most widespreadand speciose in Africa, but noted that the delimi-tation of infrageneric taxa remain problematic (seealso Van Wyk & Dedekind 1985; Verdcourt 1999).Fourteen and 35 species occur in Southeast Asiaand the Pacific, respectively (WCSP 2013).
Phyllocalyx and StenocalyxAmong the genera included as synonyms of Eugenia byMcVaugh (1968), are Stenocalyx and Phyllocalyx,characterised by peduncles with leaf-like bracts and oblongsepals proportionally larger than the flowers. In Stenocalyx,the inflorescence-axis bears normal leaves after thedevelopment of the flowers. At first glance, this arrange-ment in amore advanced stage of development (generallywhen the individual is in fruit) may resemble solitaryflowers. Barroso et al. (1984) termed this a “stenocalyx-typeinflorescence” (illustrated in Souza & Morim 2008) inallusion to the group of species of Eugenia considered inFlora Brasiliensis as a distinct genus. Most authors (Holst etal. 2003; Niedenzu 1893; Kiaerskou 1893; McVaugh 1968;Sánchez-Vindas 1990) agree that these characteristics donot allow the separation of these two genera from Eugenia,despite Kausel’s (1966) recognition of Stenocalyx as a validgenus including Phyllocalyx as its synonym.
Calycorectes and HexachlamysFifty one species have been assigned toCalycorectesO. Bergand 18 toHexachlamysO. Berg (WCSP 2013). Calycorectes isdistributed from Mexico to South America, and is morediverse in the Amazon, Southern Brazil, Paraguay andArgentina and rare in Uruguay (Legrand & Klein 1972;Romagnolo & Souza 2004; Soares-Silva 2000), whileHexachlamys is found in Bolivia, South and SoutheasternBrazil, Northern Argentina, Paraguay and Uruguay(Legrand & Klein 1977; McVaugh 1968). Both generawere considered synonyms of Eugenia by some of Berg’scontemporaries (Bentham 1868; Niedenzu 1893) whilelater authors maintained them as distinct entities(McVaugh 1968; Legrand & Klein 1977; Rotman 1982;Mattos 1983; Marchiori & Sobral 1997). Calycorectes hascalyx lobes fused in bud that tear irregularly at anthesis,whilst in Eugenia calyx lobes are discrete in bud and openregularly at anthesis. This distinction is considered bysome authors as insufficient to warrant generic recogni-tion, hence the inconsistent nomenclature.Hexachlamys ischaracterised by axillary inflorescences with mainlypentamerous (sometimes tetramerous or hexamerous)flowers. The new rank of subgenus was proposed forCalycorectes and Hexachlamys, creating Eugenia subgen.Calycorectes (O. Berg) Mattos and Eugenia subgen.Hexachlamys (O. Berg) Mattos (Mattos 1995, 2005). Inthe same works, new combinations for the specific namesof Calycorectes and Hexachlamys were made under Eugenia.
MyrcianthesMyrcianthes is essentially anAndeangenuswith 35 currentlyaccepted species (WCSP 2013) extending from Mexico toChile. Eight species are distributed in Southern andEasternBrazil, with a single species in Northern Brazil (Sobral et al.2013). According to Lucas et al. (2007), Myrcianthes is thesister-group of Eugenia, also included in the “Eugenia group”(sensu Lucas et al. 2007). It is characterised by an embryo
with two separate plano-convex cotyledons and dichasial(sometimes uniflorous) inflorescences.
Subgeneric groups in EugeniaBerg (1856) recognised eight groups in neotropicalEugenia, based on inflorescence type. They are:Eugenia sects. Uniflorae, Biflorae, Glomeratae, Umbellatae,Corymbiflorae, Racemulosae, Dichotomae and Racemosae(Table 1). Berg (1856) included the type-species ofEugenia, E. uniflora L., in Stenocalyx and as a result, thisgroup (not recognised by Berg in Eugenia) must betreated as Eugenia sect. Eugenia.
The main goal of our study is to clarify themonophyly of Eugenia and relationships between thevarious species groups as currently defined anddescribed above. Additionally, the relationships ofspecies described in groups recognised as distinctgenera by most 20th and 21st century authors, suchas Phyllocalyx (sensu Berg, nom. illegit.), Stenocalyx(sensu Berg, nom. illegit.), Calycorectes, Hexachlamysand Myrcianthes with Eugenia are examined. This studyrepresents the first attempt at understanding thephylogenetic relationships within the large and eco-nomically important genus Eugenia, particularly inSouth America where most of the species are found,and will provide a framework on which subsequentstudies of the genus will be designed.
Materials and Methods
SamplingA total of 70 samples were analysed as part of this study(see Appendix). This sampling represents the fourgenera of the “Eugenia group” (sensu Lucas et al. 2007)with Eugenia species selected to represent as much aspossible the morphological diversity and geographicalvariation found in the genus. Nine outgroup taxa wereselected from different genera belonging to four tribesof subfamily Myrtoideae (sensu Wilson et al. 2005;Appendix). In total, 153 sequences were newly pro-duced for this study.
DNA extraction, sequencing and alignmentTotal genomic DNA was extracted, amplified andsequenced according to the protocols outlined inLucas et al. (2007). Three DNA regions are used: thenuclear internal and external transcribed spacers (ITSand ETS, respectively) and the plastid intergenicspacer psbA-trnH. The ITS region was amplified usingthe primers 17SE and 26SE (Sun et al. 1994) with thefollowing PCR conditions: 2 min at 94°C followed by30 cycles of 1 min at 94°C, 1 min at 52°C, 1 min at72°C, and a final extension of 4 min at 72°C. Theprimers Myrt (Lucas et al. 2007) and 18S (Wright et al.2001) were used to amplify the ETS region with thefollowing PCR conditions: 4 min at 94°C followed by30 cycles of 1 min at 94°C, 1 min at 50°C, 1 min at72°C, and a final extension of 4 min at 72°C. Theplastid psbA-trnH was amplified using the primers psbAand trnH of Hamilton (1999) and the following PCRconditions: 4 min at 94°C followed by 30 cycles of 1min at 94°C, 1 min at 48°C, 2 min 30 seconds at 72°C,and a final extension of 4 min at 72°C. Sequenceswere produced on either an ABI 3100 or ABI3730Genetic Analyzer. Electropherograms were assembledand base-calling was verified using Sequencher™version 4.5 (Gene Codes Corporation). Sequenceswere aligned by eye in PAUP* (v 4.0b2; Swofford2002) following the guidelines of Kelchner (2000). Allsequences are deposited in GenBank (Appendix).
Phylogenetic analysesAll character transformations are treated as equallylikely and unordered (Fitch 1971). Five potentiallyinformative, non-overlapping indels were scored asseparate, binary characters in the parsimony analysis(Table 2), according to the simple method ofSimmons & Ochoterena (2000), with absence of agap coded as 0 and presence as 1. Overlapping indelswere excluded from the phylogenetic analyses as theyonly occurred in regions where it was impossible toobtain an unambiguous alignment of all taxa.
A parsimony analysis was carried out using PAUP*(4.0b2; Swofford 2002) and the heuristic search
Table 1. Subgeneric groupings of Eugenia according to Berg (1856). The most distinctive attributes are underlined.
Group according to Berg (1856) Characters
Eugenia sect. Uniflorae, Linnaea 27: 141 Pedicels axillary, lateral or subterminal, very rarely terminal; uniflorous, very rarely3-flowered.
option. Analysis consisted of 1000 replicates of ran-dom taxon entry order, tree bisection reconnection(TBR) branch-swapping with MulTrees on and saving10 trees per replicate to reduce time searchingsuboptimal islands of trees (Maddison 1991). Treesresulting from this analysis were used as starting treesfor a second analysis using TBR branch-swapping withMulTrees on and saving no more than 10,000 trees.Bootstrap (Felsenstein 1985) was used to assess sup-port for individual clades using the following heuristicsearch settings: 1000 bootstrap replicates with simpletaxon addition, TBR branch swapping with MulTreeson and saving no more than 10 trees per replicate.Bootstrap support percentages (BP) of 50 – 74 areconsidered weakly supported, 75 – 84 moderatelysupported and 85 – 100 strongly supported (Chase etal. 2000). The psbA-trnH, ITS and ETS data sets wereanalysed separately before a combined analysis wasperformed. Congruence among the three data setsand the combined analysis was evaluated, with incon-gruence recognised by the presence of inconsistenttaxon groupings with moderate to high bootstrapsupport (≥75 BP).
A Bayesian analysis was performed on the com-bined data set using the programme MrBayes (version3.1.1; Huelsenbeck & Ronquist 2001; Ronquist et al.2005). An appropriate model of nucleotide substitu-tion was selected for each sequence partition usingMrModeltest2 (version 2.2; Nylander 2004). Thefollowing models were recommended under theAkaike information criterion for ITS, ETS and psbA-
trnH, respectively; GTR+I+G, TVM+G, TVN+G. Datasets were analysed in combination, with these modelparameters fitted independently to each data parti-tion. Two independent analyses were conducted with5,000,000 generations of Monte Carlo Markov chainsand a sampling frequency of 1,000. Resultswere examined using Tracer v1.4.1 (Rambaut &Drummond 2007) to ensure that the analyses reachedconvergence and that the effective sample size of eachparameter was > 200. A consensus tree with posteriorprobabilities based on both analyses was generatedusing the “sumt” option in MrBayes and the defaultburn-in of 10% (500 trees). The maximum credibilitytree and associated posterior probabilities werevisualised using FigTree (Rambaut 2006).
ResultsStatistics from the parsimony analyses are summarised inTable 3. There was no supported incongruence (i.e., BP >75) among the three separate analyses (not shown), whichjustify the combination of all partitions in subsequentanalyses. Only the results from the combined analyses willbe discussed hereafter. The maximum credibility treeresulting from the Bayesian analysis of the combinedDNA regions is shown in Fig. 1. Bootstrap support valuesobtained the from parsimony analysis are also indicatedon the Bayesian tree. One of the 293 most parsimonioustrees produced by the parsimony analysis of the com-bined data is also presented in Fig. 2.
DiscussionWithin the ingroup, nine clades are identified asmorphologically diagnosable groups (summarised inTable 4). As observed in other molecular basedphylogenies of Myrtaceae (Lucas et al. 2007, 2011;Biffin et al. 2010) statistical support is low, in particularalong the backbone of this phylogenetic hypothesis.Studies underway (e.g. Bernardini et al. in prep.) haveadded further molecular data to similar datasets and
Table 3. Data set characteristics for ITS, ETS, psbA-trnH and combined analysis (with indels) and associated results from themaximum parsimony analysis.
Fig. 1. Bayesian consensus tree from analysis of the combined ITS (internal transcribed spacer), ETS (external transcribed spacer) andpsbA-trnH matrix. Bootstrap percentages (from parsimony analysis) greater than 50 are shown above branches; clades that receiveBayesian probabilities equal or greater than 0.95 are indicated by thicker branches. Numbered clades are discussed in the text;letters in brackets refer to Fig. 3.
Fig. 2. One of 293 most parsimonious trees generated by the maximum parsimony analysis of the combined ITS (internaltranscribed spacer), ETS (external transcribed spacer) and psbA-trnH data.
found little increase in support on the backbone ofthese groups. With less than 10% of Eugenia speciessampled here, it is possible that sampling more widelywill increase statistical support for the phylogenetichypothesis presented here. We divide these nineclades between two genera, Myrcianthes and Eugenia,and discuss them and their morphological synapo-morphies below.
1. MyrcianthesMyrcianthes is distinguished from Eugenia by mostly decid-uous and linear (or lanceolate) bracteoles andplacentation(the ovules are in one or more series around a protrudingplacenta; Grifo 1992). These characters support clade 1 assister to the rest of Eugenia. In Eugenia, the ovules mostlyarise at a single point on the septum (see Fig. 3B in Lucas etal. 2007). The results of Lucas et al. (2007) are herecorroborated, asMyrcianthes remains sister to Eugenia.
2. Eugenia s.s.Most groups (clades) in Eugenia s.s. are correlated withmorphological features of the inflorescence (Table 4;Figs 1, 3). Five clades are united by the presence ofpersistent bracteoles (Clades 5, 6, 7, 8 and 9). Thepresence of trichomes in the locular wall is anattribute shared by clades 2, 3 and 4 (but not inMyrcianthes). The presence of trichomes in the loculewalls of some groups in Myrtaceae had been discussedby Cruz et al. (2013) (illustrations in Rotman 1982).
Several of the infrageneric groups suggested byBerg (1856) are supported. Each of the eight clades ofEugenia s.s. are discussed in turn below.
2.1. Eugenia clade 2Clade 2 includes Eugenia arenosa Mattos, E. dysentericaDC. and E. tetramera (McVaugh) M. L. Kawas. & B.Holst. These species share filiform and deciduousbracteoles, and large, edible fruits. E. tetramera hasfruits and leaves with velvety indumentum. AlthoughE. dysenterica was included by Berg (1856) in hisgenus Stenocalyx, it does not emerge in this analysiswith other species from that group. It appears thatthe shared character of hairy locules is moreindicative of a shared evolutionary history than thepossession of enlarged calyx lobes. This clade is themost morphologically incongruent recovered by thepresent analysis.
2.2. Eugenia clade 3The three included species of Hexachlamys emerged asa monophyletic group within Eugenia sensu O. Berg(Fig. 1). Based on results presented here and themorphological distinction of this genus from Eugeniadiscussed in the introduction (flower buds shown inFig. 3L), we concur that Hexachlamys should berecognised as a synonym of Eugenia (Cruz et al. 2013;Landrum & Kawasaki 1997; Sobral 2003). All species ofHexachlamys have been synonymised or have newnames in Eugenia (WCSP 2013).
2.3. Eugenia clade 4The species found in this clade correspond largelywith Berg’s (1856) Eugenia sect. Dichotomae. Berg(1856) included 52 species in Eugenia sect. Dichotomae,a group characterised by flowers born in dichasia
Table 4. Informal groups of Eugenia, based on clades generated by the combined analyses (Fig. 1) and combinations ofmorphological characters useful for their circumscription.
Informal group Character combinations
Eugenia clade 9 Flowers single or arranged in clusters, fascicles, racemes; when in racemes, flowers with pedicel at least 4 timeslonger than flower internode; bracteoles usually persistent at anthesis, rarely deciduous; flowers not showy;calyx-lobes short, not showy; flowers without bracts or with very short bracts, not covering the pedicel; ovary2-locular; many ovules per locule; testa membranous or cartilaginous.
Eugenia clade 8 Flowers arranged exclusively in racemes or diplobotrys; pedicel at most twice as long as the flower internodes;calyx lobes 4, free in the flower bud; ovary 2-locular; many ovules per locule; testa membranous or cartilaginous.
Eugenia clade 7 Calyx closed in the bud, tearing irregularly, or regularly in 4 equal lobes; ovary 2-locular; many ovules per locule;testa membranous or cartilaginous.
Eugenia clade 6 Flowers single, axillary and showy; bracteoles generally persistent at anthesis; calyx lobes 4, free in the flower bud,showy, frequently foliaceous; ovary 2-locular; many ovules per locule; testa membranous or cartilaginous.Paraguay and Argentina to Northeastern Brazil.
Eugenia clade 5 Flowers precocious (appear before or with the new leaves), flowers grouped in decussate pairs surrounding theaxillary bud that grows into a leafy branch; non-leafy bracts at the basis of the pedicels often conspicuouslyimbricate, oblong and clustered (flowers born in branchlets producing normal leaves after flowering, anarrangement superficially similar to single flowers but these inflorescences have imbricate, oblong anddensely clustered basal bracts); calyx lobes 4, free in the flower bud; ovary 2-locular; many ovules per locule;testa membranous or cartilaginous.
Eugenia clade 4 Flowers in dichasia or botryoids (rachis terminates in a flower); bracteoles deciduous at anthesis; ovary 2-locular;many ovules per locule; testa membranous or cartilaginous.
Eugenia clade 3 Flowers in fascicles, rare single flowers; calyx usually 5-merous (but varying from 4 – 6-merous); ovary 2 – 3-locular;2 – 3 ovules per locule.
Eugenia clade 2 Flowers single or arranged in fascicles; bracteoles filiform and deciduous; fruits large and edible; ovary 2 – 4-locular;1 – 2 ovules per locule.
Fig. 3. Inflorescences and flower buds in Eugenia. A diplobotrys of Eugenia florida (Loureiro INPA 38829); B raceme of E.longiracemosa (Araújo 316); C botryoid of E. convexinervia (Mazine 994); D dichasium of E. pyriformis (Ziller 943); E single flower ofE. angustissima (Archer & Gert SP 36408); F cluster of E. verticillata (Cordeiro 901); G fascicle of E. cerasiflora (Moraes 2418); Hshowy flower with foliaceous calyx-lobes (Phyllocalyx) of E. speciosa (Bertoncini 877); J “Stenocalyx-type” inflorescence of E.brasiliensis (Catharino 938); K flower bud of E. subterminalis (=Calycorectes psidiiflorus) (Hatschbach 69267); L flower bud of E.myrcianthes (=Hexachlamys edulis) (Souza 2729). DRAWN BY M. ALICE REZENDE.
(Fig. 3D). From these, only fifteen species correspondeffectively to species of Eugenia s.s. (all of them areBrazilian) and 20 are species of Myrcianthes. The otherspecies described in Eugenia sect. Dichotomae belong toother genera, such as Myrceugenia and Pimenta(Landrum 1981, 1986).
The presence of hairs inside the locules of theovary and the deciduous filiform bracteoles is sharedwith Clades 2 and 3. The inflorescence of Eugenia istypically either solitary flowers or a bracteate shootwith no terminal flower present as a raceme orreduced so as to appear fasciculate. An unusualarrangement in Eugenia is a botryoid (Endress 2010;Weberling 1988), where the apex of the inflores-cence axis ends in a terminal flower (e.g. E.convexinervia D. Legrand, E. paracatuana O. Berg;Fig. 3C). This arrangement is exclusively found inClade 4, as well as other cymose arrangements suchas dichasia (e.g. E. pyriformis; Fig. 3D). According toEndress (2010), a botryoid is a racemose inflores-cence (see fig. 2 in Endress 2010). McVaugh (1956)has not used the term botryoid, but he refers to thisarrangement as an exceptional case of a raceme(see fig. 3 in McVaugh 1956, e.g. E. stipitata). Onthe other hand, Prenner et al. (2009) have defined acymose inflorescence as a ‘closed’ inflorescence inwhich all axes terminate in a flower (i.e. all axes aredeterminate), but they did not use a specific termfor what we have termed a botryoid. We presumethis is a cymose rather than racemose inflorescence;ontogenetic studies would be helpful here.
Eugenia convexinervia has glabrous ovary locules,unlike the rest of Clade 4.
Pseudomyrcianthes Kausel and Pilothecium (Kiaersk.)Kausel, both synonyms of Eugenia (Landrum &Kawasaki 1997) were described or used by Kausel(1956, 1962) to accommodate groups of species inClade 4.
A study currently underway (Faria-Jr. pers. comm.)on this group suggests that this clade is composedof c. 20 species and occurs from Central Brazil toArgentina.
2.4. Eugenia clade 5Represented in this analysis by Eugenia brasiliensis,E. neomyrtifolia Sobral, E. sulcata Spring ex Mart., E.pitanga Kiaersk. and E. uniflora (Fig. 1), the speciesformerly assigned to genus Stenocalyx emerge as amonophyletic clade within Eugenia. Stenocalyx spe-cies share inflorescence axes that begin to developleaves after the flowers, generally with oblong,densely clustered bracts at the base (Sobral 2003;Fig. 3J). The bracteoles of Stenocalyx can be linearor fimbriate, generally deciduous at some stage(before, at or after anthesis). Bracteoles are seldompersistent on fruits, but when they are, they are usuallysmall.
2.5. Eugenia clade 6The present phylogenetic analysis shows good supportfor a clade of species previously circumscribed asPhyllocalyx (BP 97). Recognition of Phyllocalyx (sensuBerg, nom. illegit.) as a genus would render Eugeniaparaphyletic (Mazine, in prep.) but the name is stillavailable at the rank of section (Eugenia sect.Phyllocalyx Nied., see Art. 58.1 of McNeill et al. 2012).Clade 6 is supported as monophyletic by sharedmorphological characters such as the presence ofshowy flowers with equally showy, frequently folia-ceous calyx lobes (Fig. 3H; Niedenzu 1893).
A study currently underway (Bünger pers. comm.)on this group suggests this clade includes approxi-mately 23 species, distributed from Northeast Brazil toArgentina.
2.6. Eugenia clade 7Eugenia brevistyla D. Legrand and E. subterminalis DC.have previously been accepted in Calycorectes [asCalycorectes australis D. Legrand and C. psidiiflorus (O.Berg) Sobral, respectively]. Traditionally, Calycorecteswas distinguished from Eugenia based on the presenceof fused calyx-lobes that tear at anthesis (Fig. 3K; Sobral2003). According to the present analysis, Calycorectesshould not be recognised as distinct from Eugenia.Results corroborate the synonymisation of Calycorectes inEugenia as proposed earlier by Landrum & Kawasaki(1997) and Sobral (2003), although the type species ofCalycorectes, C. grandifolius O. Berg (which has anavailable name in Eugenia, E. neograndifolia Mattos),has not been sampled in this analysis. The sampleEugenia sp. 4, included here, is most likely C.grandifolius, but due to lack of fertile material onthis specimen, we have not been able to provide aconfident identification and thus will not make anynomenclatural changes based on it alone.
Eugenia excelsa O. Berg, previously assigned tosect. Umbellatae and with free calyx lobes, emergesin clade 7. Species in this clade share flowers with longpedicels and multi-flowered, bracteate fascicles.
2.7. Eugenia clade 8According to Berg (1856), species of Eugenia sect.Racemosae have longer racemes, or sometimes panicles,whereas species included in Eugenia sect. Racemulosaehave shorter, fewer-flowered racemes (Table 1). Thecurrent analysis does not support this separation;species from both sections fall in a single clade withinwhich support is low (Fig. 1). It is not possible todistinguish Berg’s sections based on shared morphol-ogy as limits between diagnostic characters areblurred. The two sections, grouped together as theyare here, can be diagnosed by flowers exclusively insimple racemose inflorescences, terminating in avegetative bud (“botrys”, polytelic inflorescences, ac-cording to Weberling 1988; or “botryum”, according
to Endress 2010; Fig. 3B — vegetative bud not evidentdue to concealment between ultimate flower pair)and/or rarely compound inflorescences with partialracemose inflorescences (i.e. a panicle of racemes;“diplobotrys” or “diplobotryum”, according toWeberling (1988) and Endress (2010); Fig. 3A). Thespecies included in Clade 8 have a pedicel tointernode ratio of 2:1 or less. In other words, therachis of these racemes is conspicuous, bearingdecussate pairs of pedicellate flowers. The racemesof some species previously described in Eugeniasect. Racemosae have a terminal flower at thesummit of the axis, as in E. convexinervia and E.paracatuana for example. These latter species havemonotelic inflorescences, in which the apex of theinflorescence axis ends in a terminal flower, not atypical raceme. Those inflorescences have beentreated as “botryoids” (see Clade 4; Fig. 3C), byEndress (2010).
The vegetative bud in Fig. 3B is not evident,because it is very short and located (concealed)between the two flowers (last pair), which iscommon in these species.
Some species with racemes (e.g. Eugeniagomesiana O. Berg, E. egensis DC.) are not foundin this clade. These species have mixed racemesand fascicles on the same individual or theinconspicuous rachis is at least four times shorterthan the flower internodes giving the appearanceof a fascicle (Mazine in prep.). In such exceptionalspecies, the flowers are not distributed equallyalong the rachis and are not paired, as incommonly recognised racemes. Ontogenetic studiescould help to solve this puzzling arrangement.
Several authors (Berg 1856, 1857; de Candolle1828; Urban 1895) treated the diplobotrys (ordiplobotryum) of Eugenia sect. Racemosae as apanicle. Weberling (1988, 1992) however, distin-guishes a panicle as having the main and allsubsidiary axes ending in a terminal flower. Also,Endress (2010) distinguishes a panicle as a multiplecompound botryoid with continuously decreasingflower numbers on the branches of the second-order and continuously decreasing branching or-ders towards the apex of the inflorescence. Thusthe panicle cannot be applied to any botrysinflorescence discussed here. Landrum (1981) in atreatment of Myrceugenia, preferred to use thename “bracteate shoot” for what we consider hereas a raceme.
Two samples of Eugenia florida DC. emerge inClade 8, one from Brazil and another from FrenchGuiana. They did not emerge with a sister relation-ship in this analysis, which maybe due to low levelsof support within this clade, but also alludes to thepossible presence of cryptic species in this widespreadtaxon.
A study currently underway (Mazine, in prep.) on thisgroup suggests this clade comprises 60 species from theNeotropics.
2.8. Eugenia clade 9The species included by Berg (1856) in Eugenia sects.Uniflorae, Biflorae, Glomeratae, Umbellatae andCorymbiflorae form a single unsupported clade, withlittle supported internal resolution (Clade 9; Fig. 1).We opt, for now, to treat the many species of thisgroup as one, as opposed to multiple clades, due tothe lack of morphological consistency amongst thesespecies as arranged here.
This clade includes species with single flowers(Fig. 3E) or flowers born in glomerules (Fig. 3F) orfascicles (Fig. 3G). It also includes species withflowers arranged in racemes and fascicles on thesame branch or in racemes with pedicels muchlonger than the floral internodes (see Clade 8, sect.Racemosae, above).
As originally described, Eugenia sect. Unifloraecomprised about 60 species (including 40 Brazilian),Eugenia sect. Biflorae with 101 species (75 Brazilian),Eugenia sect. Glomeratae with 55 species (30 Brazil-ian), Eugenia sect. Umbellatae with 86 species (64Brazilian) and Eugenia sect. Corymbiflorae with 15species (12 Brazilian). This group includes morethan 317 species in total and is predominantlyfound in the Brazilian Atlantic Forest. It is the mostspeciose group in genus Eugenia. It is interesting tonote that all non-American species of Eugeniasampled in the present study are found in this largegroup, hinting that every dispersal outside SouthAmerica in Eugenia was from this group. A moreextensive sampling of Eugenia species from Africaand Asia will be necessary to confirm this.
ConclusionsThe inclusion of Calycorectes and Hexachlamys inEugenia receives good support. The position of speciespreviously included in Phyllocalyx and Stenocalyx sup-port their inclusion in Eugenia and their status assynonyms of this genus. Some sections of Eugeniadescribed by Berg (1856) now appear nomenclaturallyproblematic. Formal modifications to the infragenericcategories in Eugenia are underway (Mazine, in prep).These will serve as basis for further phylogeneticstudies, which will increase our understanding of theevolution and morphology in the large genus Eugeniaand also to provide a framework for future taxonomicrevisions.
Statistical support for the backbone of thisphylogenetic tree is low. This situation has beenreported in other studies within Myrtaceae (e.g.Syzygium, Biffin et al. 2007; Myrteae and Myrcia,Lucas et al. 2007, 2011) and within large taxa in
other families (e.g. Sapindaceae, Buerki et al.2009; Fabales, Bello et al. 2009). It has beensuggested that the underlying low genetic diversitybetween species may be the result of recent rapidspeciation in these groups (e.g. Biffin et al. 2010;Thornhill et al. 2012). Further studies on individ-ual clades using additional molecular markers areunderway; it is anticipated that increased molecu-lar data will allow detection and strengthening ofspecies relationships in this ecologically importantgroup.
AcknowledgementsThis work would not have been possible withoutsupport in the field from Ony Rodrigues de Campos,Fernando Antonio Capelo, Cassia Sakuragui, Joel daSilva, Daniela Sampaio, Wellington Forster, AndréaOnofre Araújo, and Alexandre Romariz Duarte. Weare very grateful to the following generous fieldcollectors: Bruce Holst and Daniela Zappi. We thankIBAMA for granting permits and FAPESP (Brazil)(Process 2002/01637-8), KLARF (Mellon Foundation)and the Royal Society, London for financial support.
Appendix
Vouchers for DNA samples used in the phylogenentic analysis of Eugenia. Voucher information, GenBank accession numbers andcountry of origin are provided. Names marked with an asterisk (*) are outgroup taxa.
Eucalyptus tetragona (R. Br.) F. Muell.* Australia Udovicic 177 MELU AF190364 AM489906 AF190381Eugenia arenosa Mattos Brazil Mazine 1021 ESA, K KJ187605 KJ187658 KJ469654Eugenia astringens Cambess. Brazil Mazine 782 ESA, K KJ187606 KJ187659 KJ469655Eugenia axillaris (Sw.) Willd. Turks and Caicos Hamilton 553 K, FTG KJ187607 KJ187660 KJ469656Eugenia bacopari D. Legrand Brazil Mazine 967 ESA, K KJ187608 KJ187661 KJ469657Eugenia beaurepaireana
(Kiaersk.) D. LegrandBrazil Mazine 1008 ESA, K KJ187609 KJ187662 KJ469658
Eugenia biflora (L.) DC. Brazil Mazine 1075 ESA, K KJ187610 KJ187663 KJ469659Eugenia bimarginata DC. Brazil Mazine 469 ESA, K KJ187611 KJ187664 KJ469660Eugenia membranifolia Nied. Brazil Duarte s.n.
ESA 85677ESA, K KJ187612 KJ187665 KJ469661
Eugenia brasiliensis Lam. Brazil Lucas 126 ESA, K KJ187613 KJ187666 KJ469662Eugenia brevistyla D. Legrand Brazil Mazine 993 ESA, K KJ187614 KJ187667 KJ469663Eugenia brongniartiana Guill. New Caledonia Pillon 176 K, NOU KJ187615 KJ187668 KJ469664Eugenia calycina Cambess. Brazil Mazine 1018 ESA, K KJ187616 KJ187669 KJ469665Eugenia capensis subsp. natalitia
(Sond.) F. WhiteSouth Africa Reeves 68 NBG AY463135 AY454141 …
Eugenia capparidifolia DC. Brazil Mazine 845 ESA KJ187617 … KJ469666Eugenia convexinervia D. Legrand Brazil Mazine 994 ESA, K KJ187618 KJ187670 KJ469667Eugenia cuprea (O. Berg) Nied. Brazil Mazine 950 ESA, K KJ187619 KJ187671 KJ469668Eugenia dysenterica DC. Brazil Mazine 466 ESA, K KJ187620 KJ187672 KJ469669Eugenia excelsa O. Berg Brazil Lucas 125 K KJ187621 KJ187673 KJ469670Eugenia florida DC. Brazil Mazine 965 ESA, K KJ187622 KJ187674 KJ469671Eugenia florida DC. (French Guiana) French Guiana Lucas 106 K AM234090 AM489912 AM489830Eugenia hiemalis Cambess. Brazil Mazine 970 ESA, K KJ187623 KJ187675 KJ469672Eugenia langsdorffii O. Berg Brazil Silva &
Farias 4528K AM234092 AM489914 AM489832
Eugenia latifolia Aubl. French Guiana Prévost 4707 K, CAY AM234091 AM489913 AM489831Eugenia melanogyna (D. Legrand) Sobral Brazil Mazine 969 ESA, K KJ187624 KJ187676 KJ469673Eugenia modesta DC. Brazil Mazine 854 ESA, K KJ187625 KJ187677 …Eugenia neoglomerata Sobral Brazil Mazine 461 ESA, K KJ187626 KJ187678 KJ469674Eugenia neomyrtifolia Sobral Brazil Mazine 953 ESA, K KJ187627 KJ187679 KJ469675Eugenia neoverrucosa Sobral Brazil Lucas 118 K, MBM KJ187628 KJ187680 KJ469676Eugenia nutans O. Berg Brazil Lucas 281 ESA, K KJ187629 KJ187681 KJ469677Eugenia oblongata O. Berg Brazil Mazine 804 ESA, K KJ187630 KJ187682 KJ469678Eugenia orbiculata Lam. Mauritius Page 104 MAU KJ187631 KJ187683 KJ469679Eugenia orbignyana O. Berg Brazil Mazine 1067 ESA, K KJ187632 KJ187684 KJ469680Eugenia patens Poir. French Guiana Lucas 104 ESA, K KJ187633 KJ187685 KJ469681Eugenia pisiformis Cambess. Brazil Lucas 232 K KJ187634 KJ187686 KJ469682Eugenia pitanga (O. Berg) Kiaersk. Brazil Mazine 1044 ESA, K KJ187635 KJ187687 KJ469683Eugenia pluriflora DC. Brazil Mazine 961 ESA, K KJ187636 KJ187688 KJ469684
ReferencesBarroso, G. M., Peixoto, A. L., Costa, C. G., Ichaso, C.
L. F., Guimarães, E. F. & Lima, H. C. (1984).Sistemática das Angiospermas do Brasil. Vol. 2.Universidade Federal de Viçosa.
Bello, M. A., Bruneau, M., Forest, F. & Hawkins, J. A.(2009). Elusive relationships within order fabales:phylogenetic analyses using matK and rbcl sequencedata. Syst. Bot. 34 (1): 102 – 114.
Bentham, G. (1868). Notes on Myrtaceae. J. Linn. Soc.,Bot. 10: 101 – 166.
Berg, O. (1856). Revisio Myrtacearum Americae.Linnaea 27: 1 – 472.
____ (1857). Myrtaceae. In: C. F. P. von Martius (ed.),Flora Brasiliensis 14: 1 – 655. Sumptibus J. G. Cottae,Stuttgartiae et Tubingae.
Biffin, E., Harrington, M. G., Crisp, M. D., Craven, L. A. &Gadek, P. A. (2007). Structural partitioning, paired-sitesmodels and evolution of the ITS transcript in Syzygiumand Myrtaceae.Molec. Phylogenet. Evol. 43: 124 – 139.
____, Lucas, E. J., Caven, L. A., Costa, I. R., Harrington,M. R. & Crisp, M. D. (2010). Evolution of exceptional
Eugenia prasina O. Berg Brazil Mazine 959 ESA KJ187637 KJ187689 KJ469685Eugenia prasina O. Berg Brazil Zappi 406 K AM234086 AM489908 AM489826Eugenia punicifolia (Kunth) DC. Brazil Mazine 1065 ESA, K KJ187638 KJ187690 KJ469686Eugenia pyriformis Cambess. Brazil Mazine 1028 ESA, K KJ187639 KJ187691 KJ469687Eugenia repanda O. Berg Brazil Lucas 273 K, RB KJ187640 KJ187692 KJ469688Eugenia sp. 1 Brazil Mazine 1077 ESA, K KJ187641 KJ187693 KJ469689Eugenia sp. 2 Brazil Mazine 1090 ESA, K … KJ187694 KJ469690Eugenia sp. 3 Brazil Mazine 1072 ESA, K KJ187642 KJ187695 KJ469691Eugenia sp. 4 French Guiana Holst 9435 SEL KJ187643 KJ187696 KJ469692Eugenia sphenophylla O. Berg Brazil Lucas 257 BHCB,
Eugenia subavenia O. Berg Brazil Mazine 805 ESA, K KJ187646 KJ187699 KJ469695Eugenia subterminalis DC.
(=“Calycorectes psidiiflorus”)Brazil Mazine 974 ESA, K … KJ187700 KJ469696
Eugenia sulcata Spring ex Mart. Brazil Mazine 787 ESA, K KJ187647 KJ187701 KJ469697Eugenia tetramera (McVaugh)
M. L. Kawasaki & B. K. HolstFrench Guiana Holst 9422 SEL KJ187648 KJ187702 KJ469698
Eugenia umbrosa O. Berg Brazil Mazine 1085 ESA, K KJ187649 KJ187703 KJ469699Eugenia uniflora L. Cultivated RBG
KewLucas 207 K AM234088 AM489910 AM489828
Eugenia verticillata (Vell.) Angely Brazil Duarte s.n.ESA 85679
ESA, K KJ187650 KJ187704 KJ469700
Eugenia wentii Amsh. French Guiana Holst 9421 SEL KJ187651 KJ187705 KJ469701Hexachlamys edulis (O. Berg) Kausel &
D. LegrandBrazil Mazine 1091 ESA, K KJ187652 … KJ469702
Hexachlamys hamiltonii Mattos Brazil Mazine 1033 ESA, K KJ187653 KJ187706 KJ469703Hexachlamys itatiaiensis Mattos Brazil Mazine 951 ESA, K KJ187654 KJ187707 KJ469704Lophostemon confertus (R. Br.) Peter G.
Wilson & J. T. Waterh.*Australia Udovicic 337 MELU AF048897 AM489925 AF190368
Myrcia hebepetala DC. * Brazil Lucas 64 K AM234111 AM489916 AM48934Myrcianthes fragrans (Sw.) McVaugh Cultivated,
unknownorigin
Holst 8862 SEL KJ187655 KJ187708 KJ469705
Myrcianthes pseudomato (D. Legrand)McVaugh
Brazil Beck 9667 K AM234100 AM489951 AM489868
Myrcianthes pungens (O. Berg) D.Legrand
Brazil Forster 1013 ESA KJ187656 KJ187709 KJ469706
species richness among lineages of fleshy fruitedMyrtaceae. Ann. Bot. 106: 79 – 93.
Buerki, S., Forest, F., Acevedo-Rodriguez, P.,Callmander, M. W., Nylander, J. A. A., Harrington,M., Sanmartín, I., Küpfer, P. & Alvarez, N. (2009).Plastid and nuclear DNA markers reveal intricaterelationships at subfamilial and tribal levels in thesoapberry family (Sapindaceae). Molec. Phylogenet.Evol. 51: 238 – 258.
Chase, M. W., De Bruijn, A. Y., Cox, A. V., Reeves, G.,Rudall, P. J., Johnson, M. A. T. & Eguiarte, L. E.(2000). Phylogenetics of Asphodelaceae Asparagales:an analysis of plastid rbcL and trnL-F DNA sequences.Ann. Bot. 86: 935 – 951.
Cruz, F., Turchetto-Zolet, A. C., Veto, N., Mondin, C.A., Sobral, M., Almerão, M. & Margis, R. (2013).Phylogenetic analysis of the genus Hexachlamys(Myrtaceae) based on plastid and nuclear DNAsequences and their taxonomic implications. Bot. J.Linn. Soc. doi: 10.1111/boj12036
De Candolle, A. P. (1828). Myrtaceae. In: A. P. deCandolle (ed.), Prodromus systematis naturalisregni vegetabilis 3: 207 – 296. Treuttel et Würtz,Paris.
Endress, P. K. (2010). Disentangling confusions ininflorescence morphology: patterns and diversity ofreproductive shoot ramification in angiosperms. J.Syst. Evol. 48 (4): 225 – 239.
Felsenstein, J. (1985). Confidence-limits on phyloge-nies: an approach using the bootstrap. Evolution 39(4): 783 – 791.
Fitch, W. M. (1971). Toward defining the course ofevolution: minimum change for a specific treetopology. Syst. Zool. 20: 406 – 416.
Grifo, F. T. (1992). A Revision of Myrcianthes Berg(Myrtaceae). PhD thesis. Cornell University, Ithaca.
Hamilton, M. B. (1999). Four primer pairs for theamplification of chloroplast intergenic regions withintraspecific variation. Molec. Ecol. 8: 521 – 523.
Holst, B. K., Landrum, L. & Grifo, F. (2003).Myrtaceae. In: P. E. Berry, K. Yatskievych & B.Holst (eds), Flora of the Venezuelan Guayana 7: 1 – 99.New York Botanical Garden, New York.
Huelsenbeck J. P. & Ronquist, F. (2001). MRBAYES:Bayesian inference of phylogeny. Bioinformatics 17:754 – 755.
Kausel E. (1956). Beitrage zur Systematik derMyrtaceen. Ark. Bot. 2 (3): 491 – 516.
____ (1962). Zur Systematik von Pilothecium Kiaersk.Ark. Bot. 4 (10): 401 – 405.
____ (1966). Lista de las Mirtáceas y Leptospermáceasargentinas. Lilloa 32: 323 – 368.
Kawasaki, M. L. (1984). A família Myrtaceae na Serra doCipó, Minas Gerais, Brasil. Ms diss. Instituto deBiociências, Universidade de São Paulo.
____ (1989). Flora da Serra do Cipó, Minas Gerais:Myrtaceae. Bol. Bot. Univ. São Paulo 11: 121 – 170.
Kelchner, S. A. (2000). The evolution of non-codingchloroplast DNA and its application in plant system-atics. Ann. Missouri Bot. Gard. 87 (4): 482 – 498.
Kiaerskou, H. (1893). Enumeratio Myrtacearumbrasiliensium. In: E. Warming (ed.), Symbolae adFloram Brasiliae Centralis Cognoscendam. Vidensk.Meddel. Naturhist. Foren. Kjøbenhavn 39: 1 – 200.
Landrum, L. R. (1981). A monograph of the genusMyrceugenia (Myrtaceae). Fl. Neotrop. Monogr. 29:1 – 137.
Lucas, E. J., Harris, S. A., Mazine, F. F., Belsham, S. R.,NicLughadha, E. M., Telford, A., Gasson, P. E. &Chase, M. W. (2007). Suprageneric phylogenetics ofMyrteae, the generically richest tribe in Myrtaceae(Myrtales). Taxon 56: 1105 – 1128.
____, Matsumoto, K., Harris, S. A., NicLughadha, E.M., Benardini, B. & Chase, M. W. (2011). Phyloge-netics, Morphology, and Evolution of the LargeGenus Myrcia s.l. (Myrtaceae). Int. J. Pl. Sci. 172 (7):915 – 934.
McNeill, J., Barrie, F. R., Buck, W. R., Demoulin, V.,Greuter, W., Hawksworth, D. L., Herendeen, P. S.,Knapp, S., Marhold, K., Prado, J., Prud’Homme vanHeine, W. F., Smith, G. F., Wiersema, J. H. &Turland, N. J. (2012). International Code ofBotanical Nomenclature (Melbourne Code). Reg-num Veg. 154.
Maddison, D. R. (1991). The discovery and impor-tance of multiple islands of most parsimonioustrees. Syst. Zool. 40: 315 – 328.
Marchiori, J. N. C. & Sobral, M. (1997). Dendrologia dasangiospermas: Myrtales. Editora da UFSM, Santa Maria.
Mattos, J. R. (1983). Myrtaceae do Rio Grande do Sul.Roessleria 5: 169 – 359.
____ (2005). Considerações sobre Calycorectes Berg.Loefgrenia 120: 1 – 11.
McVaugh, R. (1956). Tropical American Myrtaceae.Notes on generic concepts and description ofpreviously unrecognized species. Fieldiana, Bot. 29(3): 145 – 228.
____ (1958). Flora of Peru: Myrtaceae. Field Mus.Public. Bot. 13 (4): 569 – 818.
____ (1968). The Genera of American Myrtaceae: AnInterim Report. Taxon 17: 354 – 418.
Niedenzu, F. (1893). Myrtaceae. In: H. G. A. Engler &K. A. E. Prantl (eds), Die Natürlichen Pflanzenfamilien3 (7): 57 – 105. W. Engelmann, Leipzig.
Nylander, J. A. A. (2004). MrModeltest2. Version 2.Evolutionary Biology Centre, Uppsala University.http://www.abc.se/~nylander/.
Oliveira-Filho, A. T. & Fontes, M. A. L. (2000). Patternsof Floristic Differentiation among Atlantic Forestsin Southeastern Brazil and the Influence of Cli-mate. Biotropica 32 (4b): 793 – 810.
Prenner, G., Vergara-Silva, F. & Rudall, P. J. (2009).The key role of morphology in modeling inflores-cence architecture. Trends Plant Sci. 14: 302 – 309.
Proença, C. E. B. (1991). The reproductive biology andtaxonomy of the Myrtaceae of the Distrito Federal (Brazil).PhD diss. Department of Biology and PreclinicalMedicine, University of St. Andrews.
Rambaut, A. (2006). FigTree. http://tree.bio.ed.ac.uk/software/figtree/. Accessed March 2009.
____ & Drummond, A. J. (2007). Tracer. Version1.4.http://beast.bio.ed.ac.uk/Tracer.
Romagnolo, M. B. & Souza, M. C. (2004). Os gênerosCalycorectes O. Berg, Hexachlamys O. Berg,Myrcianthes O. Berg, Myrciaria O. Berg e Plinia O.Berg (Myrtaceae) na planície alagável do alto rioParaná, Brasil. Acta Bot. Bras. 18 (3): 613 – 627.
Ronquist, F., Huelsenbeck, J. P. & van derMark, P. (2005).MrBayes 3.1 manual. http://mrbayes.csit.fsu.edu/manual.php.
Rotman, A. D. (1982). Los géneros Calycorectes,Hexachlamys, Myrciaria, Paramyrciaria, Plinia ySiphoneugena en la flora argentina. Darwiniana 24:157 – 185.
Sánchez-Vindas, P. E. (1990). Myrtaceae. In: A.Gomez-Pompa (ed.), Flora de Veracruz 1 – 146.Instituto de Ecologia, Xalapa, Veracruz.
Simmons, M. P. & Ochoterena, H. (2000). Gaps asCharacters in Sequence-Based Phylogenetic Analy-ses. Syst. Biol. 49 (2): 369 – 381.
Soares-Silva, L. H. (2000). A família Myrtaceae: subtribosMyrciinae e Eugeniinae na bacia hidrográfica do rioTibagi, estado do Paraná, Brasil. PhD diss. Instituto deBiologia, Universidade Estadual de Campinas.
Sobral, M. (2003). A família Myrtaceae no Rio Grande doSul. Editora Unisinos, São Leopoldo.
____, Proença, C., Souza, M., Mazine, F. F. & Lucas, E.(2013). Myrtaceae. In: R. C. Forzza (ed.), Lista deEspécies da Flora do Brasil. Jardim Botânico do Riode Janeiro. (http://floradobrasil.jbrj.gov.br/2010/FB010781).
Souza, M. C. &Morim, M. P. (2008). Subtribos EugeniinaeO. Berg eMyrtinaeO. Berg (Myrtaceae) na Restinga daMarambaia, RJ, Brasil. Acta Bot. Bras. 22 (3): 652 – 683.
Sun, Y., Skinner, D. Z., Liang, G. H. & Hulbert, S. H.(1994). Phylogenetic analysis of Sorghum and relatedtaxa using internal transcribed spacers of nuclearribosomal DNA. Theor. Appl. Genet. 89: 26 – 32.
Swofford, D. L. (2002). PAUP; phylogenetic analysis usingparsimony (and other methods), version 4.0b2. Sinauer,Sunderland, Massachusets, USA.
Thornhill, A. H., Popple, L. W., Carter, R. J., Ho, S. Y.W. & Crisp, M. D. (2012). Are pollen fossils usefulfor calibrating relaxed molecular clock dating ofphylogenies? A comparative study using Myrtaceae.Molec. Phylogenet. Evol. 63: 15 – 27.
Urban, I. (1895). Additamenta ad cognitionem floraeIndiae Occidentalis. Bot. Jahrb. Syst. 19: 562 – 681.
Van der Merwe, M. M., van der Wyk, A. E. & Botha, A.M. (2005). Molecular phylogenetic analysis ofEugenia L. (Myrtaceae), with emphasis on southernAfrican taxa. Pl. Syst. Evol. 251: 21 – 34.
Van Wyk, A. E. (1982). A note on the seed morphologyof the genus Eugenia (Myrtaceae) in southernAfrica. S. African J. Bot. 46: 115 – 119.
____ (1985). The genus Eugenia (Myrtaceae) insouthern Africa: the structure and taxonomic valueof the bark. S. African J. Bot. 51: 157 – 180.
____ & Botha, R. (1984). The genus Eugenia (Myrtaceae)in southern Africa: ontogeny and taxonomic value ofthe seed. S. African J. Bot. 3: 63 – 80.
____ & Dedekind, I. (1985). The genus Eugenia(Myrtaceae) in southern Africa: morphology andtaxonomic value of pollen. S. African J. Bot. 51: 371– 378.
____ & Lowrey, T. K. (1988). Studies on the repro-ductive biology of Eugenia L. (Myrtaceae) in south-ern Africa. Monogr. Syst. Bot. Missouri Bot. Gard. 25:279 – 293.
____, Robbertse, P. J. & Kok, P. D. F. (1982). Thegenus Eugenia (Myrtaceae) in southern Africa: thestructure and taxonomic value of stomata. Bot. J.Linn. Soc. 84: 41 – 56.
Verdcourt, B. (1999). The genus Eugenia L.(Myrtaceae) in East Africa. Kew Bull. 54: 41 – 62.
Weberling, F. (1988). The architecture of inflores-cences in the Myrtales. Ann. Missouri Bot. Gard. 75(1): 226 – 310.
____ (1992). Morphology of flowers and inflorescences.Cambridge University Press, Cambridge.
Wilson, P. G., O’Brien, M. M., Heslewood, M. M. &Quinn, C. J. (2005). Relationships within Myrtaceaesensu lato based on a matK phylogeny. Pl. Syst. Evol.251: 3 – 19.
WCSP (World Checklist of Selected Plant Families)(2013). World checklist of selected plant families. http://www.kew.org/wcsp/myrtaceae. Accessed June 5, 2013.
Wright, S. D., Yong, C. G., Wichman, S. R., Dawson, J.W. & Gardner, R. C. (2001). Stepping stones toHawaii: a trans-equatorial dispersal pathway forMetrosideros (Myrtaceae) inferred from nrDNA(ITS + ETS). J. Biogeogr. 28: 769 – 774.
