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Spatial analyses of the phylogenetic diversity ofMinaria (Apocynaceae): assessing priority areas forconservation in the Espinhaço Range, BrazilPatrícia Luz Ribeiro a , Alessandro Rapini a , Uiara Catharina Soares e Silva a , TatianaUngareti Paleo Konno b , Leilton Santos Damascena a & Cássio van den Berg aa Departamento de Ciências Biológicas, Universidade Estadual de Feira de Santana, BR-116,Km 3, Av. Transnordestina s/n, Novo Horizonte, 44036-900, Feira de Santana, Bahia, Brazilb Universidade Federal do Rio de Janeiro, Campus Macaé, Núcleo de Pesquisa em Ecologiae Desenvolvimento Sócio-Ambiental de Macaé, Rua Rotary Club s/n, São José do Barreto,27910-970, Rio de Janeiro, RJ, Brazil

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To cite this article: Patrícia Luz Ribeiro, Alessandro Rapini, Uiara Catharina Soares e Silva, Tatiana Ungareti PaleoKonno, Leilton Santos Damascena & Cássio van den Berg (2012): Spatial analyses of the phylogenetic diversity of Minaria(Apocynaceae): assessing priority areas for conservation in the Espinhaço Range, Brazil, Systematics and Biodiversity, 10:3,317-331

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Systematics and Biodiversity (2012), 10(3): 317–331

Research Article

Spatial analyses of the phylogenetic diversity of Minaria (Apocynaceae):assessing priority areas for conservation in the Espinhaco Range, Brazil

PATRICIA LUZ RIBEIRO1, ALESSANDRO RAPINI1, UIARA CATHARINA SOARES E SILVA1,TATIANA UNGARETI PALEO KONNO2, LEILTON SANTOS DAMASCENA1 & CASSIO VAN DEN BERG1

1Departamento de Ciencias Biologicas, Universidade Estadual de Feira de Santana, BR-116, Km 3, Av. Transnordestina s/n,Novo Horizonte 44036-900, Feira de Santana, Bahia, Brazil2Universidade Federal do Rio de Janeiro, Campus Macae, Nucleo de Pesquisa em Ecologia e Desenvolvimento Socio-Ambiental deMacae, Rua Rotary Club s/n, Sao Jose do Barreto 27910-970, Rio de Janeiro, RJ, Brazil

(Received 9 February 2012; revised 18 May 2012; accepted 6 June 2012)

The protection of areas that shelter high evolutionary diversity represented by geographically and phylogenetically isolatedlineages is becoming an important conservation strategy. Nevertheless, the spatial distribution of this component ofbiodiversity is still unknown for most groups, which limits its application for selecting priority areas to conserve. In thepresent study, we reconstructed the phylogeny of Minaria (Apocynaceae) based on plastid (trnH-psbA, rps16, trnS-trnGand trnD-trnT) and nuclear (ITS and ETS) DNA markers and 34 morphological characters, and analysed the geographicdistribution of the phylogenetic diversity (PD) and endemism (PE) in this genus. Minaria includes 21 species that are highlyconcentrated in the Espinhaco Range, in eastern Brazil, most of which (∼75%) are narrowly distributed. The spatialanalyses of PD and PE of Minaria indicate four evolutionary relevant areas in this region. The Serra do Cipo and theDiamantina Plateau contain 10 endemic species and present the highest levels of PD. However, the two other areas alsodeserve special attention. Rio de Contas has high levels of PE, because of two endemic sister species that represent aphylogenetically isolated lineage and the Southern Espinhaco Range houses the most critically endangered species of thegenus. Most endemic species of Minaria occur in vegetation islands on rocky outcrops (campos rupestres). These low-fuelareas are less susceptible to fire, suggesting that the Espinhaco Range has served as a historical refuge for fire-sensitivelineages. Our results suggest that conservation units in the Espinhaco Range cover a great proportion of the evolutionarydiversity of Minaria and that fire management is probably an important strategy to preserve this endemic biodiversity.

Key words: Asclepiadoideae, Bahia, biogeography, campos rupestres, endangered species, endemism, fire management,Metastelmatinae, Minas Gerais, Neotropics

IntroductionThe Espinhaco Range in the states of Minas Gerais andBahia, in eastern Brazil, is widely and historically knownfor its high proportion of endemic species of angiosperms(Rapini et al., 2008). This region forms a 1000 km strip(north–south) and is located in the intersection of threecontrasting biomes, the Atlantic Forest (in the southeast),the savannah-like Cerrado (in the southwest) and the sea-sonal dry forest known as Caatinga (in the north). Althoughthis area corresponds to roughly 1% of Brazil, its vascularflora has been estimated to be 4000 species (Giuliettiet al., 1997), which corresponds to more than 10% of thespecies native to this country (32,364; Forzza et al., 2012).

Correspondence to: Patrıcia Luz Ribeiro. E-mail: patyluzribeiro@yahoo.com.br

On average, 30% of the angiosperm species from theEspinhaco Range are endemic to this region (Giulietti et al.,1987), but estimates are much higher for certain groups,such as Bromeliaceae (49%; Versieux et al., 2008) andEriocaulaceae (85%; Costa et al., 2008), and this area is animportant centre of diversity of Asclepiadoideae (Apocy-naceae). Of the 392 species of Asclepiadoideae reported forBrazil (Rapini et al., 2010a), 133 occur in the EspinhacoRange and more than 10% (42 species) are endemic tothe region (Rapini, 2010). Some groups within Asclepi-adoideae, however, present higher levels of endemism.The recently described genus Minaria is one of them, witharound 75% of its species confined to only small areas inthe Espinhaco Range of Minas Gerais (Konno et al., 2006).

In the Espinhaco Range, endemic species of angiospermsare distributed predominantly in campos rupestres (rocky

ISSN 1477-2000 print / 1478-0933 onlineC© 2012 The Natural History Museum

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fields), which usually occur above 900 m elevation, haveopen vegetation on quartzite soil, and often contain rockyoutcrops. They are concentrated in areas of the DiamantinaPlateau and Serra do Cipo, in the state of Minas Gerais(e.g. Rapini et al., 2002; Rapini, 2010; Echternacht et al.,2011), but the Chapada Diamantina, in the state of Bahia,is also an important centre of plant endemism (Conceicaoet al., 2005; Rapini, 2010). Because of the high number ofspecies with narrow distributions and the uneven floristicdata that exist for the Espinhaco Range, each area is uniqueand potentially important for conservation (Rapini et al.,2002; Conceicao et al., 2005). Thus, inventories are stillneeded to detect centres of diversity and endemism in theregion. In addition, phylogenetic and ecological studies ofgroups concentrated in the Espinhaco Range should also beencouraged because they may reveal historical processesand biological interactions responsible for the origin andmaintenance of the overall biodiversity of this region andmay contribute to its management (Rapini et al., 2008).

Although intuitive and more feasible, the number ofspecies alone is not the most adequate criterion to selectpriority areas for biological conservation (e.g. Prendergastet al., 1993; Reid, 1998), and endemic-rich areas, especiallythose under threat, have received special attention (Pimmet al., 1995; Myers et al., 2000; Lamoreux et al., 2005;Orme et al., 2005). However, species with restricted rangesthat resulted from different evolutionary histories are notbiologically equivalent. Their phylogenetic representation,which is sometimes denoted by taxonomic distinctiveness,is an important factor that should also be considered. Theextinction of a species or a group of closely related speciesrepresents the end of a lineage and the evolutionary diver-sity lost will tend to be greater for older lineages. Therefore,the recognition of areas that shelter high levels of exclusivephylogenetic diversity is an important strategy in conserva-tion (Vane-Wright et al., 1991; Williams et al., 1991; Faith,1992, 2008; O’Brien, 1994; Vasquez & Gittleman, 1998;Rosauer et al., 2009).

Phylogenetic diversity can be based on nodes (Mc-Googan et al., 2007), branch length (Faith, 2008; Rosaueret al., 2009) or ages (Sechrest et al., 2002; Forest et al.,2007). Although relevant, the distribution of phylogeneticdiversity is still not available for most groups and, there-fore, it is not broadly used as a criterion to select priorityareas for conservation. The recognition of areas with higherevolutionary diversity depends on accurate data about thegeographic distribution of species and reliable hypothesesof phylogenetic relationships (Faith, 2008). Although theEspinhaco Range is an important centre of diversity andendemism for many groups of plants (Giulietti et al., 1997;Rapini et al., 2008), as far as we know, there has beenno study designed to examine the spatial distribution of thephylogenetic diversity in this region. In this context, our pri-mary objective was to examine, for the first time, the spatialdistribution of the phylogenetic diversity and endemism of

a lineage from the Espinhaco Range. This approach willprovide an initial basis for evaluating the current distribu-tion of conservation units and may indicate key factors thatinfluence the flora, which should be managed to preservethis component of biodiversity.

The first step to achieve this objective was to obtaina complete phylogeny of a relatively well-known groupwhose diversity is concentrated in the Espinhaco Range.Minaria meets both criteria. The genus was revised re-cently (Konno, 2005; though taxonomic changes were notformally proposed in this work) and is predominantly dis-tributed in the Espinhaco Range (Konno et al., 2006). It wasinitially discovered as a separate phylogenetic lineage basedon molecular data (Rapini et al., 2006) and can be morpho-logically distinguished by its shrubby, erect habit and smallleaves (Konno et al., 2006). Minaria was not supportedbased solely on morphology (Konno, 2005), but formed astrongly supported clade based on plastid DNA markers(Rapini et al., 2003, 2006; Liede-Schumann et al., 2005).Nevertheless, at that time, only seven of the 19 speciesoriginally considered in the genus had been sampled.

Recently, phylogenetic analyses, including a larger sam-pling of Metastelmatinae (Silva et al., in press), questionedthe position of Minaria polygaloides as part of the genusand showed that two species (Barjonia harleyi and Hemi-pogon harleyi) endemic to the Espinhaco Range of Bahia,which were not originally assigned to Minaria, form a cladethat is sister to the Minaria core group. In the present study,we investigated the circumscription of Minaria and the re-lationships within the genus based on plastid and nuclearDNA markers and morphological characters. The recon-structed phylogeny of Minaria was then associated with thegeographic distribution of its species based on a databasecomprising 762 specimens. Based on this information, weevaluated whether the current system of conservation unitsin the Espinhaco Range is sufficient to properly protect theevolutionary history of this lineage.

Materials and methodsTaxon samplingForty-one species representing seven genera of Metastel-matinae were included in the analyses (Appendix 1, seesupplementary material, which is available on the Sup-plementary tab of the article’s Taylor & Francis Onlinepage at http://dx.doi/10.1080/14772000.2012.705356). Weselected genera that presented morphological affinities withMinaria and/or represented major clades in the phylogenyof the subtribe. Eighteen of the 21 species of Minaria wereincluded. Fresh material of the remaining species (M. mono-coronata, M. inconspicua and M. bifurcata) was not foundduring fieldwork and our attempts to sequence herbariummaterial of two of these species failed; therefore, they

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were analysed based only on morphological characters. Themolecular contribution of these species, however, would bemainly restricted to their terminal branches. Three popula-tions of Minaria cordata were sequenced, in an attempt tobetter represent its geographic distribution and morphologi-cal variation. Blepharodon ampliflorum was used as the out-group because it is closely related to B. lineare (and even-tually considered a synonym of this species; e.g. Morillo,1976; Fontella-Pereira et al., 1984), which was shown to besister to the remaining Metastelmatinae (Liede-Schumannet al., 2005; Rapini et al., 2006).

DNA extraction, amplification,sequencing and alignmentDNA was extracted from dehydrated leaves using the 2×CTAB protocol (Doyle & Doyle, 1987), adapted for mi-crotubes. Total DNA was used to test 25 regions suggestedas potentially useful in phylogenetic reconstructions at thespecies level (e.g. Shaw et al., 2005, 2007; Li et al., 2008),representing 16 plastid and nine nuclear regions. Fourplastid (rps16 intron: Oxelman et al., 1997; trnH-psbAintergenic spacer: Hamilton, 1999; trnS-trnG intergenicspacer: Shaw et al., 2005; and trnD-trnT intergenic spacer:Demesure et al., 1995) and two nuclear regions (ITS:Sun et al., 1994; and ETS: Beardsley & Olmstead, 2002)were selected based on their variability and number ofparsimony-informative characters (Ribeiro, 2011). Theamplification mix that achieved success for most regionsconsisted of 1 µL total DNA, 1× buffer, 2.0 mM MgCl2,0.2 mM dNTP, 0.2 mM primer, 10 ng BSA, Taq DNApolimerase (Phoneutria) – 1.25 units for the plastid regionsand 0.75 for the nuclear ones – complemented withultrapure water to 25 µL; for ITS and ETS amplification,1.0 M betaine and 2% DMSO were added to the mix.TopTaq (Quiagen) mix was used to amplify regions thatfailed with the prepared mix, following the standardprotocol in the kit manual.

PCR products were purified in EXO-SAP (AmershamBiosciences) enzymatic reactions or using polyethyleneglycol (PEG) and were sequenced directly with the sameprimers used for the PCR amplification. Sequence electro-pherograms were produced in an automatic sequencer (ABI3130XL Genetic Analyser) using a Big Dye Terminator 3.1(Applied Biosystem). They were edited using the StadenPackage (Staden et al., 2003) and aligned using BioEditSequence Alignment Editor (Hall, 1999).

Whenever we found polymorphic paralogues of ITS,which was the case in Minaria grazielae, M. hemi-pogonoides, M. polygaloides, M. semirii and Blepharodonpictum, we cloned the PCR products. For cloning, weused the pGEM R©–T kit, following the manufacturer’s(Promega) protocols. At least five colonies were sequencedfor cloned PCR product. Clones of the same species

formed supported clades (not shown), suggesting thatthey coalesce within the respective species. Therefore,only one sequence per species was included in the finalanalyses.

Morphological studyMorphological data were obtained from the literature(Rapini et al., 2001; Konno, 2005) and herbarium material.The matrix included 34 vegetative and floral parsimony-informative characters commonly used in the taxonomy ofMetastelmatinae (Appendices 2 and 3, see supplementarymaterial, which is available on the Supplementary tab ofthe article’s Taylor & Francis Online page at http://dx.doi/10.1080/14772000.2012.705356).

Phylogenetic analysesIncongruence length difference (ILD) test (Farris et al.,1994), implemented as the partition homogeneity test inPAUP, was used to evaluate the congruence among indi-vidual plastid matrices and between plastid and nucleardatasets. We performed 500 replicates using a random ad-dition heuristic search. To avoid false positives (Hipp et al.,2004), only values of P < 0.01 were considered evidence ofsignificant incongruence between datasets (Cunningham,1997). Phylograms based on results by maximum parsi-mony (MP), maximum likelihood (ML) and Bayesian in-ference (BI) analyses were examined visually and specieswith branches disproportionally longer were excluded toevaluate their effects on the analysis.

Four combined matrices were built: (1) plastid dataset;(2) nuclear dataset; (3) plastid and nuclear datasets com-bined (combined molecular dataset); and (4) molecular andmorphological datasets combined (called from now on to-tal evidence). MP, ML and BI analyses were conducted foreach matrix.

MP analyses were conducted in PAUP v. 4.0b10a(Swofford, 2000). Characters were considered unorderedand equally weighted. Heuristic searches were performedwith TBR branch swapping on 1000 random-taxon additionreplicates, saving a limit of 20 trees per replicate; trees re-tained were then subject to another search with TBR swap-ping, fixing a limit of 20,000 trees. Clades were statisticallyevaluated by non-parametric bootstrap support calculatedin PAUP through 1000 pseudo-replicates of simple-taxonaddition followed by TBR swapping, with a limit of 15 treesper pseudo-replicate.

ML analyses were conducted using RAxML (Stamatakis,2006) as implemented in the CIPRES v. 2.0 Portal (Milleret al., 2010). The GAMMA + P -Invar model was used tosearch for the most likely tree and the GTR + CAT model tocalculate the bootstrap support, using 1000 replicates underthe Rapid Bootstrap algorithm.

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For BI analyses, partitions were unlinked. The best-fittingsubstitution model for each molecular partition was inferredin MrModeltest (Nylander, 2008) and the standard discretemodel was applied for the morphological partition. Theanalyses consisted of two simultaneous independent runswith four chains each (one cold and three heated), con-ducted in MrBayes version 3.1.2 (Huelsenbeck & Ronquist,2001; Ronquist & Huelsenbeck, 2003). Chains were runfor 5 million generations, starting with random trees andsampling a tree every 1000 generations. Trees prior to thestationarity of the likelihood values were excluded (burn-in) and the Posterior Probability (PP) of each clade wasindicated by their frequency in the majority rule consensusof the remaining trees.

Spatial analyses of phylogenetic diversityThe geographic distribution of each species was obtainedbased on fieldwork and 762 specimens deposited in theprincipal herbaria of Brazil, Europe and the United States.Only specimens with accurate locality information wereincluded. GPS coordinates were confirmed or obtained withthe help of Google Earth (©2010 Google).

Indexes of phylogenetic diversity (PD; Faith, 1992) andendemism (PE; Rosauer et al., 2009) were calculated usingBiodiverse v. 0.15 (Laffan et al., 2010). They were based onthe branch lengths of the tree produced in the BI analysis oftotal evidence and the matrix of the geographic distributionof Minaria (available under request from PLR). The dis-tribution is illustrated using cells of 0.1◦, considering twoneighbour cells to calculate its diversity (Laffan & Crisp,2003). Total richness and richness of endemics were alsocalculated using the two neighbour cells in the same pro-gram.

ResultsThe plastid matrix (psbA, trnS-G, rps16 and trnD-T) con-sists of 3274 characters, 6.62% of which are variable, andthe nuclear matrix (ITS and ETS) consists of 1335 char-acters, 14.26% of which are variable (TreeBase access ID11738). Regions within the same genome are not incongru-ent according to the ILD test (P = 0.406, among plastiddatasets, and P = 0.416, between ITS and ETS), but plastidand nuclear datasets display significant incongruence (P =0.006). This incongruence seems to be mainly caused by theposition of Nephradenia filipes (Fig. 1). With the exclusionof this species, the conflict between the two data sources isnot highly significant (P = 0.016). The exclusion of specieswith longer branches did not affect the significance of theILD test. Considering only Minaria, the conflict betweenplastid and nuclear datasets was also significant accordingto the ILD test (P = 0.002). However, the ILD test performspoorly in several situations and is currently not consideredan indicator of increased accuracy when combining results

(Planet, 2006). Therefore, phylogenetic relationships in Mi-naria are mainly discussed based on the combined molecu-lar datasets (Fig. 2) and total evidence (Fig. 3) analyses.

Phylogenetic reconstructionApart from Blepharodon ampliflorum (the outgroup), theMetastelmatinae are composed of a basal polytomy, withthree species (Petalostelma martianum, Hemipogon acero-sus and Nephradenia acerosa) unresolved among Barjonia,Minaria and a clade comprising the remaining Metastel-matineae. Barjonia is supported based on plastid and nu-clear datasets. Minaria is supported in the plastid tree, butis not resolved in the nuclear tree. The remaining Metastel-matinae are divided into the Astephanopsis clade and theMetastelmatinae core group, which appear as sister groupsin the trees from the combined molecular dataset. TheAstephanopsis clade includes species currently classifiedin three genera (Blepharodon pictum, Hemipogon spru-cei and Nephradenia filipes), but is contradicted in thenuclear trees: Nephradenia filipes appears nested in theAstephanopsis clade based on plastids, but is found withNephradenia acerosa in the nuclear dataset. The Metastel-matinae core group is supported by the two molecular datasources and includes three species of Ditassa unresolvedamong two clades. One clade consists of Ditassa hispida,appearing as sister to a clade composed of Nephradeniaasparagoides and Ditassa capillaris. The other clade com-prises all sampled species of Hemipogon, except the typespecies (H. acerosus), and is predominantly distributed inthe Espinhaco Range.

The Minaria core group is strongly supported in bothplastid and nuclear trees. In the plastid and combinedmolecular dataset results, it is nested in a grade com-posed by M . polygaloides and a clade formed by M. harleyiand M. volubilis. The core group is divided in two clades,one including only comose-seed species and another includ-ing all species without coma (a tuft of long trichomes at oneend of seed). The former clade is supported in all datasets,but the latter emerged only in the total evidence analyses,although all sequenced species without coma, except M.hemipogonoides, also formed a supported clade based onthe nuclear dataset. Based on total evidence, three majorclades are within the group with coma, although only twoof them are supported in the combined molecular dataset.The three samples of Minaria cordata did not form a cladein any analysis and the specimen from Goias appears con-sistently closer to M. campanuliflora and M. ditassoidesthan to the other two specimens of M. cordata.

Spatial analysis of phylogenetic diversityThe Serra do Cipo and the Diamantina Plateau presentedthe highest richness of both total number of species andnumber of endemics. We detected four areas (Fig. 4) with

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Fig. 1. Majority rule consensus tree produced by Bayesian inference analysis based on the plastid dataset (trnH-psbA, trnS-G, trnD-Tand rps16) on the left and nuclear dataset (ITS and ETS) on the right, showing only clades with Posterior Probability ≥95%. Valuesabove branches are the PP and those below are maximum likelihood and maximum parsimony bootstrap supports (BS ML/BS MP). Thedashed square denotes Minaria, as currently circumscribed. Nephradenia filipes is in bold to highlight its conflicting position. ∗Indicatesthe Minaria core group.

higher indices of phylogenetic diversity and endemism: Riode Contas, in the state of Bahia, the Diamantina Plateau,Serra do Cipo and the Southern Espinhaco Range, in thestate of Minas Gerais. Serra do Cipo has the highest lev-els of phylogenetic diversity and endemism, followed bythe Diamantina Plateau and Rio de Contas, respectively(Table 1; Fig. 4).

DiscussionThe plastid markers tested in this study displayed low levelsof variation and, individually, they were neither sufficient

to recover Minaria nor offered relevant support for internalrelationships apart from the core group. However, the BIanalysis based on the plastid dataset presented high sup-port for Minaria. The ITS and ETS regions were morevariable than the plastid regions and presented fewer spec-imens with polymorphic paralogues when compared withthe other nuclear regions tested in this study. ITS poly-morphisms were mainly restricted to species with narrowranges (M. grazielae, M. hemipogonoides, M. magisteriana,M. polygaloides, M. refractifolia and M. semirii) and notnecessarily present in all individuals. Moreover, divergentITS paralogues coalesced within the same species (shallowparalogy sensu Bailey et al., 2003), suggesting that they

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Fig. 2. Phylogram of majority rule consensus trees produced by the Bayesian inference analysis based on combined molecular (plastid andnuclear) datasets. Values below branches are maximum parsimony (MP) bootstrap supports (BS). Asterisks indicate clades with PosteriorProbability ≥95%; arrows indicate clades without BS present in the strict consensus of MP.

appeared after speciation; in this manner, they do not affectthe species’ phylogenetic reconstructions.

This was the first phylogenetic study of Apocynaceaethat used the ETS (external transcribed spacer) region ofthe 18S−26S gene. ETS and ITS regions play similar, in-terdependent functions in rRNAs maturation and have sim-ilar substitution rates; they appear in tandem, as part ofthe same transcribed unity, and therefore are supposed toprovide congruent phylogenetic information (Baldwin &Markos, 1998; Linder et al., 2000; Calonje et al., 2009). Forangiosperms, ETS is usually more informative than ITS andits popularity in phylogenetic studies is not greater prob-ably because the region lacks a universal forward primer(Baldwin & Markos, 1998; Mitsui et al., 2008; Logacheva

et al., 2010). However, the forward primer designed forMimulus L. (Phrymaceae; Beardsley & Olmstead, 2002),in combination with the universal reverse primer (Baldwin& Markos, 1998) successfully amplified our samples. ETSwas shown to be more variable and less homoplastic thanITS; together, they were two times more variable (14.26%)than the plastid dataset (6.62%), although they are also morehomoplastic.

The conflict between plastid and nuclear data sourceswas mostly caused by the position of Nephradenia filipes(Fig. 1). Supported conflicts between gene trees are notrare (Rokas et al., 2003) and should not be a reason forabandoning species trees (Knowles, 2009). Although we arenot yet able to explain this incongruence, it is not supposed

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Fig. 3. Majority rule consensus trees produced by Bayesian Inference analysis based on total evidence (molecular and morphologicaldatasets) showing all compatible clades. Values below branches are maximum parsimony (MP) bootstrap supports (BS). Asterisks indicateclades with Posterior Probability ≥95%; arrows indicate clades without BS present in the strict consensus of MP. Species in bold areendemic to the Espinhaco Range; areas of occurrence of microendemics are between parentheses: DiP (Diamantina Plateau), RCo (Riode Contas); SCi (Serra do Cipo), and SER (Southern Espinhaco Range).

to negatively affect internal relationships in Minaria. Theincongruent position of M. parva in MP and MV analyses(not shown), on the other hand, was probably dependent onthe choice of the method of analysis, because BI analyses

recovered congruent results for this species based on plastidand nuclear datasets (Fig. 1). This suggests that the plastiddataset provided a misleading relationship for M. parvawhen analysed by either MP or ML (Ribeiro, 2011).

Table 1. Areas with higher Minaria phylogenetic diversity and endemism, indicating their geographic position and species present in thearea. Asterisks indicate microendemics (restricted to the area); the most relevant species for the phylogenetic diversity of the area are inbold.

Area (State) AbbreviationGeographic

position Species

Rio de Contas (BA) RCo 13.4S41.9W

Minaria acerosa, M. cordata ‘var. cordata’, M. cordata ‘var. virgata’,M. harleyi∗ and M. volubilis∗

Diamantina Plateau (MG) DiP 18.3S43.5W

Minaria bifurcata∗, M. campanuliflora∗, M. decussata, M.diamantinensis∗, M. ditassoides, M. grazielae∗, M. inconspicua∗,M. parva and M. refractifolia∗

Serra do Cipo (MG) SCi 19.1S43.7W

Minaria acerosa, M. decussata, M. ditassoides, M. hemipogonoides∗,M. magisteriana∗, M. parva, M. polygaloides∗ and M. semirii∗

Southern Espinhaco Range (MG) SER 20.1S43.9W

Minaria acerosa, M. decussata, M. micromeriaand M. monocoronata∗

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Fig. 4. Distribution map of Minaria phylogenetic diversity (on the left) and endemism (in detail, on the right) in the Espinhaco Range(areas above 900 m in grey), considering grids of 0.1◦ with two neighbour cells. Areas: DiP = Diamantina Plateau; RCo = Rio de Contas;SCi, Serra do Cipo; SER = Southern Espinhaco Range. Conservation Unities are shown by black outlines; numbers identify those withmore than 4000 hectares: 1. Gruta dos Brejoes Environmental Protection Area (EPA); 2. Morro do Chapeu State Park (SP); 3. MarimbusEPA; 4. Chapada Diamantina National Park (NP); 5. Serra do Barbado EPA; 6. Nascente do Rio de Contas Area of Ecological Interest;7. Contendas do Sincora National Forest; 8. Boa Nova NP; 9. Serra do Ouro EPA; 10. Caminho dos Gerais SP; 11. Montezuma SP; 12.Serra Nova SP; 13. Mata Escura Biological Reserve; 14. Grao Mogol SP; 15. Lapa Grande SP; 16. Acaua Ecological Station; 17. SempreVivas NP; 18. Serra do Cabral SP; 19. Serra Negra SP; 20. Biribiri SP; 21. Rio Preto SP; 22. Aguas Vertentes EPA; 23. Pico do ItambeSP; 24. Serra do Intendente SP; 25. Morro da Pedreira EPA; 26. Serra do Cipo NP; 27. Cartes da Lagoa Santa EPA; 28. Rio Doce SP; 29.Serra do Rola Moca SP; 30. Cachoeira das Andorinhas EPA; 31. Itacolomi SP.

Phylogenetic relationships in MinariaThe original circumscription of Minaria was recently en-larged to include M. harleyi and M. volubilis (Silva et al.,in press), two species which are endemic to the ChapadaDiamantina, in the Espinhaco Range of Bahia. Our anal-yses support Minaria as a monophyletic genus if the twospecies were included, and they place M. polygaloides assister to all species of the clade (Fig. 2). Minaria harleyiand M. volubilis are quite distinct morphologically from theother species of the lineage and also from each other, rep-resenting remnants of an ancient lineage (Ribeiro, 2011).Minaria harleyi occurs in rocky fissures while M. volubilis

grows in sandy soils, in areas with low vegetation. Theyhave glabrous branches, relatively large flowers and an elon-gated gynostegium, which is long-rostrate (c. 2.4 mm long)in M. volubilis.

The Minaria core group has been highly supported sincethe first phylogenetic analyses with molecular data (Rapiniet al. 2003, 2006, 2007; Liede-Schumann et al., 2005). Itcomprises erect, pubescent shrubs, with branches, leaves(except by M. magisteriana), pedicels and follicles cov-ered by trichomes over the whole surface. The lineages di-versified in the Espinhaco Range during the Pleistocene,presenting high niche conservatism restricted to open

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vegetation (Ribeiro, 2011). The clade is divided in two prin-cipal groups, one comprising six species, with axillary in-florescences, and few (usually 1 or 2) seeds per fruit, whichlack coma (Fig. 3), and another comprising 12 species, withsubaxillary inflorescences and many comose seeds per fruit.

The group lacking coma is rupicolous, with species thatgrow exclusively on rocky outcrops in small areas of theEspinhaco Range in Minas Gerais: three species from Serrado Cipo, two from the Diamantina Plateau and one fromthe Southern Espinhaco Range. With low concentration offlammable biomass, rocky outcrops may represent refugesfor lineages less tolerant to fire, and many other plant groupsfrom the Espinhaco Range also occur exclusively in thiskind of environment (Neves & Conceicao, 2010). The ab-sence of coma and the reduced number of seeds per fruitlimited the dispersal capability in this lineage and may havebecome established as response to habitat specialization.With a short dispersal range, their seeds tend to fall close tothe parents, being only secondarily dispersed by rainwater.Therefore, they have less chance of reaching areas outsiderocky outcrops, where the lineage is not competitive andbecomes more susceptible to negative agents, such as fire.Moreover, because these plants are perennial and rocky out-crops are arranged in patches, it is likely that most seedsdo not find suitable places to germinate, and are wasted af-ter patch saturation. Thus, limited seed-dispersal may havefavoured the maintenance of species close to the originalrocky outcrops, preventing dispersal to distant outcrops.

The group with comose seeds is broadly distributed, al-though most species are also restricted to small areas inthe Espinhaco Range of Minas Gerais. The species in thisgroup are also characterized by cordate or acerose, revoluteleaves, and usually grow on stony or sandy soil. The rupi-colous Minaria refractifolia emerges as an odd species inthis clade. It is more similar to the species lacking coma, butthe comose seeds and molecular data support its positionas a derived species within the comose clade. Therefore,similarities between M. refractifolia and the group with-out coma are probably a result of convergences because oftheir rupicolous habitat. Plants with cordate leaves forma complex of species and the three individuals of M. cor-data (or four, considering that M. abortiva was also treatedas a variety of M. cordata) sampled in our study do notform a monophyletic group. The variation comprised bythem has been treated as a single species (Fontella-Pereira,1989; Farinaccio & Mello-Silva, 2004; Konno, 2005) or asdifferent species (Rapini et al., 2001; Konno et al., 2006;Rapini, 2010). Our results, therefore, suggest that this vari-ation reflects the existence of cryptic species. Other bio-geographic analyses (Ribeiro, 2011) also suggest that thesespecies derived from a broadly distributed ancestor thatgave rise to several isolated lineages after fragmentation. Inthis case, similarities shared by this complex of species arelikely symplesiomorphic. However, more detailed studies,

including biosystematic and phylogeographic analyses, arestill required to clarify their relationships.

Although our phylogenetic analyses do not provide highsupport for all clades in Minaria, the phylogenetic uncer-tainties are mainly confined to short branches, which arenot supposed to greatly affect analyses of diversity as longas they are based on branch length. Therefore, these phylo-genetic results can be considered sufficient to provide a pre-liminary perspective on the distribution of the phylogeneticdiversity across the Espinhaco Range. Yet, the phylogeneticdiversity of other lineages concentrated in Espinhaco Rangemust be investigated to evaluate and synthesize patterns de-tected with Minaria.

Distribution of phylogenetic diversityand priority areas for conservationIn the state of Bahia, Rio de Contas stands out becauseof two microendemic species, Minaria harleyi and M.volubilis. They are remnants of an ancient lineage, as oldas the entire Minaria core group. The Chapada DiamantinaNational Park, which is the largest conservation unit in theEspinhaco Range, protects only Minaria harleyi, whereasthe Serra do Barbado Environmental Protected Area com-prises the distribution of the two species. Therefore, animportant part of the phylogenetic diversity of Minaria isrepresented within conservation units of Bahia (Fig. 4).

The central region of the Espinhaco Range in MinasGerais, which comprises the Diamantina Plateau and Serrado Cipo, is the richest in endemic species of Asclepi-adoideae (Rapini et al., 2001, 2002; Rapini, 2010) andother groups of angiosperms (Echternacht et al., 2011).Together, they contain 13 of the 21 species of Minaria, 10of which are microendemic (restricted to only one of the twoareas). Serra do Cipo houses four microendemics, whereasthe Diamantina Plateau houses six. In spite of this, Serra doCipo shows the highest levels of phylogenetic diversity andendemism. The microendemics from Serra do Cipo belongto older lineages that present narrow distributions whereashalf of the microendemics from the Diamantina Plateau be-long to a more recent, widespread lineage. Together, thetwo areas comprise the largest continuous region coveredpredominantly by rocky outcrops, which provide the finespatial heterogeneity typical of the campos rupestres for-mation and help to protect species against recurrent fires(Baker, 2009; see below). Therefore, extensive areas ofcampos rupestres in the Espinhaco Range, such as the Dia-mantina Plateau and Serra do Cipo, may represent historicalrefuges for fire-sensitive lineages, especially during periodswhen the fire regime becomes a determinant factor in thedynamics of savannah-like formations.

There are two national parks in the central part of theEspinhaco Range in Minas Gerais, but part of the Range’sflora is only in units that are not under integral protection.

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The Serra do Cipo National Park includes the range of twomicroendemic species (M. polygaloides and M. semirii)while M. hemipogonoides and M. magisteriana occur in theErmos Gerais National Heritage Protection Reserve, whichalso shelters other species with restricted ranges (Echter-nacht et al., 2010; Rapini et al., 2010b). The flora in theSempre Vivas National Park is still poorly known and thereare no reports of Minaria from this unit. Of the six speciesendemic to the Diamantina Plateau, Minaria grazielae, M.inconspicua and M. refractifolia occur in the Biribiri StatePark and M. campanuliflora in the Rio Preto State Park. Mi-naria diamantinensis, on the other hand, is known only in ar-eas that can be easily accessed, along the road Diamantina –Conselheiro Mata, and remains unprotected. Finally, Mi-naria bifurcata is known only from the type, whose localityis uncertain.

The relevance of the Southern Espinhaco Range lies inthe presence of the microendemic Minaria monocoronata,which belongs to the clade without coma. The area presentsfour species of Minaria, but only one of them is a mi-croendemic. Despite this, the area also deserves particularattention. Conservation units in the Southern EspinhacoRange are small and highly anthropized. Minaria mono-coronata is restricted to the cangas, which are ironstoneoutcrops, comprising only around 100 km2. Because theyhave been intensely exploited by the mining industry,cangas are among the most threatened habitats in theEspinhaco Range (Jacobi et al., 2007), as well as the speciesendemic to them. Serra do Rola-Moca State Park coversthe distribution area of M. monocoronata. Nevertheless,the species has not been collected in the area for more than50 years and the park was established only in 1994. The lastrecord of M. monocoronata was in the Itabirito in 2004.However, there is now a huge quarry (from iron mining)in this area. Therefore, for conservation purposes, otherattributes should also be considered in combination withphylogenetic diversity and endemism, such as abundance(Cadotte & Davies, 2010) and habitat specialization. Levelsof species threat should also be integrated into the impor-tance values of a region. In this perspective, the SouthernEspinhaco Range is historically the most disturbed areaand currently the most endangered because of mining.

There are no absolute scientific criteria for selecting pri-ority areas for conservation and different reasons can beused for this purpose. Nee & May (1997), for example,considered that conservation strategies should be basedon individual species, regardless of their phylogenetic re-lationships. Others (Vane-Wright et al., 1991; Williamset al., 1991; Faith, 1992, 2008; O’Brien, 1994; Vasquez &Gittleman, 1998; Rosauer et al., 2009), however, advocatethat evolutionary history should be considered in biodiver-sity conservation, although their perspectives on how toconsider this point might diverge. The phylogenetic sin-gularity of a region is determined either by the presenceof endemic remnants of ancient lineages (‘museums’), as

in the southwestern part of Chapada Diamantina, in theEspinhaco Range in Bahia, or by the presence of severalendemic species that belong to a lineage with a broader dis-tribution, as in Serra do Cipo and Diamantina Plateau, in theEspinhaco Range in Minas Gerais. These two patterns areproduced by different processes and their implications forconservation may also be different. Protecting areas withrare ancient lineages will preserve a great amount of exclu-sive phylogenetic diversity whereas protecting evolutionaryfronts (i.e. contemporary centres of radiation) will preserveareas of active diversification (Erwin, 1991). Phylogeneticdiversity and endemism, however, do not distinguish be-tween these two patterns clearly.

Considering the phylogenetic relationships amongspecies and their geographical distribution, phylogeneticendemism (Rosauer et al., 2009) indicates areas thatcomprise high levels of exclusive evolutionary history. Thefour areas that emerged as being relevant in the evolutionof Minaria are roughly congruent with those previouslysuggested by empirical observations and previous studiesbased on richness of endemic Asclepiadoideae (Rapini,2010). Endemic-rich areas capture a great proportion ofspecies richness and threatened species (Orme et al. 2005),and in the absence of complete phylogenetic data this canbe used as an initial reference for areas with high phylo-genetic diversity. In Minaria, endemics are concentratedin the Diamantina Plateau and Serra do Cipo, which arealso the areas with higher levels of phylogenetic diversityand endemism. However, the high level of phylogeneticendemism in Rio de Contas would not be directly inferredbased on richness of endemics, because the area containsonly two endemic species.

In spite of their differences, the four areas with highMinaria phylogenetic endemism are biologically relevantand deserve special attention in conservation programmes.Rio de Contas houses an ancient lineage represented by twoendemic species and is also broadly known by its singularfloristic diversity (Stannard, 1995), including severalmicroendemics, such as the milkweed ‘Cynanchum’morrenioides Goyder, which is also an old lineage, sisterto the remaining species of the subtribe Orthosinae(Liede-Schumann et al., 2005). Therefore, protectingthis area is essential to preserve this unique evolutionaryheritage. The Serra do Cipo and Diamantina Plateau,in the central region of the Espinhaco Range in MinasGerais, house several microendemic species, both neo-and palaeoendemics. They probably represent long-termrefuges for lineages less tolerant to fire disturbance and arehistorical centres of radiation, representing ‘cumulativerefugia’ (sensu Medail & Diadema, 2009). Protecting thesetwo areas will preserve the principal evolutionary processesresponsible for Minaria diversification. Finally, the South-ern Espinhaco Range houses the most threatened speciesof the lineage, because the strong anthropogenic pressurein this region puts its endemic flora under serious risk of

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extinction. Therefore, the establishment of more conserva-tion units in this area is needed, which should be designedto protect particularly specialized habitats like cangas.

Management of conservation unitsin the Espinhaco RangeDistinct formations respond differently to key factors andrequire different research programmes and managementstrategies (Rogers, 2003). Dry seasonal climates and poorsoils are possible determinants of savannah-like formations(Bond et al., 2005), and fire is an ancient natural distur-bance of the Cerrado (Miranda et al., 2009). The establish-ment of fire regimes in the modern Cerrado during the LateMiocene–Pliocene was favoured by a dryer climate and theexpansion of C4 grasses, which became more abundant asthe atmospheric CO2 decreased (Beerling & Osborne, 2006;Edwards et al., 2010). Lineages have responded differentlyto the establishment of fires in central South American sa-vannahs. Some groups of plants are easily adapted to fireregimes and have repeatedly colonized the Cerrado (Simonet al., 2009). Others, particularly trees and shrubs, havebeen more conservative and remain fire-sensitive (Moreira,2000; Medeiros & Miranda, 2005).

The campos rupestres in the Espinhaco Range differfrom the surrounding open formations because they occurat higher elevations and have higher concentrations ofrocky outcrops. At higher elevations, the proportion ofC4 grasses is lower and the length of the dry season isshorter and also minimized by orographic moisture. Flashdensity increases with elevation, but chances of lightningare lower, and the low-fuel rocky outcrops serve as naturalbarriers, limiting the spread of fire and favouring spatialheterogeneity (Baker, 2009). As a consequence, frequencyand extension of natural fires in campos rupestres areprobably lower than those in tropical savannahs at loweraltitudes. Therefore, areas in the Espinhaco Range arepostulated to serve as long-term refuges for fire-sensitivelineages. With increasing fire incidence, the widespreadancestral species of these lineages would become reducedto vicariant populations in mountain areas. Lineages withlimited seed dispersal, such as Minaria, are particularlyaffected by this process. Many of them became extinct,but many others went through a period of non-adaptiveradiation caused by geographic isolation (Givnish, 2010;Flenley, 2011), which might explain the richness and thehigh rates of endemics in the Espinhaco Range.

Wildfire was an important component for manyformations before humans existed (Scott, 2000) andfire-dependent ecosystems are not merely artefacts of an-thropogenic burning (Bond et al., 2005). Nevertheless, theeffects of fire on natural communities are complex and firemanagement in conservation units is still a matter of discus-sion (e.g. Whelan, 1995; Wilgen et al., 2003; Baker, 2009;

McKenzie et al., 2011). Modern humans have directly (e.g.by lighting fires) or indirectly (e.g. by cultivating grasses forpasture) interfered with the fire regimes on Earth (Bowmanet al., 2011). Human activities have changed the season, fre-quency and intensity of fires in the Cerrado for thousandsof years, but much more markedly during the last threecenturies (Dias, 2006), and more than half of the biomehas been transformed in the last four decades (Klink &Machado, 2005).

Fire is a recurrent agent in fire-prone formations(Bond et al., 2005), including the savannah-like Cerrado(Moreira, 2000) and campos rupestres (Neves &Conceicao, 2010), affecting their composition and physiog-nomy. Empirical studies (Clarke, 2002; Neves & Conceicao,2010) have shown that the effects of fires on the dynamicsof adjacent habitats can greatly vary. Species that occupythe continuous vegetation matrix (the ‘entremeios’ of thecampos rupestres, sensu Conceicao & Pirani, 2005) areusually fire-tolerant, and regenerate after fires (‘sprouters’sensu Clarke, 2002). However, many others, especiallywoody species in vegetation islands of rocky outcrops, arefire-sensitive; they are killed by fire and depend on seeddispersal and seedling recruitment (‘obligate seeders’ sensuClarke, 2002) to become established in burned areas. Insome cases, both strategies of post-fire recruitment (seedingand resprouting) can be equally affected by environmentalfactors (Vivian & Cary, 2012). Due to the nutrient-poorsoils in campos rupestres, the regeneration of vegetation isslow (Kolbeck & Alves, 2008). Therefore, increasing firefrequencies in campos rupestres may represent a seriousthreat to seeders, because they will have higher chances ofbeing burned before the new generation can produce seeds,and also to resprouters, because the costs of allocation tostorage reserves may become inefficient (Vivian & Cary,2012).

The Chapada Diamantina National Park presents one ofthe highest incidences of fire among the protected areas inBrazil, but more than 99% of fires registered in the parkhave anthropogenic origins (Berlinck et al., 2010). In spiteof this, 39% of the area has not been burned since 1973(Mesquita et al., 2011) and only 1% of the park has burnedmore than four times in the last 25 years (Goncalves et al.,2011). In the Serra do Cipo National Park, a similar sit-uation is found and the incidence of fire is also mostlythe result of human activity (Ribeiro & Figueira, 2011).This scenario suggests that the frequency of fire in mostparts of the Espinhaco Range is generally low under nat-ural conditions, and supports the idea that the region is ahistorical refuge for many fire-sensitive lineages and thatthe conservation of its endemic biodiversity may rely on ad-equate fire management. Long-term experimental researchto clarify the behaviour of fire and its impacts on the dynam-ics of campos rupestres is still needed. In this meantime,however, approaches to prevent fires caused by humansand the dissemination of exotic grasses, inside and nearby

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conservation units, must be adopted to preserve the highplant biodiversity endemic to the Espinhaco Range.

AcknowledgementsThis study is part of the Ph.D. thesis of PLR, devel-oped at PPGBot-UEFS, with a fellowship from FAPESBand CAPES. It was supported by the APR 140/2007 andPNX0014/2009 research grants from FAPESB. We thankAbel A. Conceicao for calling our attention to the impor-tance of fire for the dynamic of campos rupestres. Wealso thank IBAMA and IEF/MG for the collection per-mits for Asclepiadoideae within the conservation units. ARand CvdB are supported by Pq-2 and Pq-1D CNPq grants,respectively, UCSS by a PhD fellowship from CAPES(PNADB) and TK by FAPERJ 170836/2006.

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