molecular phylogenetics and morphological evolution of st. john's wort (hypericum; hypericaceae

Molecular Phylogenetics and Evolution 66 (2013) 1–16

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Molecular Phylogenetics and Evolution

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Molecular phylogenetics and morphological evolution of St. John’s wort (Hypericum;Hypericaceae)

Nicolai M. Nürk a,⇑, Santiago Madriñán b, Mark A. Carine c, Mark W. Chase d, Frank R. Blattner a

a Leibniz Institute of Plant Genetics and Crop Research (IPK), D-06466 Gatersleben, Germanyb Universidad de los Andes, AA 4976 Bogotá, Colombiac Natural History Museum, London SW7 5BD, UKd Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK

a r t i c l e i n f o a b s t r a c t

Article history:Received 17 February 2012Revised 18 June 2012Accepted 22 August 2012Available online 8 September 2012

Keywords:Character evolutionHypericaceaeInternal transcribed spacer (ITS)PhylogenyThorneaTriadenum

1055-7903/$ - see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.ympev.2012.08.022

⇑ Corresponding author.E-mail address: nuerk@ipk-gatersleben.de (N.M. N

Phylogenetic hypotheses for the large cosmopolitan genus Hypericum (St. John’s wort) have previouslybeen based on morphology, and molecular studies have thus far included only a few species. In this study,we used 360 sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA)for 206 species representing Hypericum (incl. Triadenum and Thornea) and three other genera of Hyper-icaceae to generate an explicit phylogenetic hypothesis for the genus using parsimony and model-basedmethods. The results indicate that the small genus Triadenum is nested in a clade within Hypericum con-taining most of the New World species. Sister to Hypericum is Thornea from Central America. WithinHypericum, three large clades and two smaller grades were found; these are based on their general mor-phology, especially characters used previously in taxonomy of the genus. Relative to the most recent clas-sification, around 60% of the sections of Hypericum were monophyletic. We used a Bayesian approach toreconstruct ancestral states of selected morphological characters, which resulted in recognition of char-acters that support major clades within the genus and a revised interpretation of morphological evolutionin Hypericum. The shrubby habit represents the plesiomorphic state from which herbs evolved severaltimes. Arborescent species have radiated convergently in high-elevation habitats in tropical Africa andSouth America.

1. Introduction

Hypericum, the largest of the nine genera in Hypericaceae (APGIII, 2009; Stevens, 2007), which is placed in the clusioid clade ofMalpighiales (Wurdack and Davis, 2009), comprises almost 500species of herbs, shrubs, and small trees. Hypericum includes morethan 80% of the described species in Hypericaceae and is, togetherwith Triadenum and the monotypic genus Lianthus, the only repre-sentative of this family occurring in temperate regions, whereasthe remainder is native to tropical or subtropical regions (Stevens,2007). Overall, Hypericum is cosmopolitan with main centers ofdiversity in Eurasia (>230 species) and Andean South America(>130 species), and smaller ones in North America (c. 40 species),Southeast Asia (c. 47 species), and Africa (c. 30 species). Typicalhabitats are rocky, sometimes calcareous, and dry to moist grass-lands or acidic fens and shallow swamps. In the tropics, Hypericumis generally confined to high-elevation habitats (Robson, 2003).

The perennial herb H. perforatum (common St. John’s wort), thesource of a mild antidepressant (Butterweck, 2003; Butterweck

ll rights reserved.

ürk).

and Schmidt, 2007; Crockett and Robson, 2011; Wurglics andSchubert-Zsilavecz, 2006), is an invasive plant now distributedglobally (except Antarctica) that has been designated a noxiousweed in the United States (Zouhar, 2004). During the past 10 years,H. perforatum has also become a subject of interest for research onapomixis (asexual seed formation; Matzk et al., 2003; Schallauet al., 2010). Apomixis is reported from at least 16 Hypericum spe-cies (Matzk et al., 2003) classified in H. sects. Ascyreia, Hirtella, andHypericum. Myers (1964) also reported apomictic embryo develop-ment in Triadenum. Whether the genetic basis is similar in all spe-cies to that reported for H. perforatum (Schallau et al., 2010) has notyet been investigated.

Classification of Hypericaceae (Stevens, 2007) above the genuslevel has been the focus of morphological and molecular studies(Nürk and Blattner, 2010; Ruhfel et al., 2011; Wurdack and Davis,2009). Relationships of genera within the three tribes – Cratoxyleae,Vismieae, and Hypericeae – are ambiguous and under investigation.For example, within Hypericeae Robson (1977, 1981, 2001) recog-nized five genera: Hypericum, Triadenum, Thornea, Lianthus, andSantomasia. Ruhfel et al. (2011) proposed a revised classificationand merged the four genera into Hypericum (Ruhfel et al., 2011;see below). Unless otherwise stated, we use the circumscriptions

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of Robson (1977, 1981, 2001) for all genera and infrageneric catego-ries, although we agree with Ruhfel et al. (2011) that several ofthese genera belong in Hypericum.

Robson (1977, 1981, 1987, 1990, 2001, 2010b) recognized 36sections within Hypericum. Based on evolutionary trends for sev-eral morphological characters (Robson, 1977: 306 ff), relationshipsbetween the sections were hypothesized and presented in a net-work-like genealogical scheme (Robson, 1981: Fig. 2). Nürk andBlattner (2010) conducted a cladistic analysis of morphologicalcharacters to test the monophyly of the sections in the genus andinterrelationships between them. In many cases, congruence be-tween Robson’s sectional classification and groups defined by thecladistic analysis of morphology was found. However, relation-ships between the sections differed. For example, Robson (1981,1985) proposed that Hypericum section Campylosporus from Africaincluded the ‘most primitive’ species of the genus, whereas in Nürkand Blattner (2010) they were nested in a derived position within aclade containing mainly Palaearctic and Indomalesian species.

During the last two decades, molecular phylogenetics has be-come a common method to evaluate relationships of taxa andhas had a major impact in our understanding of evolution (Stechand Quandt, 2010). Several molecular phylogenetic studies haveincluded Hypericum, but these were either focused on family- orgenus-level relationships (Gustafsson et al., 2002; Ruhfel et al.,2011; Wurdack and Davis, 2009) or based on non-representativesampling for the entire genus; these included few and/or relativelydistantly related outgroups (Crockett et al., 2004; Heenan, 2008;Park and Kim, 2004; Pilepic et al., 2010, 2011). Only one of thesestudies has addressed relationships of major lineages withinHypericum. Ruhfel et al. (2011), analyzing three plastid and onemitochondrial gene, provided evidence for two large clades withinHypericum and inclusion of the small genera Santomasia, Thornea,and Triadenum in one of these. However, their taxon sampling inHypericum was also narrow (21 species/accessions) because thestudy focused on the entire clusioid clade. Thus, many questionsremain unanswered, in particular relationships of major lineagesand monophyly of sections within Hypericum.

To generate a phylogenetic hypothesis of species/section rela-tionships within Hypericum, we sequenced the nuclear ribosomalDNA (nrDNA) internal transcribed spacer region (ITS), includingITS-1, 5.8S rDNA, and ITS-2 (Baldwin, 1992; Baldwin et al., 1995),of accessions representing the genus (incl. Thornea and Triadenum)and three other genera of Hypericaceae classified in Vismieae andCratoxyleae. Previous studies have demonstrated the utility of theITS region for phylogenetic inference at the species level in Hyper-icum (Crockett et al., 2004; Heenan, 2008; Park and Kim, 2004). Thepossibility of amplifying ITS-1 and ITS-2 separately using internalprimers (Blattner, 1999) allowed us to use poorly preserved planttissue from older herbarium specimens for amplification of this lo-cus, and, thus, to extend our sampling.

The main objectives of this study are to (i) provide a molecularanalysis based, wherever possible, on several accessions perspecies covering almost all sections of the genus with multipletaxa, (ii) evaluate monophyly of Hypericum, (iii) compare resultswith the current classification of the genus, and (iv) reconstructthe evolution of selected characters used in previous classificationsand identify those that putatively distinguish major groupings.

2. Material and methods

2.1. Taxon sampling

Our approach involved extensive sampling within Hypericum,with multiple accessions for as many species as possible from al-most all sections. Dense taxon sampling is generally recognized

to be important in breaking up long branches and correctly assess-ing the occurrence of homoplasy, both factors that can producemisleading results (Huelsenbeck, 1995); indeed, adding additionaltaxa seems more valuable than adding more genes to improveresolution, at least in some cases (Zwickl and Hillis, 2002). Sampleswere obtained from herbarium collections (ANDES, B, BM, GH,HEID, K, KYO, TI), freshly collected silica-gel dried material (Chaseand Hills, 1991) from Colombia and Japan, as well as from livingcollections cultivated at the Royal Botanic Gardens, Kew (K), UKNational Council for the Conservation of Plants and Gardens – Na-tional Plant Collection of Hypericum sections Androsaemum andAscyreia at Wakehurst Place (WAK), IPK Gatersleben (GAT) andUniversity of Heidelberg (HEID). Additionally, GenBank was que-ried for Hypericaceae ITS sequences. Fifty-eight selected sequencesfrom the database were included in the final alignment togetherwith 282 newly generated for this study. Twenty sequences pub-lished by Crockett et al. (2004) were re-edited from the originalchromatogram files, resulting in longer ITS sequences, which wereresubmitted to GenBank as updated versions. In total, Hypericumcurrently comprises 496 species (incl. Lianthus, Santomasia, Thor-nea and Triadenum; Norman Robson, Nat. Hist. Museum, London,pers. comm.) of which 200 (40%) were included in our analysis(missing: Santomasia and Lianthus; Robson, 1981, 2001). Of 36sections recognized by Robson (1977 onwards) within Hypericum,34 were sampled here; we were unable to sample the two mono-typic H. sects. Umbraculoides (H. umbraculoides from Oaxaca,Mexico) and Thasia (H. thasium from Greece, Bulgaria, and Turkey).Outgroup representatives of Vismia, Harungana, and Cratoxylumwere included (Appendix A).

2.2. Molecular methods

Genomic DNA was extracted from fresh material, herbariumexsiccatae or silica-gel dried samples. For DNA extraction, differentamounts of plant tissue and several extraction methods weretested. Two methods provided the best results in Hypericum: aCTAB (cetyltrimethyl ammonium bromide) approach modifiedfrom Doyle and Doyle (1987, 1990) and the Invisorb� Spin PlantMini Kit (Invitek, Berlin, Germany) following the manufacturer’sprotocol. For old and poorly preserved tissues from herbariumsheets the CTAB method, including 2% PVP40 (polyvinylpyroli-done), was used. No more than 10 mg of plant tissue was includedper extraction to avoid decrease of DNA quality and yield.

The entire ITS region was amplified with primers ITS-A and ITS-B (Blattner, 1999) in 50 ll reactions using 1 U Taq DNA polymerase(QIAGEN, Hilden, Germany), 5 ll of the supplied buffer (10�) andadditionally 5 mM MgCl2, 100 lM of each dNTP, 5 pmol of eachprimer and approximately 20 ng of total DNA. In order to weakenDNA secondary structure, Q-solution (QIAGEN) was added to thereactions with a final concentration of 20%. In case of degradedherbarium material ITS-1 and ITS-2 were amplified separatelyusing the initial amplification primers in combination with inter-nal primers (i.e. ITS-A/ITS-C and ITS-B/ITS-D) binding in the5.8S rDNA gene (Blattner, 1999). PCR profiles consisted of an initialdenaturation at 95 �C for 3 min, followed by 38 cycles of 95 �C for30 s, 53 �C for 45 s, 68 �C for 1 min and a final step at 70 �C for8 min. Amplicons were cut out and purified using the QIAquickgel extraction kit (QIAGEN). PCR products were directly sequencedon the ABI 3730xl DNA Analyzer (Applied Biosystems, Darmstadt,Germany) using ABI dye-terminator sequencing technologyaccording to the manufacturer’s protocols. Samples were se-quenced using either two nested primers, ITS-SF and ITS-SR(Blattner et al., 2001) or these primers together with the internalprimers ITS-C(F) and ITS-D(R). Forward and reverse sequencesfrom each template were manually edited and combined in singleconsensus sequences with Sequencher v4.7 (Gene Codes of Ann

Fig. 1. Bayesian tree from analysis of ITS sequence data, showing relationships of 360 accessions representing 200 Hypericum and six outgroup species. A light-graybackground indicates clades of Hypericum. Support values are given above the branches (BI|ML|MP). Habit, shrubs (in green), trees (red), perennial (dark blue) and annualherbs (light blue), is highlighted by color of branches. The four pie charts display bootstrap percentages of the character state ‘habit’ reconstructed using MCMC in 1000 treesat a certain node. Taxonomy follows Ruhfel et al. (2011). Outgroups arranged as in Wurdack and Davis (2009).

N.M. Nürk et al. / Molecular Phylogenetics and Evolution 66 (2013) 1–16 3

Arbor, MI, USA). Polymorphic positions were coded as ambiguities.To test for multiple ITS copies within individuals (intra-individualITS polymorphism; Álvarez and Wendel, 2003) PCR products of 13

selected accessions belonging to H. sects. Ascyreia, Adenosepalum,Hirtella, Hypericum, Olympia, and Brathys were cloned in thepGEM-T Easy vector (Promega, Madison, WI, USA). Criterion for

Fig. 2. (A–C) (Part A) Phylogenetic tree obtained by Bayesian analysis of ITS sequence data (360 accessions), showing in detail relationships of accessions belonging toHypericum and outgroup representatives from Vismia, Harungana and Cratoxylum. Outgroups (in dark gray), clade names within Hypericum (black) and section names (lightgray) are given. Quotations mark non-monophyletic sections. Numbers above branches indicate posterior probabilities and below bootstrap percentages (ML|MP). Taxonomyfollows Ruhfel et al. (2011) and former species names are included in parentheses. (Part B and C).

Fig. 2. (continued)

selecting samples for cloning was that they exhibited more thanone polymorphic position in the chromatogram files obtained bydirect sequencing. Between five and eight clones per individualwere sequenced with the Templi-Phi DNA Sequencing TemplateAmplification Kit (GE Healthcare Life Science, Chalfont St. Giles,UK). All cloned sequences were carefully examined for mosaicsequence (chimaeric) patterns, which may be the results of recom-bination between different ITS copies after hybridization (Brassacet al., 2012; Koch et al., 2003; Nieto Feliner et al., 2004; Robbaet al., 2005). To identify possible pseudogenes, the highlyconserved 5.8S region was visually scanned for sequences consid-erably differing in variation compared to the entire dataset.Sequences obtained by direct sequencing were deposited in theEMBL nucleotide database under accession numbers HE653396–HE653675, and the cloned sequences under accession numbersHE662695–HE662752 (Appendix A). All data sets and the resultingtrees can be obtained from TreeBASE under study ID 13339 (http://purl.org/phylo/treebase/phylows/study/TB2:S13339).

2.3. Phylogenetic inference

Sequences were aligned initially using the multiple alignmentmode implemented in ClustalX v2 (Larkin et al., 2007; Thompsonet al., 1997) and manually refined. Two data sets were aligned:the first contained only sequences obtained by direct sequencing;the second additionally included these plus the cloned sequences.Bayesian, likelihood and parsimony analyses were performed on adata set containing 360 sequences obtained by direct sequencingrepresenting 200 Hypericum species and six outgroup representa-tives (Appendix A). A likelihood analysis was performed on thedata set additionally including 56 sequences obtained by cloning(Appendix A). Two Cratoxylum species were used to root the trees,following Wurdack and Davis (2009) who showed this genus to bethe sister to the remainder of Hypericaceae. MODELTEST 3.7

Fig. 3. Morphological evolution of Hypericum – distribution of apomorphic characteroptimization of eight characters (Appendix B). In addition, the character ‘petal color’ is plHypericum pink or white flowers also occur). Stem node apomorphies of Hypericeae areplesiomorphic character states of Hypericeae are pale glands, deciduous petals, stamensgynoecium. (For interpretation of this figure in color, the reader is referred to the web v

(Posada and Crandall, 1998) was used to select the appropriatemodel of sequence evolution, and the GTR + I + C model was cho-sen according to the Akaike information criterion (AIC; Akaike,1974). Bayesian analysis was performed using the Metropolis-cou-pled Markov chain Monte Carlo algorithm implemented in MrBa-yes v3.1.2p (Ronquist and Huelsenbeck, 2003), likelihoodanalyses in RAxML v7.2.4 (Stamatakis, 2006) and parsimony anal-yses in PAUP⁄ v4.0b10 (Swofford, 2002). For Bayesian inference (BI)two simultaneous runs each with four chains and starting from arandom starting tree were performed under the GTR + I + C modelfor 14 � 106 generations, setting temperature to 0.1 and samplinga tree every 1000 generations. Likelihood values appeared station-ary after 3.5 � 106 generations, and the first 3500 trees per runwere discarded as burn-in. Posterior probabilities were calculatedon the basis of the remaining 21002 trees. For maximum likelihood(ML) analyses 1000 bootstrap replicates were performed with a ra-pid bootstrapping and a subsequent ML search (RAxML with theGTRCAT model). These settings were also used to run the ML anal-ysis for the data set including the sequences obtained by cloning.The parsimony analyses (MP) followed a two-step heuristic searchapproach modified from Blattner (2004) and described in detail inNürk and Blattner (2010) with the following modifications: thefirst analysis was conducted with 1000 random additions and 20trees held at each step, saving five trees from each repetition. Treesets of different parsimonious scores were then used as startingtrees in multiple second analyses each in a TBR search to findshortest trees that was limited to 50,000 trees. Support was inves-tigated with 100,000 bootstrap replicates (Felsenstein, 1985), usingthe ‘fast and stepwise’ procedure in PAUP⁄.

To test for the significance of topological differences betweenour results and those of Ruhfel et al. (2011) we first used theapproximately unbiased (AU) test (Shimodaira, 2002) as imple-mented in the software CONSEL (Shimodaira and Hasegawa,2001). The test was conducted in a ML framework for the entire

s on the summary tree from analysis of ITS of Hypericaceae, based on Bayesianotted onto the summary tree (note, in Triadenum and some late branching species of

derived by comparison to the outgroup (Vismieae and Cratoxyleae, not displayed);in three fascicles, three staminodes between the stamen fascicles, and a trimerousersion of this article.)

data set (360 accessions) as described above, with only one nodeconstrained to reflect the Ruhfel et al. (2011) topology (i.e. Thorneaand Triadenum, and H. sects. Elodes, Adenotrias, Brathys, Trigynobra-thys, and Myriandra constrained in a clade). Second, we reducedour dataset to a sampling similar to that used in Ruhfel et al.(2011); (i.e. 21 representative placeholder taxa and sequencesnewly aligned) and analyzed the data with BI and ML (results notshown). Visualization of results was conducted using FigTreev1.3.1 (Rambaut, 2006–2009).

2.4. Character reconstruction

We analyzed character state changes for eight selected charac-ters (Appendix B) over a posterior distribution of 1000 trees usingreversible-jump Markov chain Monte Carlo (MCMC) methods(Pagel, 1999) in the program BayesMultiState (Pagel et al., 2004)as implemented in the software package BayesTraits (http://www.evolution.reading.ac.uk/BayesTraits.html), to account forphylogenetic uncertainties. Bayesian reconstruction of ancestralcharacter states was calculated for well-supported nodes in the tree(i.e. the backbone nodes, Fig. 3) using the default settings over aposterior distribution of possible tree topologies, represented by1000 randomly sampled trees (after discarding 25% as burn-in) asobtained by the MrBayes analysis. A bootstrap analysis of 10,000replicates was performed on the p values obtained by BayesTraitsusing a Python script. Visualization of results was done in R (R Devel-opment Core Team, 2011) with density plots and pie charts (resultsshown only for analysis of growth forms to illustrate the method,

Table 1Classification of sections of Hypericum (Robson, 1977–2010), results of Nürk and Blattner[acces.] for ITS) and support of morphological and molecular analyses. The phylogenetic staaccession in ITS), p = non-monophyletic.

Classification (sensu Robson) Morphology ITS seq

Incl. spec. Support [BI|MP] Include

1. Campylosporus (Spach) R.Keller 10 0.58|– 3/92. Psorophytum (Spach) Nyman 1 1/33. Ascyreia Choisy 43 – 29/584. Takasagoya (Y.Kimura) N.Robson 5 – 1/15. Androsaemum (Duhamel) Gordon 4 0.99|51 5/126. Inodora Stef. 1 1/36a. Umbraculoides N.Robson 1 07. Roscyna (Spach) R.Keller 2 1|57 2/48. Bupleuroides Stef. 1 1/19. Hypericum 42 – 23/509a. Concinna N.Robson 1 1/19b. Graveolentia N.Robson 9 – 6/89c. Sampsonia N.Robson 2 1|57 1/29d. Elodeoida N.Robson 5 – 3/69e. Monanthema N.Robson 7 – 1/110. Olympia (Spach) Nyman 4 0.71|– 2/911. Campylopus Boiss. 1 1/212. Origanifolia Stef. 13 1|– 4/413. Drosocarpium Spach 11 0.69|– 7/1214. Oligostema (Boiss.) Stef. 6 – 2/615. Thasia Boiss. 1 016. Crossophyllum Spach 3 0.99|– 2/417. Hirtella Stef. 30 – 7/1018. Taeniocarpium Jaub. & Spach 28 – 10/1519. Coridium Spach 6 1|– 3/720. Myriandra (Spach) R.Keller 29 1|56 27/2821. Webbia (Spach) R.Keller 1 1/422. Arthrophyllum Jaub. & Spach 5 – 3/423. Triadenioides Jaub. & Spach 5 – 1/324. Heterophylla N.Robson 1 1/125. Adenotrias (Jaub. & Spach) R.Keller 3 1|96 1/426. Humifusoideum R.Keller 12 – 1/127. Adenosepalum Spach 25 – 11/2228. Elodes (Adans.) W.Koch 1 1/329. Brathys (Mutis ex L.F.) Choisy 87 – 16/2830. Trigynobrathys (Y.Kimura) N.Robson 52 – 11/15

Fig. 1). Character states reconstructed for ancestral nodes wereinterpreted to represent apomorphic traits for major clades (Fig. 3).

3. Results

The final data set included 360 ITS rDNA sequences obtained bydirect sequencing. After introducing gaps, the alignment com-prised 771 characters, of which 483 were variable and 404 werepotentially parsimony informative. No additional gap was neces-sary to include in the alignment the 56 sequences (13 species) ob-tained by cloning (Appendix A). Multiple ITS copies were found insome individuals. For example, six ITS copies were found in sevenclones of one individual of H. reflexum, and these differed by up to10 nucleotide positions. Patterns consistent with hybridization be-tween closely related species were also detected (Brassac et al.,2012), e.g., different ITS copies produced by cloning the PCRproduct from one accession grouped with different (sub-) clades(e.g., H. reflexum HE662741–HE662747; Fig. S1, Supplementaryonline material). In all cases however, cloned sequences derivedfrom one individual fell in one of the major clades defined(Fig. 1) and were resolved in a position consistent with the sec-tional placement of the species (Fig. S1). No chimaeric ITS typesconsistent with hybridization between the major clades were iden-tified. Also, we found no evidence for pseudogenes; the 5.8S genewas equally conserved across all accessions and clones sequences.

Bayesian inference (BI), maximum likelihood (ML), and maxi-mum parsimony (MP) analyses of directly sequenced ITS resultedin trees with an identical topology for the main clades and only

(2010) and results found in this study: number of included species (and accessionstus of sections is given by m = monophyletic, mt = monotypic (single species or single

uence data Phylogenetic status

d spec./acces. Support [BI|ML|MP] % Spec. sampled Morph ITS

1|99|84 30 m m1|100|99 100 mt m– 67 p p

20 p mt1|89|87 125 m m1|100|100 100 mt m

0 mt –– 100 m p

100 mt mt– 54 p p

100 mt mt– 67 p p1|100|100 50 m m– 60 m p

14 p mt1|95|96 50 m m1|100|100 100 mt m1|92|75 31 m m– 64 m p0.78| 51|59 33 p m

0 mt –1|91|89 67 m m– 23 p p– 36 p p0.63|47|– 50 m m1|72|63 93 m m1|100|99 100 mt m1|100|100 60 p m1|100|99 20 m m

100 mt mt1|100|100 33 m m

8 p mt– 44 p p1|100|100 100 mt m– 18 p p– 21 p p

minor differences at the terminals. The most parsimonious trees hada length of 2342 steps, a consistency index of 0.39, and a retentionindex of 0.92. One polytomy occurring only in the BI tree (H. sects.Adenotrias and Elodes, see below; Fig. 1) was resolved in the ML tree(with H. sects. Adenotrias and Elodes in a sister group relationshipand together as sister to the remainder; not shown) and the MPconsensus tree (with H. sect. Adenotrias as sister to the remainder;not shown), but both without bootstrap percentages P50.

Each of the three tribes of Hypericaceae – Cratoxyleae, Vismieaeand Hypericeae – is well supported (Figs. 1 and 2). Within Hyperi-ceae, a major division is evident, dividing the taxon into: (A) agrade comprising Thornea (1.00 Bayesian posterior probability(pp), 100 bootstrap support (bp) for ML, 100 bp for MP), H. sects.Adenotrias and Elodes, and a clade (1.00|93|54) that includes Triade-num as sister to the New World H. sects. Myriandra + Brathys + Trig-ynobrathys (1.00|100|97), and (B) the remainder of Hypericum in aclade (1.00|100|68). The alternative topology of Ruhfel et al.(2011), i.e. our grade A constrained as sister clade to B was not re-jected with significance by the AU test (p = 0.05).

Within A, the (mainly) New World sections of Hypericum areplaced in a clade (1.00|100|97), labeled Brathys s.l. + Myriandra,with H. sect. Myriandra (1.00|72|63) as sister to H. sects. Bra-thys + Trigynobrathys (1.00|100|100). Most accessions of H. sect.Trigynobrathys are resolved in a clade (0.99|94|85) sister to allaccessions of H. sect. Brathys plus four species of H. sect. Trigyno-brathys (1.00|100|100). The two Old World sections of Hypericumbelonging to A (H. sects. Adenotrias and Elodes) are resolved in apolytomy in the BI tree (here called Mediterranean I), branchingfrom the rest subsequent to Thornea as sister to the remainder ofHypericum (0.99|77|– [not resolved in MP]).

Group B comprises the mainly Old World sections of Hypericum(sections 1–19 and 21–24 and 26, 27; section numbers refer to Ta-ble 1). A weakly supported grade, Mediterranean II and Arthrophyl-lum, comprising the Mediterranean H. sects. Arthrophyllum,Androsaemum, Inodora, Bupleuroides, Webbia, and H. sampsonii(monotypic H. sect. Sampsonia from China). The two clades formingthis grade each received moderate to strong support (Fig. 1), butweak to moderate support for their backbone nodes (0.69| 50|–;0.95|90|–). A clade of Ascyreia s.l. + Campylosporus (1.00|100|86)contains two subclades, one that includes Afrotropical species fromH. sect. Campylosporus (1.00|99|84) and a second that contains Med-iterranean H. sect. Psorophytum together with Asian H. sects. Ascyreia,Takasagoya and Roscyna (1.00|80|50). The largest clade, core Hyperi-cum (1.00|100|99), contains the type species H. perforatum andcomprises taxa of sections 9, 9a, 9b, 9d, 9e, 10–19, 23–24, 26, and 27.

Within the named clades and grades identified in Figs. 1 and 2,internal relationships between clades and species are often re-solved, but sometimes not well supported (Fig. 2A–C). We do notdiscuss all these terminals (species relationships) in detail. Ex-tended taxon sampling and more data/DNA regions would be nec-essary to arrive at sound phylogenetic hypotheses for the specieswithin these clades. Implications for sectional classification arediscussed only in those cases in which ITS provides clear evidencefor the status of sections, i.e. for their monophyly or otherwise(summarized in Table 1).

Reconstruction of ancestral states is aimed to identify apomor-phic traits for well-supported nodes found by the ITS analysis.Although morphology is highly plastic and diverse within a genussuch as Hypericum containing almost 500 species, it is possible to de-fine characters supporting major clades using reversible MCMC opti-mization (summarized in Fig. 3). As evident in Section 4.1 anddiscussed in Section 4.2 of this study, recognition of homoplasy forcertain characters can be challenging and has to be based on anunderlying explicit phylogenetic hypothesis. As an example and toillustrate the method, reconstruction of ‘habit’ is shown in Fig. 1.

4. Discussion

4.1. Phylogenetics results for Hypericum

Although we found multiple nrITS copies within some individ-uals, no obvious chimaeric sequences could be identified thatwould introduce conflicting phylogenetic signals for larger clades.The multiple ITS copies found by cloning consistently groupedwithin the major clades, and thus we did not detect any evidencefor reticulate evolution between these clades (Fig. S1). Moreimportant for the purpose of this study is the possibility of paralo-gous ITS sequences or pseudogenes that would also confound phy-logenetic reconstruction (Álvarez and Wendel, 2003). No evidence,however, was detected in our data set that would hint at existenceof paralogous loci. Preliminary analyses of the petD region from theplastid genome (Borsch et al., unpublished data) revealed the samemajor groupings as found with the ITS data.

Relationships of tribes as revealed by the ITS analysis, with Crat-oxylum (Cratoxyleae) as outgroup (Wurdack and Davis, 2009) fol-lowed by Vismia and Harungana (Vismieae) as sister to Hypericum(Hypericeae), are generally in accordance with the recent classifi-cation of Hypericaceae (Stevens, 2007).

Within Hypericeae, the grade labeled A includes a clade here la-beled Thornea as sister to the remainder of Hypericum, followed bya grade (or a clade) Mediterranean I, followed by a clade containingTriadenum and Brathys s.l. + Myriandra as sister to the reminder ofHypericum. Although each of these clades obtained maximum sup-port in all three analyses (BI, ML, MP), relationships between cladeswere highly supported only in BI (P0.99 pp), more weakly in ML(77 and 93, respectively, see Fig. 2A), and P50 in MP. Clade B con-tains all remaining species of Hypericum and consists of anothergrade and two larger clades. The grade labeled as MediterraneanII and Arthrophyllum consists of H. sect. Arthrophyllum, followedby a clade containing H. sects. Inodora and Webbia, Bupleuroides,Androsaemum, and Sampsonia. Support is high to moderate forthe two clades, but low for relationships between them and theremainder of B (Fig. 2B).

Inclusion of Triadenum in Hypericum is in accord with a recentlypublished four-marker analysis (Ruhfel et al., 2011). However, taxarepresenting grade A (additionally including Santomasia) werepresent in the Ruhfel et al. (2011) analysis as a strongly supportedsister to B. The AU test provides weak statistical support for our MLtotal evidence topology by not rejecting the constrained topology(i.e. the topology suggested by Ruhfel et al. (2011), with Thornea,Mediterranean I, Triadenum and Brathys s.l. + Myriandra as sisterto B; p = 0.05). Analysis of a reduced data set with sampling com-parable to Ruhfel et al. (2011) was congruent with that of the en-tire dataset (Fig. 1), with the exception Thornea and MediterraneanI as unresolved in BI (results not shown). Thus, we assume that thesparse sampling of Hypericeae in Ruhfel et al. (2011) is partly con-tributing to the topological differences (although, according to ourtests sampling is not alone a sufficient reason). It is notable thatmorphology supports the position of Thornea as sister to Hypericum(Fig. 3). However, further molecular investigations with more dataare needed to clarify the exact topology in this group (i.e. whetherthey form a clade or a grade with Thornea as sister to the remainderof Hypericum). In particular, low-copy markers from the nucleargenome may be useful and reveal reticulation (Bänfer et al.,2006; Rieseberg and Soltis, 1991) or incomplete lineage sorting(Jakob and Blattner, 2006), which may explain the topologicaldifferences noted here.

The inclusion of Santomasia in this group (our grade A) byRuhfel et al. (2011) is ambiguous from a morphological perspec-tive. According to Robson’s observations (pers. comm.), Santomasiais vegetatively most similar to H. roeperianum, which belongs to

H. sect. Campylosporus (placed by ITS in Ascyreia s.l. + Campylospo-rus; Fig. 1). The cladistic analysis of morphological characters thatconsidered almost all described species of Hypericum (Nürk andBlattner, 2010) revealed Santomasia in close relation to H. sect. As-cyreia. The pentamerous gynoecium of Santomasia and Ascyreias.l. + Campylosporus might be a synapomorphy. Ruhfel et al.(2011) argues that occurrence of staminodes (i.e. vestigial fascicles,also called fasciclodes) supports a relationship of Santomasia toThornea + Mediterranean I + Triadenum + Brathys s.l. + Myriandra.However, this implies independent evolution of a pentamerousandroecium and gynoecium in Ascyreia s.l. + Campylosporus andSantomasia, the latter in a clade containing exclusively species witha trimerous gynoecium (with three staminodes in Thornea, Medi-terranean I, and Triadenum). Hence, the phylogenetic placementof this taxon needs to be investigated further. In general, presenceof staminodes in Hypericum co-occurs with ‘pseudo-tubular’ corol-las, which have been hypothesized as modifications for more spe-cialized insect pollination (Robson, 1981, p. 302). With respect tothe ITS results and, from a functional viewpoint, stellate flowerswith ‘unspecialized’ pollination are typical for the clades of Hyper-icum with much higher species-richness (14 species with stamin-odes vs >480 without). Furthermore, molecular analysesincluding Lianthus are necessary to evaluate the close relationshipbetween Triadenum and Lianthus as revealed by Nürk and Blattner(2010) and by Robson (2001), who suggested a close relationshipbetween Lianthus, Triadenum and Thornea.

Our phylogenetic tree (Fig. 1) is highly congruent with Crockettet al. (2004), but differs in several respects from Park and Kim(2004). Crockett et al. (2004), using Clusia rosea as outgroup, recov-ered the same major clades as presented here, i.e. Brathys s.l. + Myr-iandra, Ascyreia s.l. + Campylosporus and core Hypericum labeled asC, B, and A, respectively. The split of Hypericum into two mainclades in Crockett et al. (2004) as (C(AB)), is reflected in our gradeA and clade B. Clade C of Crockett et al. (2004), however, consistedof taxa belonging to H. sect. Myriandra only. The MP tree presentedin Park and Kim (2004) used the two Thornea species as outgroups,but is topologically different regarding the placement of accessionsclassified as H. sect. Hypericum (grouping together with speciesfrom H. sect. Trigynobrathys) and H. sect. Adenotrias (with speciesfrom H. sect. Roscyna). To test whether incorrect species determi-nation could have contributed to these differences, we sequencedITS for additional individuals of these species, each determinedby N. Robson (Nat. Hist. Museum, London). We found that the new-ly sequenced individuals of such ‘misplaced’ taxa grouped accord-ing to their sectional affiliation. For this reason, we excluded fromour analysis several sequences of Park and Kim (2004) and othersimilarly obvious problematic sequences available in GenBank.

A general morphological characterization of the describedclades/grades is possible, but it should be kept in mind that excep-tions exist for several characters, especially within core Hypericumand the large taxa from the New World, namely H. sect. Myriandra,Brathys and Trigynobrathys.

The two species belonging to Thornea are shrubs possessingpale glands only (dark glands absent). The corolla is pseudo-tubu-lar (i.e. unfused petals that do not open completely at anthesis).Petals are deciduous, and pink or white. Stamens are persistent,with filaments basally united in three fascicles. Between the fasci-cles three staminodes are present. The gynoecium is trimerouswith axile placentation.

Mediterranean I (H. sect. 25 and 28) contains shrubs (H. sect.Adenotrias) and herbs (H. sect. Elodes). Pale glands are present,but dark glands are present only in H. elodes at the connective ofthe anthers and sepals (described as red glands). The flower is het-erostylous in H. sect. Adenotrias. The corolla is pseudo-tubular, andpetals are persistent and yellow. Stamens are persistent in threefascicles. Filaments are united below the middle. Three staminodes

are present between fascicles. The gynoecium is trimerous, withparietal placentation in H. sect. Elodes and axial in H. sect.Adenotrias.

Triadenum species are perennial rhizomatous herbs that occurin ± aquatic habitats such as swamps and lake margins. Pale glandsare present only. The corolla is pseudo-tubular to stellate. Petalsare and pink to purple or yellowish and are deciduous after anthe-sis (i.e. the corolla does not fall off immediately, but only as thecapsule grows). Stamens are deciduous, and in three fascicles. Fil-aments are united, and three staminodes between the fascicles arepresent. The gynoecium is trimerous with axial placentation.

Brathys s.l. + Myriandra (H. sect. 20, 29 and 30) contains shrubsand herbs and a few annuals. Only pale glands are present. Thecorolla is stellate. Petals are yellow in both groups, but persistentin H. sect. Brathys and Trigynobrathys and deciduous in H. sect.Myriandra. Stamens are mostly persistent. They are arranged in abroad ring in H. sect. Myriandra and in a narrow ring or withmodifications/reductions in H. sects. Brathys and Trigynobrathys.The gynoecium is trimerous, and with parietal placentation(incompletely axile in some H. sect. Myriandra species).

Mediterranean II and Arthrophyllum (H. sect. 5, 6, 8, 9c, 21 and22) is typically characterized by a shrubby habit, but there aresome herbs. Pale glands are present with dark glands also presentin H. sects. Arthrophyllum, Sampsonia, Inodora and Bupleuroides (butonly in reproductive parts in the last two). The corolla is stellate,and petals are yellow and persistent in all members of the groupexcept H. sect. Androsaemum where they are deciduous. Stamensare similarly persistent, except in H. sect. Androsaemum where theyare deciduous. The stamens are arranged in three fascicles (five inH. sect. Androsaemum). The gynoecium is trimerous with incom-pletely axile placentation (but fully axile in H. sect. Bupleuroides).

The mainly Indomalesian Ascyreia s.l. + Campylosporus clade(H. sect. 1, 2, 3, 4 and 7) includes tropical African H. sect. Campy-losporus as sister to H. sects. Psorophytum, Ascyreia, Takasagoyaand Roscyna. It contains mostly shrubs, but trees occur in H. sect.Campylosporus. Pale glands are present, and there are dark glandsin H. sect. Campylosporus. The corolla is stellate. Petals are yellow,sometimes tinged red and they are typically deciduous, but persis-tent in H. sect. Roscyna and in some species of H. sect. Campylosp-orus, where they are described as ‘‘tardily deciduous’’. Stamens aregenerally deciduous, but persistent in H. sect. Roscyna and tardilydeciduous in some species of H. sect. Campylosporus. They are ar-ranged in five fascicles. The gynoecium is pentamerous withincompletely axile placentation.

The mainly Palaearctic crown clade, core Hypericum (H. sect. 9,9b, 9d, 9e, 10–19, 23, 24, 26 and 27) is well supported but highlyunresolved within. This clade contains most of the sections de-scribed in Hypericum and has the greatest species-richness(Fig. 2C). It consists of herbs and (secondarily) some dwarf shrubs.Both pale and dark glands are present although the latter are ab-sent from three species (H. heterophyllum, H. taygeteum, and some-times in H. saxifragum) and are found only in reproductive parts inH. sect. Coridium and in some species of H. sects. Hirtella and Tae-niocarpium. The corolla is stellate. Petals are persistent (except insome species of H. sect. Coridium) and yellow. Stamens are persis-tent and arranged in three fascicles, but in a narrow ring in H. sect.Humifusoideum. The gynoecium is trimerous with axile placenta-tion (incompletely axile in H. sects. Adenosepalum, Humifusoideum,Triadenioides).

4.2. Character evolution

Morphological support for major groupings within Hypericumindicated by analysis of ITS is limited although some apomorphiccharacters can be identified (Fig. 3). Only character state changesclearly evident in the data are indicated as apomorphic in Fig. 3.

In the following, we selected two characters to discuss: dark/paleglands, and habits. The uncertainties in the backbone topology(see Section 4.1. Phylogeny of Hypericum) do not bear much onthe interpretation of the evolution of these characters, and our con-clusions (e.g., habit evolution, see below) are not affected.

Two types of glandular tissue occur in Hypericum: (i) multicel-lular black or red nodules containing hypericin, generally termeddark glands (Curtis and Lersten, 1990; Karppinen et al., 2008)and (ii) translucent cavities in all species of Hypericum containinghyperforin and essential oils, termed pale glands. Pale and darkglands are biochemically, and to a certain extent also anatomically,different and, thus, may not be homologous structures. However,Curtis and Lersten (1990) reported ‘chimerical’ canals (elongatedglands) changing color abruptly at some point from black to trans-lucent. They concluded that dark nodules are a modification ofcommon (translucent) secretory reservoirs. Dark glands have alsobeen reported from Cratoxylum, Harungana, and Vismia. Presenceof hypericin, however, has only been confirmed in Hypericum(Crockett and Robson, 2011). Comparative studies focusing onontogeny and biochemical constitution (also in other genera ofHypericaceae), which also complement phylogenetic results, areneeded to clarify homology of these secretory structures. WithinHypericeae, dark glands do not occur in species belonging to A,with the exception of H. elodes (H. sect. Elodes) with red glandson sepals and black glands on anther connectives. According tothe phylogenetic hypothesis presented here, dark glands in repro-ductive organs (anthers) and therefore presence of hypericin arealso apomorphic for Hypericum species of clade B (but were lostagain in H. sects. Androsaemum, Ascyreia, Heterophylla, Takasagoya,and Webbia). Dark glands in vegetative organs (stem and leaves)evolved later and are apomorphic for H. sect. Arthrophyllum,Campylosporus and core Hypericum (except H. sects. Coridium andTaeniocarpium p.p.). In core Hypericum, dark glands in vegetativeorgans are typical of a clade that underwent a rapid radiation asindicated by short branch lengths in ITS. It has been suggested thathypericin plays a major role in response to herbivore attack(Sirvent et al., 2003), and it might be a key innovation for coreHypericum, triggering rapid diversification. The puzzling patternof pale with dark glands, however, exemplifies the problem ofhomology assessment in species belonging to such a large taxonas Hypericum.

Reconstruction of habit change revealed a shrubby ancestor forHypericum from which herbs evolved several times within thegroup (see Fig. 1), the latter habit being characteristic of coreHypericum. Annuals evolved only in H. sects. Brathys and Trigyno-brathys, most probably independently (e.g., H. gentianoides,H. japonicum). Trees with a single trunk over 10 m in height arereported for some tropical African species of Hypericum, namelyH. bequaertii endemic to the Rwenzori Mountains and H. revolutumfrom the Ethiopian Highlands. Such trees have been observed,however, in disturbed and open habitats. In natural habitats theyare usually tall but bushy or slender shrubs (Robson, 1993). Evolu-tion of such tall erect shrubs, which have been called tree-like bysome authors, is evidently connected to tropical montane habitats.Within Hypericum, sclerophyllous arborescent shrubs are also na-tive to the páramos of Andean South America (H. sect. Brathys)and broad-leafed arborescent shrubs to mountains of New Guinea(H. sect. Humifusoideum). The elevational range, c. 1600–4500 m, ofthese species is similar in South America, Africa and New Guinea,and so are the habitats, from the montane forest belt to shrublandsand alpine grasslands. This phenomenon may result from parallelevolution as a response to the extreme climatic conditions inthese tropical montane habitats. At least in the species belongingto H. sects. Brathys (South America) and Campylosporus (Africa),such habits have evolved in parallel. It is worthwhile pointingout the (probably) recent radiation that occurred in Andean South

America: c. 65 species and 16 subspecies are native to the páramo,a habitat that originated with the final uplift of the Andes, no ear-lier than 5 million years ago, and these belong mainly to H. sect.Brathys. In contrast, only 17 species are reported from tropicalmontane regions of Africa (belonging to H. sects. Campylosporus 8sp., Adenosepalum 4 sp., and Trigynobrathys 5 sp.).

4.3. Phylogenetics and classification

One outcome of phylogenetic research is to provide a basis forrevising existing classifications. The sectional classification inHypericum (Robson, 1977, 1981, 1987, 1990, 2001, 2010b) is basedon the ‘‘recognition and correlation of morpho-geographicaltrends’’ (Robson, 2010a) to identify sister-taxon relationships. Itis part of a monograph of Hypericum that presents a remarkable re-source for research on the genus (for a numerical list of all studies,see Carine and Christenhusz, 2010). This classification in principleis an evolutionary one as it incorporates the concept of ‘‘plesiospe-cies’’ and a priori character polarity (Robson, 2006).

Of 36 sections recognized in Robson’s classification (Table 1), 10are monotypic. Inclusion of multiple species for 20 of the remain-ing 26 sections allowed us to evaluate their monophyly. Mono-phyly of nine sections was supported (1, 5, 10, 12, 14, 16, 19, 20,22; Table 1). Of the remainder, six sections (9, 9b, 13, 17, 27, 29)were resolved as non-monophyletic but without support. Non-monophyly of section 30 is strongly supported, but sparse sam-pling in these groups mean that relationships of sections 30 and29 remain unclear with paraphyly of section 30 a possibility. Foursections (3, 7, 9d and 18) are clearly polyphyletic. The sectionalclassification developed by Robson (1977 onwards) works on apractical basis, and we believe that many researchers will use italso in future. Nevertheless, a re-evaluation of the classificationin light of our results and, in particular, status of polyphyletic sec-tions identified (Table 1) is appropriate. A summary of cladistic re-sults for morphological characters (Nürk and Blattner, 2010) andITS sequence analysis is given in Table 1. This can be used in futurestudies as a basis to select groups of interest, i.e. sections in whichspecies sampling is still too sparse to infer insights into their evo-lutionary history.

Although incongruence between sectional circumscriptions ofRobson and clades identified in this study is limited, relationshipsbetween sections differ markedly from those proposed by Robson(2003: Fig. 1.16). This revised hypothesis of phylogenetic relation-ships between larger parts within the genus (i.e. the clades/gradesdescribed above) bears on interpretation of biogeographical pat-terns and ancestral character state reconstructions. Sectionsbelonging to our Ascyreia s.l. + Campylosporus have been hypothe-sized to include ‘basal’ species (Robson, 1985). However, accordingto character state reconstructions based on ITS sequence phyloge-netic inference, all Ascyreia s.l. + Campylosporus species havecharacter states that are apomorphic within the genus, e.g., a5-numerous gynoecium (Fig. 3), which is due to the derived posi-tion of these taxa (Fig. 2B). Thus, based on morphological andnrDNA sequence phylogenetic inference H. sect. Campylosporusdoes not appear to be morphologically primitive as hypothesizedby Robson (2003).

The split of Hypericum into (mainly) New and (mainly) OldWorld clades (Brathys s.l. + Myriandra vs. clade B), as well as the po-sition of species almost exclusively distributed in the Mediterra-nean on initial splits within the ITS tree has implications forbiogeography of Hypericum. For Old World taxa (Mediterranean Iand clade B), the western Asian–pan Mediterranean region seemsto be important for diversification and dispersal (which could bereferred to as the ‘Tethys hypothesis’ for Old World Hypericum).The New World taxa belonging to A (species placed in Thornea,Triadenum, and Brathys s.l. + Myriandra) have an independent

biogeographic history as might be expected from their phyloge-netic position. More detailed conclusions, however, demand bio-geographic and molecular clock reconstructions, which are inprogress.

5. Conclusions

According to our ITS results, (1) Thornea (two species) is sisterto the remainder of Hypericum, and Triadenum (six species) isembedded within Hypericum. Although Thornea is not embeddedwithin Hypericum in our ITS analysis, we follow here for practicalreasons the Ruhfel et al. (2011) taxonomy, who proposed that thisgenus (together with Triadenum, Santomasia and Lianthus) be in-cluded in Hypericum. (2) Occurrence of three staminodes in Thor-nea, H. sects. Elodes and Adenotrias and Triadenum supports theirpositions at basal nodes if these are assumed to be plesiomorphicfor Hypericeae (five staminodes are present in Vismieae and threein Cratoxyleae, both outgroups). (3) Dark glands evolved severaltimes in the Old World, first in reproductive parts only and lateralso in vegetative parts. (4) All Hypericum species (incl. Triadenum)in the New World (and some Asian species of this group) and notpossessing dark glands form a clade. (5) Hypericum had a shrubbyancestor, and herbs evolved later in the genus. (6) Evolution ofarborescent shrubs is related to colonization of or speciation intropical montane habitats. In the New World it resulted in a burstof species in Andean páramos, a habitat not older than �5 millionyears. (7) The crown clade, core Hypericum, is characterized by aperennial habit and occurrence of dark glands in vegetative parts.(8) According to the phylogenetic hypothesis presented here, apo-mictic species alone do not form a clade. Thus, we speculate thatapomixis has evolved several times independently within Hyperi-cum (Fig. 1): conceivably once in Triadenum, once in the Ascyreias.l. clade, and independently probably twice in core Hypericum.

Acknowledgments

We are deeply indebted to Norman Robson for invaluable helpwith species determination and advice. We thank the herbaria AN-DES, B, BM, GAT, GH, HEID, K, KYO, TI, WAK for allowing us to usetheir collections. Special thanks are due to Andrea Sánches Mese-guer for offering the ITS sequence of H. geminiflorum and to BeritGehrke for the picture of H. revolutum used in the graphical ab-stract. We are grateful to the comments by Maia Gurushidze, SaraCrockett, Norman Robson, and Jonathan Brassac and two anony-mous reviewers, who have considerably contributed to improvingthis manuscript. Collection of Japanese Hypericum species was car-ried out with support from the Gemeinschaft zur Förderung derKulturpflanzenforschung Gatersleben e.V., grant to NMN, collec-tion of páramo species with support from the Facultad de Ciencias,Universidad de los Andes, Bogotá, seed grant to SM. DNA extractionand sequencing was performed by SM at Royal Botanic Gardens,Kew, with support from the Kew Latin American Research Fellow-ship Programme, and by NMN at IPK Gatersleben with supportfrom the Deutsche Forschungsgemeinschaft (DFG Grant BL 462/8).

Appendix A. List of taxa included in the study of Hypericum

Information is given in the following order: Species, Voucherspecimen or reference, GenBank accession. Species names forHypericeae follow Ruhfel et al. (2011).

Cratoxyleae: Cratoxylum formosum (Jack) Benth. & Hook.f. exDyer subsp. formosum, Larson Larson 33255 (B), HE653674;C. pruniflorum Dyer, Larson Larson Nielsen Santisuk 32141 (B),HE653675; Hypericeae: Hypericum acmosepalum N.Robson,N.M. Nürk 402 (GAT), HE653397; H. acmosepalum N.Robson,

Sino-British Expedition to Cangshan (SBEC) K052 (BM),HE653398; H. acmosepalum N.Robson, N.M. Nürk 401 (GAT),HE653396; H. addingtonii N.Robson, N.M. Nürk 348 (GAT),HE653399; H. adenotrichum Spach, Cultivated 1981-35 (Kew-Wakehurst), HE653400; H. adpressum W.P.C.Barton, AY555865.2;H. aegypticum L. subsp. maroccanum (Pau) N.Robson, S.L. Juryand B. Tahiri T.M. Upson 14264 (BM), HE653401; H. aegypticumL. subsp. marrocanum (Pau) N.Robson, Cultivated 1978-4468(Kew Wakehurst), HE653402; H. aegypticum L. subsp. webbii(Spach) N.Robson, Turland 111 (BM), HE653403; H. aegypticumL. subsp. webbii (Spach) N.Robson, E. Stamatiadou 12008 (BM),HE653404; H. aethiopicum Thunb. subsp. sonderi (Bredell) N.Rob-son, D.S. Pigott s.n. 8.11.98 (BM), HE653405; H. androsaemum L.,C. Scheriau HEID-808382 (HEID), HE653406; H. androsaemum L.,Cultivated Bed 256G (Kew-Wakehurst), HE653407; H. androsae-mum L., Cultivated 1969-31240 (Kew-Wakehurst), HE653408;H. apocynifolium Small, AY555883.2; H. asahinae Makino,AY572997; H. ascyron L., S. Fujii 11937 (KYO), HE653409; H. ascy-ron L., Wan Chow 81093 (BM), HE653410; H. ascyron L. subsp.ascyron, Hort. Bot. Acad. Sci St 1580/63/64 (GAT), HE653411;H. ascyron L. subsp. longistylum Maxim., AY573015; H. athoumBoiss. & Orph., C. Scheriau HEID-808390 (HEID), HE653412;H. athoum Boiss. & Orph., C. Scheriau HEID-801636 (HEID),HE653413; H. athoum Boiss. & Orph., Bot. Gard. Berlin-Dahlems.n. EPG 6/2001 (GAT), HE653414; H. atomarium Boiss., E. Stama-tiadou 9106 (BM), HE653416; H. atomarium Boiss., Bot. Gard.Potsdam s.n. 385/57/60 (GAT), HE653415; H. attenuatum Fisch.ex Choisy, AY572993; H. attenuatum Fisch. ex Choisy, F. BlattnerFB2010-029b (GAT), HE662751 H. attenuatum Fisch. ex Choisy,F. Blattner FB2010-032a (GAT), HE662752 H. attenuatum Fisch.ex Choisy var. confertissimum (Nakai) T.B.Lee, AY572995;H. aviculariifolium Jaub. & Spach, Ulrich s.n. (BM), HE653417;H. balearicum L., J.F.M. M.J. Cannon 3780 (BM), HE653418;H. balearicum L., AY555862; H. balearicum L., Cultivated 2002-181 (Kew-Wakehurst), HE653419; H. barbatum Jacq., N.M. Nürk410 (GAT), HE653420; H. beanii N.Robson, Alpine Garden SocietyExpedition to China 1994 (ACE) ACE 32 (BM), HE653421; H. beaniiN.Robson, Cultivated 452-81/05839 (Kew-Wakehurst), HE653422;H. beanii N.Robson, Cultivated 1996-744 (Kew-Wakehurst),HE653423; H. bellum H.L.Li, D.E. Boufford S.L Kelly R.H. Ree S.K.Wu 29827 (BM), HE653424; H. bellum H.L.Li subsp. latisepalumN.Robson, Sino-British Expedition to Cangshan (SBEC) 0424 (BM),HE653425; H. bellum H.L.Li subsp. latisepalum N.Robson, N.K.B.Robson s.n. 15.8.1995 (BM), HE653426; H. bithynicum Boiss.,N.M. Nürk 398 (GAT), HE653427; H. bithynicum Boiss., CultivatedNKB Robson (), HE653428; H. brachyphyllum (Spach) Steud.,AY555870.2; H. brevistylum Choisy, AY573019; H. buckleyiM.A.Curtis, AY555880.2; H. bupleuroides Griseb., A. Groger W. Lo-bin 113-3 (M), HE653429; H. calcicola Standl. & Steyerm.,AY573028; H. calycinum L., Bot. Gard. Frankfurt/Main s.n. EPG 7/2001 (GAT), HE653431; H. calycinum L., Cultivated 1969-16045(Kew-Wakehurst), HE653432; H. calycinum L., D. McClintock s.n.1993 (BM), HE653430; H. calycinum L., FJ694194; H. canadenseL., S. Crockett 19 (UGA), HE653433; H. canariense L., Cultivated2000-0075 (Jardin Botanico Barcelona), HE653434; H. canarienseL., FJ694195; H. canariense L. var. canariense, R. Davis 10261(BM), HE653435; H. canariense L. var. flori (Aiton) Bornm., N.M.Nürk 386 (GAT), HE653436; H. cerastioides (Spach) N.Robson,AY555884; H. cerastioides (Spach) N.Robson, voucher data notavailable, HE653437; H. chapmanii W.P.Adams, AY555869.2;H. chejuense S.-J. Park & K.-J. Kim, AY572996; H. choisyanum Wall.ex N.Robson, Ikeda et al. 20913019 (TI), HE653441; H. choisyanumWall. ex N.Robson, AY555856; H. cistifolium Lam., AY555881.2;H. concinnum Benth., D. Potter P. Lash 110612-01 (GAT),HE653442; H. confertum Choisy, Himmetoglu H22 (BM),HE653443; H. conjungens N.Robson, L.B. Mwasumbi 16191A

(BM), HE653444; H. coris L., S.L. Jury M.F. Watson D.A. Webb M.B.Wyse Jackson 6415 (BM), HE653445; H. coris L., R.E. Longton 4436(BM), HE653446; H. coris L., FJ694196; H. crux-andreae (L.) Crantz,AY555874.2; H. curvisepalum N.Robson, Cultivated 1993-3327(Kew-Wakehurst), HE653447; H. davisii N.Robson, A. Cubukcus.n. (1978) (BM), HE653448; H. decandrum Turcz., M. WeigendG. Brokamp 9102 (B), HE653449; H. delphicum Boiss. & Heldr., C.Scheriau HEID-808391 (HEID), HE653450; H. delphicum Boiss. &Heldr., C. Scheriau HEID-808395 (HEID), HE653451; H. delphicumBoiss. & Heldr., Cultivated 000-69.19158 (Kew-Wakehurst),HE653452; H. densiflorum Pursh, AY555886; H. denticulatumWalter subsp. acutifolium (Elliott) N.Robson, Kral 48272 (BM),HE653453; H. dogonbadanicum Assadi, Assadi Aboohamzeh38585 (BM), HE653454; H. dolabriforme Vent., AY555889;H. dyeri Rehder, Toshiyuki Nakaike 1797 (BM), HE653455;H. elatoides R.Keller, Boufford et al. 26156 (BM), HE653456;H. elodeoides Choisy, Miyamoto et al. 96062 (BM), HE653459;H. elodeoides Choisy, L.W. Beer C.R. Lancaster D. Morris 9492(BM), HE653457; H. elodeoides Choisy, Ikeda et al. 20911111 (TI),HE653458; H. elodes L., C. Scheriau HEID-808396 (HEID),HE653460; H. elodes L., C. Scheriau HEID-808399 (HEID),HE653461; H. elodes L., Michael F. Fay 374 (K), HE653462;H. elongatum Ledeb. var. lythrifolium, K. Sutory 112 (BM),HE653463; H. empetrifolium Willd., R.C. Lancaster 1118 (BM),HE653464; H. epigeium R.Keller, M. Veliz Gallardo Vasquez 9542(BM), HE653465; H. erectum Thunb. var. erectum, N.M. Nürk 365(BM), HE653466; H. erectum Thunb. var. erectum f. papillosum(Y.Kimura) Y.Kimura, N.M. Nürk 383 (GAT), HE653467; H. erectumThunb. var. erectum f. tateutianum (Koidz.) Y.Kimura, N.M. Nürk354 (GAT), HE653468; H. erectum Thunb. var. erectum f. vaniottii(H.Lév.) Y.Kimura, N.M. Nürk 372 (KYO), HE653469; H. ericoides L.,Stubing 25 (BM), HE653470; H. ericoides L., P.F. Cannon P.R. CraneS.R. Jury D.M. Moore (R.U. Botany Dept. Exped.) 475 (BM),HE653471; H. ericoides L., Cultivated 1985-922 (Kew-Wakehurst),HE653472; H. fasciculatum Lam., AY555868.2; H. fauriei R.Keller,N.M. Nürk 376 (GAT), HE653664; H. fauriei R.Keller, N.M. Nürk414 (GAT), HE653665; H. foliosum Aiton, H. Schaefer HS 208 (K),HE653473; H. forrestii (Chitt.) N.Robson, Sino-British Expeditionto Cangshan (SBEC) 0472 (BM), HE653474; H. forrestii (Chitt.)N.Robson, C.R. Lancaster L2032 (BM), HE653475; H. forrestii(Chitt.) N.Robson, D.S. Pigott s.n. 15.11.98 (BM), HE653476; H. fos-teri N.Robson, N.K.B. Robson s.n. 4.8.2004 (BM), HE653477; H. fra-seri (Spach) Steudel, S.R. Hill 17290 (GH), HE653663; H. fraseri(Spach) Steudel, B. Boivin A. Champagne 14188 (GH), HE653668;H. fraseri (Spach) Steudel, W. Hess N. Stoynoff 7351 (KYO),HE653662; H. frondosum Michx., AY555887; H. galioides Lam.,AY555864.2; H. garciae Pierce, S. Madriñán 2063 (ANDES),HE653478; H. geminiflorum Hemsl., HM162838; H. gentianoides(L.) Britton, Sterns & Poggenb., N.M. Nürk 384 (GAT), HE653479;H. gentianoides (L.) Britton, Sterns & Poggenb., Cultivated 2000-3136 (Kew-Wakehurst), HE653480; H. gleasonii N.Robson,S. Madriñán 2285 (ANDES), HE653481; H. gleasonii N.Robson, S.Madriñán 2011 (ANDES), HE653482; H. goyanesii Cuatrec.,C. Garcia 143 (ANDES), HE653483; H. gracillimum Koidz., N.M.Nürk 371 (BM), HE653484; H. gracillimum Koidz., N.M. Nürk 373(BM), HE653485; H. gramineum G.Forst., EU352256; H. gramine-um G.Forst., EU352257; H. grandifolium Choisy, N.M. Nürk 403(GAT), HE653487; H. grandifolium Choisy, C.E. Jarvis GibbyHumphries 411 (BM), HE653486; H. graveolens Buckley,AY555843; H. hakonense Franch. & Sav., N.M. Nürk 375 (KYO),HE653488; H. hakonense Franch. & Sav., Tomitaro Makino 33650(KYO), HE653489; H. haplophylloides Halácsy & Bald., F.K. Meyer5973 (BM), HE653490; H. henryi H.Lév. & Vaniot subsp. henryi,N.K.B. Robson s.n. 28.8.1983 (BM), HE653491; H. henryi H.Lév. &Vaniot subsp. uraloides (Rehder) N.Robson, AY555859; H. hetero-phyllum Vent., A.A. Donmez 3812 (BM), HE653492; H. hircinum

L. subsp. albimontanum (Greuter) N.Robson, C. Whitefoord 185(BM), HE653493; H. hircinum L. subsp. hircinum, G. Bocquet Iti-nera Galica 15507 (BM), HE653494; H. hircinum L. subsp. majus(Aiton) N.Robson, Turland 769 (BM), HE653495; H. hircinum L.subsp. majus (Aiton) N.Robson, J.R. Akeroyd S.L. Jury C.J. Miles F.J.Rumsey 3788 (BM), HE653496; H. hircinum L. subsp. metroi(Maire & Sauvage) Sauvage, S.L. Jury J.B. Peris G. Stubing 64 (BM),HE653497; H. hirsutum L., N.M. Nürk 390 (GAT), HE653498;H. hirsutum L., F. Dvorak 1648 (HEL), HE653499; H. hirsutum L.,M.F. Fay 350 (K), HE653500; H. hookerianum Wight & Arn., L.W.Beer C.R. Lancaster D. Morris 12316 (BM), HE653502; H. hookeria-num Wight & Arn., Sino-British Expedition to Cangshan 1981 (SBE)469 (BM), HE653501; H. hookerianum Wight & Arn., N.M. Nürk413 (GAT), HE653503; H. humboldtianum Steud., M.A. Bello 21(ANDES), HE653504; H. humifusum L., Carine Ait Lafkih RumseyRutherford 262 (BM), HE653505; H. humifusum L., K. Harris s.n.1988 (BM), HE653506; H. humifusum L., J.F. Veldkamp 8837(BM), HE653507; H. humifusum L., N.M. Nürk 381 (GAT),HE653509; H. humifusum L., A. Strid 22275 (BM), HE653508;H. hypericoides (L.) Crantz subsp. hypericoides, AY555879.2;H. hyssopifolium Vill., P. Bamps 9004 (BM), HE653511; H. hysso-pifolium Vill., B. de Retz 67577 (BM), HE653510; Hypericum xinodorum Mill., FJ694208; H. japonicum Thunb. ex Murray, Kazua-ki Masuda 3360 (KYO), HE653512; H. japonicum Thunb. ex Mur-ray, Ikeda et al. 20913073 (TI), HE653513; H. japonicum Thunb.ex Murray, GQ396674; H. jeongjocksanense S.-J. Park & K.-J. Kim,AY573023; H. jozoënse Maxim., AY573004; H. jozoënse Maxim.,FJ793046; H. juniperinum Kunth, S. Madriñán 2062 (ANDES),HE653515; H. juniperinum Kunth, S. Madriñán 2123 (ANDES),HE653514; H. kalmianum L., FJ694209; H. kamtschaticum Ledeb.,N.M. Nürk 366 (GAT), HE653516; H. kamtschaticum Ledeb.,AY572992; H. kamtschaticum Ledeb., K. Yonekura 12937 (KYO),HE653517; H. kinashianum Koidz., AY573001; H. kiusianumKoidz. var. yakusimense (Koidz.) T.Kato, N.K.B. Robson s.n.7.2006 (BM), HE653518; H. kouytchense H.Lév., J.R. Hosking P.T.Gorham 2007 (BM), HE653519; H. kouytchense H.Lév., N.M. Nürk405 (GAT), HE653520; H. kouytchense H.Lév., AY555853; H. cf.kouytchense x calycinum, N.M. Nürk 406 (GAT), HE653439;H. lagarocladum N.Robson, Cultivated 1988-3144 (Kew-Wake-hurst), HE653522; H. lagarocladum N.Robson, Sino-British Expedi-tion to Cangshan (SBEC) K149 (BM), HE653521; H. lancasteriN.Robson, Sino-British Expedition to Cangshan (SBEC) K039 (BM),HE653523; H. lancasteri N.Robson, AY555854; H. lancasteriN.Robson, Cultivated 1990-2357 (Kew-Wakehurst), HE653524;H. lanuginosum Lam., R. Ulrich s.n. 1998 (BM), HE653525; H. lari-cifolium Juss., M. Weigend G. Brokamp 9101 (B), HE653526; H. lar-icifolium Juss., C. Garcia 24 (ANDES), HE653527; H. laricifoliumJuss., S. Madriñán 2284 (ANDES), HE653528; H. laricifolium Juss.,S. Madriñán 2125 (ANDES), HE653529; H. laricifolium Juss., S.Madriñán 2113 (ANDES), HE653530; H. laxiflorum N.Robson, Ul-rich s.n. (15.6.2000) (BM), HE653531; H. leschenaultii Choisy,AY555857; H. linariifolium Vahl, N.M. Nürk 379 (GAT),HE653532; H. linarioides Bosse, P. Hein 64 (BM), HE653533; H. lis-sophloeus W.P.Adams, AY555885; H. lloydii (Svenson) W.P.Adams,AY555867.2; H. lobocarpum Gatt., AY555876.2; H. maclareniiN.Robson, C.R. Lancaster L2016 (BM), HE653534; H. maclareniiN.Robson, N.K.B. Robson s.n. 13.8.2000 (BM), HE653535; H. macul-atum Crantz, C. Scheriau HEID-811874 (HEID), HE653536; H. mac-ulatum Crantz, C. Scheriau HEID-808359 (HEID), HE653537;H. maculatum Crantz, C. Scheriau HEID-704351 (HEID),HE653538; H. maculatum Crantz subsp. obtusiusculum (Tourlet)Hayek, FJ694199; H. marginatum Woron., Davis Hedge D.32436(BM), HE653539; H. matudae Lundell, D.E. Breedlove 40408 (B),HE653661; H. matudae Lundell, AY573027; H. mexicanum L., S.Madriñán 2051 (ANDES), HE653540; H. microsepalum (Torr. &A.Gray) A.Gray ex S.Watson, AY555877.2; Hypericum x mitchellia-

num Rydb., N.M. Nürk 382 (GAT), HE653541; H. monanthemumHook.f. & Thomson ex Dyer, Sino-American Botanical Expeditionto Yunnan (SABEY) 1166 (BM), HE653542; H. monogynum L., C.R.Lancaster 1848B (BM), HE653543; H. monogynum L., N.K.B. Rob-son s.n. 7.2006 (BM), HE653544; H. montanum L., P. Hein 7504(BM), HE653545; H. montanum L., Hein 7541 (BM), HE653546;H. montanum L., C. Scheriau HEID-808415 (HEID), HE653547;H. montbretii Spach, D. McClintock s.n. 1993 (BM), HE653549;H. montbretii Spach, E. Stamatiadou 14999 (BM), HE653550;H. montbretii Spach, Stearn A.3. (BM), HE653548; Hypericum xmoserianum Luquet & ex André, N.M. Nürk 409 (GAT),HE653551; Hypericum x moserianum Luquet & ex André,AY555855; H. mutilum L., DQ006013; H. mutilum L. subsp. bore-ale, AY573026; H. myrtifolium Lam., AY555875.2; H. nakaiiH.Koidz. subsp. nakaii, N.M. Nürk 360 (GAT), HE653552; H. nakaiiH.Koidz. subsp. nakaii, N.M. Nürk 363 (KYO), HE653553; H. na-num Poir., P.H. Davis 10149 (BM), HE653554; H. nanum Poir., Cul-tivated 1945-31204 (Kew-Wakehurst), HE653555; H. nitidumLam. subsp. nitidum, AY555871.2; H. nudiflorum Michx.,AY555888; H. nummularioides Trautv., C.R. Lancaster s.n.1.viii.1979 (BM), HE653556; H. nummularium L., C.-A. Haegg-strom 7063 (BM), HE653557; H. oaxacanum R.Keller, AY573003;H. oblongifolium Choisy, FJ694226; H. oliganthum Franch. & Sav.,T. Kawahara H. Im T. Yahara 55 (TI), HE653558; H. oliganthumFranch. & Sav., Turu Sawada 236 (KYO), HE653559; H. oliganthumFranch. & Sav., AY573005; H. olympicum L. forma minus Hausskn.,Cultivated 1973-21185 (Kew-Wakehurst), HE653560; H. olympi-cum L. forma olympicum, D. McClintock s.n. 1983 (BM),HE653561; H. olympicum L. forma olympicum, W. Greuter 16146(BM), HE653562; H. olympicum L. forma uniflorum D.Jord. &Kozuharov, E. Stamatiadou 10094 (BM), HE653563; H. olympicumL. forma uniflorum D.Jord. & Kozuharov, N.M. Nürk 387 (GAT),HE653564; H. orientale L., N.M. Nürk 396 (GAT), HE653565;H. orientale L., FJ694213; H. cf. orientale L., N.M. Nürk 404 (GAT),HE653440; H. origanifolium Willd. var. origanifolium, A. CubukcuE. Yesilada A. Basaran H. Kocak 1412 (BM), HE653566; H. ovalifoli-um Koidz., AY572998; H. pallens Banks & Sol., C. Scheriau HEID-801626 (HEID), HE653567; H. pallens Banks & Sol., Cultivated1945-31202 (Kew-Wakehurst), HE653568; H. pallens Banks &Sol., AY555848; H. pamphylicum N.Robson & P.H.Davis, R. Ulrichs.n. 1998 (BM), HE653569; H. papillare Boiss. & Heldr., A. CubukcuA. Basaran A-12 (BM), HE653570; H. papuanum Ridl., Marsden 91(K), HE653571; H. patulum Thunb. ex Murray, C.R. Lancaster L.623(BM), HE653572; H. patulum Thunb. ex Murray, J.R. Hosking M.J.Williams 1845 (BM), HE653573; H. patulum Thunb. ex Murray,O.M. Hilliard B.L. Burtt 16088 (BM), HE653574; H. perfoliatum L.,S.L. Jury and M. Ait Lafkih M. El Haila R.G. Wilson 16522 (BM),HE653575; H. perfoliatum L., N.M. Nürk 385 (GAT), HE653576;H. perforatum L. subsp. chinense N.Robson, N.M. Nürk 353 (GAT),HE653577; H. perforatum L. subsp. chinense N.Robson, N.M.Nürk 370 (GAT), HE653578; H. perforatum L. subsp. perforatum,M. Wayda s.n. 2006 (BM), HE653579; H. perforatum L. subsp.veronense (Schrank) Ces., C.R. Lancaster 232 (BM), HE653580;H. perforatum L. subsp. veronense (Schrank) Ces., S. Collenette6079 (BM), HE653581; H. petiolulatum Hook.f. & Thomson exDyer, A.J.C. Grierson D.G. Long 2549 (BM), HE653582; H. petiolula-tum Hook.f. & Thomson ex Dyer subsp. yunnanense (Franch.) N.Robson, B. Bartholomew D.E. Boufford Q.H. Chen et al. 2163 (BM),HE653583; H. pibairense (Miyabe & Y.Kimura) N.Robson, TatsumiKato 3132-3 (TI), HE653584; H. pibairense (Miyabe &Y.Kimura) N.Robson, N.M. Nürk 367 (GAT), HE653585;H. podocarpoides N.Robson, J.R.I. Wood 6110 (BM), HE653586;H. podocarpoides N.Robson, Ikeda et al. 20913062 (TI),HE653587; H. polyphyllum Boiss. & Balansa, N.M. Nürk 407(GAT), HE653588; H. polyphyllum Boiss. & Balansa, N.M. Nürk388 (GAT), HE653591; H. polyphyllum Boiss. & Balansa, N.M.

Nürk 391 (GAT), HE653589; H. polyphyllum Boiss. & Balansa,N.M. Nürk 392 (GAT), HE653590; H. prolificum L., AY555873.2;H. prolificum L., FJ694217; H. prostratum Cuatrec., C. Garcia 108(ANDES), HE653592; H. przewalskii Maxim., G.S. Miehe 9215/04(BM), HE653593; H. pseudohenryi N.Robson, AY555850; H. pseud-ohenryi N.Robson, FJ694218; H. pseudolaeve N.Robson, J. Roper 68(BM), HE653594; H. pseudomaculatum Bush, Culwell Tucker s.n.12.5.1968 (BM), HE653595; H. pseudopetiolatum R.Keller,AY573002; H. pubescens Boiss., S.L. Jury with M.A. Carine M. Rej-dali F.J. Rumsey R.W. Rutherford 19630 (BM), HE653596; H. pubes-cens Boiss., Davis 52932 (BM), HE653597; H. pulchrum L., S.L. JuryM.F. Watson 6219 (BM), HE653598; H. pulchrum L., Cubr 39891(B), HE653599; H. pulchrum L., M.F. Fay 298 (K), HE653600;H. punctatum Lam., AY555844; H. punctatum Lam., D.E. BouffordE.W. Wood 23250 (BM), HE653601; H. punctatum Lam., K.G. SikesJ. Stone 24 (BM), HE653602; H. quartinianum A.Rich., J.C. LovettC.J. Kayombo 4922 (BM), HE653603; H. quartinianum A.Rich.,T.R.I. Wood 2817 (BM), HE653604; H. quartinianum A.Rich., Nkho-ma Changwe 2032 (BM), HE653605; H. quitense R.Keller, M. Weig-end G. Brokamp 9100 (B), HE653606; H. reflexum L., F. BlattnerFRB-2008-004 (GAT), HE653607; H. reflexum L., Cultivated 1999-00370 (Jardin Botanico Barcelona), HE653608; H. reflexum L.,FJ694221; H. reptans Hook.f. & Thomson ex Dyer, Cultivated1972-6301 (Kew-Wakehurst), HE653609; H. revolutum Vahl, S.Chaudhary 3901 (BM), HE653610; H. revolutum Vahl, I.F. LaCroix3098 (BM), HE653611; H. revolutum Vahl, Cultivated 1972-3163(Kew-Wakehurst), HE653612; H. richeri Vill., Cultivated 1993-1024 (Kew-Wakehurst), HE653613; H. rigidum A.St.-Hil.,AY573021; H. roeperianum G.W.Schimp. ex A.Rich., W.T. Stearns.n. 1977 (BM), HE653614; H. roeperianum G.W.Schimp. exA.Rich., AY555863; H. roeperianum G.W.Schimp. ex A.Rich., Culti-vated 1982-2124 (Kew-Wakehurst), HE653615; H. rumeliacumBoiss., D. McClintock s.n. 28.5.1983 (BM), HE653616; H. rumelia-cum Boiss. subsp. rumeliacum, A.O. Chater 21 (BM), HE653617;H. ruscoides Cuatrec., Amalia Diaz 13 (ANDES), HE653618;H. sabiniforme Trevir., Favio Gonzalez 3838 (ANDES), HE653619;H. sampsonii Hance, unknown 16917 (KYO), HE653620; H. sam-psonii Hance, AY573011; H. saxifragum N.Robson & Hub.-Mor.,R. Ulrich s.n. 6.10.1997 (BM), HE653621; H. scabroides N.Robson& Poulter, C. Scheriau HEID-808410 (HEID), HE653622; H. scabro-ides N.Robson & Poulter, C. Scheriau HEID-808412 (HEID),HE653623; H. scabrum L., K. Sutory 110 (BM), HE653624; H. sca-brum L., Cultivated 1995-3560 (Kew-Wakehurst), HE653625;H. scouleri Hook., R. Halse 5427 (BM), HE653626; H. scruglii Bac-ch., Brullo & Salmeri, D. Metzing s.n. 30.9.2010 (OLD), HE653627;H. senanense Maxim., N.M. Nürk 362 (GAT), HE653628; H. senan-ense Maxim. mutiloides (R.Keller) N.Robson, N.M. Nürk 361 (GAT),HE653629; H. seniawinii Maxim., Xiao Bai-Zhong 3778 (BM),HE653630; H. setosum L., AY573020; H. sikokumontanum Maki-no, AY572999; H. sinaicum Hochst. ex Boiss, A. Danin 962609(BM), HE653631; Hypericum spec., AY555866.2; H. sphaerocar-pum Michx., AY555878.2; H. spruneri Boiss., N.M. Nürk 408(GAT), HE653632; H. strictum Kunth, Maria Angelica Bello 97 (AN-DES), HE653633; H. strictum Kunth, S. Madriñán 2048 (ANDES),HE653634; H. subsessile N.Robson, Alpine Garden Society Expedi-tion to China 1994 (ACE) 2526 (BM), HE653635; H. subsessileN.Robson, Cultivated 1981-5841 (Kew-Wakehurst), HE653636;H. suffruticosum W.P.Adams, S. Crockett 156 (UGA), HE653637;H. taygeteum Quézel & Contandr., W. Greuter H. Merxmüller17233 (BM), HE653638; H. tenuicaule Hook.f. & Thomson ex Dyer,F. Miyamoto M. Amano H. Iked C.M. Joshi K. Arai T. Komatsu9596032 (BM), HE653639; H. tenuifolium Pursh, AY555872.2;H. ternum A.St.-Hil., AY573022; H. terrae-firmae Sprague & Riley,Rees 221 (BM), HE653640; H. tetrapetalum Lam., AY555882.2;H. tetrapterum Fr., Cultivated 1983-533 (Kew-Wakehurst),HE653641; H. tetrapterum Fr., FJ694224; H. tetrastichum

Cuatrec., S. Madriñán 2039 (ANDES), HE653642; H. tetrastichumCuatrec., S. Madriñán 2010 (ANDES), HE653643; H. thesiifoliumKunth, M. Weigend G. Brokamp 9119 (B), HE653644; H. thymbrifo-lium Boiss. & Noë, A. Cubukcu 3 (BM), HE653645; H. thymifoliumBanks & Sol., R. Ulrich 0/52 (BM), HE653646; H. thymifolium Banks& Sol., Ulrich 0/52 (BM), HE653647; H. tomentosum L., S.L. Jury andL.S. Springate M. Ait Lafkih 11271 (BM), HE653648; H. tomentosumL., N.M. Nürk 412 (GAT), HE653649; H. tosaense Makino, TamikiKobayashi 41978 (KYO), HE653650; H. triquetrifolium Turra, JR.Akeroyd S.L. Jury F.J.Rumsey 3572 (BM), HE653651; H. triquetrifo-lium Turra, Cultivated 1990-100 (Kew-Wakehurst), HE653653;H. triquetrifolium Turra, Tomkinson 29 (BM), HE653652; H. tubu-losum Walter, J.D. Ray 5412 (GH), HE653666; H. undulatumSchousb. ex Willd., C. Scheriau HEID-808335 (HEID), HE653654;H. undulatum Schousb. ex Willd. subsp. undulatum, D.J. GoyerS.L. Jury 545 (BM), HE653655; H. vacciniifolium Hayek & Siehe, R.Ulrich s.n. 2000 (BM), HE653656; H. virginicum L., R.S. Mitchel J.Focht 8507 (GH), HE653667; H. walteri J.F.Gmel., R. Kral R.K.Godfrey 5921 (GH), HE653669; H. wardianum N. Robson, N.K.B.Robson s.n. 7.2006 (BM), HE653657; H. wilsonii N.Robson, N.K.B.Robson s.n. 7.2006 (BM), HE653658; H. xylosteifolium (Spach)N.Robson, C. Scheriau HEID-808337 (HEID), HE653659; H. xylost-eifolium (Spach) N.Robson, Cultivated 1979-6434 (Kew-Wake-hurst), HE653660; H. cf. xylosteifolium (Spach) N.Robson, N.M.Nürk 411 (GAT), HE653438; Vismieae: Harungana madagascari-ensis Poir., A.J.M. Leeuwenberg 8143 (B), HE653670; Vismia cay-ennensis (Jacq.) Pers., Mori Lokova Keeley 25662 (B), HE653671;V. guianensis (Aubl.) Choisy, Jansen-Jacobs Lilwah RaghoenandanScheplitz Vermeer 5501 (B), HE653672; V. macrophylla Kunth, Jan-sen-Jacobs Welle James Andrew 4920 (B), HE653673.

Cloned sequences: Hypericum choisyanum Wall. ex N.Robson,Ikeda et al. 20913019 (TI), HE662695, HE662696, HE662697,HE662698, HE662699; H. davisii N.Robson, A. Cubukcu s.n.(1978) (BM), HE662731, HE662732, HE662733, HE662734,HE662735, HE662736, HE662737; H. gentianoides (L.) Britton,Sterns & Poggenb., Cultivated 2000-3136 (Kew-Wakehurst),HE662750; H. juniperinum Kunth, S. Madriñán 2062 (ANDES),HE662749; H. lagarocladum N.Robson, Sino-British Expedition toCangshan (SBEC) K149 (BM), HE662700, HE662701, HE662702,HE662703, HE662704, HE662705, HE662706; H. lancasteri N.Rob-son, Sino-British Expedition to Cangshan (SBEC) K039 (BM),HE662707, HE662708, HE662709, HE662710, HE662711; H. nakaiiH.Koidz. subsp. nakaii, N.M. Nürk 360 (GAT), HE662712,HE662713, HE662714; H. polyphyllum Boiss. & Balansa, N.M.Nürk 407 (GAT), HE662726, HE662727, HE662728, HE662729,HE662730; H. reflexum L., F. Blattner FRB-2008-002, HE662741,HE662742, HE662743, HE662744, HE662745, HE662746,HE662747; H. scabroides N.Robson & Poulter, C. Scheriau HEID-808412 (HEID), HE662738, HE662739, HE662740; H. scouleriHook., R. Halse 5427 (BM), HE662715, HE662716, HE662717,HE662718, HE662719, HE662720; H. strictum Kunth, S. Madriñán2048 (ANDES), HE662748; H. undulatum Schousb. ex Willd. subsp.undulatum, D.J. Goyer S.L. Jury 545 (BM), HE662721, HE662722,HE662723, HE662724, HE662725.

Appendix B. Character coding for MCMC reconstruction

Habit: tree (0), shrub (1), herb (2). Dark glands in vegetativeparts: absent (0), present (1). Dark glands in reproductive parts:absent (0), present (1). Petals: persistent (0), deciduous (1). Sta-mens: persistent (0), deciduous (1). Arrangement of stamens: 5free fascicles (0), 3 (2 + 2 + 1) free fascicles (1), narrow continuousring (2), broad continuous ring (3), 5 obscure fascicles (4), 3 ob-scure fascicles (5), 5 single stamens (6). Staminodes (sterile fasci-cles, ‘fasciclodes’): absent (0), present (1). Placentation: parietal

(0), incompletely axile (1), axile (2). The data matrix can be ob-tained from the corresponding author or from TreeBASE study ID13339.

Appendix C. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ympev.2012.08.022.

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molecular phylogenetics and morphological evolution of st. john's wort (hypericum; hypericaceae

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