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Huerteales sister to Brassicales plus Malvales, and newly circumscribed to include , Gerrardina, Huertea, , and Tapiscia

Article in Taxon · May 2009 DOI: 10.1002/tax.582012

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Huerteales sister to Brassicales plus Malvales, and newly circumscribed to include Dipentodon, Gerrardina, Huertea, Perrottetia, and Tapiscia

Andreas Worberg1, Mac H. Alford2, Dietmar Quandt1,3 & Thomas Borsch1,4

1 Nees-Institut für Biodiversität der Pflanzen, Rheinische Friedrich-Wilhelms-Universität Bonn, Meckenheimer Allee 170, 53115 Bonn, Germany. [email protected] (author for correspondence) 2 Department of Biological Sciences, University of Southern Mississippi, 118 College Drive 5018, Hattiesburg, Mississippi 39406, U.S.A. 3 Institut für Botanik, Technische Universität Dresden, Zellescher Weg 20b, 01217 Dresden, Germany 4 Current address: Botanischer Garten und Botanisches Museum Berlin-Dahlem und Institut für Biologie, Freie Universität Berlin, Königin Luise-Str. 6–8, 14195 Berlin, Germany

Sequence data from the matK gene, the trnK group II intron, the trnL group I intron and the trnL-F spacer were analysed for a broad sampling of the and other . For the first time all putative genera of and (Dipentodon, Huertea, Perrottetia, Tapiscia), as well as the recently de- scribed Gerrardinaceae were included in a molecular phylogenetic dataset. All genera were found in a well supported Huerteales clade. Moreover, with the rapidly evolving and non-coding plastid sequence data we were able to resolve the Huerteales clade to branch after Sapindales, and to be sister to a Brassicales-Malvales clade. Increased resolution and support among the malvids underscore the potential of plastid introns and spacers as well as the matK gene as phylogenetic markers in rosids.

KEYWORDS: Huerteales, matK, rosids, trnK, trnL-F

per carpel. However, analyses of rbcL and atpB INTRODUCTION datasets remained inconclusive, and Gerrardina could not The large scale phylogenetic analysis of angiosperms be placed unambiguously in Huerteales. In the latest up- based on rbcL, atpB, and 18S sequence data of Soltis & date of their angiosperm classification Thorne and Reveal al. (2000) included one that was unresolved in ei- (Thorne, 2007) grouped all three families into Huerteales ther Brassicales, Malvales, or Sapindales in the eurosid II within the newly described superorder named Huerteanae. (malvid) clade. This genus, Tapiscia, was later inferred to No study, though, has included all of the genera potentially be sister to Dipentodon (Dipentodontaceae), a relationship placed in Huerteales, thus underscoring the need for an strongly supported (94% JK) based on combined analyses analysis with complete taxon sampling. of matR, rbcL, and 18S DNA data (Peng & al., 2003) and Pre-phylogenetic placements of genera that are now matR alone (Zhu & al., 2007). The order Huerteales was candidates as members of a monophyletic order Huerteales thus described by Doweld (2001) to include the genus varied considerably among diverse angiosperm orders. Di- Dipentodon (Dipentodontaceae) and the two small gen- pentodon sinicus Dunn is a small native to southern era Tapiscia and Huertea (Tapisciaceae), formerly placed , Burma and northern India and can be recognized together as subfamily Tapiscioideae of . Wu by stipulate, serrate , umbelliform , & al. (2002) later recognized the Dipentodontales as a and essentially free-central placentation (Takhtajan, new order in their classification system, 1997). This taxon was formerly included in consisting of Dipentodon sinicus alone. All three genera (Cronquist, 1981) because of its placentation or in Viola- are poorly known, but toothed margins, 1–2 ovules les (now Malpighiales) because of perianth morphology, per carpel, and relatively small embryos seem to be shared wood anatomy, and pollen (Dahlgren, 1980; Thorne, 1992; morphological characters (Stevens, 2001 onwards). Re- Takhtajan, 1997). Dunn (1911) originally placed Dipent- cently, an analysis based on several plastid and nuclear odon in , whereas a series of subsequent au- genes by Zhang & Simmons (2006) also placed Perrotte- thors hypothesized it to be a member of tia (formerly Celastraceae) with Tapiscia and Dipentodon. (including Samydaceae) mostly based on floral features Later, an analysis based on rbcL and atpB by Alford (Sprague, 1925; Fischer, 1941; Loesener, 1942; Metcalfe (2006) indicated that the genus Gerrardina (formerly Fla- & Chalk, 1950; Lobreau, 1969). Tapisciaceae consist of courtiaceae) was unplaced in the eurosids II (malvids) but the two genera Tapiscia Ruiz & Pav. and Huertea Oliv. potentially related to the Huerteales based on having two (Takhtajan, 1987). Tapiscia sinensis is native to China,

468 TAXON 58 (2) • May 2009: 468–478 Worberg & al. • Phylogenetic position of Huerteales

and the four known Huertea are distributed from support of phylogenetic hypotheses, for example, in early the West Indies and Central America through the Andes branching angiosperms (Borsch & al., 2005; Müller & to Peru. They comprise small with spirally arranged al., 2006) or early branching eudicots (Worberg & al., compound leaves that are odd-pinnate or trifoliolate and 2007). have serrate margins (Krause, 1942; Stevens, 2001 on- Our approach was therefore to first combine matK se- wards). Whereas Tapiscia was originally described in quence data with trnK group II intron sequence data. The Sapindaceae by Oliver (1890), Huertea and Tapiscia were matK gene is located within domain V of the trnK intron, then mostly included together in Staphyleaceae (Bean, and therefore both markers can be co-amplified easily. In 1909; Diels, 1909; Schneider, 1912: p. 1026 and Fig. 607; the context of work aimed at understanding the mutational Dickison, 1986) and placed in Sapindales (Cronquist, dynamics and phylogenetic signal in non-coding plas- 1981). Takhtajan (1987) later described a separate fam- tid regions in eudicots, a large dataset is currently being ily Tapisciaceae that remained classified in Sapindales. assembled for the rosids, a clade which comprises more Perrottetia comprises about 15 species and is distributed than 70,000 species in ca. 140 families. One of the most in Asia from China to Malesia and southwards to north- prominent results is that we found strong support for the eastern Australia but also occurs in the Neotropics from Huerteales clade in a well resolved eurosid II ( = malvid) Mexico to Peru. The floral structure of Perrottetia was clade, based on a combined matK/trnK dataset. Moreover, studied in some detail by Matthews & Endress (2005), trnL-intron and trnL-F spacer data confirmed the mono- whereas that of the other genera is poorly known. All spe- phyly of Huerteales and provided molecular evidence for cies of Perrottetia share two carpels per and spirally the inclusion of Huertea, for which any kind of sequence arranged leaves (Stevens, 2001 onwards), characters also data were previously unavailable. present in Dipentodon and Tapiscia. Gerrardina Oliv. Considering the still unclear phylogenetic position consists of two species, Gerrardina eylesiana and Ger- of Gerrardinaceae (Alford, 2006) and the hitherto un- rardina foliosa, both of which are small trees or shrubs resolved position of Dipentodontaceae and Tapisciaceae native to southern Africa (Malawi and Tanzania south to (see APG II, 2003), our study aims at contributing to the the Eastern Cape Province of South Africa). Species of ongoing discussion by first clarifying the composition of Gerrardina have simple, alternate, stipulate leaves with the Huerteales clade, and by second resolving the phylo- serrate margins, opposite the , and two api- genetic relationships of Gerrardinaceae and Huerteales, cal ovules per carpel. Formerly Gerrardina was placed respectively. in Flacourtiaceae (including Samydaceae; Oliver, 1870; Warburg, 1894; Gilg, 1925). Sequences of rapidly evolving plastid spacers and introns have been very successfully used in phyloge- MATERIAL AND METHODS netic analyses to infer deep relationships among families Taxon sampling. — The dataset comprises 54 taxa and orders of angiosperms (Borsch & al., 2003, 2005; from core eudicots with three representatives of Gun- Löhne & Borsch, 2005; Müller & al., 2006; Wanke & nerales as outgroup. Representatives for all rosid orders al., 2007; Worberg & al., 2007; Korotkova & al., 2009), recognized by APG II (2003) were sampled with a focus and they were expected to provide some resolution to the on the eurosids II (malvid) clade to establish the frame- above-mentioned phylogenetic ambiguity. Well known work for positioning Huerteales. Moreover, the sampling examples of such rapidly evolving genomic regions that of this study represents major lineages also found in a also can be amplified using universal primers are the more extensive combined trnK/matK plus trnL-F rosid so-called trnT-trnF-region (composed of the trnT-L and analyses by the authors ( > 180 taxa, not shown here). All trnL-F spacers and the group I intron in trnL; Taberlet genera with putative affinities to Huerteales are included & al., 1991; Borsch & al., 2003; Quandt & al., 2004), and in this study. Taxa with complete matK sequences already the group II introns within trnK (Johnson & Soltis, 1994; available (Worberg & al., 2007) were completed by adding Müller & Borsch, 2005a, b), rpl16 (Kelchner, 2002) and the trnK intron, what could easily be achieved through petD (Löhne & Borsch, 2005; Korotkova & al., 2009). further sequencing reactions on already existing ampli- In addition to high phylogenetic structure per nucleotide cons. In addition, some species with published completely sequenced (Müller & al., 2006) these non-coding data- sequenced plastid genomes were added. Material for fur- sets yield additional indel matrices that usually further ther taxa came from the living collections at the Botani- improve resolution and support of the reconstructed trees cal Gardens Bonn, Dresden, and Berlin (FU-BGBM). A (Worberg & al., 2007; Borsch & al., 2007). Combining DNA extraction of Gerrardina foliosa was provided from such intron and spacer sequence data with sequences of the DNA Bank of the Royal Botanic Gardens, Kew, U.K. the rapidly evolving matK gene (Hilu & al., 2003) has A list of all sampled taxa, their origin and voucher infor- been shown to lead to further improved resolution and mation is given in Appendix 1.

469 Worberg & al. • Phylogenetic position of Huerteales TAXON 58 (2) • May 2009: 468–478

DNA isolation, amplification and sequencing. regions adequately (Simmons & al., 2007). These align- — DNA was isolated from fresh or silica gel-dried plant ment rules are based on recognizing sequence motifs that material, using a CTAB method with three extractions result from microstructural changes (Kelchner & Clark, (Borsch & al., 2003), designed to yield high amounts of ge- 1997; Kelchner, 2000, 2002) rather than globally applying nomic DNA. Fresh leaves were generally dried in silica gel fixed gap costs. Inclusion of ambiguously alignable regions before extraction. Dry tissue was ground to a fine powder into character matrices (Lutzoni & al., 2000; Aagesen, using a mechanical homogenizer (Retsch MM200) with 2004) were not pursued here. Mutational hotspots with 5 mm beads at 30 Hz for 2 min. To identify mutational unclear primary homology (Borsch & al., 2003) were ex- hotspots complete sequences of spacers and introns are cluded from tree inference in order to achieve maximum necessary. We amplified with primers that were located accuracy. To utilize indel characters we applied the simple sufficiently far away from the actual region under study. indel coding method of Simmons & Ochoterena (2000) via Sequencing was performed with either the universal SeqState v1.36 (Müller, 2005b). The resulting indel matrix primers already used for amplification or with additional was directly combined with the nucleotide-sequence ma- internal primers, some of which were newly designed us- trix for parsimony and Bayesian Inference, as an increase ing SeqState v1.36 (Müller, 2005b). The matK gene was of resolution and support by adding indel information from amplified within the trnK intron, either entirely or in two matK/trnK could be expected based on previous evidence overlapping halves. Primers annealing to the trnK exons (Müller & Borsch, 2005b; Worberg & al., 2007). were trnKFbryo forward (Wicke & Quandt, in press) and To infer most parsimonious trees we used the Par- trnK2R (Johnson & Soltis, 1994). To amplify two overlap- simony Ratchet (Nixon, 1999) as implemented in the ping fragments, internal primers with higher taxon speci- program PRAP (Müller, 2004). Ratchet settings were 20 ficity primers were placed roughly 600 nt (reverse) and random addition cycles of 200 ratchet replicates and up- 450 nt (forward) downstream from the matK-start codon. weighting 25% of the characters. If more than one shortest Internal primers for respective families were either auto- tree was found, strict consensus trees were created. Nodes matically selected from the eudicot matK primer database were evaluated by jackknifing in PAUP* 4b10 (Swofford, (www.eudicots.de) or designed newly with SeqState v1.36. 2001) with 36.79% deletion of characters and 10,000 rep- The trnL intron and the trnL-F spacer (in the following licates saving only one tree per replicate. This approach called trnL-F region) were co-amplified using primers c follows studies on the reliability of jackknife percent- and f of Taberlet & al. (1991) and sequenced with primers ages (Freudenstein & Simmons, 2004; Müller, 2005a). d of Taberlet & al. (1991) and trnL460F (Worberg & al., Bayesian inference (BI) was performed using the program 2007) that is a forward primer universal for angiosperms MrBayes v3.1 (Ronquist & Huelsenbeck, 2003) applying annealing within the trnL intron. All primers used in this the GTR + Γ + I model for sequence data and the adaption study are listed in Appendix 2 (in the online version of of the restriction site model (“F81”) for presence/absence this article). for the indel matrix. Four runs (106 generations each) with Amplification and sequencing reactions were per- four chains each were run simultaneously, starting from formed in a T3 Thermocycler (Biometra, Göttingen, random trees. Chains were sampled every 10th genera- Germany). Amplicons were purified with an Avegene gel tion. Stationarity of the runs was reached within the first extraction kit (Avegene) after running them out on a 1.2% 250,000 generations (burn-in). Calculations of the con- agarose gel and excising the bands. The BeckmannCoulter sensus tree and the posterior probability (PP) of clades DTCS QuickStart Reaction kit was used for direct sequenc- were done based upon the trees sampled after the burn-in. ing. Temperature profiles and PCR reaction conditions for Only PP’s of 90 and higher were considered significant trnK/matK followed Müller & Borsch (2005a) and Borsch (alpha = 0.1). Trees were drawn using TreeGraph (Müller & al. (2003) for trnL-F. Extension products were run on & Müller, 2004). BeckmannCoulter CEQ 8000 automated sequencers in Bonn or Dresden. Alternatively, sequences were generated on ABI377 autosequencers. Sequences were edited manu- ally with PhyDE v0.995 (Müller & al., 2005). RESULTS Alignment, indel coding and phylogenetic analy- The combined trnK and matK dataset, excluding sis. — The presence of microstructural changes, such as mutational hotspots, comprises 3,334 substitution char- deletions, single sequence repeats, other insertions, and in- acters, of which 1,402 were parsimony-informative, 757 versions, necessitates special attention to the alignment of variable but not parsimony-informative, and 1,175 constant, sequences. Alignment was carried out by eye using PhyDE and an additional 348 indel characters. The parsimony v0.995, applying the rules outlined in Borsch & al. (2003) ratchet analysis yielded four most parsimonious trees with and Löhne & Borsch (2005), as there are still no automated a length of 8,697 steps (consistency index, CI = 0.420; alignment algorithms that can handle length-variable DNA retention index, RI = 0.410; rescaled consistency index,

470 TAXON 58 (2) • May 2009: 468–478 Worberg & al. • Phylogenetic position of Huerteales

RC = 0.172; homoplasy index, HI = 0.580). The strict two species of Perrottetia sampled here (100% JK, 100 consensus tree is depicted in Fig. 1 and displays a topol- PP). The Huerteales are a highly supported clade (100% ogy with Gerrardina (Gerrardinaceae) well supported as JK, 100 PP). They are well positioned within the euro- part of the Huerteales clade (100% JK, 100 PP). Gerrar- sids II ( = malvids), which are overall highly supported dina is then sister to Tapisciaceae plus Dipentodontaceae (99% JK, 100 PP). Within malvids, Sapindales are sister (100% JK, 100 PP), Tapisciaceae, including Huertea and to Huerteales + Brassicales + Malvales (94% JK, 100 PP), Tapiscia (100% JK, 100 PP) are sister to Dipentodonta- and Huerteales are sister to Brassicales + Malvales (94% ceae (99% JK, 100 PP), and Dipentodon is sister to the JK, 100 PP). Separate analyses of trnK and matK (trees not

100 BIX Bixa 53 100 CIST Cistus 51 MUNT Muntingia 100 Malvales 100 100 MALV Gossypium 50 100 BOMB Durio 94 - THYM Daphne 94 68 TROP Tropaeolum 100 54 CARIC Vasconcellea Brassicales 100 100 RESED Reseda 94 100 BRASS Lobularia 93 100 DIPEN Perrottetia long. 99 100 DIPEN Perrottetia ovat. 100 99 DIPEN Dipentodon Huerteales 100 100 TAP Tapiscia malvids 99 100 100 98 100 TAP Huertea GERR Gerrardina 54 RUTA Citrus 100 - MELI Melia 100 99 SIMA Ailanthus Sapindales 100 66 SAPIN Acer - ANAC Schinus 100 PARN Parnassia 97 100 CELAS Salacia 96 OXAL 99 99 77 SALIC Populus 100 76 PASS Passiflora Malpighiales 100 69 EUPH Euphorbia rosids 97 69 RHIZO Kandelia 89 100 CORI Coriaria Cucurbitales 50 100 CUCUR Cucumis fabids 69 51 JUG 57 100 ROSAC Malus 54 93 100 - ROSAC Spiraea Rosales 77 URTI Urtica 100 FAB Glycine Fabales 67 100 POLY Polygala 64 90 GERAN Erodium Geraniales 75 ZYGO Larrea Zygophyllales 58 MELAS Melastoma 55 65 ONAG Oenothera Myrtales 100 61 COMBR Combretum 99 56 MYRT Eucalyptus 57 100 STACH 100 100 VIT Vitis Vitales 100 LEEA Leea 70 AQUI Ilex 100 70 BALS Impatiens 98 100 API Daucus 51 Asterids 100 ARALI Panax 72 51 70 SAX Chrysosplenium AEX Aextoxicon 100 MYRO Myrothamnus flab. 100 MYRO Myrothamnus mosc. Gunnerales Outgroup GUNN Gunnera

Fig. 1. Combined strict consensus tree based on substitutions and indels of trnK and matK, inferred with maximum par- simony. Bold values above branches are Jackknife percentages with indels coded, plain ones below are Jackknife values without simple indel coding applied.

471 Worberg & al. • Phylogenetic position of Huerteales TAXON 58 (2) • May 2009: 468–478

shown) found identical topologies as depicted here but with significance in the Bayesian analyses if information ob- generally lower node support. The Bayesian Analysis (Fig. tained from indels was included in the analyses. More- 2) yielded nearly the same topology as Maximum Parsi- over, two strong supported clades appear: (1) Malpighia- mony. Minor differences concern the unsupported position les, Oxalidales and Celastrales, and (2) Rosales, Fabales, of the Geraniales and Zygophyllales, i.e., the monophyly Cucurbitales, and Juglans (Fagales), with the former being of the fabids (compare Figs. 1 and 2). Whereas Geraniales resolved with high support under parsimony as well. To and Zygophyllales are resolved within the fabid clade and further clarify the composition of Huerteales we sampled share a moderate supported sister group relation under trnL-F data for a smaller dataset (26 taxa). Considering parsimony, the position is unresolved under a Bayesian the high statistical confidence of the matK/trnK trees with framework. However, the remaining fabid clade reaches larger sampling, we restricted the analysis of trnL-F data

100 BIX Bixa Fig. 2. Bayesian phylo- CIST Cistus gram based on the com- THYM Daphne 100 Malvales bined trnK + matK matrix. 100 MALV Gossypium Posterior probabilities BOMB Durio are depicted above 100 MUNT Muntingia TROP Tropaeolum branches. * indicates that the respective node did 100 CARIC Vasconcellea Brassicales 100 RESED Reseda not reach significance 100 BRASS Lobularia ( > 90) in the analysis after 100 DIPEN Perrottetia long. exclusion of the indel 100 DIPEN Perrottetia ovat. matrix. 100 DIPEN Dipentodon Huerteales TAP Tapisci malvids 100 100 100 TAP Huertea GERR Gerrardina RUTA Citrus 100 SIMA Ailanthus 100 MELI Melia Sapindales SAPIN Acer 100* ANAC Schinus 100 PARN Parnassia Celastrales CELAS Salacia 100 OXAL Oxalis Oxalidales 100 SALIC Populus 98 100 PASS Passiflora Malpighiales EUPH Euphorbia rosids 100 92* RHIZO Kandelia 100 CORI Coriaria Cucurbitales CUCUR Cucumis 100 ROSAC Malus 100 ROSAC Spirea Rosales 100 URTI Urtica 100 FAB Glycine Fabales POLY Polygala JUG Juglans Fagales ZYGO Larrea Zygophyllales GERAN Erodium Geraniales 99 MELAS Melastoma 100 MYRT Eucalyptus Myrtales COMBR Combretum ONAG Oenothera 100 STACH Stachyurus Crossosomatales 100 VIT Vitis Vitales LEEA Leea 100 API Daucus 100 ARALI Panax 100 AQUI Ilex Asterids 100 BALS Impatiens AEX Aextoxicon SAX Chrysosplenium 100 MYRO Myrothamnus flab. MYRO Myrothamnus mosc. Gunnerales GUNN Gunnera 0.1

472 TAXON 58 (2) • May 2009: 468–478 Worberg & al. • Phylogenetic position of Huerteales

to a malvid subset that included all six putative Huerteales genera (Fig. 3). The matrix comprised 1,231 characters of DISCUSSION which 274 were parsimony-informative, and an additional Although morphological features and previous 183 indel characters. Parsimony analysis yielded six short- analyses of DNA sequence data could not confidently est trees of 1,168 steps (CI = 0.622, RI = 0.440, RC = 0.273). place Gerrardina (Alford, 2006), this study using matK/ The Huerteales clade was resolved with 88% JK, again trnK sequence data provides unambiguous support for a rendering Gerrardina as sister to the remaining genera. placement of Gerrardinaceae sister to a Dipentodonta- Huertea was found as sister to Tapiscia (Fig. 3). Notably, cae + Tapisciaceae clade (100% JK, 100 PP). We have thus the rather small trnL-F dataset also yielded good support enlarged the Huerteales to include Gerrardina. Moreover, for the support within all other malvid orders, Brassicales, phylogenetic analysis of trnK/matK and trnL-F sequence Malvales, and Sapindales. data shows for the first time with molecular data that Huertea is the sister group of Tapiscia. Furthermore, our results provide strong evidence for the interrelationships MALV Gossypium 100 of the orders of the eurosids II (malvids). Brassicales and 100 64 BOMB Bombax Malvales are sister, which are then sister to Huerteales, 76 89 BIX Bixa which are then sister to Sapindales. This branching pattern 90 has strong support, regardless of the analytical approach. MUNT Muntingia Malvales 58 Our results are a further example of the effectiveness 76 53 CIST Cistus of matK/trnK and trnL-F as phylogenetic markers at deeper - CARIC Vasconcellea levels in eudicots. High performance of partial matK was 82 80 earlier shown by Hilu & al. (2003) for angiosperms. In 97 BRASS Lobularia addition to larger quantities of informative sites in such 94

TROP Tropaeolum Brassicales rapidly evolving and non-coding genomic regions, Müller & al. (2006) found increased phylogenetic signal per in- TAP Tapiscia 99 formative character of rapidly evolving or non-coding 96 75 TAP Huertea markers like matK or trnT-F in contrast to slow evolving 77 regions like rbcL. Complete matK sequences alone (tree malvids 87 81 DIPEN Dipentodon 78 76 not shown) inferred a topology similar to the complete DIPEN Perrottetia long. 99 angiosperm analysis made by Hilu & al. (2003; Huerteales

88 Huerteales 100 85 DIPEN Perrottetia ovat. not included) with high support for most nodes. This study is the first to apply trnK group II intron sequences GERR Gerrardina at deeper level in angiosperms. Similar to the addition of RUTA Citrus 100 petD group II intron sequence data to a matK + trnL-F ma- 100 MELI Melia trix in eudicots (Worberg & al., 2007), the addition of trnK drastically improved the signal, especially for deeper nodes 100 ANAC Schinus 100 99 (e.g., raising the JK support for rosids from 88% to 97%). 100 SIMA Brucea Sapindales The results presented here were based on analyses of all putative genera of the Huerteales clade. Gerrardina is SAPIN Acer consistently inferred as sister to all other genera (Figs. 1–3) ZYGO Larrea 62 whereas the detailed relationships within the clade com- 77 GERAN Erodium prised of Perrottetia, Dipentodon, Huertea and Tapiscia await additional sequence data. Both Huertea and Tapiscia MYRT Eucalyptus share a large number of indels in trnL-F and trnK/matK CORI Coriaria that further underscores their close relationship. Whereas

CELAS Salacia Outgroup trnK/matK depict Dipentodon and Perrotetia as sisters with 99% JK (100 PP) support, trnL-F signal is inconsis- ROSAC Malus tent (75% JK) and therefore not conclusive to this question. STACH Stachyurus Perrottetia, which previously had been classified within Celastraceae, does not belong in this any- Fig. 3. Combined strict consensus tree based on substitu- more. Matthews & Endress (2005) found substantial floral tions and indels of trnL-intron and trnL-F intergenic spacer structural differences between Perrottetia and Celastrales. region for a smaller sampling, inferred with MP. Bold val- ues above branches are Jackknife percentages with indels While reconstructing Celastrales phylogeny, Zhang & coded, plain ones below are Jackknife values without sim- Simmons (2006) found Perrottetia among the outgroup ple indel coding applied. taxa of their taxon set. Zhang & Simmons (2006) further

473 Worberg & al. • Phylogenetic position of Huerteales TAXON 58 (2) • May 2009: 468–478

investigated the position of Perrottetia by including newly Huerteales has been modified drastically in its short life- generated 18S, atpB and rbcL sequences into the 567-taxon span, phylogenetic studies utilizing morphological data dataset of Soltis & al. (2000) and found Perrottetia as sister or mapping morphological features onto molecular trees to the monotypic Tapiscia with 100% JK support. Our study have not incorporated all of the relevant genera (cf. Soltis supports these results, placing the two Perrottetia species & al., 2005; Ronse de Craene & Haston, 2006). with high statistical confidence into the Dipentodontaceae The highly supported resolution resulting from this clade (JK 98%, 100 PP) sister to Dipentodon sinicus. Alford study now permits the formation of preliminary hypoth- (2006) recovered Gerrardinaceae in the malvid clade using eses about the evolution of the order and highlights types rbcL+atpB sequences from Soltis & al. (2000) with the of data that may be useful in further clarifying morpho- addition of Gerrardina foliosa and Dipentodon sinicus to logical evolution within the eurosids II. Table 1 summa- the sampling. Although Gerrardinaceae were recovered in rizes the current state of knowledge on morphology and a clade of Huerteales + Malvales + Brassicales (with 70% anatomy of Huerteales taxa and is the basis for our discus- JK), its relationships among those orders were unresolved. sion. Within Huerteales, Dipentodon and Perrottetia are While ndhF-sequences (3′ end only), for which less com- sister, a relationship suspected even in morphological and parative data are available, showed an affinity to Tapiscia, pre-phylogenetic works (e.g., Dunn, 1911; Fischer, 1941; Dipentodon, Brassicales, and Malvales, analyses of atpB Liu & Cheng, 1991). They both have stipulate, toothed and rbcL sequences did not unambiguously reveal the rela- leaves, poorly differentiated perianth ( and petals tionship of Gerrardinaceae within the eurosids II. similar), a nectary disk, and similar wood anatomy. Zhang Phylogenetic relationships in the malvids ( = eurosids & Simmons (2006) recognize them together in the family II) are depicted in our study as Brassicales being sister Dipentodontaceae. Huertea and Tapiscia are not obvi- to Malvales, with Huerteales being sister to the Brassi- ous relatives of Dipentodon and Perrottetia morphologi- cales + Malvales clade. Finally, Sapindales are shown to cally, but they do share scalariform perforation plates in be sister of all remaining malvids. The latter topology the vessels (Carlquist & Hoekman, 1985), septate fibers was already hypothesised by partial matK sequences (lacking in Perrottetia), number equal to the (Hilu & al., 2003), although no member of the Huerteales number, and stamens alternating with the petals. Unlike was represented. The three gene analysis of Soltis & al. Dipentodontaceae, Huertea and Tapiscia have compound (2000) could not resolve relationships among Malvales, leaves, a calyx tube, and clearly differentiated calyx and Sapindales, Brassicales and Tapiscia using maximum par- corolla. Tapiscia also lacks a nectary disk. Previously rec- simony. Huerteales were represented by Tapiscia alone in ognized as a subfamily of the Staphyleaceae, Huertea and Soltis & al. (2000). However, in their subsequent Bayes- Tapiscia are now recognized as the family Tapisciaceae. ian analysis of the same dataset, Soltis & al. (2007) ar- Rarely, the two genera have been placed in monogeneric rived at a different malvid topology, with Tapiscia sister families, Tapisciaceae and Huerteaceae (Doweld, 2001). to Brassicales (unsupported), and this clade being sister to The final member of Huerteales is the monogeneric fam- a well-supported Malvales + Sapindales clade. Similarly, ily Gerrardinaceae. Like other Huerteales, Gerrardina the recent addition of mitochondrial matR sequences to a has toothed leaves, cymose inflorescences, a rbcL + atpB + 18SrDNA dataset (Zhu & al., 2007) indicated (short in Tapiscia and Huertea), a unilocular with a position of Tapiscia sister to Brassicales, albeit without 1–2 ovules per carpel, and small embryos. Although not support. The presented analyses, however, clearly resolve present in all members of Huerteales, Gerrardina also the Huerteales sister to Malvales and Brassicales irrespec- has stipules (lacking in Huertea), mucilage cells (lacking tive of the optimization criterion with strong support. in Dipentodon), and a nectary disk (lacking in Tapiscia). As a taxonomic order, Huerteales is a recent creation Gerrardina differs from the other members of Huerteales based almost entirely on analyses of DNA sequence data in having stamens opposite the petals. (Soltis & al., 2000; Doweld, 2001; Peng & al., 2003). The Comparison of the features of Huerteales and the other taxa of Huerteales, in general, are poorly known and have orders of the eurosids II (Sapindales, Malvales, Brassicales) been considered as peripheral members of various fami- may provide clues for uncovering potential synapomorphies lies, often shuffled among notoriously diverse families for Huerteales. Wood anatomy seems a good place to start. such as Celastraceae and Flacourtiaceae. Much of the de- Sapindales, Malvales, and Brassicales usually have simple tailed morphological and anatomical work for adequate perforation plates and axial parenchyma in the wood, but comparisons, however, remains to be done. In particular, Huerteales have scalariform plates (rarely simple in Per- Gerrardina is lacking study of wood anatomy, and data rottetia ; unknown for Gerrardina) and lack axial paren- on seed anatomy and secondary chemistry for all of the chyma (except Perrottetia, where it is paratracheal; again, genera are scarce. Thus, one would not be surprised to unknown for Gerrardina). Although nectary disks are com- learn that morphological synapomorphies for Huerteales mon in Sapindales, they are not usually associated with a are not yet clear. Furthermore, since the composition of hypanthium (albeit short in Tapisciaceae), as in Huerteales.

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Table 1. Representative anatomical and morphological features of the taxa included in Huerteales, as well as predominant states for the related orders Sapindales, Malvales, and Brassicales. Feature Gerrardina Tapiscia Huertea Dipentodon Perrottetia Sapindales Malvales Brassicales Vessel perforation plates ? Scalari - Scalari- Scalari- Scalari- Simple Simple Simple form form form form (rarely simple) Septate fibers ? Present Present Present Absent Often Absent Absent or present present (then plesiomorphic) Axial parenchyma in wood ? Absent Absent Absent Present Present Present Present Mucilage cells Present Present Present Absent Present Sometimes Present Absent present (except Cistaceaee) Leaf complexity Simple Compound Compound Simple Simple Simple or Simple or Simple or compound compound compound Stipules Present Present Present Present Present Absent Present Usually (modified)/ absent absent aestivation Imbricate Imbricate +/– valvate Valvate Open to Imbricate Valvate Mostly valvate imbricate Hypanthium (floral cup) Present Present Present Present Present Usually Absent Usually (but short) absent absent Nectary disk Present Absent Present Present Present Present Absent Absent Ovary Unilocular Unilocular Unilocular Unilocular Unilocular Multi- Usually Usually (with basal (with apical locular multi - multi- septum) septum) locular locular Carpels 2 22322–5(–6) 2–5(–many) 2–6(–12) Ovules/carpel 2 11221–21–∞ 1–2(–∞) Placentation Apical Basal Axile/ Free-central Basal and Axile, Axile Axile or basal-axile lateral basal-axile, parietal apical-axile Embryo Straight Straight Straight Straight ? Straight or Folded Straight or curved curved General information compiled from Metcalfe & Chalk (1950), Cronquist (1981), Takhtajan (1997), Stevens (2001 onwards), and Ronse de Craene & Haston (2006). Specific data for Gerrardina from Alford (2006); for Tapiscia and Huertea from Krause (1942), Carlquist & Hoekman (1985), and Dickison (1986); for Dipentodon from Dunn (1911), Merrill (1941), Liu & Cheng (1991), and Zhang & Gao (1995); and for Perrottetia from Matthews & Endress (2005).

The nectary disks in Huerteales are non-vascularized or the order Huerteales undoubtedly as a monophyletic only supplied by phloem (Dickison, 1986; Matthews & group of families with ordinal level that has a distinct Endress, 2005). Huerteales also share unilocular ovaries, position within the malvid grade with high support. although this feature may occasionally occur in the other Thus we support an angiosperm classification system orders. Some characters show remarkable diversity within with the order Huerteales included into the rosids. Huerteales. Leaf complexity, presence/absence of stipules, and position of stamens have already been mentioned, but the order also includes taxa with imbricate, valvate, and open sepal aestivation, uniseriate and homocellular to mul- ACKNOWLEDGEMENTS tiseriate and heterocellular wood rays, and apical, basal, We acknowledge support by the Deutsche Forschungs- partially axile, and free-central placentation. gemeinschaft (DFG) for the project “Mutational dynamics of APG (2003) treat Tapisciaceae as an unplaced fam- non-coding genomic regions and their potential for reconstruct- ily in malvids. In addition to insufficient tree resolution, ing eudicot evolution” (grants BO1815/2 to T.B. and QU153/2 earlier analyses were also incomplete in taxon sampling, to D.Q.). Most of the material was provided by the living col- thereby hindering the development of a strongly sup- lections of the Botanical Gardens of Bonn University and ported ordinal classification. We therefore recognise Dresden Technical University. The authors like to thank Peter

475 Worberg & al. • Phylogenetic position of Huerteales TAXON 58 (2) • May 2009: 468–478

Stevens for helpful comments on the manuscript. For vari- Dunn, A. 1911. Dipentodon, a new genus of uncertain system- ous kinds of support and helpful discussions we are grateful atic position. Bull. Misc. Inform. Kew 1911: 310–313. to Wilhelm Barthlott (Bonn), Mark W. Chase (K), Jay Horn Fischer, C.E.C. 1941. Contributions to the flora of Burma: XVIII. Kew Bull. 1940: 282–294. (FTG), Nadja Korotkova (Bonn), Xia Lei (Cornell University), Freudenstein, J.V. & Simmons, M.P. 2004. Relative effects of Anna-Magdalena Barniske (Dresden), Kai Müller (Bonn). We increasing genetic distance on alignment and phylogenetic would like to express our sincere thanks to Armando Urquiloa analysis. Cladistics 20: 83–83. (Jardín Botánico de Pinar del Río), Ramona Oviedo (HAC) Gilg, E. 1925. Flacourtiaceae. Pp. 377–457 in: Engler, A. & and Werner Greuter (B) in organizing to collect fresh material Prantl, K. (eds.), Die natürlichen Pflanzenfamilien, ed. 2, of Huertea cubensis, and to Angela T. Leiva Sánchez, Jardín vol. 21. Duncker & Humblot, Berlin. Botánico Nacional, Havana, for arranging the permission to Hilu, K.W., Borsch, T., Müller, K., Soltis, D.E., Soltis, P.S., Savolainen, V., Chase, M., Powell, M., Alice, L.A., export material. Helpful comments of three anonymous reviews Evans, R., Sauquet, H., Neinhuis, C., Slotta, T.A., Ro- are gratefully acknowledged. T.B. acknowledges a Heisenberg hwer, J.G., Campbell, C.S. & Chatrou, L. 2003. Angio- scholarship by the Deutsche Forschungsgemeinschaft (DFG) sperm phylogeny based on matK sequence information. that greatly facilitated his work on angiosperm phylogenetics. Amer. J. Bot. 90: 1758–1776. This study is in partial fulfilment for the requirements to obtain Johnson, L.A. & Soltis, D.E. 1994. MatK DNA sequences and a Ph.D. from Bonn University by the first author. phylogenetic reconstruction in Saxifragaceae sensu stricto. Syst. Bot. 19: 143–156. Johnson, L.A. & Soltis, D.E. 1995. 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Appendix 1. Taxa and GenBank/EMBL/DDBJ accession numbers of the sequences used in this study. Species in alphabetical order and their corresponding family following APG II (2003), voucher information, garden or field origin and GenBank/ EMBL/DDBJ accession numbers of matK, trnL-F and trnK with corresponding citations. New sequences generated in this study are labeled with “this study”. Dashes indicate missing data. Acer campestre L., Sapindaceae, EMBL/GenBank, – , AJ438793 Bittkau & Mueller-Starck (2002), AF401189 Tian & al. (2001), AJ438793 Bittkau & Mueller- Starck (2002); Aextoxicon punctatum Ruiz & Pav., Aextoxicaceae, T. Borsch 3459 (BONN), BG Bonn, DQ182342 Müller & al. (2006), – , DQ182342 this study; Ailanthus altissima Swingle, Simaroubaceae, A. Worberg 025 (BONN), BG Bonn, FM179922 this study, – , FM179922 this study; Bixa orellana L., Bixaceae, A. Worberg 040 (BONN), BG Bonn, FM179929 this study, FM179540 this study, FM179929 this study; Bombax malabaricum DC., Bombacaceae, EMBL/GenBank, – , – , AY328149 Yuan & al. (2003), – ; Brucea javanica (L.) Merr., Simaroubaceae, EMBL/GenBank, – , – , AB365015, AB365024 Tanaka & al. (2007), – ; Chrysosplenium alternifolium L., Saxifragaceae, T. Borsch s.n. (BONN), Germany, AM396496 Worberg & al. (2007), – , AM396496 this study; Cistus ladanifer L., Cistaceae, A. Worberg s.n. (BONN), BG Bonn, FM179939 this study, FM179538 this study, FM179939 this study; Citrus sinensis Osbeck, Rutaceae, EMBL/GenBank, – , NC_008334 Bausher & al. (2006), NC_008334 Bausher & al. (2006), NC_008334 Bausher & al. (2006); Combretum molle R. Br. ex G. Don, Bombacaceae, IPEN-xx-0-B-1560480, BG Berlin, FM179938 this study, – , FM179938 this study; Coriaria myrtifolia L., Coriari- aceae, T. Borsch 3415 (BONN), BG Bonn, AF542600 Worberg & al. (2007), AM397179 Worberg & al. (2007), AF542600 this study; Cucumis sativus L., Cucurbitaceae, EMBL/GenBank, – , DQ119058 Kim & al. (2006), – , DQ119058 Kim & al. (2006); Daphne bholua Buch.-Ham. ex D. Don, Thymeleaeceae, A. Worberg 026 (BONN), BG Bonn, FM179927 this study, – , FM179927 this study; Daucus carota L., Apiaceae, EMBL/GenBank, – , DQ898156 Ruhl- man & al. (2006), – , DQ898156 Ruhlman & al. (2006); Dipentodon sinicus Dunn, Dipentodontaceae, EMBL/GenBank, – , AJ429397 Bremer & al. (2002), AJ430865 Bremer & al. (2002), – ; Durio zibethinus Murr., Bombacaceae, EMBL/GenBank, – , AY321188 Nyffeler & al. (2005), – , AY321188 Nyffeler & al. (2005); Erodium cicutarium (L.) L’Hér, Geraniaceae, T. Borsch 3483 (BONN), Germany, Eifel, AM396500 Worberg & al. (2007), AM397178 Worberg & al. (2007), AM396500 this study; Eucalyptus globulus Labill., Myrtaceae, EMBL/GenBank, – , NC_008115 Steane (2005), NC_008115 Steane (2005), NC_008115 Steane (2005); Euphorbia milii Desmoul., Euphorbiaceae, A. Worberg 002 (BONN), BG Bonn, FM179936 this study, – , FM179936 this study; Gerrardina foliosa Oliv., Gerrardinaceae, Balkwill & al. 11983, KEW, FM179924 this study, FM179535 this study, FM179924 this study; Glycine max Merr., Fabaceae, EMBL/GenBank, – , DQ317523 Saski & al. (2005), – , DQ317523 Saski & al. (2005); Gossypium hirsutum L., Malvaceae, EMBL/GenBank, – , NC_007944 Lee & al. (2006), NC_007944 Lee & al. (2006), NC_007944 Lee & al. (2006); Gunnera tinctoria (Molina) Mirb., Gunneraceae, N. Korotkova 50 (BONN), BG Bonn, AM396506 Worberg & al. (2007), – , AM396506 this study; Huertea cubensis Griseb., Tapisciaceae, R. Oviedo & al. s.n. (B, HAC), Cuba, FM179926 this study, FM179539 this study, FM179926 this study; Ilex aquifolium L., Aquifoliaceae, T. Borsch 3419 (BONN), BG Bonn, AF542607 Worberg & al. (2007), – , AF542607 this study; Impatiens noli-tangere L., Balsaminaceae, T. Borsch 3485 (BONN), BG Bonn, AF542608 Worberg & al. (2007), – , AF542608 this study; Juglans microcarpa Berland., , EMBL/GenBank, – , AF118034 Stanford & al. (2000), – , AF118034 Stanford & al. (2000); Kandelia candel Druce, Rhizophoraceae, EMBL/GenBank, – , AF105090 Shi & al. (1999), – , – ; Larrea tridentata Coult., Zygophyllaceae, A. Worberg 012 (BONN), BG Bonn, AM396502 Worberg & al. (2007), AM397180 Worberg & al. (2007), AM396502 this study; Leea coccinea Planch., Leeaceae, T. Borsch 3418 (BONN), BG Bonn, AM396497 Worberg & al. (2007), – , AM396497 this study; Lobularia maritime L. (Desv.), Brassicaceae, EMBL/GenBank, – , AP009375 Hosouchi & al. (2007), AP009375 Hosouchi & al. (2007), AP009375 Hosouchi & al. (2007); Malus trilobata C.K. Schneid., Rosaceae, EMBL/ GenBank, – , DQ860463 Campbell & al. (2006), DQ863235 Campbell & al. (2006), DQ860463 Campbell & al. (2006); Melastoma septemnervium Jacq., Melastomataceae, T. Borsch s.n. (BONN), BG Bonn, FM179940 this study, – , FM179940 this study; Melia azedarach L., Meliaceae, A. Worberg 035 (BONN), BG Bonn, FM179921 this study, FM179536 this study, FM179921 this study; Muntingia calabura L., Muntingiaceae, EMBL/GenBank, – , – , AY328166 Yuan & al. (2003), – ; Muntingia calabura L., Muntingiaceae, D. Quandt s.n. (BONN), BG Bonn, FM179930 this study, – , FM179930 this study; Myrothamnus flabellifolia Welw., Myrothamnaceae, A. Worberg 011 (BONN), BG Bonn, AM396507 Worberg & al. (2007), – , AM396507 this study; Myrothamnus mos- chata Baill., Myrothamnaceae, E. Fischer & W. Höller s.n. (BONN), BG Bonn, AF542591 Worberg & al. (2007), – , AF542591 this study; Oenothera elata Kunth, Onagraceae, EMBL/GenBank, – , NC_002693 Hupfer & al. (2000), – , NC_002693 Hupfer & al. (2000); Oxalis hedysaroides Kunth, , N. Korotkova 55 (BONN), BG Bonn, FM179935 this study, – , FM179935 this study; Panax ginseng C.A. Mey., Araliaceae, EMBL/GenBank, – , NC_006290 Kim & Lee (2004), – , NC_006290 Kim & Lee (2004); Parnassia palustris L., Parnassiaceae, EMBL/GenBank, – , AY935911 Zhang & Simmons (2006), – , – ; Passiflora quadrangularis L., Passifloraceae, N. Korotkova 56 (BONN), BG Bonn, FM179937 this study, – , FM179937 this study; Perrottetia ovata Hemsl., Dipentodontaceae, EMBL/GenBank, – , AY935916 Zhang & Simmons (2006), AY935771 Zhang & Simmons (2006), – ; Perrottetia longistylis Rose, Dipent- odontaceae, EMBL/GenBank, – , AY935915 Zhang & Simmons (2006), AY935770 Zhang & Simmons (2006), – ; Polygala californica Nutt. ex Torr. & A. Gray, Polygalaceae, EMBL/GenBank, – , AY386842 Wojciechowski & al. (2004), – , – ; Populus alba L., , EMBL/GenBank, – , AP008956 Okumura & al. (2005), – , AP008956 Okumura & al. (2005); Reseda lutea L., Resedaceae, A. Worberg 027 (BONN), BG Bonn, FM179932 this study, – , FM179932 this study; Salacia lehmbachii Loes., Celastraceae, T. Borsch 3549 (BONN), BG Bonn, AF542599 this study update, FM179534 this study, AF542599 this study; Schinus molle L., Anacardiaceae, EMBL/GenBank, – , – , AY640463 Yi & al. (2004), – ; Schinus molle L., Anacardiaceae, A. Worberg s.n. (BONN), BG Bonn, FM179923 this study, – , FM179923 this study; Stachyurus chinensis Franch., Stachyuraceae, A. Worberg s.n. (BONN), BG Bonn, AM396501 Worberg & al. (2007), – , AM396501 this study; Stachyurus chinensis Franch., Stachyuraceae, EMBL/GenBank, – , – , AB066335, Ohi & al. (2003), – ; Spiraea thunbergii Siebold ex Blume, Rosaceae, A. Worberg s.n. (BONN), BG Bonn, FM179934 this study, – , FM179934 this study; Tapiscia sinensis Oliver, Tapisciaceae, T. Borsch s.n. (BONN), BG Bonn, FM179925 this study, FM179541 this study, FM179925 this study; Tropaeolum majus L., Tropaeolaceae, A. Worberg s.n. (BONN), BG Bonn, FM179931 this study, – , FM179931 this study; Urtica cannabina L., Urticaceae, A. Worberg s.n. (BONN), BG Bonn, FM179933 this study, – , FM179933 this study; Vasconcellea parviflora A. DC., Caricaceae, A. Worberg 038 (BONN), BG Bonn, FM179928 this study, FM179537 this study, FM179928 this study; Vitis riparia A. Gray, Vitaceae, T. Borsch 3458 (BONN), BG Bonn, AF542593 Worberg & al. (2007), – , AF542593 this study.

478 TAXON 58 (2) • May 2009: E1 Worberg & al. • Phylogenetic position of Huerteales

Appendix 2. List of primers used in this study.

Primers used for the amplification of matK along with their sequences and the taxa for which they were designed. References are given for primers that were not designed for this study. Primer name Sequence Taxa Reference MG15 ATC TGG GTT GCT AAC TCA ATG Angiosperms Liang & Hilu (1996) MG1 AAC TAG TCG GAT GGA GTA GAT Angiosperms Liang & Hilu (1996) trnKFbryo GGG TTG CTA ACT CAA TGG TAG AG Land plants Wicke & Quandt (in press) trnK3914Fdi GGG GTT GCT AAC TCA ACG G Angiosperms Johnson & Soltis (1995) trnK2R AAC TAG TCG GAT GGA GTA G Angiosperms Johnson & Soltis (1995) psbA-R CGC GTC TCT CTA AAA TTG CAG TCA T Angiosperms Steele & Vilgalys (1994) NYmatK480F CAT CTG GAA ATC TTG STT C Angiosperms Borsch (2000) ARmatK660R AYG GAT TCG CAT TCA TA Angiosperms Wanke & al. (2006a) ARmatK1200F TTC CAA AGT CAA AAG AGC G Angiosperms Wanke & al. (2006a) ARmatK2400R ATT TTC TAG CAT TTG ACT CC Angiosperms Wanke & al. (2006a) ASmatK460F TAC TTC CCT TTT T(ACT)G AGG Angiosperms Wanke & al. (2006a) ACmatK873R ATA TAC TCC TGA AAG AGA AGT GG Eudicots Müller & Borsch (2005) ACmatK100F CTC GAC TGT ATC AAC AGA ATC Proteaceae Müller & Borsch (2005) ACmatK1400R GGA TTC GTA TTC ACA TAC A Aquifoliaceae Müller & Borsch (2005) ACmatK634R ATA GGA ACA AGA ATA ATC T Basal eudicots Müller & Borsch (2005) ACmatK1746F CTT CAA AAG GGA CAT CTC TTC TG Aextoxicaceae Müller & Borsch (2005) ACmatK2231R TCT TCC AAA AAT TCT GAA CCT Cercidiphyllaceae Müller & Borsch (2005) ACmatK499F CCT CKT CTT TGC ATT TAT TAC G Stachyuraceae Müller & Borsch (2005) PImatK1060F ACT T(AG)T GGT CTC AAC (CT)G Trochodendraceae Wanke & al. (2006b) PImatK1480F TCG TAA ACA (CT)AA AAG TAC Papaveraceae Wanke & al. (2006b) ROSmatK655R GGA TTC GTA TTC ACA TAC AT Rosids Worberg & al. (2007) ROSmatK530F AGA TGC CTC TTC TTT GC Rosids Worberg & al. (2007) DIDYmatK1035R CGA ACT CGT AAA GAC TCG A Didymelaceae Worberg & al. (2007) DIDYmatK1107F CAA TTA CTC CTC TGA TTG GAT C Didymelaceae Worberg & al. (2007) DIDYmatK570F CGA GTA TCA GAA CTG GAG Didymelaceae Worberg & al. (2007) EUPTmatK1006F GGC TAT CTT TCA AGT GTA CG Eupteleaceae Worberg & al. (2007) NYmatK980R GGT TAG AAT CAT TAG CRG Rosids Worberg & al. (2007)

Primers used for the amplification of trnK along with their sequences and the taxa for which they were designed. References are given for primers that were not designed for this study. Primer name Sequence Taxa Reference trnK-f bryo GGG TTG CTA ACT CAA TGG TAG AG Angiosperms Wicke & Quandt (in press) trnK-2R AAC TAG TCG GAT GGA GTA G Angiosperms Johnson & Soltis (1995) MG15 ATC TGG GTT GCT AAC TCA ATG Angiosperms Liang & Hilu (1996) psbAR CGC GTC TCT CTA AAA TTG CAG TCA T Angiosperms Steele & Vilgalys (1994)

Primers used for the amplification of trnL-F region along with their sequences and the taxa for which they were designed. References are given for primers that were not designed for this study. Primer name Sequence Taxa Reference trnTc CGA AAT CGG TAG ACG CTA CG Angiosperms Taberlet & al. (1991) trnTf ATT TGA ACT GGT GAC ACG AG Angiosperms Taberlet & al. (1991) trnTd GGG GAT AGA GGG ACT TGA AC Angiosperms Taberlet & al. (1991) trnL110R GAT TTG GCT CAG GAT TGC CC Angiosperms Borsch & al. (2003) trnL460F GAG AAT AAA GAT AGA GTC C Angiosperms Worberg & al. (2007)

E1

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