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A comprehensive phylogenetic analysis of : Evidence from nuclear (ITS) and plastid (psbA-trnH and trnL-F) DNA sequences

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A comprehensive phylogenetic analysis of Eriocaulaceae: Evidence from nuclear (ITS) and plastid (psbA-trnH and trnL-F) DNA sequences

Maria José Gomes de Andrade,1 Ana Maria Giulietti,1 Alessandro Rapini,1 Luciano Paganucci de Queiroz,1 Adilva de Souza Conceição,1,2 Paulo Ricardo Machado de Almeida1 & Cássio van den Berg1

1 Programa de Pós-Graduação em Botânica and Laboratório de Sistemática Molecular de Plantas, Departamento de Ciências Biológicas, Universidade Estadual de Feira de Santana, Av. Transnordestina s/n. Novo Horizonte, 44.036-900, Feira de Santana, Bahia, Brazil 2 Departamento de Ciências, Centro de Ensino Superior de Paulo Afonso, Universidade do Estado da Bahia, Rua do Gangorra 503, CHESF, 48.620-410, Paulo Afonso, Bahia, Brazil Author for correspondence: Maria José Gomes de Andrade, [email protected]

Abstract The Eriocaulaceae are easily recognized because of the small unisexual flowers in long-pedunculate heads and spiraperturate pollen grains. Their monophyly has never been disputed but internal relationships within the family have not been broadly explored and genera are typically distinguished by few floral characters. Here, we present the first comprehensive phylogenetic study of Eriocaulaceae based on individual and combined molecular datasets, including the plastid psbA-trnH and trnL-F and the nuclear ITS. Results are largely congruent among DNA regions and support the internal dichotomy between the two subfamilies: Eriocauloideae and Paepalanthoideae. Eriocaulon and Leiothrix are strongly supported as monophyletic, whereas , Blastocaulon, and Syngonanthus are not monophyletic. The phylogenetic nature of Actinocephalus is not resolved and Lachnocaulon, Mesanthemum, Philodice, Rondonanthus, and Tonina (monospecific) are represented in our analyses by a single species each. Based on our results, we suggest two principal generic realignments in the family. The first is the division of Syngonanthus in two genera: Syngonanthus s.str., including Philodice (conserving the former name against the latter), and Comanthera, which is being reinstated to include two sections segregated from Syngonanthus: S. sect. Eul- epis and S. sect. Thysanocephalus. The second realignment suggested is the amalgamation of Actinocephalus, Blastocaulon, Lachnocaulon, and Tonina into Paepalanthus s.l. Three ‘stat. nov.’ within Paepalanthus are published (P. ser. Leptocephali, P. ser. Rosulati, and P. ser. Dimeri).

Keywords molecular phylogeny; Paepalanthus; Syngonanthus; systematics; taxonomy

INTRODUCTION with isostemonous flowers and eglandular petals, including: Blastocaulon Ruhland, Lachnocaulon Kunth, Leiothrix Ruh- The Eriocaulaceae are a monophyletic family classified in land, Paepalanthus Mart., Philodice Mart., Syngonanthus Ruh- (Chase & al., 1995; Linder & Kellogg, 1995; Stevenson land, and Tonina Aubl. Six other genera have been proposed & Loconte, 1995; Givnish & al., 1999; Giulietti & al., 2000; after Ruhland’s treatment: Moldenkeanthus P. Morat (sy nony- Bremer, 2002; APG III, 2009). They are easily characterized mized to Paepalanthus ; Stützel, 1987), Wurdackia Moldenke by graminoid leaves, small unisexual flowers grouped in dense (synonymized to Rondonanthus Herzog; Hensold & Giulietti, capitula, a 3- or 2-locular ovary (with a single pendulous ovule 1991), Comanthera L.B. Sm. and Carptotepala Moldenke per locule), and having spiraperturate pollen grains (Giulietti & (synonymized to Syngonanthus; Giulietti & Hensold, 1991), al., 1995, 2000). The family includes eleven genera and about Rondonanthus and Actinocephalus (Körn.) Sano. 1400 species occurring primarily in the Neotropics, especially Phylogenetic studies in Eriocaulaceae have been previ- in mountain areas or high plateaus. The Eriocaulaceae are more ously carried out based on morphological (Giulietti & al., diverse in savanna and “campos rupestres” vegetation in Brazil 1995, 2000) and unpublished molecular data (Unwin, 2004; and in “tepuis” in Guayana shield of Venezuela and Guyana. Andrade, 2007). Giulietti & al. (1995) undertook a preliminary The inter- and infrageneric relationships in Eriocaulaceae cladistic analysis based on 29 morphological characters and are not well resolved and the traditional generic circumscription eleven taxa representing eight genera, which was broadened remains based on few floral characters. These include: (1) de- to 49 morpho-anatomical characters and 25 taxa in Giulietti gree of petal union in pistillate flowers, (2) presence of glands & al. (2000). Unwin (2004) performed a phylogenetic analy- on petals, (3) number of stamens, and (4) number of pollen-sacs sis of Eriocaulaceae using sequences of trnL intron (58 taxa), per anther (Ruhland, 1903; Hensold, 1988; Giulietti & al., 1995, trnL-F spacer (58), and the gene ndhF (21), but Mesanthemum, 2000; Stützel, 1998). The last complete revision of the family Rondonanthus, and Philodice were not sampled and sampling (Ruhland, 1903) recognized two subfamilies: Eriocauloideae, within more diverse genera, such as Paepalanthus and Syngo- with diplostemonous flowers and glandular petals, including nanthus, was thin. Andrade (2007) performed the phylogenetic Eriocaulon L. and Mesanthemum Körn.; and Paepalanthoideae, analysis of Neotropical genera of the family. Therefore, the

379 Andrade & al. • Phylogenetics of Eriocaulaceae TAXON 59 (2) • April 2010: 379–388

present study aims at re-evaluating Eriocaulaceae classification were purified by enzymatic treatment using the EXOSAP-IT based on comprehensive molecular phylogenetic analyses using kit (GE Healthcare, USB, Cleveland, Ohio, U.S.A.). Sequenc- the nuclear ITS (ITS 1, 5.8S rRNA and ITS 2) and the plastid ing reactions were performed using the Big Dye Terminator regions trnL-F (trnL intron and trnL-trnF intergenic spacer) kit, version 3.1 (Applied Biosystems, Foster City, California, and psbA-trnH spacer. U.S.A.). Samples were sequenced in both directions using a SpectruMedix SCE2410 automatic sequencer according to the protocols supplied by the manufacturer. MATERIALS AND METHODS Sequence alignment and matrices. — Forward and re- verse electropherograms were superimposed and edited using Taxa sampled. — A total of 81 species (82 samples) was the STADEN Package software (Staden & al., 1998). The se- included in the analyses; all were sequenced for this study. The quences of psbA-trnH and trnL-F were subsequently aligned outgroup was composed of Abolboda pulchella Bonpl., Xyris using CLUSTAL X v.1.81 (Thompson & al., 1997). The initial anceps Lam., X. jupicai L.C. Rich., and X. lagoinhae Kral & alignment was then corrected manually according to the crite- L.B. Sm. (Xyridaceae). The 77 species of the ingroup represent ria of Kelchner (2000) for plastid regions. ITS alignment was all recognized genera of Eriocaulaceae and a great part of the in- performed exclusively by eye. Indels were considered missing frageneric taxa, according to Ruhland (1903). The rate of species data and regions with dubious alignment were excluded from sampled per genus was: Actinocephalus (6 species sampled/28 analyses. total species), Blastocaulon (3/5), Eriocaulon (5/479), Lachno- Phylogenetic analyses. — Maximum parsimony (MP) caulon (1/7), Leiothrix (9/64), Mesanthemum (1/15), Paepalan- analyses were performed using PAUP* v.4.0b10 (Swofford, thus (38/420), Philodice (1/2), Rondonanthus (1/6), Syngonanthus 2002) with Fitch parsimony (equal weights, unordered; Fitch, (11/199), and Tonina (1/1) (total species number according Go- 1971) as the optimality criterion. We performed individual vaerts, 2006) (Appendix; see this material for taxa authorship (psbA-trnH, trnL-F, ITS) and combined analyses (combined and three new series names in Paepalanthus). plastid and combined plastid + nuclear). For MP analyses, a Extraction, amplification, and sequencing of DNA. — heuristic search with 1000 replicates using random taxon-ad- DNA was extracted from silica-gel–dried leaves (5–15 mg) dition and tree bisection reconnection (TBR) swapping were or, in a few cases, fresh leaves or herbarium material using a performed, saving no more than 15 trees per replicate to pre- modified version of 2× CTAB (Doyle & Doyle, 1987) protocol. vent extensive swapping on large suboptimal islands. Trees The region psbA-trnH was amplified in a final reaction volume in memory were then used for another set of TBR swapping of 50 μL containing 1 μL of total DNA, 1× reaction buffer, with a limit of 50,000 trees. Non-parametric bootstrap support 2.5 mM MgCl2, 1 mM dNTPs, 0.5 μM of each primer, 1 μg (BS; Felsenstein, 1985) was estimated from 1000 bootstrap BSA, and 1 unit of Taq DNA polymerase (Phoneutria Ltda, replicates using simple addition sequences and TBR swapping. Belo Horizonte, Brazil). The region trnL-F was amplified in a Bayesian inference was perfomed using MRBAYES v.3.1 final reaction volume of 50 μL containing 1 μL of total DNA (Ronquist & Huelsenbeck, 2003), with the fittest evolutionary with PCR 1× reaction buffer, 2.5 mM MgCl2, 1 mM dNTPs, models as estimated by MrModeltest v.2.2 (Nylander, 2004); 0.5 μM of each primer, 0.5 μg BSA, and 0.25 units of Taq DNA data was partitioned in combined analyses. MRBAYES was polymerase. Most ITS amplifications were performed in a final run for 1,000,000 (psbA-trnH, trnL-F, ITS, and combined plas- reaction volume of 50 μL containing 1 μL of total DNA with tid) and for 1,500,000 (combined plastid + nuclear) generations PCR 1× reaction buffer, 2.5 mM MgCl2, 1 mM dNTPs, 0.5 μM with two sets of four chains, sampling a tree every 100 genera- of each primer, 1 μg BSA, 1.0 M betaine, 2% DMSO, and 0.25 tions. Instead of choosing burn-in by plotting the likelihood units of Taq DNA polymerase. Table 1 presents the reference scores against the number of generations, we discarded all trees for PCR primers and profiles of the three regions. PCR products obtained before the standard deviations between sets of chains

Table . PCR primers and profiles. Region Primers Reference psbA-trnH psbAF and trnHR Sang & al. (1997) trnL-trnF C and F Taberlet & al. (1991) ITS 75 and 92; 17SE and 26SE Desfeaux & al. (1996); Sun & al. (1994) PCR profile Initial denaturation Denaturation Annealing Extension Final extension Region temp./time temp./time temp./time temp./time temp./time No. of cycles psbA-trnH 94°C/3 min 94°C/30 s 53°C/40 s 72°C/40 s 72°C/5 min 40 trnL-trnF 94°C/3 min 94°C/60 s 50°C–52°C/60 s 72°C/2 min 72°C/5 min 35 ITS 75/92 94°C/3 min 94°C/45 s 56°C & 58°C/60 s 72°C/1 min 72°C/5 min 40 ITS 17/26 94°C/4 min 94°C/30 s 53°C/40 s 72°C/40 s 72°C/7 min 28

380 TAXON 59 (2) • April 2010: 379–388 Andrade & al. • Phylogenetics of Eriocaulaceae

reached a value < 0.01. This is a more strict way of guaranteeing Carphocephalus) form a clade (100/100) sister to Philodice that chains achieved stability. The majority rule consensus of (93–95/100). Actinocephalus, Blastocaulon, Tonina, and Lach- the remaining trees was calculated in PAUP*, with frequencies nocaulon are nested in Paepalanthus (J; 99–100/100). Clade J is of groups representing posterior probabilities. divided into two main lineages: Clade K (85–89/100), including Actinocephalus, Tonina, and Lachnocaulon, with the species of Paepalanthus subg. Thelxinoë and P. subg. Paepalanthus and RESULTS Clade N (89–99/100), including the species of Paepalanthus subg. Paepalanthus, P. subg. Xeractis, and P. subg. Platycaulon General features of DNA regions are summarized in Ta- plus the biphyletic Blastocaulon. ble 2. For Bayesian analysis, we excluded 400 trees (burn-in stage) from ITS analysis and 500 trees from the others (psbA- trnH, trnL-F, combined plastid, and combined plastid + nu- DISCUSSION clear). After visual inspection of single region analyses, we noticed that the position of Paepalanthus myocephalus, P. ses- Stützel (1985) proposed a third subfamily in Eriocaulaceae, siliflorus, and P. bifidus was incongruent and for this reason Syngonanthoideae, defined by syngonanthoid corolla fusion in they were not included in combined analyses. Most results female flowers and including Mesanthemum, Philodice, Syn- and discussion are based on the combined analyses (Figs. 1 gonanthus, and Wurdackia. Syngonanthoideae and Paepalan- and 2; see also Figs. S1–S3 in the Electronic Supplement to thoideae (petals free in female flowers; including Blastocaulon, this article). Lachnocaulon, Leiothrix, Paepalanthus, Rondonanthus, and The monophyly of Eriocaulaceae (clade A; BS 100/PP Tonina) were considered to be independently derived from 100) and its subfamilies Eriocauloideae (B; 99–100/100) and Eriocauloideae (including only Eriocaulon, with free petals Paepalanthoideae (C; 100/100) are strongly supported. In of both sexes of flowers). Hensold & Giulietti (1991) included Eriocauloideae, Mesanthemum radicans appears as a sister species with female flowers with free petals or with syngonan- to Eriocaulon (100/98). In Paepalanthoideae, the position of thoid corolla in Rondonanthus. They did not accept Syngonan- Rondonanthus capillaceus is uncertain, appearing as sister thoideae since it would require parallel evolution of the highly to the rest of the subfamily (C; 50/96) in the combined plastid modified gynoecia with stylar appendages (branched style with analysis, but nested in it (70/82). It also appears as being sister both stigmatic and nectariferous portions; Rosa & Scatena, to clade J (99–100/100) in combined plastid + nuclear analysis. 2007) and independent reductions of androecial whorls. They The monophyly of Leiothrix (I; 100/100) is strongly supported also stressed that evolution of free petals in Paepalanthus from as well as for the clade composed of Tonina f luviatilis and a species with syngonanthoid fusion would be simpler than Lachnocaulon anceps (L; 80–100/99–100). Syngonanthus is assuming several parallel evolutions to the reverse state. Our not monophyletic in any analysis: S. vernonioides (S. sect. Thy- results cannot unambiguously demonstrate how petal fusion sanocephalus) is sister to S. sect. Eulepis (100/100), whereas evolved in Eriocaulaceae, but they explicitly refute the mono- S. arenarius (S. sect. Syngonanthus) and S. caulescens (S. sect. phyly of Syngonanthoideae (sensu Stützel, 1985).

Table . Dataset and parsimony-based tree statistics for psbA-trnH, trnL-F and ITS analyses of Eriocaulaceae. Combined Plastid Nuclear + Plastid Characteristic psbA-trnH trnL-F ITS (psbA-trnH, trnL-F) (ITS, psbA-trnH, trnL-F) No. taxa 82 75 73 79 71 Aligned length (bp) 820 1418 1039 2238 3277 Characters included 688 1319 874 2007 2881 Ingroup minimum length (bp) 579 [Phh*] 626 [Roc*] 639 [Pax*] Ingroup maximum length (bp) 683 [Pae*] 1075 [Erl*] 877 [Pas*] Constant characters 342 756 236 1113 1410 Variable characters 346 563 638 894 1471 Parsimony informative characters 182 (26.4%) 360 (27.3%) 536 (61.3%) 526 (26.2%) 1023 (35.5%) # trees obtained 50,000 50,000 246 50,000 16 Tree length 588 1209 2575 1746 4005 CI 0.76 0.68 0.51 0.71 0.60 RI 0.88 0.84 0.81 0.85 0.83 * Codes for taxa: Erl: Eriocaulon ligulatum; Pae: Paepalanthus erigeron; Pas: Paepalanthus sessiliflorus; Pax: Paepalanthus extremensis; Phh: Philodice hoffmannseggii; Roc: Rondonanthus capillaceus.

381 Andrade & al. • Phylogenetics of Eriocaulaceae TAXON 59 (2) • April 2010: 379–388

OUTGROUPS XY jupicai AB pulchella XY lagoinhae XY anceps ME radicans 100 ER cinereum B 99 100 ER linearifolium 98 88 ER decangulare 88 100 ER ligulatum 92 ER modestum RO capillaceus A 100 SY vernonioides sect. Thysanocephalus 100 100 SY aciphyllus E 100 SY nitidus 100 SY bisulcatus 100 SY elegans sect. Eulepis 100 SY harleyi 100 99 SY curralensis C 100 82 82 SY hatschbachii SY mucugensis 100 PH hoffmannseggii 100 100 PH hoffmannseggii H 93 100 SY arenarius sect. Syngonanthus 100 SY caulescens sect. Carphocephalus D 96 G 100 LT angustifolia 50 100 100 LT flavescens 100 100 71 LT distichoclada I 96 Leiothrix 100 LT sp. nov. 100 LT curvifolia 98 99 LT crassifolia 65 100 LT flagellaris 100 100 LT arrecta Stephanophyllum 100 LT vivipara 99 LC anceps L 80 TO fluviatilis 100 PP lamarckii 100 100 100 PP tortilis ser. Leptocephali F K 85 100 PP obtusifolius Paepalanthus PP stannardii subsect. Polyactis PP repens M 100 93 100 PP exiguus sect. Conodiscus 96 100 PP leucocephalus 99 PP scleranthus Thelxinoë PP elongatus ser. Dimeri 100 100 100 PP giganteus sect. Diphyomene Paepalanthus 98 100 PP extremensis subsect. Polyactis 82 PP trichophyllus Sect. Diphyomene AC bongardii AC ramosus Genera: 100 AC aff. ramosus J 100 99 PP glareosus 100 subsect. Aphorocaulon Mesanthemum PP aff. bahiensis Paepalanthus Eriocaulon 100 AC brachypus 86 AC ciliatus Rondonanthus AC stereophyllus PP macrocaulon subsect. Polyactis Syngonanthus PP neglectus ser. Paepalanthus Philodice 89 PP spathulatus PP cinereus subsect. Polyactis Paepalanthus Leiothrix PP erigeron 100 PP regalis ser. Paepalanthus 98 PP pulchellus subg. of Leiothrix 100 98 62 N 89 PP pulvinatus ser. Rosulati Lachnocaulon 100 BL albidum 100 Bl rupestre Tonina 100 PP sphaerocephalus sect. Conodiscus Paepalanthus 100 99 PP eriophaeus ser. Paepalanthus Actinocephalus 89 BL scirpeum PP superbus Blastocaulon 89 Xeractis 76 PP argenteus Paepalanthus PP silveirae ser. Paepalanthus Paepalanthus 100 PP comans Xeractis subg. of Paepalanthus 100 64 PP bryoides ser. Leptocephali Paepalanthus 90 100 PP planifolius sect. Conferti Platycaulon 74 PP tuberosus sect. Divisi 100 PP cf. acantholimon ser. Rosulati 91 PP almasensis ser. Dimeri 100 PP implicatus subsect. Polyactis Paepalanthus 99 PP canescens subsect. Actinocephaloides

Fig. . Strict consensus of the 50,000 most parsimonious trees based on combined plastid data (psbA-trnH and trnL-F) (1746 steps, CI 0.71, RI 0.85). Numbers below lines are bootstrap support values and above lines are Bayesian posterior probabilities. Infrageneric classification based on Ruhland (1903). Genera: AB, Abolboda; AC, Actinocephalus; BL, Blastocaulon; ER, Eriocaulon; LC, Lachnocaulon; LT, Leiothrix; ME, Mesanthemum; PH, Philodice; PP, Paepalanthus; RO, Rondonanthus; SY, Syngonanthus; TO, Tonina; XY, Xyris. Major clades denoted in capital letters (A, B, etc.) are those discussed in the text.

382 TAXON 59 (2) • April 2010: 379–388 Andrade & al. • Phylogenetics of Eriocaulaceae

AB pulchella XY lagoinhae Outgroup 85 XY jupicai ER cinereum 100 ER linearifolium B 100 98 Eriocaulon 99 100 ER ligulatum 100 ER modestum SY vernonioides 100 SY aciphyllus E 100 100 98 SY curralensis Comanthera A 100 SY hatschbachii 100 100 PH hoffmannseggii 100 PH hoffmannseggii H 100 Syngonanthus 95 100 SY arenarius 100 SY caulescens LT angustifolia 100 LT flavescens C 100 100 100 100 100 93 100 LT distichoclada 88 I 100 LT sp. nov. LT crassifolia 100 Leiothrix 100 100 LT curvifolia 83 100 LT flagellaris 100 100 LT arrecta 100 LT vivipara RO capillaceus Rondonanthus 100 L 100 LC anceps Lachnocaulon 94 100 TO fluviatilis Tonina 100 PP lamarckii 100 100 100 PP tortilis K 89 100 PP obtusifolius PP elongatus 100 PP almasensis M 77 PP stannardii 82 PP repens Paepalanthus 70 PP giganteus 100 100 PP extremensis 99 98 PP trichophyllus 100 PP exiguus 100 100 PP leucocephalus 100 PP scleranthus PP aff. bahiensis 100 AC bongardii J 100 99 AC ramosus Actinocephalus 100 99 AC aff. ramosus 75 PP glareosus Paepalanthus Paepalanthus AC brachypus 100 AC ciliatus Actinocephalus 69 AC stereophyllus 100 PP cinereus 76 97 PP macrocaulon 100 63 PP neglectus 79 PP spathulatus 100 100 PP pulchellus 82 100 97 PP pulvinatus 69 100 PP regalis 89 100 PP erigeron 100 N 99 BL albidum 100 Bl rupestre PP argenteus Paepalanthus 100 100 PP superbus 97 100 PP comans 100 58 100 PP planifolius 57 96 PP tuberosus 100 PP sphaerocephalus 100 PP eriophaeus PP silveirae 92 100 PP implicatus 59 100 99 PP canescens 83 PP cf. acantholimon 63 92 BL scirpeum 82 PP bryoides

Fig. . Strict consensus of the 16 most parsimonious trees resulting from combined analysis of plastid (psbA-trnH, trnL-F) and nuclear (ITS) data (4005 steps, CI 0.60, RI 0.83). The generic classification in black is suggested by our results; clades in bold are part of the taxonomic implica- tions. For further explanation see Fig. 1.

383 Andrade & al. • Phylogenetics of Eriocaulaceae TAXON 59 (2) • April 2010: 379–388

Phylogenetic studies using morphological data (Giulietti & to the State of Minas Gerais. It is characterized by pistilate al., 1995) already confirmed the monophyly of Eriocauloideae flowers with styles forming a column, having stigmatic and and Paepalanthoideae, but analyses based on morphological, nectariferous portions departing at different levels, basifixed anatomical and chemical characters (Giulietti & al., 2000) anthers, and striated seeds (Giulietti & al., 2000). Two of the and on sequences of rbcL/atpB (Bremer, 2002) had rejected four subgenera recognized by Ruhland (1903) were sampled— this dichotomy. Phylogenetic studies with ndhF and trnL-F L. subg. Leiothrix (= L. subg. Calycocephalus) and L. subg. (Unwin, 2004) resolved Eriocaulon as sister to Paepalanthoi- Stephanophyllum. The monophyletic nature of Leiothrix was deae with high support, but Mesanthemum was not included strongly supported, confirming earlier phylogenetic analysis in these analyses. The dichotomy between Eriocauloideae and based on morphology and anatomy (Giulietti & al., 1995, 2000). Paepalanthoideae is therefore strongly supported here for the Previous molecular analysis (Unwin, 2004) obtained similar first time based on molecular data, confirming the distinct results with a distinct set of species, sampling the L. subg. morphology found in the two subfamilies. Whereas Eriocau- Leiothrix and the monospecific L. subg. Rheocaulon. loideae are characterized by diplostemonous flowers, glandular Leiothrix was considered related to Syngonanthus based petals and three simple styles with stigma in carinal position, on the presence of a smooth inner wall of floral trichomes, the Paepalanthoideae have isostemonous flowers and two or three slightly papillate style appendices (= stigmatic portion), and the branched styles with a stigmatic portion alternating with a nec- presence of flavones only in these two genera (Dokkedall & tariferous portion. Since there is no consensus yet as to which Salatino, 1992; Giulietti & al., 1995, 2000; Ricci & al., 1996). families are more closely related to Eriocaulaceae, these con- Unfortunately, our results are not conclusive concerning the trasting character states cannot be unambiguously polarized to position of Leiothrix. Plastid regions support Leiothrix as sister safely establish synapomorphies for subfamilies. Below we will group of a clade composed of Philodice and part of Syngonan- discuss the phylogenetic results relative to individual genera. thus, but ITS supports a closer relationship between Leiothrix Eriocaulon. — Includes more than 400 species and is the and Rondonanthus. only genus occuring in all continents. It is characterized by Syngonanthus. — With approximately 200 species, the diplostemonous flowers, four or six stamens, petals with glands genus occurs disjunctly in the Americas and Africa and it is and staminate and pistillate flowers with free petals. The mono- characterized by pistillate flowers with petals united in the phyly of Eriocaulon was strongly supported in all analyses. middle and free at the base and apex (sygonanthoid corolla). Mesanthemum. — Restricted to the African continent, Ruhland (1903) recognized five sections: S. sect. Syngonan- the genus includes 15 species. Like Eriocaulon, it has diplo- thus (= S. sect. Dimorphocaulon), sect. Carphocephalus, sect. stemonous flowers, four or six stamens and petals with glands. Eulepis, sect. Thysanocephalus, and sect. Chalarocaulon However, it has staminate flowers with united petals and pistil- including only S. macrocaulon Ruhland. This species was late flowers with petals united in the middle but free at the base synonymized in S. anomalus (Körn.) Ruhland and included and apex. Despite the fact that Mesanthemum is represented in sect. Carphocephalus (Hensold, 1999). All sections were by the psbA-trnH sequence only, its position as sister group of sampled in the present study and, contrary to analyses based Eriocaulon is strongly supported. Since only one species of the on morphology (Giulietii & al., 1995, 2000) and molecular genus was sampled, the phylogenetic status of Mesanthemum ndhF (Unwin, 2004), our results clearly refute the monophyly remains to be studied. of Syngonanthus. Syngonanthus sect. Thysanocephalus and Rondonanthus. — Endemic to the Guayana shield of Ven- S. sect. Eulepis form a clade, a relationship previously indicated ezuela, Guyana, and Brazil, the genus includes approximately by morphological and anatomical data (Giulietti & al., 2000; six species and is characterized by the presence of long linear Parra, 2000; Scatena & al., 2005), and also by the presence of staminodes. Petals in both types of flowers can be free (as in C-glycoside flavones as derivatives of luteoline (Ricci & al., R. roraimae (Oliv.) Herzog, type of the genus, and in R. capil- 1996). Based on a cladistic analysis with morphological data, laceus) or with sygonanthoid corolla in pistillate flowers (Hen- Parra (2000) considered the following synapomorphies for the sold & Giulietti, 1991). It was putatively considered the most clade comprising S. sect. Eulepis and sect. Thysanocephalus: basal genus in Paepalanthoideae (Hensold & Giulietti, 1991; malpighiaceous trichomes appear on the scapes, the petals Stützel, 1998). In the morphological analyses (Giulietti & al., in the pistilate flowers are longer than sepals and narrowly 2000), Rondonanthus presented an unstable position and its spathulate, and the seeds have rugose surfaces. The presence monophyly was questioned because some species present pistil- of cyathiform capitula is a synapomorphy of S. sect. Thysano- late flowers with free petals as in Paepalanthus and Leiothrix, cephalus, whereas, malpighiaceous trichomes on the leaves and whereas other species have the petals fused in the middle like an increasing in length of fruiting pedicels before dispersal are Syngonanthus. Unfortunately, only one species of Rondonan- synapomorphic in S. sect. Eulepis. thus was sampled here and its position in Paepalanthoideae A second clade of Syngonanthus brings together species was erratic. For example, the combined plastid analysis sug- of S. sect. Syngonanthus and sect. Carphocephalus, which gests that Rondonanthus diverged at the base of the subfamily, is strongly supported as sister to Philodice. Together, these whereas the ITS analysis suggests that it is sister to Leiothrix. two sections are characterized by having pistillate flowers Leiothrix. — The genus includes 64 species distributed with petals smaller than sepals. However, they are fleshy and throughout South America, but with its center of diversity in the urceolate in S. sect. Carphocephalus (Ruhland, 1903), while Espinhaço mountain range; more than 40 species are endemic staminate flowers have membranaceous gamopetalous corolla

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in S. sect. Syngonanthus. Chemically, the two sections pro- clades K and N, also demonstrating that internal arrangement duce flavonoids with 6-hydroxy-luteoline derivatives (Ricci in Paepalanthus is quite artificial). As in our study but with a & al., 1996). larger sample of Lachnocaulon, Unwin (2004) found Lachno- Philodice. — The genus occurs in Central and South Amer- caulon and Tonina f luviatilis forming a highly supported clade, ica and includes only two species: P. hoffmannseggii (type), characterized by a reduced perianth in pistillate flowers. Tonina an annual herb from humid savanna areas, and P. cuyabensis fluviatilis is distributed from Central America to southeastern (Bong.) Körn., known only from the type material collected Brazil while Lachnocaulon is restricted to North America. by Riedel near Cuiabá, of Mato Grosso state, Brazil. Morpho- Actinocephalus was segregated from Paepalanthus based logical cladistic analysis (Giulietti & al., 2000) supported it as on morphological study (Sano, 2004). The genus contains 28 sister to Tonina based on roots with exodermis and air storage species and is characterized mainly by inflorescences com- in the cortex and anthers bisporangiate. Molecular data clearly posed of capitula in umbels and in having a geographic range refute this hypothesis, suggesting independent origins of these restricted to Brazil. The analyses presented here are inconclu- traits. According to our study, Philodice is sister to part of sive about the monophyly of Actinocephalus but unambigu- Syngonanthus (above), sharing the syngonanthoid fusion in ously support the genus as being closely related to Paepalan- pistillate flowers with that genus (Stützel, 1985), but differing thus subsect. Aphorocaulon. This causes Paepalanthus to be in having bisporangiate (vs. tetrasporangiate) anthers. paraphyletic, a condition earlier revealed by morphological Paepalanthus and allied genera (Actinocephalus, Blasto- (Giulietti & al., 2000) and molecular (Unwin, 2004) analyses. caulon, Lachnocaulon, Tonina). — Paepalanthus (420 species) A putative synapomorphy of the Actinocephalus–P. subsect. occurs disjunctly in the Americas and Africa, and is a quite Aphorocaulon clade is the presence of paraclades. heterogeneous genus, both in vegetative and reproductive fea- Paepalanthus subg. Thelxinoë is exclusively composed of tures. This is reflected in Ruhland’s (1903) taxonomic system, the two species analyzed (P. leucocephalus, P. scleranthus) which recognized 22 infrageneric taxa, and is expressed in and is easily recognized by the lack of floral scape, a condition the high level of homoplasy (CI = 0.38) in the morphologi- which is rare outside the group. Paepalanthus exiguus (P. sect. cal analysis (Giulietti & al., 2000). The same complexity is Conodiscus) appeared sister to P. subg. Thelxinoë, a relation- found in chemical features due to the occurrence of different ship supported by the dimerous flowers. secondary compounds, such as flavonoids, coumarins, and Clade N, the second main clade of Paepalanthus, includes waxes (Salatino & Salatino, 1998). The genus according to representatives of the P. subg. Paepalanthus and P. subg. Xe- Ruhland (1903) includes six subgenera: P. subg. Paepalanthus ractis plus a monophyletic P. subg. Platycaulon and a biphy- (= subg. Paepalocephalus), subg. Platycaulon, subg.Thelxinoë, letic genus Blastocaulon. Unwin (2004) found Blastocaulon subg. Xeractis, subg. Psilandra, and subg. Bostrychophyllum. to form a clade closely related to P. subg. Xeractis in most The latter subgenus (based on P. capillaceus Klotzsch) was analyses. None of our analyses, however, recovered Blasto- synonymized in Rondonanthus (Hensold & Giulietti, 1991) caulon as monophyletic. The only difference between Blasto- and included in this paper. Representatives of all subgenera, caulon and Paepalanthus is the number of pollen sacs, 4 and except the monospecific P. subg. Psilandra (known only from 2, respectively. Therefore, reduction of the number of pollen the type specimen) and P. subg. Monosperma Hensold (23 spe- sacs must have occurred more than once in the evolution of cies, all in the Guayana shield; Hensold, 1991), were included Eriocaulaceae. in the present work. Taxonomic implications. — A phylogenetic hypothesis Morphological (Giulietti & al., 2000) and molecular allows several alternative taxonomic arrangements. Here, we (Unwin, 2004) analyses refuted the monophyly of Paepalanthus. suggest two basic generic realignments in Eriocaulaceae in or- This is not different in our study, since Actinocephalus, Blasto- der to arrive at a classification based on meaningful monophy- caulon, Lachnocaulon, and Tonina are nested in Paepalanthus. letic genera: (1) division of Syngonanthus into two genera, and Paepalanthus myocephalus, P. sessiliflorus, and P. bifidus pre- (2) inclusion of Actinocephalus, Blastocaulon, Lachnocaulon, sented incongruent relationships among individual regions and and Tonina in Paepalanthus s.l. were not included in combined analyses. They are annual herbs Syngonanthus is not monophyletic, with S. sect. Syngo- with short life cycles and the lack of congruence can be due to nanthus and sect. Carphocephalus being more closely related higher nucleotide substitution rate (Smith & Donoghue, 2008). to Philodice than to S. sect. Eulepis and sect. Thysanocepha- Apart of this, the only incongruence observed in our study was lus. Therefore, Comanthera L.B. Sm. is being reinstated to the position of P. almasensis which appeared nested in clade N name the clade composed by the species of the latter two sec- according to plastid datasets, but in clade M according to ITS tions (Parra & al., in press). Syngonanthus sect. Syngonanthus (and combined plastid + nuclear). Excluding P. myocephalus, and sect. Carphocephalus together form the sister group of P. sessiliflorus, and P. bifidus, clade J can be divided in two Philodice. Since the latter genus consists of only two species principal lineages, clades K and N. and is morphologically similar to the former, merging the two Clade K groups all representatives of Actinocephalus, into a single genus will simplify the taxonomy of the group Lachnocaulon, and Tonina with species of a monophyletic Pae- without any loss of relevant information. Philodice (Martius, palanthus subg. Thelxinoë and many representatives of a poly- 1834), however, was published before Syngonanthus (Ruhland, phyletic Paepalanthus subg. Paepalanthus (note that P. sect. 1900); because of this, conservation of Syngonanthus against Conodiscus, P. subsect. Polyactis, and P. ser. Dimeri appear in Philodice has been proposed (Giulietti & al., 2009).

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Our results also indicate the need to include Actinocephalus Chase, M.W., Stevenson, D.W., Wilkin, P. & Rudall, P.J. 1995. Mono- (28 species), Blastocaulon (5), Lachnocaulon (7), and Tonina (1) cot systematics: A combined analysis. Pp. 685–730 in: Rudall, P.J., in Paepalanthus s.l. This change will add 41 species to the 420 Cribb, P.J., Cutler, D.F. & Humphries, C.J. (eds.), : Systematics and evolution. Kew: Royal Botanic Gardens. species currently recognized in Paepalanthus s.str. The genus Desfeaux, C., Maurice, S., Henry, J.P., Lejeune, B. & Gouyon, P.H. Paepalanthus thus circumscribed includes species with reduced 1996. The evolution of reproductive system in the genus Silene. petals in pistillate flowers (a condition from Tonina and Lach- Proc. Roy. Soc. London, ser. B, Biol. Sci. 263: 409–414. nocaulon) and bisporangiate anthers (from Blastocaulon). The Dokkedall, A.L. & Salatino, A. 1992. Flavonoids of Brazilian Leio- inflorescences with paracladia (from Actinocephalus) also occur thrix Ruhland (Eriocaulaceae). Biochem. Syst. Ecol. 20: 31–32. Doyle, J.J. & Doyle, J.L. 1987. A rapid DNA isolation procedure for in Paepalanthus subsect. Aphorocaulon. Current and previous small amounts of fresh leaf tissue. Phytochem. Bull. 19: 11–15. molecular analyses (Unwin, 2004) indicate that these traits may Felsenstein, J. 1985. Confidence limits on phylogenies: An approach be homoplastic and thus of reduced phylogenetic importance. to using bootstrap. Evolution 39: 783–791. Despite the advances presented here to improve our un- Fitch, W.M. 1971. Toward defining the course of evolution: Minimum derstanding of the relationships in Eriocaulaceae, much work change for a specific tree topology. Syst. Zool. 20: 406–416. remains to be done. Studies should be undertaken to improve Giulietti, A.M., Amaral, M.C.E. & Bittrich, V. 1995. Phylogenetic analysis of inter- and infrageneric relationships of Leiothrix Ruh- our understanding of the early evolution of Eriocaulaceae land (Eriocaulaceae). Kew Bull. 50: 55–71. within the Poales and to establish the relationships of the fam- Giulietti, A.M., Andrade, M.J.G., Parra, L.R., van den Berg, C. & ily within this order. Eriocaulon remains poorly investigated, Harley, R.M. 2009. (1902) Proposal to conserve the name Syngo- and the relationships within Paepalanthus still need attention. nanthus against Philodice (Eriocaulaceae). Taxon 58: 1008–1009. Stützel (1998: 202) stated that “Paepalanthus is a conglomer- Giulietti, A. M. & Hensold, N. C. 1991. Synonimization of the genera ate of several, probably not closely related groups of species Comanthera and Carptotepala with Syngonanthus (Eriocaula- ceae). Ann. Missouri Bot. Gard. 78: 460–464. …” but this does not hold when considering Paepalanthus s.l. Giulietti, A.M., Scatena, V.L., Sano, P.T., Parra, L.R., Queiroz, L.P., (clade J). Our understanding of the relationships within the Harley, R.M., Menezes, N.L., Benko-Yseppon, A.M., Salatino, enlarged genus Paepalanthus is still very incomplete and fu- A., Salatino, M.L., Vilegas, W., Santos, L.C., Ricci, C.V., Bon- ture molecular phylogenetic work on neotropical Eriocaulaceae fim, M.C.P. & Miranda, E.B. 2000. Multidiciplinary studies might therefore focus on the relationships within this group. on Neotropical Eriocaulaceae. Pp. 580–589 in: Wilson, K.L. & Morrison, D.A. (eds.), Monocots II: Systematics and evolution. Melbourne: CSIRO. Givnish, T.J., Evans, T.M., Pires, J.C. & Sytsma, K.J. 1999. Poly- ACKNOWLEDGEMENTS phyly and convergent morphological evolution in Commelinales and Commelinidae: Evidence from rbcL sequence data. Molec. We would like to thank Ricardo Vilas-Bôas, Alessandra Schna- Phylog. Evol. 12: 360–385. Govaerts, R. 2006. World checklist of Eriocaulaceae. Royal Botanic delbach, and Cristiano Villela for technical support; Maria das Graças Gardens, Kew. http://www.kew.org/wcsp/, accessed 16 October L. Wanderley for identification of Xyridaceae species; Wayt Thomas 2009. and Douglas H. Goldman for providing material; Bruno Andrade Hensold, N.C. 1988. Morphology and systematic of Paepalanthus sub- for help with GenBank sequence submission; three anonymous refer- genus Xeractis. Syst. Bot. Monogr. 23: 1–150. ees for providing valuable comments on the manuscript and linguistic Hensold, N.C. 1991. Revisionary studies in Eriocaulaceae of Venezuela. assistance; Richard Lyndsay for linguistic revision of the manuscript; Ann. Missouri Bot. Gard. 78: 424–440. Hensold, N.C. 1999. Eriocaulaceae. Pp. 1–57 in: Berry, P.E., Yatski- and the Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB, evych, K. & Holst, B.K. (eds.), Flora of Venezuelan Guayana, vol. # 251/04), Instituto do Milênio do Semi-Árido (IMSEAR), Programa 5. St. Louis: Missouri Botanical Garden Press. de Pesquisa em Biodiversidade do Semi-Árido (PPBio), and the Con- Hensold, N.C. & Giulietti, A.M. 1991. Revision and redefinition of selho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) the genus Rondonanthus Herzog (Eriocaulaceae). Ann. Missouri for grants supporting this work. MJGA received a fellowship (Bolsa Bot. Gard. 78: 441–459. doutorado) from Coordenação de Aperfeiçoamento de Pessoal de Nível Kelchner, S.A. 2000. The evolution of noncoding chloroplast DNA and its application in plant systematics. Ann. Missouri Bot. Gard. Superior (CAPES) and received a fellowship (Bolsa Pós-doutorado) 87: 482–498. from CNPq. AMG, AR, LPQ, and CVDB are supported by grants Linder, H.P. & Kellogg, E.A. 1995. Phylogenetic patterns in the Com- from CNPq (Bolsa de Produtividade – PQ 1A, PQ 2, PQ 1D, and PQ melinid clade. Pp. 473–496 in: Rudall, P.J., Cribb, P.J., Cutler, 1D, respectively). D.F. & Humphries, C.J. (eds.), Monocotyledons: Systematics and evolution. Kew: Royal Botanic Gardens. Martius, C.F.P. 1834. Les Eriocaulées considerées comme une famille distincte, par le D. Martius. Ann. Sci. Nat., Bot., ser. 2, 2: 25–43. LITERATURE CITED Nylander, J.A.A. 2004. MrModeltest, version 2. Evolutionary Biology Centre, Uppsala University: Program distributed by the author. Andrade, M.J.G. 2007. Filogenia e taxonomia em Eriocaulaceae neo- http://www.abc.se/~nylander/. tropicais. Ph.D. Thesis, Universidade Estadual de Feira de Santana, Parra, L.R. 2000. Redelimitação e revisão de Syngonanthus sect. Eu- Feira de Santana, Brazil. lepis (Bong. ex Körn.) Ruhland – Eriocaulaceae. Ph.D. Thesis, APG III. 2009. An update of the Angiosperm Phylogeny Group clas- Universidade de São Paulo, São Paulo. sification for the orders and families of flowering : APG III. Parra, L.R., Giulietti, A.M., Andrade, M.J.G. & van den Berg, C. Bot. J. Linn. Soc. 161: 105–121. In press. 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Appendix. Taxa analysed; classification based on Ruhland (1903). Voucher specimens are deposited in herbarium HUEFS (Universidade Estadual de Feira de Santana, Brazil) except for Lachnocaulon anceps (BH), Mesanthemum radicans and Eriocaulon decangulare (NY), and Paepalanthus repens (FLAS). * = DNA bank sample at LAMOL (Univ. Estadual de Feira de Santana, Brazil); an n-dash (–) indicates sequences not available. All samples where country is not specified are from Brazil. Taxon: voucher information; geographic origin; *FSA; GenBank accession numbers ITS, psbA-trnH, trnL-F. INGROUP SPECIES: ERIOCAULOIDEAE: Eriocaulon L.: Eriocaulon decangulare L.: R Kral 85389; Choctaw/Alabama, U.S.A.; *6900; GU078711, EU924353, –. Eriocaulon linearifolium Körn.: AM Giulietti 2366; Mucugê/BA; *297; EU924277, EU924349, EU924429. Eriocaulon ligulatum (Vell.) L.B. Sm: AM Giulietti 2368; Mucugê/BA; *298; EU924278, EU924350, EU924430. Eriocaulon modestum Kunth: MJG Andrade 445; Rio de Contas/BA; *780; EU924279, EU924351, EU924431. Eriocaulon cinereum R. Br.: AM Giulietti 2582; Nossa Senhora do Livramento/MT; *6875; EU924280, EU924352, EU924432. Mesanthemum Körn.: Mesanthemum radicans (Benth.) Körn.: JM & B Reitsma 688; Sabrliére/Gabon; *6874; –, EU924348, –. PAEPALANTHOIDEAE: Actinocephalus (Wikstr.) Sano: Actinocephalus brachypus (Bong.) Sano: MJG Andrade 521; Diamantina/MG; *1938; EU924281, EU924354, EU924433. Actinocephalus bongardii (A. St.-Hil.) Sano: MJG Andrade 501; Itacambira/MG; *1927; EU924282, EU924355, EU924434. Actinocephalus ciliatus (Bong.) Sano: MJG Andrade 544; Diamantina/MG; *1956; EU924283, EU924356, EU924435. Actinocephalus ramosus (Wikstr.) Sano: BRN Araújo 78; Rio de Contas/ BA; *1798; EU924284, EU924357, EU924436. Actinocephalus stereophyllus (Ruhland) Sano: MJG Andrade 514; Diamantina/MG; *1933; EU924285, EU924358, EU924437. Actinocephalus aff. ramosus (Wikstr.) Sano: AM Giulietti 2235; Rio de Contas/BA; *1425; EU924286, EU924359, EU924438. Blastocaulon Ruhland: Blastocaulon albidum (Gardner) Ruhland: MJG Andrade 541; Diamantina/MG; *1953; EU924287, EU924360, EU924439. Blastocaulon rupestre (Gardner) Ruhland: MJG Andrade 542; Diamantina/MG; *1954; EU924288, EU924361, EU924440. Blastocaulon scirpeum (Mart.) Giul.: JR Pirani 4162; Congonhas do Norte/MG; *6876; EU924289, EU924362, EU924441. Lachnocaulon Kunth: Lachnocaulon anceps Morong: D. Goldman s/n; Florida, U.S.A.; *6877; GU078712, EU924363, EU924442. Leiothrix Ruhland: Leiothrix subg. Leiothrix (=L. subg. Calycocephalus Ruhland): Leiothrix angustifolia (Körn.) Ruhland: R Harley 54660; Rio de Contas/BA; *607; EU924290, EU924364, EU924443. Leiothrix flavescens (Bong.) Ruhland: MJG Andrade 440; Rio de Contas/BA; *775; EU924291, EU924365, EU924444. Leiothrix crassifolia (Bong.) Ruhland: MJG Andrade 516; Diamantina/MG; *6817; EU924292, EU924366, EU924445. Leiothrix curvifolia (Bong.) Ruhland: MJG Andrade 553; Datas/MG; *1964; EU924293, EU924367, EU924446. Leiothrix distichoclada Herzog: MJG Andrade 458; Rio de Contas/BA; *792; EU924294, EU924368, EU924447. Leiothrix sp. nov: MJG Andrade 461; Rio de Contas/BA; *795; EU924295, EU924369, EU924448. Leiothrix subg. Stephanophyllum Guill.: Leiothrix arrecta Ruhland: AM Giulietti 2496; Santana do Riacho/MG; *6872; EU924296, EU924370, EU924449. Leiothrix flagellaris (Guill.) Ruhland: MJG Andrade 485; Grão Mogol/MG; *1920; EU924297, EU924371, EU924450. Leiothrix vi- vipara (Bong.) Ruhand: AM Giulietti 2503; Santana do Riacho/MG; *7415; EU924298, EU924372, EU924451. Paepalanthus Mart.: Paepalanthus subg. Paepalanthus: Paepalanthus sect. Paepalanthus: Paepalanthus subsect. Paepalanthus: Paepalanthus ser. Leptocephali (Ruhland) Giul. stat. nov. (= P. [unranked] Leptocephali Ruhland in Engler, Pflanzenr. IV. 30: 123. 1903): Paepalanthus bryoides Kunth: FN Costa 263; Diamantina/MG; *6878; EU924299, EU924373, EU924452. Paepalanthus bifidus (Schrad.) Kunth: MJG Andrade 489; Grão Mogol/MG; *1924; EU924300, EU924374, EU924453. Paepalanthus myocephalus Mart.: MJG Andrade 613; Feira de Santana/BA; *6879; EU924301, EU924375, EU924454. Paepalanthus tortilis (Bong.) Mart.: MJG Andrade 479; Grão Mogol/MG; *1914; EU924302, EU924376, EU924455. Paepalanthus lamarckii Kunth: DS Carneiro-Torres 461; Lagartos/SE; *6880; EU924303, EU924377, EU924456. Paepalanthus obtusifolius (Steud.) Körn.: R Harley 54802; Rio de Contas/BA; *1321; EU924304, EU924378, EU924457. Paepalanthus sessiliflorus Mart. ex Körn.: JG Jardim 2237; Maraú/BA; *6881; EU924305, EU924379, EU924458. Paepalanthus subsect. Paepalanthus: Paepalanthus ser. Paepalanthus (= P. [unranked] Variabiles Ruhland): Paepalanthus erigeron Mart. ex Körn.: AA Ribeiro-Filho 107; Lençóis/BA; *6882; EU924306, EU924380, –. Paepalanthus eriophaeus Ruhland: MJG Andrade 504; Itacambira/MG; *6811; EU924307, EU924381, EU924459. Paepalanthus neglectus Körn.: BRN Araújo 85; Rio de Contas/BA; *1805; EU924308, EU924382, EU924460. Paepalanthus pulchellus Herzog: AM Giulietti 2423; Rio de Contas/BA; *1409;

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Appendix. Continued. EU924309, EU924383, EU924461. Paepalanthus regalis Mart.: R Harley 54640; Mucugê/BA; *660; EU924310, EU924384, EU924462. Paepalanthus silvei- rae Ruhland: MJG Andrade 568; Tiradentes/MG; *1976; EU924311, EU924385, EU924463. Paepalanthus spathulatus Körn., R Harley 55476, Itaberaba/BA; *6883; EU924312, EU924386, EU924464. Paepalanthus subsect. Paepalanthus: Paepalanthus ser. Rosulati (Ruhland) Giul. stat. nov. (= P. [unranked] Rosulati Ruhland in Engler, Pflanzenr. IV, 30: 123. 1903): Paepalanthus pulvinatus N.E. Br.: R Harley 54634; Mucugê/BA; *638; EU924313, EU924387, EU924465. Paepalanthus cf. acantholimon Ruhland: MJG. Andrade 522; Diamantina/MG; *1939; –, EU924388; EU924466. Paepalanthus subsect. Paepal- anthus: Paepalanthus ser. Dimeri (Ruhland) Giul. stat. nov. (= P. [unranked] Dimeri Ruhland in Engler, Pflanzenr. IV, 30: 123. 1903): Paepalanthus elon- gatus (Bong.) Körn.: MJG Andrade 572; Tiradentes/MG, *6884; EU924314, EU924389, EU924467. Paepalanthus almasensis Moldenke: MJG Andrade 429, Rio de Contas/BA, *768; EU924315, EU924390, EU924468. Paepalanthus subsect. Polyactis: Paepalanthus cinereus Giul & L.R. Parra: AM Giulietti s/n; Rio de Contas/BA; *1293; EU924316, EU924391, EU924469. Paepalanthus macrocaulon Silveira: MJG Andrade 431; Rio de Contas/BA; *766; EU924317, EU924392, EU924470. Paepalanthus extremensis Silveira: MJG Andrade 491; Grão Mogol/MG; *1926; EU924318, EU924393, EU924471. Paepalanthus implicatus Silveira: MJG Andrade 550; Datas/MG; *1961; EU924319, EU924394, EU924472. Paepalanthus stannardii Giul. & L.R. Parra: MJG Andrade 438; Rio de Contas/BA; *773; EU924320, EU924395, EU924473. Paepalanthus repens Körn.: R Abbott 21006; *7416; EU924321, EU924396, EU924474. Paepal- anthus subsect. Aphorocaulon Ruhland: Paepalanthus glareosus (Bong.) Kunth: MJG Andrade 548; Datas/MG; *1959; EU924322, EU924397, EU924475. Paepalanthus aff. bahiensis (Bong.) Kunth: MJG Andrade 437; Rio de Contas/BA; *772; EU924323, EU924398, EU924476. Paepalanthus subsect. Actino- cephaloides Ruhland: Paepalanthus canescens (Bong.) Körn.: MJG Andrade 536; Diamantina/MG; *1950; EU924324, EU924399, EU924477. Paepalanthus sect. Diphyomene Ruhland: Paepalanthus giganteus Sano: MJG Andrade 527; Diamantina/MG; *1943; EU924325, EU924400, EU924478. Paepalanthus trichophyllus (Bong.) Körn.: MJG Andrade 439; Rio de Contas/BA; *774; EU924326, EU924401, EU924479. Paepalanthus sect. Conodiscus Ruhland: Pae- palanthus sphaerocephalus Ruhland: MJG Andrade 456; Rio de Contas/BA; *790; EU924327, EU924402, EU924480. Paepalanthus exiguus (Bong.) Körn.: E Guarçoni 710; Caparaó/MG; *6885; EU924328, EU924403, EU924481. Paepalanthus subg. Xeractis Mart.: Paepalanthus comans Silveira: MJG Andrade 540; Diamantina/MG; *6820; EU924329, EU924404, EU924482. Paepalanthus superbus Ruhland: AM Giulietti 2504; Santana do Riacho/MG; *6858; EU924330, EU924405, EU924483. Paepalanthus argenteus (Bong.) Körn.: MJG Andrade 539; Diamantina/MG; *1952; EU924331, EU924406, EU924484. Paepalanthus subg. Platycaulon Mart.:Paepalanthus sect. Conferti (Ruhland) Tissot-Squalli (= P. [unranked] Conferti Ruhland): Paepalanthus planifolius (Bong.) Körn.: MJG Andrade 526; Diamantina/MG; *1942; EU924332, EU924407, EU924485. Paepalanthus subg. Platycaulon: Paepalanthus sect. Divisi (Ruhland) Tissot-Squalii (=P. [unranked] Divisi Ruhland): Paepalanthus tuberosus (Bong.) Kunth: C van den Berg 1364; Conceição do Mato Dentro/MG; *1641; EU924333, EU924408, EU924486. Paepalanthus subg. Thelxinoë Ruhland: Paepalanthus leucocephalus Ruhland; MJG Andrade 620; Rio de Contas/ BA; *6886; EU924334, EU924409, EU924487. Paepalanthus scleranthus Ruhland: MJG Andrade 537; Diamantina/MG; *1951; EU924335, EU924410, EU924488. Philodice Mart.: Philodice hoffmannseggii Mart.: AM Giulietti 2483; Nossa Senhora do Livramento/MT; *6887; EU924336, EU924411, EU924489; and CG Lopes 157; Campo Maior/PI; *6888; EU924337, EU924412, EU924490. Rondonanthus Herzog: Rondonanthus capillaceus (Körn.) Hensold & Giul.: C van den Berg 1792; without locality, Roraima, Brazil; *6889; EU924338, EU924413, GU078716. Syngonanthus Ruhland: Syngonanthus sect. Eulepis (Bong.) Ruhland: Syngonanthus aciphyllus (Bong.) Ruhland: MJG Andrade 532; Diamantina/MG; *1948; EU924339, EU924414, EU924491. Syngonanthus cur- ralensis Moldenke: MJG Andrade 595; Rio de Contas/BA; *6890; EU924340, EU924415, EU924492. Syngonanthus hatschbachii Moldenke: AC Pereira 122; Morro do Chapéu/BA; *6891; EU924341, EU924416, EU924493. Syngonanthus mucugensis Giul. subsp. riocontensis A.C.S.Pereira & Giul.: AC Pereira 4; Rio de Contas/BA; *6892; –, EU924417, EU924494. Syngonanthus nitidus (Bong.) Ruhland: MJG Andrade 545; Diamantina/MG; *1957; –, EU924418, EU924495. Syngonanthus bisulcatus (Körn.) Ruhland: MJG Andrade 497; Itacambira/MG; *6805; –, EU924419, EU924496. Syngonanthus harleyi Moldenke: AC Pereira 172; Umburanas/BA; *6893; –, EU924420, EU924497. Syngonanthus elegans (Bong.) Ruhland: PT Sano 3047; Diamantina/MG; *6894; –, EU924421, –. Syngonanthus sect. Syngonanthus: Syngonanthus arenarius (Gardner) Ruhland: MJG Andrade 493, Itacambira/MG; *6802; EU924342, EU924422, EU924498. Syngonanthus sect. Thysanocephalus (Körn.) Ruhland: Syngonanthus vernonioides (Kunth) Ruhland: AM Giulietti 2185; Rio de Contas/BA; *220; EU924343, EU924423, EU924499. Syngonanthus sect. Carphocephalus (Körn.) Ruhland: Syngonanthus caulescens (Poir.) Ruhland: MJG Andrade 452; Rio de Contas/BA; *786; EU924344, EU924424, EU924500. Tonina Aubl.: Tonina fluviatilis Aubl.: MJG Andrade 616; Recife/PE; *6895; EU924345, EU924425, EU924501. OUTGROUP SPECIES: Xyridaceae: Abolboda pulchella Humb. & Bonpl.: W Ganev 340; Abaíra/BA; *6897; –, EU924427, – and R. Harley 55931; Rio de Contas/BA; *9500; GU078713, –, –. Xyris jupicai L.C. Rich.: MJG Andrade 615; Recife/PE; *6898; EU924347, GU078714, –. Xyris anceps Lam.: C van den Berg 1718; Vera Cruz/BA; *6899; –, EU924428, –. Xyris lagoinhae Kral & L.B. Sm.: R. Harley 55926; Rio de Contas/BA; *9501; –, GU078715, GU078717.

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